Investigation on the Pharmacoprinciples of a Pergularia daemia (Forssk.) Chiov.
Thesis Submitted to the BHARATHIDASAN UNIVERSITY, TIRUCHIRAPPALLI for the award of the Degree of
DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY
By P. Vinoth Kumar, M.Sc. (Reg. No. 021817/Ph.D. 2/Biotechnology/Full-time/January 2008)
Under the Guidance of Dr. N. Ramesh, M.Sc., M.Phil., Ph.D.
DEPARTMENT OF BIOTECHNOLOGY J.J. COLLEGE OF ARTS AND SCIENCE (AFFILIATED TO BHARATHIDASAN UNIVERSITY) PUDUKKOTTAI 622 422, TAMIL NADU
AUGUST 2013
DECLARATION
I do hereby declare that the Thesis entitled “Investigation on the Pharmacoprinciples of a Pergularia daemia (Forssk.) Chiov. ” submitted to Bharathidasan University, Tirucharapalli, Tamil Nadu, has been carried out by me under the supervision of Dr. N. Ramesh, Assistant Professor, Department of Biotechnology, J.J.College of Arts and Science, Pudukkottai for award of Degree of DOCTOR OF PHILOSOPHY IN BIOTECHNOLOGY. I also declare that this Thesis is a result of my own effort and has not been submitted earlier for the award of any Diploma, Degree, Associateship, Fellowship or other similar title to any candidate other university.
Place: Pudukkottai (P. VINOTH KUMAR)
Date:
ACKNOWLEDGEMENT
I prostrate before the God for his blessing, which guided me in taking up this project and gave me the confidence and ability to complete it successfully.
I express my personal indebtness and greatfulness to my Research Guide Dr. N. Ramesh, Asst. Prof., Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai, for his sustained guidance and encouragement throughout the course of this project.
I would like to express my sense of gratitude to Mr. N. Subramanian, Secretary, J.J. College of Arts and Science, Dr. Kavitha Subramanian, Management Trustee, J.J. College of Arts and Science and Dr. J. Parasuraman, Principal, J.J. College of Arts and Science, Pudukkottai for allowing me to under take this project work.
I extend my sincere gratitude to my Doctoral Committee Member Dr. M.B. Viswanathan, Prof. Department of Plant Science, Bharathidasan University, Tirchurappalli, for valuable suggestions and encouragement.
I am grateful to Mr. B. Venkatesh, Head, Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai Mr. G. Manigandan, Asst. Prof., Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai, Mr. V. Jeyaraman, Asst. Prof., Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai and Ms. P. Priyadharshini, Asst. Prof., Department of Biotechnology, J.J. College of Arts and Science, Pudukkottai for their continued support. My deep since of gratitude is extended Prof. S. Navaneethan, Department of English, J.J. College of Arts and Science, Pudukkottai, and Dr.S. Siva Subramaniyan, Head, Department of Botany, J.J. College of Arts and Science, Pudukkottai for timely helpful and valuable suggestions during the course of this study.
I deem it as a great previlage to thank sincerely and whole heartedly to the research scholars Mr. R. Ravi Kumar, Mr. A. Kalidhass, Mr. P. Muthu, Mr. A. Venkata Ramana for their timely help and constant encouragement throughout the course of this study.
I thank the Lab assistants Ms. S. Usha, Ms. K. Thangamani, Ms. P. Hemalatha and Ms. R. Rajeswari for their help.
I owe special thanks to my family and my Friends for successful completion of this thesis.
P. Vinoth Kumar
CONTENT
Chapter-1 INTRODUCTION 1
1.1 Background of the Study 1
1.2 Diabetes Mellitus 3
1.3 Classification of Diabetes Mellitus 3
1.4 Demand for Medicinal Plants 4
1,5 Micropropagation and Callus Induction 5
1.6 Pergularia daemia – an Ethnomedicinal Plant 6
1.7 Ethnomedicinal uses 6
1.8 Epidemiological Studies 7
1.9 Pharmacological Studies of Pergularia daemia 8
1.10 Objectives and Scope of Research work 11
Chapter-2 REVIEW OF LITERATURE 12
2.1 Trials for Anti-diabetes Potential of Medicinal Plants 13
2.2 Phytochemical and Pharmacology of Pergularia species 14
2.2.1 Pergularia daemia Root- Phytochemical Constituents 14
2.2.1.1 Pergularia daemia Root-Biological Activity 14
2.2.2 Pergularia daemia Stem-Phytochemical Constituents 14
2.2.2.1 Pergularia daemia Stem-Biological Activity 15
2.2.3 Pergularia daemia Leaves-Biological Activity 15
2.2.4 Pergularia daemia Aerial Part-Phytochemical Constituents 15
2.2.4.1 Pergularia daemia Aerial Part-Biological Activity 16
2.2.5 Pergularia daemia Whole Plant-Phytochemical Constituents 16
2.2.5.1 Pergularia daemia Whole Plant-Biological Activity 16
2.2.5.2 Pergularia daemia Whole Plant-Phytochemical Constituents and 17 Biological Activity
2.2.6 Pergularia daemia Latex-Biological Activity 17
2.2.7 Pergularia pallida Whole Plant-Phytochemical Constituents 17 2.2.8 Pergularia tomentosa Whole Plant-Phytochemical Constituents 17
2.2.9 Pergularia tomentosa Whole Plant-Biological Activity 17
Chapter-3 MATERIALS AND METHODS 18
3.1 Plant Material 18
3.2 Plant Description 18
3.3 Strategy of Work Plan 19
3.4 Micropropagation and Callus Induction 19
3.4.1 Explant Collection 19
3.4.2 Sterilization of Explants 19
3.4.3 Preparation of Medium 20
3.4.4 Preparation of Stock Solution 20
3.4.5 Culture Medium and Conditions for Plant Regeneration 20
3.4.6 Effects of Basal Medium Strength on Multiple Shoot Induction 21
3.4.7 Effect and Concentration of Auxin for In Vitro Rooting 21
3.4.8 Hardening of In Vitro Grown Plant 21
3.5 Phytochemistry 22
3.5.1 Qualitative Analysis on Phytochemical Constituents 22
3.5.1.1 Test for Tannins 22
3.5.1.2 Test for Flavonoids 22
3.5.1.3 Test for Terpenoids - Puncal D test 22
3.5.1.4 Test for Alkaloids - Dragendorff’s test 23
3.5.1.5 Test for Glycosides 23
3.5.1.6 Test for Steroids 23
3.5.1.7 Test for Carbohydrates 23
3.5.2 Gas Chromatography –Mass Spectroscopic Analysis 23
3.6 Pharmacology 24
3.6.1 Animals 24
3.6.2 Chemicals 24 3.6.3 Acute Toxicity Study 24
3.6.4 Experimental Induction of Diabetes 25
3.6.5 Experimental Design 25
3.6.6 Preparation of Erythrocyte Lysate 26
3.6.7 Preparation of Tissue Homogenate 26
3.6.8 Biochemical Estimation 26
3.6.8.1 Determination of Blood Glucose 26
3.6.8.2 Estimation of Total Cholesterol 27
3.6.8.3 Estimation of Triglycerides 27
3.6.8.4 Estimation of Phospholipids 28
3.6.8.5 Estimation of Plasma Insulin 29
3.6.8.6 Estimation of Glycosylated Haemoglobin 29
3.6.8.7 Assay of Hexokinase 30
3.6.8.8 Assay of Glucose 6-phosphatase 30
3.6.8.9 Assay of Fructose 1, 6-bisphosphatase 31
3.6.8.10 Estimation of Tissue Protein 31
3.6.8.11 Estimation of Haemoglobin 32
3.6.8.12 Estimation of Albumin 32
3.6.8.13 Estimation of Urea 33
3.6.8.14 Estimation of Uric Acid 33
3.6.8.15 Estimation of Creatinine 34
3.6.8.16 Estimation of Glutathione Peroxidase 34
3.6.8.17 Estimation of Reduced Glutathione 34
3.6.8.18 Estimation of Glutathione-S-Transferase 35
3.6.8.19 Assay of Superoxide Dismutase 35
3.6.8.20 Estimation of Catalase 36
3.6.8.21 Estimation of Hydroperoxides 37
3.6.8.22 Estimation of Thiobarbituric Acid Reactive Substances 37
3.6.8.23 Estimation of Ascorbic Acid 38 3.6.8.24 Estimation of α–Tocopherol 39
3.6.8.25 Assay of Alkaline Phosphatase 39
3.6.8.26 Assay of Acid Phospatase 40
3.6.8.27 Assay of Aspartate Aminotransferase 40
3.6.8.28 Assay of Alanine Aminotransferase 41
3.6.9 Histopathological Studies 42
3.6.10 Statistical Analysis 42
Chapter-4 RESULTS AND DISCUSSION 43
4.1 Micropropagation and Callus Induction 43
4.2 Phytochemistry 47
4.3 Pharmacology 80
Chapter-5 SUMMARY 107
REFERENCES 112
PUBLICATIONS
ABBREVIATIONS
BAP _ 6-Benzylamino purine
CAT _ Catalase
D.W _ Dry weight
F.W _ Fresh weight
GPX _ Glutathione peroxidase
GSH _ Reduced glutathione
GST _ Glutathione-s-transferase
H Hour
H2O2 _ Hydrogen peroxide
Hb _ Haemoglobin
HbA1c _ Glycosylated haemoglobin
IAA _ Indole 3-acetic acid
IBA _ Indole 3-butyric acid
IDDM _ Insulin dependent diabetes mellitus
KIN _ Kinetin
MS _ Murashige and Skoog Medium
Min _ Minutes
NIDDM _ Non Insulin dependent diabetes mellitus
PGR _ Plant growth regulators
SOD _ Superoxide dismutase
STZ _ Streptozotocin
TBARS _ Thiobarbituric acid reactive substances
2,4-D _ 2,4-Dichlorophenoxcy acetic acid
α-NAA _ Naphthyl acetic acid
Chapter 1 INTRODUCTION
1.1. Background of the Study
Medicinal plants have been in use in traditional health care systems since ancient times and are still the most important health care source for the vast majority of the population around the world (Heinrich, 2000; Leaman, 2006; Teklehaymanot and
Giday, 2007). At present, 80% of the population in developing countries rely largely on plant based drugs for their primary health care needs, and the world market for herbal products based on traditional knowledge was estimated to be worth US $ 60 million (WHO, 2002). Thirty percent of the drugs sold worldwide contain compounds derived from plant material.
Among the 4,00,000 plant species on the earth, only a small percentage have been phytochemically investigated and the fraction submitted to biological or pharmacological screening is even smaller (Hostettmann et al., 1998). Only 1,00,000 secondary plant products have so far been characterized (Harborne and Williams,
2000). The plant kingdom thus represents an enormous reservoir of pharmacologically valuable materials to be discovered (Potterat and Hostettmann, 1995). Fourty percent of herbal medicines are derived from natural sources or synthesized from chemical blue prints provided by a plant or animal (Anonymous, 1989). In 1986, the Japanese developed about 40% of new medicines from natural products. About 25% of prescribed medicines are of plant origin and some 120 plant–derived compounds from about 90 plant species are used in modern therapy. In the United States of America more than 60 % of the anticancer drugs approved between 1983 and 1994 are of natural origin.
1 Through millennia of trial and error, indigenous people have gained substantial knowledge of medicinal plants which has been transmitted from generation to generation as part of oral traditions (Blouin, 2003). However, concerns are being raised about the loss of native knowledge and the possible extinction of medicinal plant resources due to disruptions to traditional ways of life induced by colonial forces (Borins, 1995). Hence, proper documentation of traditional knowledge regarding plant use, along with conservation and sustainable management of key habitats, could contribute to safeguarding this heritage (Hamilton, 2004). Many today consider this more valuable than the information generated by large-scale clinical trials. Many of the lead compounds derived from ethnobotanical research exhibit potent antiviral, antifungal, anticancer or antibacterial properties that are solely needed in the pharmaceutical arsenal (Table 1). Some outstanding medicinal drugs developed from the ethnomedicinal uses of plants include: vinblastine and vincristine from
Catharanthus roseus (Periwinkle) used for treating acute lymphoma, acute leukaemias, etc, reserpine from Rauvolfia serpentina (Indian snake root) used for treating hypertension, aspirin from Salix purpurea (Willow) used for treating inflammation, pain and thrombosis and quinine from Cinchona pubescens (Cinchona) used for treating malaria (Williams, 2006).
Medicinal and aromatic plants (MAP’s) play a significant role in meeting the demands of the traditional medicinal markets both domestic and overseas. Traditional medicines, like Traditional Chinese medicine (TCM), Indian Ayurveda and Arabic
Unani Medicine and various forms of Indigenous Medicine, and alternative medicines utilized in industrialized countries, are becoming credible in many parts of the world.
The percentage of people using traditional medicines in developed countries is such as
40-50% in Germany, 42% in the USA, 48% in Australia and 49% in France (Titz,
2004).
2 Table 1. Drugs of traditional/ethnobotanical origin with their active compounds and clinical uses Therapeutic categories in medical Plant name Compound(s) Traditional medicinal use science Alangium chinense Nicotinic acid Neuromuscular blocking Muscle relaxant Artemesia annua Ginkgolides Malaria Malaria Ammi visnaga Khellin Angina pectoris and bronchial asthma Asthma Atropa belladonna Atropine Anticholinergic Dilate pupil of eye Berberis vulgaris Berberine Antibacterial Gastric ailments Abortifacient, cuts, injuries Camellia sinensis Theophylline Open bronchial passage and foul smell from mouth Carica papaya Chymopapain and papain Proteolytic and mucolytic Digestant Anticancer, pediatric leukemia and Catharanthus roseus Vinblastine and vincristine Cancer Hodgkins’s disease Cinchona ledgeriana Quinine Antimalarial Malaria Cinchona officinalis Quinine Antimalarial Malaria Cinchona pubescens Quinine Antimalria Malaria Datura metel Scopolamine Sedative Sedative Jaundice, stomach complaints Datura stramonium Scopolamine Motion sickness and toothache
Ephedrine and Nasal congestion and chronic Ephedra sinica Chronic bronchitis psedudoephedrine bronchitis Glycyrrhiza glabra Glycyrrhizin Sweetener Sweetener Homalanthus nutans Prostratin HIV infection Yellow fever Hyoscyamus niger Hyoscyamine Anticholinergic Sedative Morus alba Miglitol Diabetes Diabetes Ocimum sanctum Eugenol Cancer Cancer Codeine, morphine and Papaver somniferum Analgaesic, cough, pain and sedative Analgaesic and sedative noscapine (narcotine) Phyllanthus amarus Phyllanthin Liver disorder Jaundice Pilocarpus jaborandi Pilocarpine To reduce eye pressure Poison Migraine headaches and acute Przewalskia tangutica Anisodine Anaesthetic preparations paralysis To lower blood pressure and antidote Rauvolfia serpentina Reserpine Antidote for snakebite for snakebite Rauvolfia verticillata Spegatrine and verticillatine Hypotensive Hypotensive Rauvolfia yunnanensis Spegatrine and verticillatine Hypotensive Hypotensive Silybum marianum Silymarin Antihepatotoxic Liver disorders Taxus brevifolia Taxol Ovarian cancer Cancer Urginea maritima Scillaren A Cardiotonic Cardiotonic Valeriana officinalis Valepotriates Sedative Sedative
The last three decades have seen substantial growth in herb and herbal product markets across the world. Rapidly rising exports of medicinal plants during the past decade are the result of worldwide interest in these products as well as in traditional health systems (Scherr et al., 2004). Trade in medicinal plants is growing in volume and in exports. It is estimated that the global trade in medicinal plants is US $ 800 million year-1. The botanical market, inclusive of herbs and medicinal plants, in the
USA, was estimated approximately at US $ 1.6 billion annum-1. China with exports of over 120,000 tones annum-1 and India with some 32,000 tones annum-1 dominate the international market. It was estimated that Europe, annually, import about 400,000 tones of medicinal plants with an average market value of US $ one billion from Africa and Asia (Khan et al., 2012).
1.2. Diabetes Mellitus
Diabetes mellitus is a chronic metabolic disorder that affects human body in terms of physical, psychological and social health. It is defined as a group of disorders characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and proteins (Patel et al., 2011; Warjeet Singh, 2011). It is becoming the third “killer” of the health of mankind along with cancer, cardiovascular and cerebrovascular diseases
(Chauhan et al., 2010). The prevalence of diabetes mellitus is expected to reach up to
4.4% in 2030, and the occurrence was found to be high in India, China, and USA.
1.3. Classification of Diabetes Mellitus
Diabetes mellitus (DM) is predominantly characterized by abnormal insulin secretion that leads to elevated glucose (Flaws and Sionneau, 2002). In 2007, the
International Diabetes Federation (IDF) estimated that in the South-East Asia region,
54 million people were diabetic and an additional 63 million adults had Impaired
Glucose Tolerance (IGT). At the regional level, the number of people with diabetes is expected to increase by 71% between 2007 and 2025 (WHO, 2009). There are two
3 main types of diabetes mellitus, Type 1 or insulin-dependent diabetes mellitus (IDDM) and Type II or non insulin-dependent diabetes mellitus (NIDDM) (Flaws and Sionneau,
2002). There are many clinical and pathophysiological differences between Type 1 and
Type 2 diabetes mellitus (DM) and the contrasting features are clearly seen in Table 2
(Mohan, 2005).
1.4. Demand for Medicinal Plants
The industrial requirement for the medicinal plant resources has been on the rise due to the worldwide hopefulness in the herbal sector engaged in the production of herbal health care formulations; herbal based cosmetic products and herbal nutritional supplements. In India, nearly 9,500 registered herbal industries and a multitude of unregistered cottage-level herbal units depend upon the continuous supply of medicinal plants for manufacture of herbal medical formulations based on Indian Systems of
Medicine. In addition to the industrial consumption, significant quantities of medicinal plant resources are consumed in the country under its traditional health care practices at the household level, by traditional healers and by practitioners of Indian Systems of
Medicine. Whereas, more than 6,000 higher plant species are estimated to be used in the codified and folk healthcare traditions in the country, the quantum of their consumption has remained a matter of guess. The fallout of the lack of reliable species- wise demand estimates - so very important for sustainable management of medicinal plant resources - has been a responsible for inadequate focus on the management of medicinal plants in the country. In fact, wild populations of many medicinal plant species, forming the major resource base for the herbal industry, are reported to be facing a serious threat of extinction due to indiscriminate harvesting (Ved and Goraya,
2007).
4 Table 2. Contrasting features of Type 1 and Type 2 diabetes mellitus (Mohan, 2005)
Type 2 Diabetes S.No Feature Type 1 Diabetes mellitus mellitus
1 Frequency 10-20% 80-90%
2 Age at onset Early (below 35 years) Late (after 40 years)
3 Type of onset Abrupt and severe Gradual and insidion
4 Weight Normal Obese/non-obese
5 Family history <20% About 60%
6 Genetic locus Unknown Chromosome 6
Insuline resistance, Autoimmune destruction 7 Pathogenesis impaired insulin of β-Cells secretion
8 Islet cell antibodies Yes No
Normal or increased 9 Blood insulin level Decreased insulin insulin
No insulitis, later fibrosis 10 Islet cell changes Insulitis, β-cell depletion of islets
Diet, exercise, oral 11 Clinical management Insulin and diet drugs, insulin
12 Acute complications Ketoacidosis Hyperosmolar coma
1.5. Micropropagation and Callus Induction
Tissue culture, an important area of biotechnology can be used to improve the productivity of planting material through enhanced availability of identified planting stock with desired traits. Plant researches have increased all over the world and a large body at evidence has been collected to show the immense potential of medicinal plants used in various traditional systems (Dhanukar et al., 2000). Plants have long been used in perfumery as a source of essential oils and aroma compounds. These aromatics are usually secondary metabolites produced by plants as protection against herbivores, infections, as well as to attract pollinators. Plants are by far the largest source of fragrant compounds used in perfumery (Edwards, 2006). Plant tissue culture is a practice used to propagate plants under sterile conditions, often to produce clones of a plant, which relies on the fact that many plant cells have the ability to regenerate a whole plant (totipotency). Single cells, plant cells without cell walls (protoplasts), pieces of leaves, roots, or rhizomes can often be used to generate a new plant on culture media given the required nutrients and plant hormones. In tissue-culturing of plant cells, plant growth regulators are used to produce callus growth, multiplication, and rooting (Badr Din and Jean Pierre, 1995; Yonova and Guleva, 1997; Hutchinson et al.,
2004; Kumar et al., 2007). Among the different plant growth hormones so far studied,
IAA and BAP are considered to be more important for in vitro propagation of plants through tissue culture techniques (Prakash et al., 1999; Krishnamurthy et al., 2001).
Plant cells synthesize IAA from tryptophan. It has many different effects, as all auxins do, such as inducing cell elongation and cell division with subsequent results on plant growth and development. A recent application of plant tissue culture is used for the production of economically valuable chemicals (Nhut et al., 2002; Shweta and Nagar,
2005).
5 1.6. Pergularia daemia – an Ethnomedicinal Plant
Pergularia daemia (Forssk.) Chiov. Syn. - Daemia extensa R. Br. (Family-
Asclepiadaceae) is distributed throughout the hotter parts of India. It is also seen in the
Himalayas up to an altitude of 3500m in the hills of Assam and Bihar and is also found in Sri Lanka and Afghanistan (Chopra et al., 1956; Gamble, 1957).
1.7. Ethnomedicinal Uses
Pergularia daemia has a vast of applications in different folk medicines including the Ayurveda system and are believed to increase defence against various diseases. It is used as pungent, cooling, anthelmintic, laxative and antipyretic agents. It cures biliousness, asthma, ulcers. It is useful in eye complaints, urinary discharges, leucoderma, uterine complaints, inflammation and facilitates parturation. Decoction and juice of the leaves are reputed to be a cure for snake bite (Kirtikar and Basu, 1999).
The whole plant is used as an anthelmintic, emmenagogue, antivenom, antiseptic, emetic and expectorant (Burkill, 1985). Extract of this plant is taken orally for gastric ulcers, uterine and menstrual complaints (Singh et al., 2002). The leaf are useful in treating leprosy and haemorrhoids (Thatoi et al., 2008). The fresh, pulped leaves are applied as a poultice to relieve carbuncles. Leaf juice is used in treating amenorrhea, dysmenorrheal, infantile diarrhea and catarrhal infections (Dalziel, 1937;
Oliver, 1960; Watt and Breyer Brandwijk, 1962). In addition dried leaf are used in curing, antirheumatic, asthma, amenorrhea, dysmenorrheal, bronchitis, whooping cough, heal cuts and wounds and to facilitate parturition (Ndukwu and Ben Nwadibia,
2005). The stem bark of this plant, is also used to treat malaria and the twig is used as an antipyretic and leaf of this plant are a good remedy for cold (Dokosi, 1998). The latex of the plant is used for treating boils, sores and venereal diseases (Van Damme et al., 1922). Dried roots are used as an abortifacient, emetic, bronchitis and treated in curing cough, asthma, constipation and Venereal diseases (Royen et al., 2001).
6 In Ayurvedic System of Medicine, this plant is used for delayed child birth, amenorrhea, asthma, snake bite, rheumatic swellings and also to treat post-partum hemorrhage. The decoction of the plant (10-20 ml) is also applied on white spots
(leucoderma). Leaf decoction is a uterine tonic and is taken orally up to 20 ml day-1.
The stem and root bark extract is taken against fever and diarrhea in infants (Ndukwu and Ben Nwadibia, 2005). The leaves are specially used as a condiment in soup and porridge yam. Fruits are digestive and thermogenic (Thatoi et al., 2008).
1.8. Epidemiological Studies
In Nigeria the plant has been used occasionally for flavoring food and the young leaf shoots are eaten like spinach. In Ghana, the hairy stems with fire resistant properties are knit together and used as binding in situations that might be exposed to fire. In Arabia, hides have been smeared with the sap and then buried in the ground to remove hair. The leaves have had veterinary applications in Ghana as a remedy for young turkeys suffering from diarrhea - and leaf juice is used in India to treat eye problems in Cattle. Medicinally, P. daemia is used locally for treating worms and period and other female disorders - and the sap (or a leaf poultice) is applied to boils.
Ghanaian medicine were recommend that roasted stems be chewed to ease bronchitis and coughs (Nigerians use the leaves and bark) and some Ghanaian women take a leaf soup after childbirth. Local Nigerian medicine also turns to the leaves and stem bark for a treatment to easing rheumatic pain. In Botswana and South Africa, the leaves are eaten as wild Spinach. The latex or decoction of the roots is used in many countries as medicine to treat several illnesses such as, venereal diseases, arthritis, muscular pains, asthma, rheumatism, snake bite, etc, the latex may also be used as fish poison.
Mainly in South Africa, the young leaves and shoots are taken as soup and spinach the soup in particular is given to newly delivered women (Irvine, 1952). In
7 Ghana and Tanganyika, the sap from the leaf is used to cure sore eyes. In Nigeria, the plant is used in combination with others for treatment of fever. The latex or a poultice of the leaves is applied to maturate abscesses, a usage known in India. In Ivory Coast, the latex is applied to maturate abscesses.
1.9. Pharmacological Studies of Pergularia daemia
Pharmacology can allow one to examine how drugs act on the organs of the living system and affect them and how they stay in them. In addition, pharmacology deals with how medicines interact with cells and genes that affects the function of the cells and the body.
Although P. daemia has been prescribed for various complications including diabetes, diarrhea, dysentery, etc., in folklore and traditional system of medicine.
P. daemia possesses various pharmacological actions as hypolipidemic, antioxidant, antiulcerogenic, hepatoprotective, antidiabetic, gastroprotective, antivenom and chemopreventive agents.
Golam Sadik et al. (2001a) demonstrated that the ethanol extract of P. daemia and its steroidal fraction have antifertility activity. The alkaloidal fraction of the ethanol extract was observed for its antifertility activity. Oral administration of the alkaloidal fraction at a dose of 200 mg/kg b.w showed significant activity in preimplantation stage of female mice. The activity of the alkaloid fraction, when compared with the steroidal fraction, was found to be more pronounced since the former inhibited not only the fertility of the female mice but also take a short period to return the oestrous cycle to normal, in 4 to 6 days of drug treatment while the steroidal fraction treated mice returned to normal in 6 to 8 days.
8 Bhaskar and Balakrishnan (2009) reported that the highest percentage of inhibition was observed for ethanol extracts of P. daemia (61%) at a dose of 100 mg/kg, after 3 h of administration. The highest percentage of inhibition was observed for ethanol extract of C. carandas (63%) at a dose of 100 mg/kg. The patterns of anti- inflammatory, analgesic and antipyretic activities exhibited by these extracts were similar to that of aspirin, which suggests that activity of the plant may be mediated by cyclooxygenase I and II inhibition. The observation that both plants increased pain threshold of animals could be due to inhibition of sensitization of pain receptors by prostaglandins at the inflammation site. After administration of acetic acid, several mediators such as cytokines, eicosanoids and arachidonic acid are liberated from the membrane after phospholipase A2 activity leading to production of prostaglandins and leukotrienes. The analgesic activity of the ethanol and aqueous extracts of P. daemia and C. carandas may be due to inhibition of phospolipase A2 or even blocking cyclooxygenase (COX-1 and/or COX-2). Carrageenan induced edema has been commonly used as an experimental animal model for acute inflammation and is believed to be biphasic. The second phase is caused by the release of bradykinin, proteases, prostaglandins and lysosomes. The prostaglandins play a major role in the development of second phase of reaction that is measured at 3 h. These mediators take part in the inflammatory response and are able to stimulate nociceptors and thus induce pain. Carrageenan induced edema model in rats is known to be sensitive to cyclooxygenase inhibitors and is used to evaluate the effect of non-steroidal anti- inflammatory agents, that is inhibition of cyclooxygenase in prostaglandin synthesis.
Based on these reports, it may be concluded that the inhibitory effect of the ethanol and aqueous extracts of P. daemia and C. carandas (100 mg/kg and 200 mg/kg b.w) on carrageenan induced inflammation in rats could be due to the inhibition of
9 cyclooxygenase. These experimental results have established pharmacological evidence for the folklore claim of the drugs to be used as an analgesic, anti-inflammatory and antipyretic agent.
Hebbar et al. (2004) surveyed the Dharwad district of Karnataka in Southern
India and revealed that 35 plants belonging to 26 families are being used to treat different types of oral ailments like toothache, plaque and caries, pyorrhea and aphthae.
Sixteen of these plants were new claims for the treatment of oral ailments not previously reported in the ethnomedicinal literature of India. Basella alba, Blepharis repens, Capparis sepiaria, Oxalis corniculata and Ricinus communis are used for the treatment of aphthae. Azima tetracantha, Caesalpinia coriaria, Cleome gynandra,
Gossypium herbaceum, Leucas aspera, Merremia chryseides, Pergularia daemia,
Prosopis juliflora and Solanum nigrum are used to treat tooth ache and Cassia hirsuta and Cassia tora are used in the treatment of plaque and caries.
Pergularia daemia has a general reputation as an expectorant, emetic and also to infantile diarrhea (Sathyavathi et al., 1976). It is commonly used among the tribes of sherveroy hills of Tamil Nadu as a substitute for Gymnema sylvestre in the treatment of diabetes (Raghunathan and Mitra, 1982). But, its antidiabetic activity is yet to be experimented on researched for its full potential (Wahi et al., 2002). Therefore, it was thought worthwhile to evaluate its antidiabetic effects using Streptozotocin (STZ) induced experimental rats and screen, the phytochemicals behind this activity.
P. daemia is an important plant described in the Ayurveda. This plant is used in the treatment of a number of ailments like urinary disorders and cardiac problems. More quantity of the plant is needed to meet the medicinal requirement and for genetic stability. So, a standard protocol is needed for in vitro propagation of the plant to help its conservation.
10
1.10. Objectives and Scope of Research work
• To develop the standard protocol for in vitro propagation of Pergularia daemia;
• Study the mass of callus induction using different phytohormones as
accelerated;
• Screening of the phytochemicals with chloroform, alcohol, aqueous extracts of
field grown plant and alcohol extracts of callus; and
• To study the antidiabetic activity, hepatoprotective and toxicological effects of
chloroform, alcohol, aqueous extracts of field grown plant and alcohol extracts
of callus.
11 Chapter 2 REVIEW OF LITERATURE
In India, medicinal plants have been in use as natural medicine since the days of
Vedic glory. Many of these medicinal plants and herbs are a part of our diet as spices, vegetables and fruits. Historically, in ‘Atharva-Veda’ (about 200 B.C.), a description of medicinal plants was made under a separate chapter ‘Ayurveda’. Sushruta (about 400
B.C.) compiled a classification of 700 herbal drugs under 37 classes in ‘Sushruta
Samhita’ (A Compendium of Ancient Indian Medicine). Charak (about 600 B.C.) made a scientific classification of herbal drugs based on remedial properties in his renowned treatise ‘Charaka Samhita’ (A Compendium of General Medicine). In which, he described 50 classes of herbal remedies comprising 500 crude drugs (Mukherjee et al.,
1988; Saxena et al., 2006). The medicinal values of plants have been tested by trial and error method for a long time by different workers. Even today, great opportunities are still open for scientific investigations of herbal medicines for cure of diabetes and its complications (Gupta et al., 2005).
Diabetes is a chronic metabolic disorder that posses a major challenge worldwide. Currently in India, the number of people with diabetes is around 40.9 million and is expected to rise to 69.9 million by 2025. India has emerged as the diabetic capital of the world. Unless urgent preventive steps are taken, it will become a major health problem. The Indian Diabetes Federation (IDF) estimated 3.9 million diabetic deaths for the year 2010, which represented 6.8% of the total global mortality
(International Diabetes Federation, 1994).
Diabetes Mellitus is the most severe metabolic pandemic of the 21st century, affecting essential biochemical activities in almost every cell in the body and increasing
12 the risk of cardiac problems. It is estimated that in the year 2000, 171 million people had diabetes, and this is expected to double by year 2030 (Boon et al., 2006).
Conventionally, insulin-dependent diabetes mellitus is treated with exogenous insulin
(Felig et al., 1995) and noninsulin-dependent diabetes mellitus with synthetic oral hypoglycemic agents like sulphonylureas and biguanides (Rosac, 2002). However, the hormone fails as a curative agent for complications of diabetes (Mukherjee and
Mukherjee, 1966) and synthetic oral drugs produce adverse health effects (Raheja,
1977). Different medicinal systems are using, active plant constituents identified as natural hypoglycemic medicine, through traditional knowledge. Herbal drugs are considered free from side-effects. They are less toxic, relatively cheap and popular
(Momin, 1987).
Traditional anti-diabetic plants might provide new oral anti-diabetic compounds, which can counter the high cost and poor availability of the current medicines for many rural populations in developing countries. Plant drugs are frequently considered to be less toxic and free from side-effects than synthetic ones. In
India, indigenous remedies have been used in the treatment of diabetes mellitus since the time of Charaka and Sushruta (6th century B.C.). The World Health Organization
(WHO) has listed 21,000 plants which are used for medicinal purposes around the world. Where in 2500 species are in India. India is the largest producer of medicinal herbs endowed with a wide diversity of agro-climatic conditions and is called as botanical garden of the world. Pharmacological and clinical trials of medicinal plants have shown anti-diabetic effects and repair of β-cells of islets of Langerhans
(Chakravarthy et al., 1980).
2.1. Trials for Anti-diabetes Potential of Medicinal Plants
Recently, plants and herbs are being used as decoctions or in other extracted forms for their blood sugar lowering potential. There are some useful reviews on Indian
13 Table 3. Anti-diabetic plants and their active principles
Plant name Family Compound(s) isolated References
Acosmium panamense Fabaceae Desmethylyangonine Wiedenfeld and derivatives Andrade Cetto, 2003
Allium sativum Liliaceae Allyl propyl disulphide, Kimbaris et al., 2006 allicin
Angylocalyx pynaertii Fabaceae Sugar-mimic Yasuda et al., 2002
Artemisia pallens Asteraceae Davana Ether Thomas and Pitton, 1971
Bauhinia forficate Fabaceae Astragalin, Kaempferitrin Viana et al., 1999
Beta vulgaris Chenopodiaceae Phenolics, Betalains Kujala et al., 2001
Camellia sinensis Theaceae Caffeine, Catechins Yang et al., 2009
Capparis deciduas Capparaceae Luteolin-7-O-D- Saxena and Goutam, glucopyranoside 2008
Combretum micranthum Combretaceae Polyphenols Muhammad and Amusa, 2005
Elephantopus scaber Asteraceae Terpenoid, 2,6,23 Sezik et al., 2005 Trienolide
Gymnema sylvestre Asclepiadaceae Dihydroxygymnemic Mhasker and Caius, triacetate 1930
Liriope spicata Asparagaceae β-sitosterol, stigmasterol Chen et al., 1998
Lobelia chinensis Campanulaceae Palmitinic acid Jiang et al., 2009
Myrcia multiflora Myrtaceae Myrciacitrins I and II, Yoshikawa et al., Myrciaphenones A and B 1998
Myrciaria dubia Myrtaceae Ellagic acid derivatives Ueda et al., 2004
Nicandra physalodes Solanaceae Calystegine Griffiths et al., 1996
Paeonia lactiflora Paeoniaceae Paeoniflorin, 8- Kim et al., 2009 debenzoylpaeoniflorin
Phyllanthus amarus Phyllanthaceae Ellagitannins Londhe et al., 2009
Pterocarpus marsupium Fabaceae Pterostilbene Joshi et al., 2012
Punica granatum Punicaceae Ursolic acid, Fl-Sitosterol Rehana Ahmed et al., 1995 Ricinus communis Euphorbiaceae Ricinolic acid Dhar et al., 1968
Salacia oblonga Celastraceae Salacinol Augusti et al., 1995
Salacia chinensis Hippocrateaceae Friedelane-type triterpene Deokate and Khadabadi, 2012
Smallanthus sonchifolius Asteraceae Sonchifolin, Uvedalin, Zheng et al., 2010 Enhydrin, Luctuanin
Stevia rebaudiana Asteraceae Fangchinoline Tsutsumi et al., 2003
Swertia punicea Gentianaceae Methyl swertianin, Chhetri et al., 2005 Bellidifolin
Tecoma stans Bignoniaceae Tecomine, 5b Costantino et al., 2003 hydroxyskitanthine, boschniakine
Trigonellafoenum graecum Fabaceae Trigonelline, Nicotinic acid Moorthy et al., 2010
Withania somnifera Solanaceae Somniferine, Withananine, Khajuria et al., 2004 Cuscohygrine
medicinal plants having blood sugar lowering potentials (Mukherjee et al.,
1981; Grover et al., 2002; Saxena and Vikram, 2004; Mukherjee et al., 2006). Many
useful herbs introduced in pharmacological and clinical trials have confirmed their
blood sugar lowering effect, and their potency in the repair of β-cells of islets of
Langerhans. Details of some potent Indian herbs, their recently reported
pharmacological and clinical hypoglycemic efficacies and active chemical constituents
are given (Table 3).
2.2. Phytochemical and Pharmacology of Pergularia species
Although P. daemia has been prescribed for various complications including
diabetes, diarrhoea and dysentery in folklore and traditional systems of medication. It
possesses various pharmacological actions as hypolipidemic, antioxidant,
antiulcerogenic, hepatoprotective, antidiabetic, gastroprotective, antivenom and
chemopreventive agent.
Phytochemical/biological studies or both are mentioned based on availability.
2.2.1 Pergularia daemia Root- Phytochemical Constituents
Phytochemical constituents for root include calotoxin, calotropagenin, dihydro calotropagenin, calotropin, uscharidin by Mittal et al. (1962); calactin, calotropin, corotoxigenin, sucrose (Seshadri and Vydeeswaran, 1971).
2.2.1.1 Pergularia daemia Root-Biological Activity
Biological activity for root include abortifacient (Kokwaro, 1981); whooping
Cough (Nagaraju and Rao, 1990); gonorrhea (Samuelsson et al., 1991).
2.2.2 Pergularia daemia Stem-Phytochemical Constituents
Phytochemical constituents for stem include hyperoside (flavonol) by Sinha and
Dogra (1985).
14 2.2.2.1 Pergularia daemia Stem-Biological Activity
Biological activities reported for stem include antimalarial activity by Kohler et
al. (2002).
2.2.3 Pergularia daemia Leaves-Biological Activity
Biological activities reported for leaves include emetic and bronchitis (Mittal et
al., 1962); fish poison, cough, asthma, and constipation (Watt and Breyer Brandwijk,
1962); abortifacient (Kokwaro, 1976); healing cuts and wounds (Pushpangadan and
Atal, 1984); catarrhal infections, infantile diarrhea (Elango et al., 1985); whooping
cough (Reddy et al., 1989); antirheumatic (Kakrani and Saluja, 1994); antisecretory,
gastric transmt time, wound healing (Karthikeyan et al., 2007); hemorrhages, fever,
colitis, skin infection (Tambekar and Khante, 2010).
Antifertility activity has been reported by several authors such as Prakash et al.
(1978) and Runnebaum et al.(1984).
Amenorrhea, dysmenorrheal activity has been reported by several authors such
as Elango et al. (1985); Dutta and Ghosh (1947 b); De Laszlo and Henshaw (1954).
Body pain has been reported by several authors such as John (1984) and Reddy
et al. (1988).
Antimicrobial activity reported by Ogunlana and Ramstad (1975) and
Palavesam et al. (2006).
2.2.4 Pergularia daemia Aerial Part-Phytochemical Constituents
Phytochemical constituents reported for aerial part includes flavonoids,
saponins by Sankara Subramanian and Nair (1968); flavonoids, tannin, carbohydrates,
alkaloids, steroids, terpenoids (Deepika Thenmozhi, 2011).
15 2.2.4.1 Pergularia daemia Aerial Part-Biological Activity
Biological activity reported for aerial part includes antipyretic, appetizer by Gill
and Akinwumi (1986); snakebite (Selvanayagam et al., 1995).
Hepatoprotective activity has been reported by several authors such as Suresh
Kumar and Mishra (2008a); Suresh Kumar and Mishra (2008b).
2.2.5 Pergularia daemia Whole Plant-Phytochemical Constituents
Phytochemical constituents reported for whole plant include α, β-amyrin and its
acetate (Raman and Barua, 1958); lupeol acetate, betaine, hentriacontane,
pentacosanoic acid (Rakhit et al.,1959); lupeol (Seshadri and Vydeeswaran, 1971);
lupeol-3-beta trans crotonate, oleanolic acid acetate, β-sitosterol (Anjaneyulu et al.,
1998); kaempferol 3-O-galactoside, isorhamnetin 3-O-malonyl hexoside (Heneidak et
al., 2006).
2.2.5.1 Pergularia daemia Whole Plant-Biological Activity
Biological activity reported for whole plant include insecticide (Heal et al.,
1950); uterine stimulant effect (Rakhit et al., 1959); delayed child birth, post-partum,
hemorrhage (Ghatak and De, 1961); emmenagogue (Berhault, 1971); expectorant
(Seshadri and Vydeeswaran, 1971); cytotoxic (Dhar et al., 1973); asthma, snakebite,
rheumatic swellings (Singh et al.,1980); antiseptic (Arseculeratne et al., 1985); emetic
(Elango et al., 1985); antifungal (Qureshi et al., 1997).
Antimicrobial activity has been reported by several authors such as Elango et al.
(1985); Valsaraj et al. (1997); Perumal Samy and Ignacimuthu (2000) and Srinivasan et
al. (2001).
16 2.2.5.2 Pergularia daemia Whole Plant-Phytochemical Constituents and Biological
Activity
Phytochemical constituents and biological activity for whole plant include reported polypeptide, glucoside and uterine stimulant effect (Dutta and Ghosh, 1947a); inorganic salts-Kcl and KNO3 and uterine stimulant effect (Dutta and Ghosh, 1947b).
2.2.6 Pergularia daemia Latex-Biological Activity
Biological activity for latex include boils and sores by Girach et al.(1994).
2.2.7 Pergularia pallida Whole Plant-Phytochemical Constituents
Phytochemical constituents for whole plant include tylophorinidine, pergularinine, desoxypergularinine, unidentified base (M+ 409) by Mulchandani
Venkatachalam (1976) and sarcogenin (Khare et al., 1986).
2.2.8 Pergularia tomentosa Whole Plant-Phytochemical Constituents
Phytochemical constituents for whole plant include cardenolides (Piacente et al., 2009); pergularine A, pergularine B, oleic acid, (9Z, 12Z)-octadecadienoic acid, α- amyrin, 3-acetyltaraxasterol, 3-taraxasterol, 16α-hydroxytaraxasterol-3-acetate, 3-epi- micromeric acid (Babaamer Zohra et al., 2012).
2.2.9 Pergularia tomentosa Whole Plant-Biological Activity
Biological activity for whole plant include molluscicidal activity (Hussein et al.,
1999).
17 Chapter 3 MATERIALS AND METHODS
3.1. Plant Material
Pergularia daemia (Forssk.) Chiov. in Res. Sci. Somalia Ital. 1:115. 1916.
Pergularia daemia (Forssk.) Blaff, & Mc Cann in J. Bombay Nat. Hist. Soc. 36:528.
1933. Asclepias daemia Forssk., Fl. Aegypt.-Arab. 51. 1775. Daemia extensa (Jacq.) R.
Br. in Ait. Hort. Kew-ed. 2.2:76. 1811; Wight, Ic. t. 596. 1842; Hook. f., Fl. Brit. India
4:20. 1883. Cyananchum extensum Jacq., Misc. 2: 353. 1781. Pergularia extensa
(Jacq.) N.E. Br. in Dyer, Fl. Cap. 4: 758. 1908; Gamble, Fl. Pres. Madras 2:588. 1957
(repr. ed.).
Family : Asclepiadaceae
Vernacular Name
Tamil : Seendhal kodi, Uttamani, Velipparuthi
Sanskrit : Uttaravaruni
Hindi : Utranajutuka
3.2 Plant Description
Pergularia daemia is a slender, hispid, fetid-smelling perennial climber. The stem bears milky juice and is covered with longer stiff erect hairs upto 1mm long.
Leaves opposite, membranous, 3-9 cm long and about as wide, broadly ovate, orbicular or deeply cordate, acute or short-acuminate at apex, pubescent beneath, petioles 2-9 cm long. Flowers greenish-yellow or dull white tinged with purple, borne in axillary, double white corona at the base of a stamina column, long-peduncled, umbellate or corymbose clusters tinged with purple. Fruits paired with follicles, lanceolate, long-
18 pointed, about 5 cm long and 1 cm in diameter, covered with soft spines. Seeds are pubescent, broadly ovate. Flowering: August to January; Fruting : October to February.
In central Indian deciduous forests, the stems typically die down in February and reappear with the onset of the rainy season.
3.3 Strategy of Work Plan
The present study covers 3 aspects:
1. Micropropagation and Callus Induction;
2. Phytochemistry; and
3. Pharmacology.
3.4. Micropropagation and Callus Induction
Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.
3.4.1 Explant Collection
Pergularia daemia was collected from the Southern parts of Pudukkottai district, Tamil Nadu, India, and planted in the J.J. College Botanical Garden. The plants were raised in pots containing mixture of soil and farmyard manure in the ratio of 1:1.
Small disease free tender twigs, leaf and stem were collected from 5-6 months grown plants, cut into 0.5-1.0 cm segments and used as explants for the induction of multiple shoots; leaf and stem for callus induction.
3.4.2. Sterilization of Explants
The explants were washed thoroughly under running tap water for 15 min then washed with 2% (v/v) Tween 20 (mild detergent) solution for 10 min, then washed with doubled distilled water thrice. Then, the explants were sterilized within the sterilized chamber. The explants were submerged in 70% ethanol for 60 sec and rinsed with
HgCl2 (0.1%) for 2 min and washed thrice with sterile distilled water.
19 3.4.3. Preparation of Medium
For preparing the MS medium (Murashige and Skoog, 1962), all the stock solutions were taken in appropriate proportions (Table 4) and the final volume was made up to required quantity by adding double distilled water. Stock solutions of plant growth regulators were also prepared and kept at 4±1°C. 3% (w/v) sucrose was added to the medium as source of carbon. Various concentrations and combinations of growth regulators were added to the medium before adjusting the pH to 5.8 using 0.1 N NaOH or 0.1 N HCl and gelled with 0.8% agar and melting the agar in a water bath. The media with all necessary ingredients were dispensed into culture tubes, conical flasks and wide mouthed bottles and were plugged with non-absorbent sterile cotton plugs and autoclaved at 121°C. The autoclaved media were kept in inoculation room until use.
3.4.4. Preparation of Stock Solution
The optimized MS medium (Murashige and Skoog, 1962) was used for the present investigation. For the preparation of the basal MS medium, separate stock solution of macro nutrients, micro nutrients, iron supplements, vitamins and amino acid were prepared by dissolving required amounts of chemicals in double distilled water
(Table 5) and were stored at 4±1°C in the Refrigerator. Individual growth regulators such as BAP and Kinetin or in combinations with different auxins such as NAA, IAA and IBA were also prepared and kept at 4±1°C.
3.4.5. Culture Medium and Conditions for Plant Regeneration
The Murashige and Skoog (1962) medium was adjusted to pH 5.8 and 0.8% agar was added. The medium was boiled to milky appearance for complete dissolving of agar in medium. About 15 ml of the medium was dispersed in each culture bottle and sealed with cotton plug before autoclaving at 121°C for 15 min under pressure of
15 Psi. The media in culture bottles were left to cool a slant in the culture room until use. Under Laminar Air Flow Chamber, the explants were inoculated aseptically on MS
20 Table 4. Composition of MS medium (Murashige and Skoog, 1962)
Stock ingredients Molecular formula Concentration (mg/l) Macronutrients
Ammonium nitrate NH4NO3 1650
Potassium nitrate KNO3 1900
Calcium chloride CaCl2.2H2O 440
Magnesium sulphate MgSO4.7H2O 370
Potassium dihydrogen KH2PO4 170 ortho phosphate Micronutrients
Ferrous sulphate FeSO4. 7H2O 27.6
Disodium ethylene diamine Na2EDTA 37.4 tetra acetic acid
Boric acid H3BO3 6.2 Potassium iodide KI 0.83
Manganese sulphate MnSO4. 4H2O 22.3
Zinc sulphate ZnSO4. 7H2O 8.6
Sodium molybdate Na2MoO4. 2H2O 0.25
Cobalt chloride CoCl2. 6H2O 0.025
Copper sulphate CuSO4. 5H2O 0.025 Vitamins Nicotinic acid 100 Pyridoxine HCl 0.5 Thiamine HCl 0.5 Myo-inositol 0.1 Amino acids Glycine 2.0 pH 5.8 Sucrose 3%
Table 5. Composition of stock solution of MS medium (Murashige and Skoog, 1962)
Stock ingredients Quantity (mg) Volume of stock /l of medium (ml) Stock A (20X) 500 ml
NH4NO3 16500
KNO3 19000 50
CaCl2.2H2O 4400
MgSO4.7H2O 3700 Stock B (100X) 100 ml
MnSO4. 4H2O 223
ZnSO4. 7H2O 86
H3BO3 62 KI 8.3 10
Na2MoO4. 2H2O 2.5
CuSO4. 5H2O 0.25
CoCl2. 6H2O 0.25 Stock C (100X) 200 ml Nicotinic acid 10 Pyridoxine HCl 10 10 Thiamine HCl 2 Myo-inositol 40 Stock D (100X) 100 ml
KH2PO4 1700 10 Stock E (200 X) 100 ml
FeSO4. 7H2O 556 5
Na2EDTA 746
media and suspension media (without addition of agar), supplemented with different concentrations (1.0 - 3.0 mg/l) of α-NAA, 2,4-D as auxins and BAP, KIN as cytokinins were added into the MS media to study the effect on callus formation. The cultures were incubated under fluorescent lights with 1500-2000 lux for 16 h at 25±1°C and
80±10 relative humidity. Each experiment had 20 replicates and was repeated thrice.
The grown callus masses were observed on 6th week and the data were processed using statistical tools.
The suspension cultured calli were harvested at 2, 4, 6, 8, 10, 12, 14, 16 and 18 days, using Whatman’s filter paper and fresh and dry weights of calli were measured.
Fresh weights of cells/calli were taken after removing the excess of moisture on the surface using blotting paper. The dry weight of callus was determined by drying in a
Hot air oven at 60°C for 24 hr.
3.4.6. Effects of Basal Medium Strength on Multiple Shoot Induction
To evaluate the ability of different basal media to support shoot culture, full and half strength of MS media supplemented with 0.5 mg/l BAP in combination with 1.0 mg/l NAA were used.
3.4.7. Effect and Concentration of Auxin for In Vitro Rooting
For root induction, in vitro developed shoots were excised and cultured in half strength MS media supplemented with different concentrations (0.5-2.0 mg/l) of IBA,
IAA and NAA.
3.4.8. Hardening of In Vitro Grown Plant
Rooted plantlets (9-10 cm length) were taken out from the culture tubes and washed to remove adhered agar and traces of medium. Plantlets were then transferred to 8.0 cm diameter plastic pot containing soil and vermiculture mixture in the ratio of
3:1. These plants were maintained inside the growth chamber for two weeks and
21 irrigated gently once a day for acclimatization. Afterwards, the plants were grown under Mist Chamber conditions until transfer to the field.
3.5. Phytochemistry
The leaves and calli were dried under shade, coarsely powdered, and extracted
with chloroform (60-80°C) followed by alcohol, and then water using Soxhlet
apparatus. The extracts so collected were distilled off on a Water bath at atmospheric
pressure and the last traces of the solvents were removed in vacuo (Merlin et al.,
2009).
3.5.1. Qualitative Analysis on Phytochemical Constituents
The solvent extracts (chloroform, ethanol and aqueous) of the plant materials
were tested through preliminary phytochemical screening (Harborne, 1976) and the
observations were recorded.
3.5.1.1. Test for Tannins
A few ml of ferric chloride were added to the extract. Formation of a dark blue or greenish black colour indicates the presence of tannins.
3.5.1.2. Test for Flavonoids
A few ml of ammonia added to the sample. It leads to fluorescence which indicates the presence of flavanoids under UV-visible light.
3.5.1.3. Test for Terpenoids - Puncal D test
A few ml of Puncal D reagent (Ammonium Heptamolybedate+Ceric sulphate in concentrated sulphuric acids) were added to the extract, heated. Formation of blue colour indicates the presence of terpenoids.
22 3.5.1.4. Test for Alkaloids - Dragendorff’s test
To 1 ml of extract, 1 ml of Dragendorff’s reagent (Potassium bismuth iodide solution) was added. Formation of an orange red precipitate indicates the presence of alkaloids.
3.5.1.5. Test for Glycosides
One ml of concentrated H2SO4 was taken in a test tube. One ml of extract from each plant sample was mixed with 2 ml of glacial acetic acid containing 1 drop of
FeCl3. The above mixture was carefully added to 1 ml of concentrated H2SO4 so that the concentrated H2SO4 was underneath the mixture. If glycoside is present in the sample, a brown ring will appear, indicating the presence of the glycoside constituent.
3.5.1.6. Test for Steroids
A few ml of concentrated sulphuric acid solution were added to the extract. The formation of green colour indicates the presence of steroids.
3.5.1.7. Test for Carbohydrates
A few ml of concentrated sulphuric acid solution were added to the extract and heated that leads to the formation of charring which indicates the presence of carbohydrates.
3.5.2. Gas Chromatography –Mass Spectroscopic Analysis
The GC-MS analysis was carried out on a GC Clarus 500 Perkin Elmer system and Gas Chromatograph interfaced to a Mass spectrometer (GC-MS) instrument employing the following conditions: Column Elite-1 fused Silica Capillary Column (30 mm×0.25 mm ID ×1 μMdf, composed of 100% Dimethyl Poly siloxane), operating in electron impact mode at 70 eV; Helium (99.999%) used as carrier gas at a constant flow of 1 ml/min and an injection volume of 2 μl was employed (split ratio of 10:1);
Injector temperature 250°C; Ion-source temperature 280°C. The oven temperature was
23 programmed from 110°C (isothermal for 2 min), with an increase of 10°C/min, to
200°C, then 5°C/min to 280°C, ending with a 9 min. isothermal at 280°C. Mass spectra were taken at 70 eV; a scan interval of 0.5 sec and fragments from 45 to 450 Da.
3.6. Pharmacology
3.6.1. Animals
In the present study healthy, matured male albino rats (wistar strain) were used.
Rats weighing 180-230g were obtained from the Periyar College of Pharmaceutical
Sciences, Tiruchirapalli, Tamil Nadu, India and kept in plastic animal cages with 12 h
light and dark cycle in the institutional animal house. The animals were fed with
standard rodent diet and provided water ad libitum. After one week of acclimatization
the animals were used for the further experiments. Approval from the Institutional
Animal Ethical Committee for the usage of animals in the experiments was obtained
as per the Indian CPCSEA guidelines (Registration Number: 265/CPCSEA).
3.6.2. Chemicals
Streptozotocin (STZ) and glibenclamide were purchased from Sigma Aldrich,
St. Louis, MO, USA. All other chemicals and solvents used were of Analytical Grade
obtained from E-Merck and Himedia, Mumbai, India.
3.6.3. Acute Toxicity Study
Acute toxicity studies were carried out using Acute Toxic Class Method as per
OECD-423 Guideliness (OECD, 1996). Chloroform leaf extract, ethanol leaf extract, aqueous leaf extract and ethanol leaf callus extract of P. daema were administered at a starting dose of 2000 mg/kg b.w of orally to 4 male rats. The animals were observed for mortality and behavioral changes during 48 h.
24 3.6.4. Experimental Induction of Diabetes
Diabetes was induced in a group of rats after an overnight fast by single intraperitoneal injection of STZ, which was freshly dissolved in 0.1M citrate buffer (pH
4.5). The dose was 40 mg/kg b.w. STZ treated animals were allowed to drink 5% glucose solution overnight to overcome drug induced hypoglycemia. After 48 h of STZ administration, the blood glucose ranges above 200-300 mg/dl was considered as diabetic rats and used for the experiment.
3.6.5. Experimental Design
In the experiment, a total of 162 rats were used, randomly divided into 27 groups of 6 animals each and treatments continued in an aqueous solution daily using an intragastric tube for 21 days.
Group-I : Normal rats received 3% gum acacia
Group-II, III, IV : Leaf chloroform extract (100, 200, 300 mg/kg b.w.)
Group-V, VI, VII : Leaf ethanol extract (100, 200, 300 mg/kg b.w.)
Group-VIII, IX, X : Leaf aqueous extract (100, 200, 300 mg/kg b.w.)
Group-XI, XII, XIII : Leaf ethanol callus extract (100, 200, 300 mg/kg b.w.)
Group-XIV : Streptozotocin (STZ) 40 mg/kg b.w. (Diabetic control)
Group-XV, XVI, XVII : STZ+Leaf chloroform extract (100, 200, 300 mg/kg b.w.)
Group-XVIII, XIX, XX : STZ+Leaf ethanol extract (100, 200, 300 mg/kg b.w.)
Group-XXI, XXII, XXIII : STZ+Leaf aqueous extract (100, 200, 300 mg/kg b.w.)
Group-XXIV, XXV, XXVI : STZ+Leaf ethanol callus extract (100, 200, 300 mg/kg b.w.)
25 Group-XXVII : STZ+Glibenclamide (600 µg/kg b.w.)
After the termination of the experiment, all the animals were anaesthetized using ketamine chloride (24 mg/kg b.w.) and sacrificed by cervical dislocation after an overnight fast. Blood was collected and tissues (liver and kidney) were immediately removed, blotted and kept at -20°C until use. Plasma, serum and tissue homogenates were separated after centrifugation and used for various biochemical estimations.
3.6.6. Preparation of Erythrocyte Lysate
After the separation of plasma, the buffy coat was removed and the packed erythrocytes were washed thrice with cold physiological saline. A known volume of the erythrocyte was lysed with cold hypotonic phosphate buffer at pH 7.4. The hemolysate was separated by centrifugation at 2000 rpm for 10 min and the supernatant was used for the estimation of enzymatic antioxidants.
3.6.7. Preparation of Tissue Homogenate
A known volume of liver and kidney tissues was homogenized in Tris-
HCl/Phosphate buffer pH 7.0 using potter-Elvehjam homogenizer with Teflon pestle.
The homogenates were centrifuged at 1000 rpm for 10 min. The supernatant was separated and used for various biochemical estimations.
3.6.8. Biochemical Estimation
3.6.8.1. Determination of Blood Glucose
Blood glucose was determined using the modified reagent of Sasaki et al.
(1972). The volume of 0.1 ml of freshly drawn blood was immediately mixed with 1.9 ml of 10% TCA to precipitate the protein and then centrifuged. One ml of the supernatant was mixed with 4 ml of o-toluidine reagent and kept in boiling water bath
26 for 15 min. The green colour developed was read Spectrophotometrically at 620 nm. A set of standard glucose (20-100 μg) was treated simultaneously with reagent blank.
Glucose concentration was expressed as mg/dl of blood.
3.6.8.2. Estimation of Total Cholesterol
Total cholesterol in the plasma, erythrocytes and tissues was estimated by the enzymic method as described by Allain et al. (1974). Cholesterol esters were hydrolyzed by cholesterol esterase to free cholesterol and free fatty acids. The free cholesterol produced and pre-existing ones were oxidized by cholesterol oxidase to cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide formed reacts with
4-aminoantipyrine and phenol in the presence of peroxidase to produce red coloured quinoneimine dye. The intensity of colour produced was proportional to the cholesterol concentration.
To 10 µl of plasma or 10 µl of lipid extract, 1.0 ml of enzyme reagent was added, mixed well and kept at 37°C for 5 min. Ten µl of cholesterol standard and distilled water (blank) were also processed similarly. The absorbance was measured at
510 nm.
Cholesterol concentration was expressed as mg/dl of plasma.
3.6.8.3. Estimation of Triglycerides
Triglycrides were estimated by the method of Van Handel (1961). About 0.1 ml of the lipid extract was mixed with 1.0 ml of chloroform-methanol mixture, added 50 mg of activated silicic acid, shaken vigorously, allowed to stand for 30 min and centrifuged. To 0.5 ml of the supernatant, as well as standard and blank, 0.5 ml of alcoholic potassium hydroxide was added and the mixture was saponified in a Water bath at 60-70°C for 20 min. To this 0.5 ml of 0.2 N sulphuric acid was added, and
27 boiled in a Water bath for 30 min. After cooling the tubes, 0.1 ml of sodium meta periodate was added and allowed to stand for 10 min.
The excess periodate was reduced by the addition of 0.1 ml of sodium meta arsenite. Then 0.5 ml of chromotrophic acid was added, mixed thoroughly, and boiled in a Water bath for 30 min. After cooling 0.5 ml of thio urea solution was added and the colour developed was read at 570 nm using Spectrophotometer.
The triglycerides level was expressed as mg/dl of plasma.
3.6.8.4. Estimation of Phospholipids
Phospholipids in the plasma, erythrocytes and tissues were estimated by the method of Zilversmit and Davis (1950). Phospholipids were digested with concentrated sulphuric acid to liberate the lipid bound inorganic phosphorus. Ammonium molybdate was added to form phosphomolybdic acid which was treated with 1-amino-2-naphthol-
4-sulphonic acid to form a stable blue colour. The intensity of the colour was proportional to the amount of phospholipids in the sample.
The volume of 0.5 ml of lipid extract was evaporated to dryness. One ml of 5.0
N sulphuric acid was added and digested till the appearance of light brown colour.
Then 2 to 3 drops of concentrated nitric acid were added and the digestion was continued till the solution became colourless. After cooling, 1 ml of water was added and heated in a Water bath for about 5 min. Then, 1.0 ml of ammonium molybdate and
0.1 ml of ANSA were added. The volume was then made up to 10 ml with distilled water and the absorbance was measured at 680 nm within 10 min. Standards in the concentration range of 2-8 mg were treated in a similar manner. The values obtained were multiplied with a factor 25 to convert inorganic phosphorus to its phospholipid equivalents.
28 The amount of phospholipids was expressed as mg/dl of plasma.
3.6.8.5. Estimation of Plasma Insulin
Plasma insulin was assayed by the solid phase system amplified sensitivity immunoassay using Reagent Kits obtained from Medgenix-INS-ELISA, Biosource,
Europe S.A., Belgium (Burgi et al., 1988). The assay was based on the oligoclonal system in which several monoclonal antibodies (Mabs) directed against distinct epitopes of insulin were used.
Standards and samples containing insulin react with captive antibodies coated on a plastic well and with monoclonal antibodies labelled with horseradish peroxidase
(HRP). Sufficient strips were selected to accommodate standards, controls and all test samples. The strips were fitted to the holding frame. Fifty μl of each standard, control or samples was dispensed into the appropriate wells. Time between distribution of first standard and last sample was kept minimum. Fifty μl of antiserum HRP conjugate was dispended into all wells and incubated for 30 min at room temperature on a horizontal shaker set at 700 rpm. The plates were washed after aspirating the liquid from the well.
Then 0.4 ml of washing solution was dispensed into each well and the contents were aspirated. This was repeated twice for complete washing. Two hundreard μl of the freshly prepared revelation solution was added into each well 15 min after washing.
Then the plate was incubated for 15 min on a horizontal shaker set at 700 rpm at room temperature, avoiding direct sunlight and 50 μl of arresting reagent was added into each well. The absorbance was read within 1h at 450 nm.
The insulin concentration was expressed as μU/ml of plasma.
3.6.8.6. Estimation of Glycosylated Haemoglobin (HbA1C) Glycosylated haemoglobin in the blood was estimated by the method of
Sudhakar Nayak and Pattabiraman (1981). The volume of 0.5 ml of saline washed
29 erythrocytes was lysed with 5.5 ml of water, mixed and incubated at 37°C for 15 min.
The contents were centrifuged and the supernatant was discarded, then 0.5 ml of saline was added, mixed and processed for estimation. To 0.2 ml of aliquot, 4 ml of oxalate hydrochloric solution was added, mixed, heated at 100°C for 4 h, cooled and precipitated with 2 ml of 40% TCA. The mixture was centrifuged. To a 0.5 ml of supernatant, 0.05 ml of 80% phenol and 3.0 ml of concentrated sulphuric acid were added. The colour developed was read at 480 nm after 30 min.
The concentration of glycosylated Hb was expressed as mg/g of haemoglobin.
3.6.8.7. Assay of Hexokinase
Hexokinase activity was assayed by the method of Brandstrup et al. (1957). The reaction mixture in a total volume of 5.0 ml contained 1.0 ml of glucose solution, 0.5 ml of ATP solution, 0.1 ml of magnesium chloride solution, 0.3 ml of potassium dihydrogen phosphate, 0.3 ml of potassium chloride, 0.3 ml of sodium fluoride and 2.5 ml of Tris-HCl buffer (pH:8.0). The mixture was pre-incubated at 37°C for 5 min. The reaction was initiated by the addition of 2.0 ml of tissue homogenate; 1.0 ml of the reaction mixture was immediately removed to the tubes containing 1.0 ml of 10% TCA which was considered as zero time. A second aliquot was removed after 30 min incubation at 37°C.
The enzyme activity was expressed as µmol of glucose phosphorylated/h/mg protein.
3.6.8.8. Assay of Glucose 6-phosphatase
Glucose 6-phosphatase was assayed by the method of Koide and Oda (1959).
The incubation mixture containing 0.3 ml buffer, 0.5 ml glucose 6-phosphate and 0.2 ml tissue homogenate was incubated at 37°C for 1 h. One ml 10% TCA was added to the tubes to terminate the enzyme activity, then centrifuged and the phosphate content
30 of the supernatant was estimated by the Fiske and Subbarow (1925) method. To a 1 ml of the aliquot of supernatant, 1 ml of ammonium molybdate and 0.4 ml ANSA reagent were added. After 20 min the blue colour developed was read at 620 nm. A tube devoid of the enzyme served as control. A series of standards containing 8-40 μg of phosphorus was treated similarly along with a blank containing only the reagent.
The enzyme activity was expressed as µmole of phosphorous liberated/min/mg protein.
3.6.8.9. Assay of Fructose 1, 6-bisphosphatase
Fructose 1, 6-bisphosphatase was assayed by the method of Gancedo and
Gancedo (1971). The assay medium in a final volume of 2.0 ml containing 1.0 ml buffer, 0.4 ml of substrate, 0.1 ml of magnesium chloride, 0.2 ml potassium chloride,
0.1 ml of EDTA and 0.2 ml of enzyme source was incubated at 37˚C for 15 min. The reaction was terminated by the addition of 1.0 ml of 10% TCA. The suspension was centrifuged and the phosphorus content of the supernatant was estimated by Fiske and
Subbarow (1925) method. To a 1 ml of an aliquot of the supernatant, 0.3 ml of distilled water and 0.5 ml of ammonium molybdate were added. After 10 min, 0.2 ml of ANSA reagent was added. The tubes were shaken well, kept aside for 20 min and the blue colour developed was read at 620 nm.
The values were expressed as µmole of phosphorous liberated/min/mg of protein.
3.6.8.10. Estimation of Tissue Protein
Protein in the tissues was determined after trichloro acetic acid precipitation by the method of Lowry et al. (1951). The volume of 0.5 ml of tissue homogenate was mixed with 0.5 ml of 10% TCA and centrifuged for 10 min. The precipitate was dissolved with 1.0 ml of 0.1 N NaOH. From this, an aliquot was taken, and 4.5 ml of
31 alkaline copper reagent was added and allowed to stand at room temperature for 10 min, added 0.5 ml of Folin’s phenol reagent after 20 min, read the blue colour developed at 640 nm. A standard curve was obtained with standard bovine albumin and used to assay the tissue protein level for enzyme activity.
Values were expressed as g/dl of blood
3.6.8.11. Estimation of Haemoglobin
Haemoglobin in the blood was estimated by the method of Drabkin and Austin
(1932). The dilution of blood in an alkaline solution containing potassium cyanide and potassium ferricyanide forms the basis of this method. Haemoglobin gets oxidized forming cyanmethaemoglobin whose absorbance was then measured at 540 nm.
To a 0.02 ml of blood, 5.0 ml of Drabkin’s reagent was added, mixed well, allowed to stand for 10 min and read at 540 nm together with the standard solution against a reagent blank.
Values were expressed as g/dl of blood.
3.6.8.12. Estimation of Albumin
Albumin in serum was estimated by Biuret method (Reinhold, 1953). A 0.5 ml of the sample was layered onto 9.5 ml of sodium sulphate in a centrifuge tube and it was inverted to mix. Two ml of the mixture was taken immediately and marked as total protein. The rest of the solution was allowed to stand for 10-15 min for precipitation of globulin and filtered using Whattman filter paper. The filtrate contains the albumin and
2 ml of filtrate was taken and marked as total albumin. The contents of all the tubes were made up to 2.5 ml of Biuret reagent, mixed and kept for 10 min. A series of standard were prepared and treated as the test. The purple or violet colour developed was read Colorimetrically at 540 nm.
32 The serum albumin levels were expressed as g/dl.
3.6.8.13. Estimation of Urea
The urea was estimated by diacetylmonoaxime method of Natelson (1957).
Urea reacts with hot acidic diacetylmonoxime, in the presence of thiosemincarbazide and produces a rose purple coloured complex, which gives an absorption maximum at
525 nm.
To 2.5 ml of diluted urea reagent, 0.01 ml of serum was added and mixed well.
A 0.5 ml of diacteyl monoxime (DAM) reagent was added to the above mixture and mixed again. The samples were kept in a Water bath exactly for 10 min and cooled by running water for 5 min. Optical density was read at 525 nm. Calculation was done using 30% urea as standard.
Urea were expressed as mg/dl of blood.
3.6.8.14. Estimation of Uric Acid
The uric acid was estimated by uricase method of Fossati and Prencipe (1980).
Uric acid is the end product of purine metabolism and is excreted to a large degree by the kidneys and to a smaller degree in the intestinal tract by microbial degradation.
Increased levels are found in impaired renal function.
Three test tubes labeled as blank, standard and test were taken and one ml of working reagent (equal amount of buffer reagent and enzyme reagent) was added in to the three test tubes. Then 0.02 ml of distilled water was added in to the blank test tube,
0.02 ml of serum in to the test tubes and 0.02 ml of uric acid standard was added. All the tubes were mixed well and incubated at room temperature for 15 min and then the absorbance of the standard and test were measured against the blank within 30 min.
Uric acid were expressed as mg/dl of blood.
33 3.6.8.15. Estimation of Creatinine
The creatinine was estimated by the modified jaffe’s kinetic method of Bowers and Wong (1980). To the volume of 0.5 ml of picric acid reagent, 0.5 ml of buffer reagent was taken in two test tubes labeled as standard and test. A 0.1 ml of creatinine was added into the standard and 0.1 ml of serum in to the test tube. All the test tubes were stirred and absorbance was read at 520 nm after exactly 30 sec. The observation was repeated exactly after 60 sec. The changes in absorbance in both the standard and test were calculated.
Creatinine were expressed as mg/dl of blood.
3.6.8.16. Estimation of Glutathione Peroxidase (GPx)
The activity of glutathione peroxidase was measured by the method of Rotruck et al. (1973). A known amount of enzyme preparation was allowed to react with H2O2 in the presence of GSH for a specified time period. Then the remaining GSH content was measured.
The tissue was homogenized using Tris buffer. To 0.2 ml of Tris buffer, 0.2 ml of EDTA, 0.1 ml of sodium azide and 0.5 ml of tissue homogenate or hemolysate were added. To the mixture, 0.2 ml of GSH followed by 0.1 ml of H2O2 was added. The contents were mixed well and incubated at 37°C for 10 min, along with a control containing all reagents except homogenate. After 10 min, the reaction was arrested by the addition of 0.5 ml of 10% TCA.
Glutathione Peroxidase was expressed as µmole of glutathione utilized/min.
3.6.8.17. Estimation of Reduced Glutathione (GSH)
Reduced glutathione was estimated by the method of Ellman (1959). This method was based on the formation of 2-nitro-5-thiobenzoic acid (a yellow colour
34 compound) and then added the 5,5-dithio-bis (2-nitrobenzoic acid) (DTNB) to compounds containing sulphydryl groups.
A known weight of tissue was homogenized in phosphate buffer (0.1 M pH
7.0). A 0.5 ml of the homogenate of plasma was pipetted out and precipitated with 2.0 ml of 5% TCA. Two ml of supernatant was taken after centrifugation and 1.0 ml of
Ellman’s reagent and 4.0 ml of 0.3 M disodium hydrogen phosphate were added. The yellow colour developed was read in a Spectronic 20D at 412 nm. A series of standards
(20–100 μg) was treated in a similar manner along with a blank containing 1.0 ml of buffer.
Reduced glutathione was expressed as mg/dl of plasma or mg/100 mg of protein for tissues.
3.6.8.18. Estimation of Glutathione-S-Transferase (GST)
The activity of glutathione-S-transferase was determined by the method of
Habig et al. (1974). To a 1.0 ml of phosphate buffer, 0.1 ml of CDNB, 17 ml of water and 0.1 ml of enzyme source were added. After 5 min of incubation at 37°C, 0.1 ml of
GSH was added and the change in optical density was measured immediately in a UV-
Vis Spectrophotometer with an interval of 1 min, for 3 min at 340 nm. A complete assay mixture without enzyme was used as control.
The activity of glutathione-S-transferase was expressed as µmole of CDNB-
GSH conjugate formed/min/mg/protein.
3.6.8.19. Assay of Superoxide Dismutase (SOD)
Superoxide dismutase in the tissues was assayed by the method of Kakkar et al.
(1984). The assay is based on the inhibition of the formation of NADH- phenazinemethosulphate, nitroblue tetrazolium formazon. The reaction was initiated by
35 the addition of NADH. After incubation for 90 sec, glacial acetic acid was added to stop the reaction. The colour developed at the end of the reaction was extracted into n- butanol layer and measured in a Spectronic 20D at 520 nm.
The tissue was homogenized by using sodium pyrophosphate buffer (0.025 M, pH 8.3). A 0.5 ml of tissue homogenate or 0.5 ml of serum was diluted to 1.0 ml with water followed by addition of 2.5 ml of ethanol and 1.5 ml of chloroform (chilled reagents were added). This mixture was shaken for 90 sec at 4°C and then centrifuged.
The enzyme activity in the supernatant was determined as follows.
The assay mixture contained 1.2 ml of sodium pyrophosphate buffer, 0.1 ml of phenazine methosulphate, and 0.3 ml of nitroblue tetrazolium and appropriately diluted enzyme preparation in a total volume of 3 ml. The reaction was started by the addition of 0.2 ml NADH. After incubation at 30°C for 90 sec, the reaction was stopped by the addition of 1 ml glacial acetic acid. The reaction mixture was stirred vigorously shaken with 4 ml n-butanol; allowed to stand for 10 min and centrifuged. The n-butanol layer was separated. The colour density of the chromogen in n-butanol was measured in at
510 nm. A system devoid of enzyme served as control. The enzyme concentration required to inhibit the chromogen produced, by 50% in 1 min, under standard conditions, was taken as one unit.
Superoxide dismutase activity of the enzyme was expressed as Unit/min/mg of protein for tissues.
3.6.8.20. Estimation of Catalase (CAT)
The activity of catalase was determined by the method of Sinha (1972).
Dichromate in acetic acid was converted to perchromic acid and then to chromic acetate, when heated in the presence of H2O2. The chromic acetate formed was measured at 620 nm. The catalase preparation was allowed to split H2O2 for various
36 periods of time. The reaction was stopped at different time intervals by the addition of dichromate-acetic acid mixture and the remaining H2O2 as chromic acetate was determined Colorimetrically.
The tissue homogenate was prepared by using phosphate buffer (0.01 M, pH
7.0). To 0.9 ml of phosphate buffer, 0.1 ml of tissue homogenate or 0.1 ml of hemolysate and 0.4 ml of hydrogen peroxide were added. The reaction was arrested after 15, 30, 45 and 60 sec by adding 2.0 ml of dichromate-acetic acid mixture. The tubes were kept, boiled in a Water bath for 10 min and cooled and the colour developed was read at 620 nm. Standards in the concentration range of 20-100 μM were taken and processed for testing.
Catalase activity was expressed as μmole H O utilized/min/mg of protein for 2 2 tissues.
3.6.8.21. Estimation of Hydroperoxides (HPx)
Hydroperoxides were estimated by the method of Jiang et al. (1992). Oxidation of ferrous ion (Fe2+) under acidic conditions in the presence of xylenol orange leads to the formation of a chromophore with an absorbance maximum at 560 nm.
The volume of 0.9 ml Fox reagent was added to 0.1 ml of the sample, incubated for 30 min at room temperature and the absorbance read in a Spectronic 20 D at 560 nm.
Hydroperioxdes were expressed as µmole/ml of plasma or µmole/mg of protein for tissues.
3.6.8.22. Estimation of Thiobarbituric Acid Reactive Substances (TBARS)
Thiobarbituric acid reactive substances was estimated by the method of Niehaus and Samuelson (1968). In this method, malondialdehyde and other thiobarbituric acid
37 reactive substances (TBARS) react with thiobarbituric acid in an acidic condition to generate pink colour chromophore which was read at 535 nm.
The volume of 0.5 ml of sample was diluted with 0.5 ml of double distilled water and mixed well, and then 2.0 ml of TBA-TCA-HCl reagent was added. The mixture was kept and bolied in a Water bath for 15 min. After cooling the tubes were centrifuged at 1000 rpm for 10 min and the supernatant was estimated. A series of standard solutions in the concentration of 2-10 nm was treated in a similar manner. The absorbance of the chromophore was read at 535 nm against reagent blank.
Thiobarbituric acid reactive substances were expressed as nmole/dl of plasma or nmole/mg of protein.
3.6.8.23. Estimation of Ascorbic Acid
Ascorbic acid (vitamin C) was estimated by the method of Roe and Kuether
(1943). The ascorbic acid was converted to dehydroascorbic acid by mixing with nitric acid and then coupled with 2,4 dinitrophenyl hydrazine (DNPH) in the presence of thiourea, a mild reducing agent. The coupled dinitrophenyl hydrazine was converted into a red coloured compound when treated with sulphuric acid.
To a 0.5 ml of the sample, 1.5 ml of 4% TCA was added, allowed to stand for 5 min and centrifuged. To the supernatant, 0.3 ml of nitric acid was added, shaken vigorously and filtered. This converts ascorbic acid to dehydroascorbic acid. A 0.5 ml of the filtrate was taken and 0.5 ml of DNPH was added, stoppered, placed in Water bath at 37°C for 3 h and then placed in ice-cold water and 2.5 ml of 85% sulphuric acid was added drop by drop. The contents of each of the tubes were mixed well and allowed to stand at room temperature for 30 min. A set of standards containing 20-100
μg of ascorbic acid were taken and processed similarly along with a blank containing
2.0 ml of 4% TCA. The colour developed was read at 540 nm.
38 Ascorbic acid were expressed as mg/dl of plasma or μg/mg of protein for tissue.
3.6.8.24. Estimation of α–Tocopherol
α–tocopherol (Vitamin E) in the plasma, erythrocytes and tissues was estimated by the method of Baker et al. (1980). The method involves the reduction of ferric ions to ferrous ions by α–tocopherol and the formation of a red coloured complex with 2,2- dipyridyl. Absorbance of the chromophore was measured at 520 nm.
To a 0.5 ml of the sample, 1.5 ml of ethanol was added, mixed and centrifuged.
The supernatant was evaporated and to the precipitate, 3.0 ml of petroleum ether, 0.2 ml of 2,2- dipyridyl solution and 0.2 ml of ferric chloride solution were added, mixed well and kept in the dark for 5 min. An intense red colour was developed. Four ml of n- butanol was added to all the tubes and mixed well. Standard tocopherol in the range of
10-100 μg was taken and treated similarly along with a blank containing only the reagent. The colour in the n-butanol layer was read at 520 nm.
α–Tocopherol were expressed as mg/dl for plasma or μg/mg protein for tissue.
3.6.8.25. Assay of Alkaline Phosphatase (ALP)
Alkaline phosphatase was assayed by the method of King (1965) using disodium phenyl phosphatase as the substrate. Disodium phenyl phosphate is hydrolysed by alkaline phosphatase with the liberation of phenol, which reacts under alkaline condition with Folin-phenol reagent to form blue colour, which is estimated
Colorimetrically at 680 nm.
An incubation mixture containing 150 µM of bicarbonate buffer and 10 µmoles of substrate in 2.9 ml distilled water was preincubated at 37°C for 10 min. A 0.2 ml of serum was added to this and incubated at 37°C for 15 min. The reaction was arrested by the addition of 1.0 ml of Folin-phenol reagent. The suspension was centrifuged and 2.0
39 ml of 10% sodium carbonate was added to the supernatant. The solution was incubated at 37°C for 10 min. Standard phenol solution (2.5 µg-10.0 µg) was also treated with
Folin-phenol reagent and sodium carbonate. The blue colour developed was read at 680 nm.
Alkaline Phosphatase were expressed as IU/I of serum.
3.6.8.26. Assay of Acid Phospatase (ACP)
The activity of acid phosphatase was estimated by the method of Gutman and
Gutman (1980). About 1.0 ml of the buffered substrate was pipetted out into two tubes containing 0.1 ml of serum and 0.2 ml of distilled water was added to the blank. The tubes were placed in a water bath at 37°C. Then to the test, 0.2 ml of NAD was added and shaken well, the tubes were incubated at 37°C for 15 min. Exactly after the time of incubation, the reaction was arrested by adding 1.0 ml of DNPH reagent followed by the addition of NAD to the control tubes. It was left at 37°C for 15 min. Five ml of 0.4
N NaOH was added. The colour developed was read at 540 nm using
Spectrophotometer.
Acid Phospatase activity was expressed as IU/I.
3.6.8.27. Assay of Aspartate Aminotransferase (AST)
Serum aspartate aminotransferase was estimated by the method of Reitman and
Frankel (1957). AST catalyses the transfer of amino group from L-aspartate to α- ketoglutarate with the formation of oxaloacetate and glutamate. The amount of oxaloacetate was measured by converting it into pyruvate by treating with aniline citrate and then reacting the pyruvate with 2,4-dinitrophenylhydrazine to form 2,4- dinitrophenylhydrazone derivative which is brown coloured in alkaline medium. The absorbance of this hydrazone derivative is correlated to AST activity.
40 The volume of 0.5 ml of buffered substrate was added to 0.1 ml of serum and placed in a Water bath at 37°C. To the blank tubes, 0.1 ml distilled water was added instead of serum. Exactly an hour later, two drops of aniline citrate reagent and 0.5 ml of DNPH reagent were added and kept at room temperature for 20 min. Finally, 5 ml of
0.4 N sodium hydroxide was added. A set of standards were also treated in the same manner and read using Spectrophotometer at 520 nm after 10 min.
Aspartate aminotransferase were expressed as IU/I of serum.
3.6.8.28. Assay of Alanine Aminotransferase (ALT)
Serum alanine aminotransferase was estimated by the method of Reitman and
Frankel (1957). ALT catalyses the transfer of amino group from L-alanine to α- ketoglutarate with the formation of pyruvate and glutamate. The pyruvate so formed, is allowed to react with 2,4-dinitrophenylhydrazine to produce 2,4- dinitrophenylhydrozone derivative which is brown coloured in alkaline medium. The absorbance of this hydrazone derivative is correlated to ALT activity.
The volume of 0.5 ml of buffered substrate was added to 0.1 ml of serum and placed in a Water bath at 37°C. To the blank tubes, 0.1 ml distilled water was added instead of serum. Exactly an hour later, two drops of aniline citrate reagent and 0.5 ml of DNPH reagent were added and kept at room temperature for 30 min. Finally, 5 ml of
0.4 N sodium hydroxide was added. A set of standards were also treated in the same manner and read using Spectrophotometer at 520 nm after 10 min.
Alanine aminotransferase expressed as IU/I of serum.
41 3.6.9. Histopathological Studies
A portion of liver and kidney were removed after sacrificing the animal and rapidly placed in 10% phosphate buffered-formalin for histological examination.
Tissues were dehydrated in alcohol, embedded in paraffin wax, sectioned in 5 µm and stained with haematoxylin and eosin for Light Microscopy. The sections were examined at 40x magnification.
3.6.10. Statistical Analysis
Statistical analysis was performed using SPSS Software Package, version 11.5.
The values were analyzed by One Way Analysis of Variance (ANOVA) followed by
Duncan’s Multiple Range Test (DMRT). All these results were expressed as mean ±SD for six rats in each group, p-values <0.05 were considered as significant (Duncan,
1957).
42 Chapter 4 RESULTS AND DISCUSSION
4.1. Micropropagation and Callus Induction
In order to establish an efficient in vitro micropropagation system, the leaf explants were inoculated on MS solid medium supplemented with varying concentrations of either BAP/Kinetin alone and in combination with NAA respectively.
Leaf segments cultured on MS basal medium (without growth regulators) did not show any growth response. However, on MS basal medium supplemented with various concentrations of cytokinin alone or in combination with auxin swelled in their size after 1-2 weeks of culture and differentiated auxillary shoots in another 2 weeks (Table
6 & fig. 1a, b).
The combination of BAP (0.5 mg/l) and NAA (1.0 mg/l) positively affected the multiplication rate of the plant compared with BAP alone. Successful results of shoot multiplication from leaf explants cultured on MS medium were obtained. The multiplication (at the rate of 95% explants) produced shoots with an average of 73 explants of the plant compared with BAP and Kinetin alone and Kinetin combination with NAA (Fig. 1c).
The regenerated shoots were excised and placed on the half strength MS medium supplemented with different concentrations 0.5-1.5 mg/l of NAA, IAA and
IBA alone for rooting (Table 7). IBA is clearly more effective in promoting root induction than NAA and IAA. The optimum rooting efficiency for shoots (95%) as well as the best root number per shoot were obtained on MS media supplemented with 0.5 mg/l and 1.0 mg/l IAA respectively (fig. 1d). Plantlets on MS medium fortified with
43
Table 6. Effects of different concentrations of plant growth regulators on in vitro shoot proliferation from the leaf explants of Pergularia daemia after 4 weeks of culture
Plant growth regulators (mg/l) No. of shoots/explants Regeneration BAP NAA KIN (mean±SE) efficiency (%) 0.5 0 0 3.3±0.83 64.0 1.0 0 0 2.0±0.33 52.0 2.0 0 0 3.0±0.58 39.0 4.0 0 0 3.0±1.00 42.0 0.5 0.5 0 1.7±0.67 56.0 1.0 0.5 0 1.3±0.33 61.0 2.0 0.5 0 3.1±0.76 67.0 4.0 0.5 0 3.2±0.31 53.0 0.5 1.0 0 7.3±0.88 95.0 1.0 1.0 0 5.6±0.22 79.0 2.0 1.0 0 4.8±0.11 82.0 4.0 1.0 0 3.9±0.10 65.0 0 0 0.5 3.0±0.58 69.0 0 0 1.0 2.7±0.88 55.0 0 0 2.0 2.3±0.33 52.0 0 0 4.0 5.0±0.28 65.0 0 0.5 0.5 4.3±0.22 52.0 0 0.5 1.0 3.4±0.11 59.0 0 0.5 2.0 2.8±0.14 48.0 0 0.5 4.0 2.3±0.33 49.0 0 1.0 0.5 3.3±0.22 57.0 0 1.0 1.0 3.2±0.88 42.0 0 1.0 2.0 4.0±0.33 61.0 0 1.0 4.0 2.9±0.31 63.0
Table 7. Effects of auxins for rooting of in vitro derived shoots of Pergularia daemia after 4 weeks of culture
Number of shoots Number of roots per Auxin (mg/l) Rooting (%) cultured shoot (mean±SE)
NAA
0.5 30 4.8±0.10 49.0
1.0 30 6.1±0.12 45.0
1.5 30 2.3±0.33 58.0
2.0 30 3.0±0.58 63.0
IBA
0.5 30 9.38±0.82 92.0
1.0 30 3.9±0.63 71.0
1.5 30 5.5±0.97 69.0
2.0 30 2.8±0.11 46.0
IAA
0.5 30 5.23±0.11 43.0
1.0 30 6.8±0.27 66.0
1.5 30 5.6±1.30 55.0
2.0 30 3.5±1.63 69.0
high concentration of auxin at 1.5 mg/l grew slowly, turned yellow in colour, thick, short with callus forming capacity at the basal cut end.
The hardening process was carried out by transferring 9-10 cm length rooted plantlets to 8.0 cm diameter plastic pot containing mixture of soil and vermicompost mixture in the ratio of 3:1. Hardening of potted plants for 2 weeks in a Growth
Chamber (fig. 1e). The survival percentage of the plantlets was 90% after transplantation to soil and sand mixture. Plants transferred to the Net House have established well in the soil, appeared to be morphologically uniform and successfully adapted to Net House mist conditions (Fig. 1f).
Callus induction as initiated from stem and leaf explants on Murashige and
Skoog (1962) medium containing different concentrations of 2,4-D (1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) with BAP (0.5 mg/l); 2,4-D (1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) with Kinetin
(0.5 mg/l); 2,4-D (1.0, 1.5, 2.0, 2.5 and 3.0 mg/l) with α-NAA (1.0 mg/l); 2,4-D (1.0,
1.5, 2.0, 2.5 and 3.0 mg/l) with α-NAA (1.0 mg/l) and BAP (0.5 mg/l). The maximum induction rate was recorded as 94.2% in leaf explants of 2,4-D 2.0 mg/l combination with 0.5mg/l BAP and 1.0 mg/l α-NAA and 87.1% in stem explants on MS medium with 2.0 mg/l 2,4-D and 0.5 mg/l BAP and 1.0 mg/l α-NAA. The callus nature was delicate with pale green in colour (Table 8).
Stem and leaf explants were cultured on MS medium which was free of growth regulators did not produce any callus. However, MS medium with single BAP induced callus in both stem and leaf explants, the callus was turned brown. A high concentration of 2,4-D in combination with BAP and α-NAA in medium was relatively more suitable for subculture. The well grown callus was selected for subculture on MS medium with
2.0 mg/l 2,4-D in combination with 0.5 mg/l BAP and 1.0 mg/l α-NAA to test their growing state.
44 In 2,4-D with BAP supplemented medium, the callus was pale, compact and hard for stem (Fig. 2) and leaf (Fig. 3). In 2,4-D with Kinetin supplemented medium, the callus was compact, hard and granular for stem (Fig. 4) and leaf (Fig. 5). In 2,4-D with-NAA supplemented medium, the callus was yellow, loose and sponge for stem
(Fig. 6) and leaf (Fig. 7). But in combination with 2,4-D, α-NAA and BAP supplemented medium the callus was pale green, loose and compact for stem (Fig. 8) and leaf (Fig. 9). In BAP medium, more biomass yield was achieved than in Kinetin.
The yield of fresh biomass was high on medium with 2,4-D 141.25 gfw/l; 8.02 gdw/l followed by α-NAA 114.64 gfw/l; 7.00 gdw/l and IBA 85.5 gfw/l; 5.21 gdw/l and BAP
136.10 gfw/l; 7.5 gdw/l and KIN 94.21 gfw/l; 6.21 gdw/l (Table 9). The biomass yield was higher in combination of auxins 2,4-D and α-NAA with BAP. About 2.0 g of actively growing calli were inoculated in conical flasks, each containing 30 ml of solid medium. The combination of 2,4-D, α-NAA and BAP showed more callus biomass yield than the combinations of auxins with Kinetin. The yield of fresh biomass in BAP supplemented medium was about three times more than that in Kinetin supplemented medium. Dry biomass was about double in BAP than Kinetin supplemented medium.
On the basis of yield of both fresh and dry biomass, BAP is seen to have greater effect than Kinetin. Based on the above results, it is found that the PGR combinations of 2,4-
D - 2.0 mg/l, α-NAA-1.0 mg/l and BAP-0.5mg/l yielded maximum biomass. These calli were used for phytochemical screening.
The development of an efficient and reproducible regeneration protocol from cells or tissues holds tremendous potential for the production of high quality plant based medicines (Murch et al., 2000). Plant regeneration from leaf segments is considered to be one of the most promising ways for multiplying a selected variety showing the same agronomic characteristics. It is evident from the results that
P. daemia can be easily clonally mass prospected in vitro using leaf segments as
45 Table 8. Effect of different concentrations of plant growth regulators on callus induction and growth in stem and leaf explants of Pergularia daemia
Rate of callus PGR (mg/l) induction Growing State S.No %±S.D 2,4-D BAP KIN α-NAA Stem Leaf Stem Leaf Pale, compact, Pale, compact, 1 1.0 0.5 0 0 15.5±2.1 16.8±2.2 hard hard Pale, compact, Pale, compact, 2 1.5 0.5 0 0 50.8±1.8 55.8±2.0 hard hard Pale, compact, Pale, compact, 3 2.0 0.5 0 0 26.2±2.0 31.1±3.2 hard hard Pale, compact, Pale, compact, 4 2.5 0.5 0 0 28.3±2.5 30.8±3.8 hard hard Yellow, loose, Yellow, loose, 5 1.0 0 0 1.0 78.5±1.7 76.7±2.5 sponge sponge Yellow, loose, Yellow, loose, 6 1.5 0 0 1.0 67.5±2.0 53.8±4.1 sponge sponge Yellow, loose, Yellow, loose, 7 2.0 0 0 1.0 37.5±3.5 40.0±1.8 sponge sponge Yellow, loose, Yellow, loose, 8 2.5 0 0 1.0 49.2±1.8 61.7±2.5 sponge sponge Compact, Compact, hard, 9 1.0 0 0.5 0 52.1±2.8 58.2±1.6 hard, granular granular Compact, Compact, hard, 10 1.5 0 0.5 0 48.2±1.1 50.1±0.5 hard, granular granular Compact, Compact, hard, 11 2.0 0 0.5 0 36.3±1.7 46.5±1.4 hard, granular granular Compact, Compact, hard, 12 2.5 0 0.5 0 49.2±2.7 52.1±1.9 hard, granular granular Pale green, Pale green, 13 1.0 0.5 0 1.0 70.5±1.2 73±2.2 loose, compact loose, compact Pale green, Pale green, 14 1.5 0.5 0 1.0 68.2±2.0 71.6±0.8 loose, compact loose, compact Pale green, Pale green, 15 2.0 0.5 0 1.0 87.1±2.1 94.2±1.5 loose, compact loose, compact Pale green, Pale green, 16 2.5 0.5 0 1.0 52.1±1.1 57.3±1.6 loose, compact loose, compact
Table 9. The effect of auxins and cytokinins on Pergularia daemia callus culture
Auxins Cytokinins
2,4-D α-NAA IBA BAP KIN PGR
mg/l f.w d.w f.w d.w f.w d.w f.w d.w f.w d.w
1.0 120.56 8.72 79.16 5.12 68.5 4.41 109.70 6.4 90.89 2.21
1.5 132.10 9.01 91.89 6.18 80.21 6.72 121.60 4.3 96.59 3.81
2.0 141.25 8.02 114.64 7.00 85.51 5.21 136.10 7.5 94.21 6.21
2.5 110.57 6.54 81.26 5.29 79.60 4.81 110.70 2.3 105.7 5.8
3.0 97.52 3.28 76.56 4.39 71.5 3.21 106.20 8.5 98.20 3.4
explants. In vitro micropropagation of P. daemia has not been reported, even though many medicinal plants, it has been reported in plant regeneration from shoot and leaf meristems has yielded encouraging results in medicinal plants like Solanum nigrum,
Cunila galioides and Rauwolfia micrantha (Sudha and Seeni, 1996; Fracaro and
Echeverrigaray, 2001; Ugandhar et al., 2010).
The results of the present study show that BAP in combination with NAA was more effective in shoot multiplication than KIN alone or KIN in combination with
NAA. The effects of auxins and cytokinins on shoot multiplication and in vitro rooting of various medicinal plants were reported by Wawrosch et al. (1999).
BAP alone and with NAA induces a high rate of shoot proliferation in
Hemidesmus indicus and Gymnema sylvestre. The number of shoots explant -1 depends on the concentration of the growth regulators (Sairam Reddy et al., 1998; Saha et al.,
2003). This investigation supports the finding in P. daemia that the MS medium supplemented with BAP 0.5mg/l and NAA 1.0 mg/l leads to maximum shoot induction and IBA 0.5 mg/l leads to greater root induction.
The objective of the suspension culture is to obtain well grown calli. Auxins and cytokinins are the most widely used plant growth regulators in plant tissue culture
(Gang et al., 2003). Subsequently plant growth regulators were added into the MS medium to test their effect on callus formation of cotyledon and hypocotyls explants of
P. daemia. The results reveal that auxins play an important role in the callus induction and different types of auxins showed various effects (Baskaran et al., 2006). The 2,4-D is superior than α-NAA in callus induction in P. daemia.
Further more, the cytokinins facilitated the effect of auxins in callus induction
(Rao et al., 2006). The leaf and stem responded differently to auxins especially to the single α-NAA supplemented medium of P. daemia. The callus induced from medium
46
Table 10. Phytochemical screening of various solvents extracts of Pergularia daemia
Phytochemicals Chloroform Ethanol Aqueous Ethanol extract extract extract callus extract
Alkaloids + + + ++
Flavonoids + + + ++
Glycosides + + - ++
Carbohydrates - + + ++
Tannins + + - ++
Terpenoids - + - +
Steroids + + + ++
‘++’ indicates more, ‘+’ indicates present, ‘-’ indicates absent.
with 2,4-D and BAP was light compact, but when they were transferred to new medium with α-NAA and BAP they become yellow, loose and more suitable for suspension culture. Numerous research reports exist in the literature about the effects of plant growth regulators on secondary metabolites through in vitro cultures, e.g. enhancement of berberine alkaloid production in Coptis japonica (Nakagawa et al., 1986).
4.2. Phytochemistry
The results of the preliminary phytochemical investigation revealed the presence of alkaloids, flavonoids and steroids in all the solvent extracts, and glycosides and tannins in chloroform and ethanol extracts, carbohydrates in chloroform and ethanol extracts and terpenoids in ethanol extracts. However, callus ethanol extract showed the presences of all these compounds (Table 10). The GC-MS analysis of the various extracts and callus ethanol extract are given below with parameters such as
Retention time (RT), Molecular formula (MF), Molecular weight (MW) and Peak area (%).
Five compounds (Fig. 10 & Table 11) such as benzeneethanamine 2,5-difluoro-
β,3,4-trihydroxy-N-methyl (fig.11); 2-naphthalenecarboxylic acid 1,2,3,4-tetrahydro-3- hydroxy-8-methoxy-ethyl ester (fig.12); l-gala-l-ido-octose (fig.13); 17- (1,5- dimethylhexyl)- 10, 13-dimethyl-3 styryl hexadecahydrocyclopenta [a] phenanthren-2- one (fig.14); 7,8-Epoxylanostan-11-ol,3-acetoxy (31.5%) (fig.15) were identified in the chloroform leaf extract.
47 Fig. 10. Chromatogram of chloroform leaf extract of Pergularia daemia
Table 11. Compounds identified from the chloroform leaf extract of Pergularia daemia
Peak Molecular Molecular S.No. RT Name of the compound Area formula weight % Benzeneethanamine,2,5-difluoro-β,3,4 25.7% 1. 3.1 C9H11F2NO3 699 trihydroxy-N-methyl 2-Naphthalenecarboxylic acid, 1,2,3,4- 2. 12.2 C H O 580 7.43% tetrahydro-3-hydroxy-8-methoxy-ethyl ester 14 18 4
3. 24.0 l-Gala-l-ido-octose C8H16O8 671 34.7% 17-1,5-Dimethylhexyl-10,13-dimethyl-3- styrylhexadecahydrocyclopenta[a]phenanthren- 4. 27.7 C35H52O 661 30.7% 2-one
5. 27.8 7,8-Epoxylanostan-11-ol,3-acetoxy C32H54O4 664 31.5%
48 Fig. 11. Benzeneethanamine (2,5-difluoro-β,3,4-trihydroxy-N-methyl)
Name : Benzeneethanamine (2,5-difluoro-β,3,4-trihydroxy-N- methyl)
Molecular formula : C9H11F2NO3
Molecular weight : 699
CAS Registry : 471651 Number
Synonyms : 3,6-Difluoro-4-[1-hydroxy-2-(methylamino)ethyl]-1,2- benzenediol
Lib. main; ID : 14052
49 Fig. 12. 2-Naphthalenecarboxylic acid (1, 2, 3, 4-tetrahydro-3-hydroxy-8- methoxy-ethyl ester)
Name : 2-Naphthalenecarboxylic acid; 1, 2, 3, 4-tetrahydro-3- hydroxy-8- methoxy-ethyl ester
Molecular formula : C14H18O4
Molecular weight : 580
CAS Registry : 101637-69-8 Number
Synonyms : -
Lib. main; ID : 118457
50 Fig. 13. l-Gala-l-ido-octose
Name : l-Gala-l-ido-octose
Molecular formula : C8H16O8
Molecular weight : 671
CAS Registry : 6291-04-9 Number
Synonyms : D-Erythro-L-Galacto-Octose
Lib. main; ID : 6311
51 Fig. 14. 17-1,5-Dimethylhexyl-10,13 dimethylstyrylhexadecahydrocyclopenta[a] phenanthren- 2-one
Name : 17-1,5-Dimethylhexyl-10,13-dimethyl-3 styrylhexadecahydrocyclopenta[a]phenanthren-2-one
Molecular formula : C35H52O
Molecular weight : 661
CAS Registry : 4519-58-05 Number
Synonyms : 3-[(Z)-2-Phenylvinyl]cholestan-2-one
Lib. main; ID : 50364
52 Fig. 15. 7,8-Epoxylanostan-11-ol, 3-acetoxy
Name : 7,8-Epoxylanostan-11-ol, 3-acetoxy
Molecular formula : C32H54O4
Molecular weight : 664
CAS Registry : 6701-13-9 Number
Synonyms : Decamethylene Glycol
Lib. main; ID : 13958
53 Six compounds (fig.16 & Table 12) such as phenol 2,6-bis(1,1-dimethylethyl)- 4-methyl-methylcarbamate (fig. 17); 1-octanol 2,7-dimethyl- (fig. 18); 3,7,11,15- tetramethyl-2-hexadecen-1-ol (fig. 19); 1,10-decanediol (fig. 20); 1-hexadecyne (fig. 21); phytol (fig. 22) were identified in the ethanol leaf extact.
Fig. 16. Chromatogram of ethanol leaf extract of Pergularia daemia
Table 12. Compounds identified from the ethanol leaf extract of Pergularia daemia
Molecular Molecular Peak S.No. RT Name of the compound formula weight Area % Phenol, 2,6-bis(1,1-dimethylethyl)-4- 1. 7.93 C H NO 277 0.40 methyl-, methylcarbamate 17 27 2
2. 11.20 1-Octanol, 2,7-dimethyl C10H22O 158 2.81 3,7,11,15-Tetramethyl-2-hexadecen- 3. 11.78 C H O 296 63.86 1ol 20 40
4. 12.09 1,10-Decanediol C10H22O2 174 7.23
5. 12.31 1-Hexadecyne C16H30 222 13.65
6. 15.56 Phytol C20H40O 296 12.05
54 Fig. 17. Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl-,methylcarbamate
205 100
HN
O O 57 220 50
41
91 105 77 115 145 161 65 128 177 189 0 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290
Name : Phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl, methylcarbamate
Molecular formula : C17H27NO2
Molecular weight : 277
CAS Registry : 1978-11-2 Number
Synonyms : Carbamic acid, methyl-2,6-di-tert-butyl-p-tolyl ester; Azak; Terbutol; Terbucarb; 2,6-Ditert-butyl-4-methylphenyl methylcarbamate
Lib. main; ID : 113173
55 Fig. 18. 1-Octanol, 2,7-dimethyl
43 100
57
50 HO
69
29 85 45 53 67 97 112 0 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 ilib)1O l2 di hl
Name : 1-Octanol, 2,7-dimethyl
Molecular formula : C20H22O
Molecular weight : 158
CAS Registry : 15250-22-3 Number
Synonyms : 2,7-Dimethyl-1-octanol
Lib. main; ID : 6737
56 Fig. 19. 3,7,11,15-Tetramethyl-2-hexadecen-1-ol
81 100 43 95 123 55 71
OH 50
109 29 137 278 151 179 193 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
Name : 3,7,11,15-Tetramethyl-2-hexadecen-1-ol
Molecular formula : C10H40O
Molecular weight : 296
CAS Registry : 102608-53-7 Number
Synonyms : 2E-3,7,11,15-Tetramethyl-2-hexadecen-1-ol
Lib. main; ID : 37978
57 Fig. 20. 1,10-Decanediol
55 100
68 OH 41 HO 82
50
31 95
110
126 138 0 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180
Name : 1,10-Decanediol
Molecular formula : C10H22O2
Molecular weight : 174
CAS Registry : 112-47-0 Number
Synonyms : Decamethylene glycol; Decamethylenediol; Decane-1,10 diol; 1,10-Decamethylene diol; 1,6-Bis(2- hydroxyethyl)hexane; 1,10- Decamethylene glycol; a,e- Decanediol
Lib. main; ID : 16672
58 Fig. 21. 1-Hexadecyne
81 100
43 67 55
50 95
29
109
123 137 0 10 30 50 70 90 110 130 150 170 190 210 230
Name : 1-Hexadecyne
Molecular formula : C16H30
Molecular weight : 222
CAS Registry : 629-74-3 Number
Synonyms : Tetradecylacetylene
Lib. main; ID : 37583
59 Fig. 22. Phytol
71 100
HO
50 43 57
81 123 95 29 111 137 151 179 196 278 296 0 10 30 50 70 90 110 130 150 170 190 210 230 250 270 290 310
Name : Phytol
Molecular formula : C20H40O
Molecular weight : 296
CAS Registry : 150-86-7 Number
Synonyms : 2-Hexadecen-1-ol; 3,7,11,15-tetramethyl-[R-[R*,R*-(E)]]; Trans-phytol; 3,7,11,15-Tetramethyl-2-hexadecen-1-ol; (2E)-3,7,11,15- Tetramethyl-2-hexadecen-1-ol
Lib. main; ID : 29451
60 Six compounds (fig. 23 & Table 13) such as methyl salicylate (fig. 24); diethyl phthalate (fig. 25); n-hexadecanoic acid (fig. 26); hexadecanoic acid ethyl ester (fig. 27); phytol (fig. 28); 9,12,15-octadecatrienoic acid ethyl ester, (Z,Z,Z)- (fig. 29) were identified in the aqueous leaf extract.
Fig. 23. Chromatogram of aqueous leaf extract of Pergularia daemia
Table 13. Compounds identified from the aqueous leaf extract of Pergularia daemia
Molecular Molecular Peak S.No. RT Name of the compound formula weight Area %
1. 3.9 Methyl salicylate C8H8O3 960 40.5%
2. 8.4 Diethyl Phthalate C12H14O4 936 81%
3. 12.1 n-Hexadecanoic acid C16H32O2 806 72.3%
4. 12.5 Hexadecanoic acid, ethyl ester C18H36O2 852 89.7%
5. 13.6 Phytol C20H40O 851 71% 9,12,15-Octadecatrienoic acid, ethyl ester, 6. 14.1 C H O 895 52.5% (Z,Z,Z)- 20 34 2
61 Fig. 24. Methyl salicylate
Name : Methyl salicylate
Molecular formula : C8H8O3
Molecular weight : 947
CAS Registry : 119-36-8 Number
Synonyms : Synthetic wintergreen oil; Methyl hydroxybenzoate; Betula oil; O- hydroxybenzoic acid; Methyl ester; Gaultheria oil; Methyl sweet birch oil; O- hydroxybenzoate; 2-(Methoxycarbonyl) Phenol; 2- carbomethoxyphenol; Linsal; Methylester kyseliny salicylove (Czech); Salicylic acid; Methyl ester; O-Anisic acid
Lib. main; ID : 16283
62 Fig. 25. Diethyl phthalate
Name : Diethyl phthalate
Molecular formula : C12H14O4
Molecular weight : 936
CAS Registry : 84-66-2 Number
Synonyms : 1,2-Benzenedicarboxylic acid; Diethyl ester; o- Benzenedicarboxylic acid diethyl ester; o- Bis(ethoxycarbonyl) benzene; Diethyl 1,2- benzenedicarboxylate; Diethyl o-phenylenediacetate; Diethyl o-phthalate; Di-n-ethyl phthalate; DPX-F5384; Ethyl phthalate; Phthalic acid; Diethyl ester
Lib. main; ID : 110716
63 Fig. 26. n-Hexadecanoic acid
Name : n-Hexadecanoic acid
Molecular formula : C16H32O2
Molecular weight : 806
CAS Registry : 57-10-3 Number
Synonyms : Hexadecanoic acid; n-Hexadecoic acid; Palmitic acid; Pentadecanecarboxylic acid; 1-Pentadecanecarboxylic acid; Cetylic acid; Emersol 140; Emersol 143; Hexadecylic acid; Hydrofol; Hystrene 8016; Hystrene 9016; Industrene 4516; Glycon P-45; Prifac 2960; NSC 5030; Palmitinic acid; Kortacid 1695; Hexadecanoic acid (palmitic acid)
Lib. main; ID : 6723
64 Fig. 27. Hexadecanoic acid, ethyl ester
Name : Hexadecanoic acid, ethyl ester
Molecular formula : C18H36O2
Molecular weight : 852
CAS Registry : 628-97-7 Number
Synonyms : Palmitic acid, ethyl ester; Ethyl hexadecanote; Ethyl palmitate; Ethyl n-hexadecanote; Ethyl hexadecanote (Ethyl palmitate)
Lib. main; ID : 49485
65 Fig. 28. Phytol
Name : Phytol
Molecular formula : C20H40O
Molecular weight : 296
CAS Registry : 150-86-7 Number
Synonyms : 2-Hexadecen-1-ol; 3,7,11,15-tetramethyl- [R-[R*,R*- (E)]]-; Trans- phytol; 3,7,11,15-Tetramethyl-2-hexadecen- 1-ol; (2E)-3,7,11,15- Tetramethyl-2-hexadecen-1-ol
Lib. main; ID : 29451
66 Fig. 29. 9,12,15-Octadecatrienoic acid, ethyl ester,(Z,Z,Z)
Name : 9,12,15-Octadecatrienoic acid, ethyl ester,(Z,Z,Z)
Molecular formula : C20H34O2
Molecular weight : 895
CAS Registry : 1191-41-9 Number
Synonyms : Linolenic acid; Ethyl ester; Ethyl cis, Cis-9,12,15- octadecatrienoate; Ethyl linolenate; Ethyl (9Z,12Z,15Z)- 9,12,15-octadecatrienoate
Lib. main; ID : 9680
67 Ten compounds (fig. 30 & Table 14) such as Propane, 1,1,3-triethoxy- (fig. 31); phytol (fig. 32); Hexadecanoic acid, ethyl ester (fig. 33); 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- (fig. 34); Octadecanoic acid, ethyl ester (fig. 35); 1,2- Benzenedicarboxylic acid, diisooctyl ester (fig. 36); 5α-Androstan-16-one, cyclic ethylene mercaptole (fig. 37); Methyl 3-oxours-12-en-23-oate (fig. 38); 2(1H)Naphthalenone, 3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1-methylethenyl)- (fig. 39); Decanoic acid, ethyl ester (fig. 40) were identified in the ethanol leaf callus extract.
Fig. 30. Chromatogram of ethanol leaf callus extract of Pergularia daemia
68 Table 14. Compounds identified from the ethanol leaf callus extract of Pergularia daemia
Peak Molecular Molecular S.No. RT Name of the compound Area formula weight %
1. 2.51 Propane, 1,1,3-triethoxy- C9H20O3 176 1.80
2. 11.05 Phytol C20H40O 296 2.08
3. 12.79 Hexadecanoic acid, ethyl ester C18H36O2 284 6.47
4. 14.95 9,12,15-Octadecatrienoic acid, methyl ester, C19H32O2 292 2.86 (Z,Z,Z)-
5. 15.28 Octadecanoic acid, ethyl ester C20H40O2 312 0.50
6. 19.96 1,2-Benzenedicarboxylic acid, diisooctyl ester C24H38O4 390 1.69
7. 30.82 5α-Androstan-16-one, cyclic ethylene C21H34S2 350 1.61 mercaptole
8. 32.46 Methyl 3-oxours-12-en-23-oate C31H48O3 468 9.47
9. 33.32 2(1H)Naphthalenone, 3,5,6,7,8,8a-hexahydro- C H O 218 6.17 4,8a-dimethyl-6-(1-methylethenyl)- 15 22
10. 34.65 Decanoic acid, ethyl ester C12H24O2 200 58.71
69 Fig. 31. Propane, 1,1,3-triethoxy-
Name : Propane, 1,1,3-triethoxy-
Molecular formula : C9H20O3
Molecular weight : 176
CAS Registry : 7789-92-6 Number
Synonyms : β-Ethoxypropionaldehyde diethyl acetal; 3- Ethoxypropionaldehyde diethyl acetal; Propionaldehyde, 3-ethoxy-, diethyl acetal; 1,3-Triethoxypropane; Propane, 1,3,3-triethoxy-; 1,3,3-Triethoxypropane
Lib. main; ID : 6581
70 Fig. 32. Phytol
Name : Phytol
Molecular formula : C20H40O
Molecular weight : 296
CAS Registry : 150-86-7 Number
Synonyms : 2-Hexadecen-1-ol; 3,7,11,15-tetramethyl-[R-[R*,R*-(E)]]; Trans-phytol; 3,7,11,15-Tetramethyl-2-hexadecen-1-ol; (2E)-3,7,11,15- Tetramethyl-2-hexadecen-1-ol
Lib. main; ID : 29451
71 Fig. 33. Hexadecanoic acid, ethyl ester
Name : Hexadecanoic acid, ethyl ester
Molecular formula : C18H36O2
Molecular weight : 852
CAS Registry : 628-97-7 Number
Synonyms : Palmitic acid, ethyl ester; Ethyl hexadecanote; Ethyl palmitate; Ethyl n-hexadecanote; Ethyl hexadecanote (Ethyl palmitate)
Lib. main; ID : 49485
72 Fig. 34. 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-
Name : 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-
Molecular formula : C19H32O2
Molecular weight : 292
CAS Registry : 301-00-8 Number
Synonyms : Linolenic acid, methyl ester; Methyl all-cis-9,12,15- octadecatrienoate; Methyl linolenate; Methyl (9Z,12Z,15Z)-9,12,15-octadecatrienoate
Lib. main; ID : 9699
73 Fig. 35. Octadecanoic acid, ethyl ester
Name : Octadecanoic acid, ethyl ester
Molecular formula : C20H40O2
Molecular weight : 312
CAS Registry : 111-61-5 Number
Synonyms : Stearic acid, ethyl ester; Ethyl n-octadecanoate; Ethyl octadecanoate; Ethyl stearate; Radia 7185
Lib. main; ID : 11146
74 Fig. 36. 1,2-Benzenedicarboxylic acid, diisooctyl ester
Name : 1,2-Benzenedicarboxylic acid, diisooctyl ester
Molecular formula : C24H38O4
Molecular weight : 390
CAS Registry : 27554-26-3 Number
Synonyms : Diisooctyl phthalate; Hexaplas M/; Isooctyl phthalate; Corflex 880; DIOP; Flexol plasticizer diop; Morflex 100; Palatinol D10; Phthalic acid, bis(6-methylheptyl) ester
Lib. main; ID : 19911
75 Fig. 37. 5α-Androstan-16-one, cyclic ethylene mercaptole
Name : 5α-Androstan-16-one, cyclic ethylene mercaptole
Molecular formula : C21H34S2
Molecular weight : 350
CAS Registry : 2759-86-6 Number
Synonyms : -
Lib. main; ID : 16385
76 Fig. 38. Methyl 3-oxours-12-en-23-oate
Name : Methyl 3-oxours-12-en-23-oate
Molecular formula : C31H48O3
Molecular weight : 468
CAS Registry : 20475-86-9 Number
Synonyms : Urs-12-en-24-oic acid, 3-oxo-, methyl ester; Urs-12-en- 24-oic acid, 3-oxo-, methyl ester, (+)-
Lib. main; ID : 117667
77 Fig. 39. 2(1H)Naphthalenone, 3,5,6,7,8,8a-hexahydro-4,8a-dimethyl-6-(1- methylethenyl)-
Name : 2(1H)Naphthalenone, 3,5,6,7,8,8a-hexahydro-4,8a- dimethyl-6-(1-methylethenyl)-
Molecular formula : C15H22O
Molecular weight : 218
CAS Registry : - Number
Synonyms : -
Lib. main; ID : 117367
78 Fig. 40. Decanoic acid, ethyl ester
Name : Decanoic acid, ethyl ester
Molecular formula : C12H24O2
Molecular weight : 200
CAS Registry : 110-38-3 Number
Synonyms : Capric acid, ethyl ester; Ethyl caprate; Ethyl caprinate; Ethyl decanoate; Ethyl decylate; n-Capric acid ethyl ester; Ethyl ester of Decanoic; Ethyl n-decanoate
Lib. main; ID : 11057
79 4.3. Pharmacology
The acute toxicity study revealed the non-toxic nature of the chloroform, ethanol, aqueous and ethanol callus extracts at the tested concentrations. No lethal toxic reactions were observed until the end of the experiment.
The biochemical parameters were recorded from the test animals. The blood glucose levels (Table 15 & Fig. 41) of the normal animals were 89.12 mg/dl on the
0thday and 89.06 mg/dl on the 21st day. The streptozotocin treated control showed
88.40 mg/dl on the 0th day and 348.21 mg/dl on the 21st day. Diabetic animals treated with 300 mg/kg of the chloroform leaf extract showed 82.02 mg/dl on the 0th day and
160.68 mg/dl on the 21st day. The scores of the diabetic animals treated with 300 mg
/kg of the ethanol extract were 87.22 mg/dl on the 0th day and 132.61 ml/dl on the 21st day. Diabetic animals treated with 300 mg /kg of the aqueous extract showed 89.33 mg/dl on the 0th day and 152.80 mg/dl on the 21st day. Diabetic animals treated with
300 mg /kg of the ethanol extract of callus showed 83.30 mg/dl on the 0th day and
123.26 mg/dl on the 21st day. Diabetic animal treated with 600 µg /kg of glibenclamide
(standard diabetic drug) registered 86.23 mg/dl on the 0th day and 194.6 mg/dl of blood glucose on the 21st day.
The serum cholesterol level too increases in diabetic animals. It was 215.42 mg/dl on the 21st day for diabetic animals for normal animals it was 97.79 mg/dl on the
21st day (Table 16 & fig. 42). The levels of cholesterol were found lower (142.4 mg/dl) in animals treated with 300 mg/kg of chloroform leaf extract compared with untreated ones. The same dose required serum cholesterol on the 21st day such as 121.80 mg/dl to ethanol extract and 131.11 mg/dl to aqueous extract. Groups treated with 300 mg/kg of ethanol callus extract showed 113.02 mg/dl of serum cholesterol, where as 600 µg/kg of glibenclamide produced 146.08 mg/dl on the 21st day.
80 Table 15. Changes in the blood glucose level of normal and experimental animals
0th day 7th day 14th day 21th day Control & Treatment Groups (mg/dl) (mg/dl) (mg/dl) (mg/dl) Normal I 89.12±4.12 90.46±2.12 90.98±2.52 89.06±2.55 100 mg/kg II 90.50±1.25 90.99±0.43 87.48±0.31 90.46±2.16 Chloroform 200 mg/kg III 90.89±1.48 87.74±0.71 87.76±0.31 88.28±1.49
300 mg/kg 1V 85.64±2.73 88.95±0.22 84.61±0.39 84.51±1.91 100 mg/kg V 90.40±5.01 92.44±4.13 91.63±3.49 91.52±3.09 Ethanol 200 mg/kg VI 91.60±8.40 93.06±6.23 94.72±4.87 90.65±8.14 300 mg/kg VII 89.15±4.19 90.82±2.39 92.76±3.78 90.82±8.96 100 mg/kg VIII 98.41±1.25 86.6±1.97 84.3±5.11 81.97±2.68 Aqueous 200 mg/kg IX 88.44±1.79 82.67±1.20 87.67±1.60 89.31±2.54 300 mg/kg X 83.51±1.79 84.66±1.65 81.25±2.36 89.16±1.37 100 mg/kg XI 89.00±4.36 90.00±0.25 85.66±1.90 88.65±1.06 Ethanol callus 200 mg/kg XII 85.52±4.33 92.15±2.37 90.33±2.49 89.17±2.35 300 mg/kg XIII 80.25±0.31 90.11±2.25 85.17±2.86 91.33±2.24 Diabetic (STZ) 40mg/kg XIV 88.40±4.17* 320.5±4.86* 337.9±4.50* 348.21±3.23* 100 mg/kg XV 89.72±5.46# 255.49±5.24# 220.31±6.87# 172.28±2.24# STZ + 200 mg/kg XVI 89.90±6.56# 251.49±5.43# 215.8±5.72# 165.01±5.73# Chloroform 300 mg/kg XVII 82.02±5.32# 248.34±5.36# 212.8±5.31# 160.68±7.16# 100 mg/kg XVIII 84.12±0.47# 241.04±0.52# 202.10±0.2# 149.4±0.61# STZ + Ethanol 200 mg/kg XIX 86.65±0.39# 236.3±3.38# 200.68±1.35# 141.78±0.84# 300 mg/kg XX 87.22±1.01# 233.15±5.23# 194.23±1.42# 132.61±1.46# 100 mg/kg XXI 84.16±1.39# 246.63±5.41# 210.32±5.85# 158.56±1.36# STZ + 200 mg/kg XXII 85.43±1.01# 244.16±5.94# 205.36±5.72# 153.42±1.82# Aqueous 300 mg/kg XXIII 89.33±1.56# 243.15±1.39# 203.17±6.97# 152.80±2.86# 100 mg/kg XXIV 83.01±1.92# 230.0±1.57# 186.31±2.21# 128.62±1.46# STZ + Ethanol 200 mg/kg XXV 85.16±1.86# 230.06±1.18# 185.80±0.42# 125.15±1.44# callus 300 mg/kg XXVI 83.30±2.16# 223.98±0.79# 177.84±1.03# 123.26±1.13# STZ + 600 µg/kg XXVII 86.23±1.08# 210.97±5.04# 204.05±3.06# 194.6±6.41# Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 41. Changes in the blood glucose level of normal and experimental animals
400
350
300
250
200
150 0th day (mg/dl)
100 7th day (mg/dl) 50
0 14th day (mg/dl)
Normal 21th day (mg/dl) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 100 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Diabetic (STZ Diabetic (STZ 40mg/kg) Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Ethanol callus 100 mg/kg + Ethanol STZ callus 200 mg/kg + Ethanol STZ callus 300 mg/kg + Ethanol STZ + Glibenclamide 600 µg/kg STZ
Table 16. Changes in levels of total cholesterol, triglycerides, and phospholipids in serum of normal and experimental animals
Total Triglycerides Phospholipids Control & Treatment Groups cholesterol (mg/dl) (mg/dl) (mg/dl) Normal I 97.79 ±1.82 69.35±1.92 72.16±1.46 100 mg/kg II 107.32±1.54 73.27±8.91 80.93±4.05 Chloroform 200 mg/kg III 108.20±1.56 75.48±3.55 73.61±1.63
300 mg/kg 1V 112.27±1.02 79.20±1.75 77.85±1.8 100 mg/kg V 109.45±2.55 79.46±2.05 80.40±2.81 Ethanol 200 mg/kg VI 109.23±2.61 77.02±5.12 73.12±1.92 300 mg/kg VII 111.83±4.71 78.63±5.29 72.97±1.65 100 mg/kg VIII 101.75±1.21 71.58±1.36 77.65±2.70 Aqueous 200 mg/kg IX 109.36±1.05 68.29±1.33 80.31±1.77 300 mg/kg X 110.22±1.55 72.85±1.38 80.90±1.73 100 mg/kg XI 99.70±2.76 75.60±2.66 72.70±1.56 Ethanol callus 200 mg/kg XII 104.4±2.61 74.70±1.55 73.00±2.67 300 mg/kg XIII 105.5±2.57 72.6±01.58 73.40±2.58 Diabetic (STZ) 40mg/kg XIV 215.42±2.67* 147.82±2.92* 146.71±2.34* 100 mg/kg XV 145.8±0.82# 135.51±1.63# 131.40±1.82# STZ + 200 mg/kg XVI 144.82±1.43# 128.82±1.12# 121.9±1.19# Chloroform 300 mg/kg XVII 142.4±2.81# 121.3±1.27# 119±1.47# 100 mg/kg XVIII 128.25±1.32# 91.16±1.82# 102.5±3.35# STZ + Ethanol 200 mg/kg XIX 125.14±1.24# 85.26±1.64# 94.43±1.62# 300 mg/kg XX 121.80±1.27# 84.30±1.16# 90.81±1.25# 100 mg/kg XXI 137.82 ±1.08# 94.12±1.97# 109.3±1.18# STZ + Aqueous 200 mg/kg XXII 135.56±1.84# 93.16±0.08# 107.8±1.24# 300 mg/kg XXIII 131.11±1.24# 92.6±2.47# 106.1±1.43# 100 mg/kg XXIV 119.4±1.23# 83.44±2.40# 87.13±1.32# STZ + Ethanol 200 mg/kg XXV 114.51±1.65# 82.41±2.42# 85.25±1.91# callus 300 mg/kg XXVI 113.02±2.62# 82.02±1.32# 84.01±1.32# STZ + 600 µg/kg 146.8±4.08# 99.16±5.08# 140.6±6.33# XXVII Glibenclamide Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 42. Changes in levels of total cholesterol, triglycerides, and phospholipids in serum of normal and experimental animals
250
200
150
100 Total cholesterol (mg/dl)
50
0 Triglycerides (mg/dl) Normal Phospholipids (mg/dl) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ + Glibenclamide 600 µg/kg STZ
The concentration of triglycerides was found significantly higher in diabetic animals. It was 147.82 mg/dl more or less double the normal of 69.35 mg/dl. But the triglycerides levels were lower in diabetic animals treated with 300 mg/kg of chloroform extract (121.30 mg/dl), in the ethanol extract treated groups it was (84.30 mg/dl), in the aqueous extract treated groups it was (92.60 mg/dl) and in the ethanol callus extract treated groups 82.02 mg/dl. It was 99.16 mg/dl in the groups treated with glibenclamide at a dose of 600 µg/kg (Table 16 & fig. 42).
The phospholipids content was higher in diabetic rats (146.71 mg/dl) almost double the normal values of 72.16 mg/dl on the 21st day. After treatment with 300 mg/kg chloroform leaf extract it was 119 mg/dl; with the diabetic animals treated with
300 mg/kg of ethanol extract it was 90.81 mg/dl; with doses of 300 mg/kg of aqueous extract 106.1 mg/dl. Diabetic animal treated with 300 mg/kg of ethanol extract of callus showed 84.01 mg/dl of phospholipids on the 21st day. Diabetic animal treated with 600
µg/kg of glibenclamide showed 140.6 mg/dl of phospholipids (Table 16 & fig. 42).
Table 17 & Fig. 43 show the plasma insulin and glycosylated haemoglobin levels with the normal and experimental groups. The plasma insulin level in normal animals was 19.46 μU/ml and with the streptozotocin treated control group it was 8.62
μU/ml on the 21st day. Diabetic animals treated with 300 mg/kg of the chloroform leaf extract showed 11.16 μU/ml; while animals treated with 300 mg /kg of the ethanol extract showed 13.25 μU/ml of plasma insulin. Diabetic animals treated with 300 mg/kg of the aqueous extract had only 12.04 μU/ml of plasma insulin. Diabetic animals treated with 300 mg/kg of the ethanol extract of callus showed a higher rate (15.41
μU/ml) and diabetic animal treated with 600 µg/kg of glibenclamide showed a plasma insulin content of 12.68 μU/ml.
81 Table 17. Changes in the levels of plasma insulin and glycosylated haemoglobin in normal and experimental animals
Glycosylated Plasma insulin Control & Treatment Groups haemoglobin (μU/ml ) (mg/g HB) Normal I 19.46±2.5 3.02±0.58 100 mg/kg II 20.11±3.10 2.55±0.52 Chloroform 200 mg/kg III 21.80±3.40 2.12±0.77
300 mg/kg 1V 21.70±1.86 2.67±0.18 100 mg/kg V 20.49±2.80 2.51±0.54 Ethanol 200 mg/kg VI 20.40±2.50 2.66±0.16 300 mg/kg VII 24.20±2.43 2.63±0.88 100 mg/kg VIII 22.31±1.94 2.49± 0.30 Aqueous 200 mg/kg IX 24.43±2.40 2.97±0.84 300 mg/kg X 23.75±1.87 2.99±0.75 100 mg/kg XI 22.14±2.63 3.45±0.55 Ethanol callus 200 mg/kg XII 20.18±2.99 3.12±0.86 300 mg/kg XIII 21.09±1.81 3.76±0.23 Diabetic (STZ) 40mg/kg XIV 8.62±1.87* 16.34±0.72* 100 mg/kg XV 10.41±0.32# 15.96±0.53# STZ + 200 mg/kg XVI 10.47±0.91# 15.04±0.82# Chloroform 300 mg/kg XVII 11.16±0.41# 14.88±0.69# 100 mg/kg XVIII 12.48±0.21# 12.79±0.95# STZ + Ethanol 200 mg/kg XIX 13.12±0.44# 12.28±0.67# 300 mg/kg XX 13.25±0.26# 11.17±0.19# 100 mg/kg XXI 11.18±0.64# 14.20±0.48# STZ + Aqueous 200 mg/kg XXII 11.63±0.48# 13.99±0.17# 300 mg/kg XXIII 12.04±0.76# 13.12±0.55# 100 mg/kg XXIV 14.21±0.31# 8.91±0.20# STZ + Ethanol 200 mg/kg XXV 14.50±0.23# 8.29 ±0.52# callus 300 mg/kg XXVI 15.41±0.51# 7.15±0.45# STZ + 600 µg/kg XXVII 12.68±0.49# 11.12±0.72# Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 43. Changes in the levels of plasma insulin and glycosylated haemoglobin in normal and experimental animals
30
25
20 Plasma insulin 15 (μU/ml )
10
5 Glycosylated 0 haemoglobin (mg/g HB) Normal Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
The glycosylated haemoglobin level in normal animals was 3.02 mg/g HB. The streptozotocin treated control group showed 16.34 mg/g HB on the 21st day. Diabetic animals treated with 300 mg/kg of the chloroform leaf extract showed 14.88 mg/g HB and diabetic animals treated with 300 mg/kg of the ethanol extract 11.17 mg/g HB.
Diabetic animals treated with 300 mg/kg of the aqueous extract had 13.12 mg/g HB on the 21st day. Diabetic animals treated with 300 mg/kg of the ethanol extract of callus had a higher proportion (7.15 mg/g HB). Diabetic animals treated with 600 µg/kg of glibenclamide had 11.12 mg/g HB on the 21st day.
The Morphological behaviour, body weight, feed and fluid intake of the control and experimental animals were observed at regular intervals (Table 18 & Fig. 44).
The body weight of the normal animals was 202 g on 0thday and 206 g on the
21st day. The streptozotocin treated control group showed 174 g on the 0th day and 145 g on the 21st day. Diabetic animals treated with 300 mg/kg of the chloroform leaf extract showed 187 g and 188 g on the 0th day and 21st days respectively. Diabetic animals treated with 300 mg/kg of the ethanol extract showed 197 g and 195 g on the
0th day and 21st days respectively. Diabetic animals treated with 300 mg/kg of the aqueous extract showed 197 g and 194 g on the 0th day and 21st day. Diabetic animals treated with 300 mg/kg of the ethanol extract of callus gained greater weight 193 g on the 0th day and 196 g on the 21st day. Diabetic animal treated with 600 µg/kg of glibenclamide weighed 192 g on the 0thday and 196 g on the 21st day.
There was a significant decrease in the food take of normal rats. At the start of the experiment it was 15.4 g/day it fell to 14.9 g/day on the 21st day. The streptozotocin treated control group took 33.3 g/day on the 0th day and 31.1 g/day on the 21st day.
Diabetic animals treated with 300 mg/kg of the chloroform leaf extract took 35.4 g/day on the 0th day and 33.7 g/day on the 21st day. Diabetic animals treated with 300 mg /kg
82 Table 18. Changes in body weight, food intake and fluid intake in normal and experimental animals
Control & Treatement Groups Body weight (g) Food Intake (g/day) Fluid Intake (ml/day) Initial Final Initial Final Initial (g) Final (g) (g/day) (g/day) (ml/day) (ml/day) Normal I 202±19.41 206±6.4 15.4±0.99 14.9±1.3 92.6±10.2 91.9±9.4 100 mg/kg II 199±18.44 207±1.81 17.2±1.01 12.4±1.8 94.2±11.8 91.5±10.6 Chloroform 200 mg/kg III 198±15.25 201±6.33 18.9±2.44 12.6±1.5 93.8±5.70 92.6±12.8
300 mg/kg 1V 205.4±12.9 199±3.54 16.7±1.72 13.7±2.8 91.0±7.38 90.3±11.6 100 mg/kg V 201±12.13 206±17.8 15.2±0.81 11.5±1.5 91.6±10.4 92.2±12.4 Ethanol 200 mg/kg VI 200±12.89 207±15.6 14.4±0.84 11.9±2.6 95.8±6.87 96.4±13.7 300 mg/kg VII 202±18.65 205±13.2 16.6±1.83 12.5±1.9 94.3±8.44 92.8±13.8 100 mg/kg VIII 202±12.52 208±9.92 15.3±0.95 13.1±1.2 91.9±10.5 93.6±13.50 Aqueous 200 mg/kg IX 199±15.5 203±5.30 16.9±1.6 11.9±1.5 91.9±5.4 93.4±12.54 300 mg/kg X 197±14.6 202±3.85 16.4±1.4 12.4±1.4 93.6±9.7 93.5±12.75 100 mg/kg XI 205±9.56 206±13.9 15.9±1.95 12.01±2.7 95.7±7.36 95.4±12.9 Ethanol callus 200 mg/kg XII 199±11.41 208±14.1 13.4±1.33 11.98±1.0 95.2±5.42 97.36±13.5 300 mg/kg XIII 201±13.5 206±13.4 16.8±1.26 11.76±1.3 96.1±3.39 94.19±13.9 Diabetic (STZ) 40mg/kg XIV 174±8.29 145± 9.4* 33.3±3.2 31.1±1.8* 119.6±10.3 137.6±15.2* # # # STZ + 100 mg/kg XV 181±2.2 179±2.2 33.6±1.8 32.9±1.6 129.7±11.9 131.3±7.2 Chloroform 200 mg/kg XVI 189±3.45 186±3.21# 34.1±2.0 33.5±1.9# 137.2±1.03 135.6±12.0# 300 mg/kg XVII 187±2.98 188±7.30# 35.4±3.41 33.7±1.7# 131.9±1.45 135.7±9.42# 100 mg/kg XVIII 192.65±1.13 193.12±1.72# 34.89±1.39 33.39±1.19# 119.23±1.02 115.06±1.86# STZ + Ethanol 200 mg/kg XIX 192.13±1.5 193.71±1.12# 34.81±1.18 35.12±0.21# 117.96±1.43 114.17±1.32# 300 mg/kg XX 197.21±1.12 195±1.93# 33.26±1.74 37.17±1.23# 117.78±1.18 113.2±1.16# 100 mg/kg XXI 196±6.18 187± 3.21# 35.4±1.8 33.4±1.9 121.5±12.8 117.9±6.4 STZ + 200 mg/kg XXII 199±8.20 193±7.10# 34.8±1.6 32.9±2.2# 123.6±9.46 119±11.4# Aqueous 300 mg/kg XXIII 197±6.42 194±2.46# 36.3±1.2 32.6±3.48# 121.7±11.41 120±10.9# 100 mg/kg XXIV 188±3.40 195.7±6.42# 33.2±1.9 32.6±1.8# 119.3±7.2 101±11.40# STZ + Ethanol 200 mg/kg XXV 195.7±6.42 195.3±6.15# 35.9±2.1 33.2±2.7# 125.3±8.44 101.6±11.1# callus 300 mg/kg XXVI 193±5.09 196±2.10# 36.4±1.7 33.9±1.9# 121.4±5.41 103.9±10.6# STZ + 600 µg/kg XXVII 192.1±6.42 196.10±19.9# 37.2±3.4 36.8±3.4# 120.9±10.2 127.4±12.4# Glibenclamide
Values are expressed as mean±SD (n=6).
Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal.
Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control.
Fig. 44. Changes in body weight, food intake and fluid intake in normal and experimental animals
250 Body weight Initial (g) 200
150 Body weight Final (g) 100
50 Food Intake Initial (g/day) 0
Food Intake Final
Normal (g/day)
Fluid Intake Initial
Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg (ml/day) Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ
STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ Fluid Intake Final
STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ Final (ml/day) STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ + Glibenclamide 600 µg/kg STZ
of the ethanol extract had an intake of 33.26 g/day on the 0th day and 37.17 g/day on the
21st day. Diabetic animals treated with 300 mg /kg of the aqueous extract recorded a food intake of 36.3 g/day on the 0th day and 32.6 g/day on the 21st day. Diabetic animals treated with 300 mg /kg of the ethanol extract of callus recorded a higher value
36.4 g/day on the 0th day and 33.9 g/day on the 21st day. Diabetic animal treated with
600 µg /kg of glibenclamide took 37.2 g on the 0th day and 36.8 g on the 21st day.
The fluid intake of normal rats was 92.6 ml/day on 0th day and 91.9 ml/day on the 21st day. The streptozotocin treated control group took 119.6 ml/day on the 0th day and 137.6 ml/day on the 21st day. Diabetic animals treated with 300 mg/kg of the chloroform leaf extract showed an intake of 131.9 ml/day on the 0th day and 135.6 ml/day on the 21st day. Diabetic animals treated with 300 mg/kg of the ethanol extract took 117.78 ml/day on the 0th day and 113.2 ml/day on the 21st day. Diabetic animals treated with 300 mg/kg of the aqueous extract consumed 121.7 ml/day on the 0th day and 120 ml/day on the 21st day. Diabetic animals treated with 300 mg/kg of the ethanol extract of callus showed a higher consumption level of 121.4 ml/day on the 0th day and
103.9 ml/day on the 21st day and diabetic animal treated with 600 µg/kg of glibenclamide took 120.9 ml/day on the 0th day and 127.4 ml/day on the 21st day.
The concentration of Hexokinase was found significantly lower in the liver of diabetic animals. In diabetic animals it was 0.13 (µmol glucose phosphorylated/h/mg protein) and in normal animal it was 0.35 µmol on the 21st day. But the Hexokinase level was higher in diabetic animals treated with the three types of extract. On administration of 300 mg/kg of the respective extracts the values were 0.19 µmol with chloroform leaf extract, 0.25µmol with the ethanol extract and 0.22 µmol in the group treated with aqueous extract. With a similar dose of ethanol callus extract the recorded value was 0.28µmol. In the group treated with glibenclamide it was 0.23 µmol (Table
19 & fig. 45).
83 Table 19. Changes in the level of liver hexokinase, glucose-6-phosphatase and fructose 1, 6 bisphosphatase in normal and experimental animals
Fructose 1,6 - Glucose-6– Hexokinase bis phopatase phospatase (μmol glucose (µmole of Control & Treatment Groups (µmole of phosphorylated phosphorous phosphorylated/ /h/mg protein) liberated/min min/mg protein) /mg protein) Normal I 0.35±0.04 0.37±0.002 0.430±0.03 100 mg/kg II 0.35±0.09 0.25±0.009 0.433±0.07 Chloroform 200 mg/kg III 0.38±0.02 0.40±0.001 0.421±0.02 300 mg/kg 1V 0.35±0.03 0.28±0.003 0.427±0.42 100 mg/kg V 0.36±0.08 0.30±0.016 0.433±0.17 Ethanol 200 mg/kg VI 0.37±0.09 0.32±0.001 0.431±0.14 300 mg/kg VII 0.35±0.05 0.38±0.014 0.422±0.11 100 mg/kg VIII 0.35±0.05 0.33±0.004 0.431±0.01 Aqueous 200 mg/kg IX 0.38± 0.07 0.29±0.001 0.425±0.18 300 mg/kg X 0.36±0.02 0.27±0.003 0.429±0.01 100 mg/kg XI 0.35±0.07 0.30±0.012 0.427±0.19 Ethanol callus 200 mg/kg XII 0.38±0.01 0.38±0.042 0.421±0.16 300 mg/kg XIII 0.35±0.08 0.36±0.063 0.429±0.17 Diabetic (STZ) 40mg/kg XIV 0.13±0.06* 0.72±0.010* 0.695±0.09* 100 mg/kg XV 0.15±0.006# 0.70±0.019# 0.557±0.02# STZ + 200 mg/kg XVI 0.18±0.027# 0.68±0.012# 0.548±0.03# Chloroform 300 mg/kg XVII 0.19±0.012# 0.65±0.015# 0.545±0.03# 100 mg/kg XVIII 0.23±0.075# 0.59±0.047# 0.501±0.02# STZ + Ethanol 200 mg/kg XIX 0.24±0.084# 0.55±0.004# 0.495±0.01# 300 mg/kg XX 0.25±0.012# 0.54±0.001# 0.477±0.09# 100 mg/kg XXI 0.20±0.014# 0.64±0.018# 0.540±0.31# STZ + Aqueous 200 mg/kg XXII 0.22±0.017# 0.63±0.005# 0.535±0.84# 300 mg/kg XXIII 0.22±0.159# 0.59±0.004# 0.525±0.02# 100 mg/kg XXIV 0.27±0.43# 0.52±0.014# 0.464±0.05# STZ + Ethanol 200 mg/kg XXV 0.27±0.72# 0.50±0.008# 0.459±0.07# callus 300 mg/kg XXVI 0.28±0.014# 0.48±0.002# 0.456±0.02# STZ + 600 µg/kg XXVII 0.23±0.011# 0.57±0.020# 0.615±0.83# Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 45. Changes in the level of liver hexokinase, glucose-6-phosphatase and fructose 1, 6 bisphosphatase in normal and experimental animals
0.8 0.7 0.6 Hexokinase (μmol 0.5 glucose phosphorylated 0.4 /h/mg protein) 0.3
0.2 Glucose-6– 0.1 phospatase (µmole of phosphorylated/ 0 min/mg protein)
Normal Fructose 1,6 - bis phopatase (µmole of phosphorous liberated/min /mg
Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg protein) Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
The level of glucose-6-phosphatase in liver was found significantly increased in diabetic animals. It was 0.72 (µmol of phosphorylated/min/mg protein). In the normal groups it was 0.37 µmol. The glucose-6-phosphatase level was lower in diabetic animals treated with the three types of extracts. On a dose of 300 mg/kg, groups treated with chloroform leaf extract recorded 0.65 µmol on the 21st day. The scores for the ethanol extract treated groups, the aqueous extract treated groups and ethanol callus extract treated groups were 0.54, 0.59, 0.48 µmol respectively. On the administration of glibenclamide at the rate of 600 µg/kg of the quantum of glucose-6-phosphatase were
0.57 µmol phosphorylated/min/mg protein (Table 19 & fig. 45).
Similarly, fructose 1,6-bis phosphatase in the liver was found significantly higher in diabetic animals. It was 0.695 (µmol of phosphorous liberated/min/mg of protein). In normal groups the values was 0.430 µmol. The fructose 1,6-bis phosphatase levels were lower in diabetic animals treated with three types of extracts at the rate of
300 mg/kg. The values were 0.545 µmol in case of chloroform leaf extract, 0.477µmol in the ethanol extract treated groups, 0.525 µmol in the aqueous extract treated groups and 0.456 µmol in the ethanol callus extract treated groups. When glibenclamide was administrated at the rate of 600 µg/kg the value was as high as 0.615 µmol of phosphorous liberated/min/mg of protein (Table 19 & fig. 45).
The level of hexokinase was found significantly lower in the kidney of diabetic animals. It was 0.023 (µmol glucose phosphorylated/h/mg protein). With normal animals it was 0.091 µmol. But the Hexokinase levels were higher in diabetic animals treated with 300 mg/kg of extracts tried out with the chloroform leaf extract group it was 0.055 µmol. The value in the ethanol extract treated groups it was 0.065 µmol, in the aqueous extract treated groups it was 0.062 µmol, in the ethanol callus extract treated groups it was 0.086 µmol. The administration of glibenclamide at the rate of
84 600 µg/kg resulted in a score of 0.071 µmol glucose phosphorylated/h/mg protein
(Table 20 & fig. 46).
The level of glucose-6-phosphatase in kidney was significantly higher in diabetic animals. It was 0.256 (µmol of phosphorylated/min/mg protein). In the normal group it was 0.145 µmol. The glucose-6-phosphatase levels were lower in diabetic animals treated with all the three extracts at the rate of 300 mg/kg. The values were
0.239 µmol in the chloroform leaf extract treated group. 0.174 µmol in the ethanol extract treated groups, 0.187 µmol in the aqueous extract treated groups and 0.161
µmol in the ethanol callus extract treated groups. The administration of glibenclamide at the rate of 600 µg/kg resulted in a score of 0.205 µmol phosphorylated/h/mg protein
(Table 20 & fig. 46).
Similarly, the level of fructose 1,6-bis phosphatase in kidney was found significantly higher in diabetic animals. It was 0.520 (µmol of phosphorous liberated/min/mg of protein). In the normal groups it was 0.301 µmol. The fructose 1,6- bis phosphatase levels were lower in diabetic animals treated with 300 mg/kg of all the three extracts. It was 0.480 µmol with chloroform leaf extract, 0.046 µmol in the ethanol extract treated groups, 0.427 µmol in the aqueous extract treated groups and
0.395 µmol in the ethanol callus extract treated groups. The administration of glibenclamide at the rate of 600 µg/kg resulted in 0.502 µmol of phosphorous liberated/min/mg of protein (Table 20 & fig. 46).
The serum protein concentration decreased in diabetic animals. It was 3.98 g/dl.
With the normal group it was 8.11 g/dl on 21st day (Table 21 and fig. 47). The level of protein increased in leaf extract treated animals 4.63 g/dl protein was recorded in the
300 mg/kg of chloroform leaf extract treated animals. In the ethanol extract treated specimen it was 5.62 g/dl; in the aqueous extract treated group it was 4.88 g/dl and in
85 Table 20. Changes in the level of kidney hexokinase, glucose-6-phosphatase and fructose 1, 6 bisphosphatase in normal and experimental animals
Fructose 1,6 - Glucose-6– Hexokinase bis phopatase phospatase (μmol glucose (µmole of Control & Treatment Groups (µmole of phosphorylated phosphorous phosphorylated/ /h/mg protein) liberated/min min/mg protein) /mg protein) Normal I 0.091±0.014 0.145±0.013 0.301±0.014 100 mg/kg II 0.094±0.018 0.128±0.015 0.299±0.012 Chloroform 200 mg/kg III 0.095±0.013 0.126±0.021 0.300±0.032
300 mg/kg 1V 0.092±0.015 0.125±0.007 0.302±0.066 100 mg/kg V 0.093±0.014 0.148±0.121 0.303±0.019 Ethanol 200 mg/kg VI 0.095±0.019 0.147±0.164 0.305±0.025 300 mg/kg VII 0.094±0.016 0.133±0.110 0.306±0.087 100 mg/kg VIII 0.092±0.015 0.129±0.018 0.305±0.021 Aqueous 200 mg/kg IX 0.094±0.022 0.127±0.044 0.298±0.024 300 mg/kg X 0.091±0.033 0.141±0.031 0.301±0.066 100 mg/kg XI 0.090±0.017 0.144±0.115 0.302±0.091 Ethanol callus 200 mg/kg XII 0.095±0.014 0.131±0.169 0.305±0.087 300 mg/kg XIII 0.095±0.019 0.137±0.154 0.304±0.082 Diabetic (STZ) 40mg/kg XIV 0.023±0.005* 0.256±0.031* 0.520±0.029* 100 mg/kg XV 0.051±0.010# 0.248±0.022# 0.514±0.047# STZ + 200 mg/kg XVI 0.054±0.019# 0.245±0.010# 0.490±0.033# Chloroform 300 mg/kg XVII 0.055±0.019# 0.239±0.015# 0.480±0.026# 100 mg/kg XVIII 0.063±0.002# 0.179±0.098# 0.422±0.069# STZ + Ethanol 200 mg/kg XIX 0.064±0.007# 0.177±0.085# 0.422±0.014# 300 mg/kg XX 0.065±0.010# 0.174±0.061# 0.406±0.039# 100 mg/kg XXI 0.059±0.021# 0.220±0.082# 0.479±0.028# STZ + Aqueous 200 mg/kg XXII 0.061±0.004# 0.199±0.042# 0.434±0.085# 300 mg/kg XXIII 0.062±0.005# 0.187±0.054# 0.427±0.084# 100 mg/kg XXIV 0.067±0.009# 0.169±0.039# 0.402±0.051# STZ + Ethanol 200 mg/kg XXV 0.069±0.002# 0.165±0.42# 0.399±0.086# callus 300 mg/kg XXVI 0.086±0.009# 0.161±0.020# 0.395±0.047# STZ + 600 µg/kg XXVII 0.071±0.006# 0.205±0.079# 0.502±0.034# Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 46. Changes in the level of kidney hexokinase, glucose-6-phosphatase and fructose 1, 6 bisphosphatase in normal and experimental animals
0.6
0.5 Hexokinase (μmol 0.4 glucose phosphorylated /h/mg protein) 0.3
0.2 Glucose-6–phospatase (µmole of 0.1 phosphorylated/ min/mg protein) 0 … … … … … … … Fructose 1,6 - bis phopatase (µmole of
Normal phosphorous liberated/min /mg protein) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg STZ + Chloroform 100 STZ STZ + Chloroform 200 STZ + Chloroform 300 STZ Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol callus 200 STZ + Ethanol callus 300 STZ STZ + Ethanol callus 100 STZ STZ + Glibenclamide 600 STZ STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ
Table 21. Changes in the level of serum protein, albumin and haemoglobin in normal and experimental animals
Serum Albumin Haemoglobin Control & Treatment Groups protein (g/dl) (g/dl) (g/dl) Normal I 8.11±0.87 6.98±0.85 14.31±5.78 100 mg/kg II 8.85±0.55 7.45±0.89 14.55±3.81 Chloroform 200 mg/kg III 8.90±0.59 7.62±0.67 14.15±0.63
300 mg/kg 1V 8.52±0.44 7.86±0.31 14.88±0.35 100 mg/kg V 8.72±0.49 7.77±0.72 13.85±4.13 Ethanol 200 mg/kg VI 8.56±0.78 8.53±0.14 14.75±0.54 300 mg/kg VII 8.53±0.26 7.21±0.14 13.87±0.36 100 mg/kg VIII 8.91±0.45 6.55±0.61 14.81±4.07 Aqueous 200 mg/kg IX 8.74±0.62 7.96±0.22 14.30±0.87 300 mg/kg X 8.78±0.36 8.53±0.23 14.92±0.95 100 mg/kg XI 8.61±0.54 7.62±0.08 13.33±0.17 Ethanol callus 200 mg/kg XII 8.43±0.72 7.92±0.31 13.54±0.58 300 mg/kg XIII 8.17±0.54 7.73±0.39 14.13±0.21 Diabetic (STZ) 40mg/kg XIV 3.98±0.61* 2.15±0.46* 8.81±3.71* 100 mg/kg XV 4.02±1.08# 3.02±0.25# 9.02±2.18# STZ + 200 mg/kg XVI 4.58±0.91# 3.13±0.61# 9.74±1.27# Chloroform 300 mg/kg XVII 4.63±0.22# 3.65±0.37# 9.89±1.35# 100 mg/kg XVIII 4.95±1.58# 4.77±0.22# 11.89±1.07# STZ + Ethanol 200 mg/kg XIX 5.09±0.13# 4.81±0.52# 12.05±1.18# 300 mg/kg XX 5.62±0.25# 4.95±0.83# 12.60±1.23# 100 mg/kg XXI 4.64±0.41# 4.22±0.43# 10.65±1.29# STZ + Aqueous 200 mg/kg XXII 4.78±0.85# 4.61±0.45# 10.66±1.79# 300 mg/kg XXIII 4.88±0.36# 4.63±0.86# 11.59±2.23# 100 mg/kg XXIV 7.10±0.35# 4.97±0.32# 13.12±3.58# STZ + Ethanol 200 mg/kg XXV 8.18±0.06# 5.02±0.62# 13.45±1.36# callus 300 mg/kg XXVI 8.84±0.14# 5.45±0.72# 13.55±1.19# STZ + 600 µg/kg XXVII 5.10±0.24# 5.19±0.88# 12.81±1.48# Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 47. Changes in the level of serum protein, albumin and haemoglobin in normal and experimental animals
16 14 12 10 8 Serum protein (g/dl) 6 4 Albumin (g/dl) 2 0
Haemoglobin (g/dl) Normal Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
the ethanol callus extract treated group it was 8.84 g/dl. In the 600 µg/kg of glibenclamide treated group, the scores was 5.10 g/dl.
The albumin level decreased in diabetic animals. It was 2.15 g/dl. In normal animal it was 6.98 g/dl on 21st day (Table 21 & fig. 47). The levels of albumin increased in animals treated with all the three extracts. When the extracts were administrated at the rate of 300 mg/kg, the groups recorded the albumin levels varying from 5.45 to 3.65g/dl. The minimum level was recorded in the group treated with chloroform leaf extract 3.65g/dl. The ethanol extract treated group recorded 4.95 g/dl.
The group treated with aqueous extract recorded 4.63 g/dl and the group treated with ethanol callus extract had the high value (5.45 g/dl). In the group treated with glibenclamide at a dose of 600 µg/kg the albumin level 5.19 g/dl on the 21st day.
The concentration of hemoglobin content decreased in diabetic animals. It was
8.81 g/dl. In normal animals it was 14.31 g/dl on 21st day (Table 21 & fig. 47). The level of haemoglobin increased in leaf extracts treated animals. It was 9.89 g/dl in the chloroform leaf extract treated animals. The ethanol extract treated sample registered
12.60 g/dl. In the case of the aqueous extract treated animals it was 11.59 g/dl; while the ethanol callus extract treated groups showed 13.55 g/dl of hemoglobin. In the glibenclamide (600 µg/kg) treated groups it was 12.81 g/dl.
Table 22 & Fig. 48 show the urea, uric acid, creatinine levels in the normal and experimental groups. The urea level in normal animals was 23.46 mg/dl. The streptozotocin treated control group showed 61.13 mg/dl on the 21stday. Diabetic animals treated with 300 mg/kg of the three extracts showed varying levels of urea. It was 53.40 mg/dl with chloroform leaf extract treated animals; 38.04 mg/dl with animals treated with ethanol extract; 42.48 mg/dl with the animals treated with aqueous extract and 34.05 mg/dl with the animals treated with ethanol extract of callus. With the
86 Table 22. Changes in the level of urea, uric acid and creatinine in normal and experimental animals
Urea Uric acid Creatinine Control & Treatment Groups (mg/dl) (mg/dl) (mg/dl) Normal I 23.46±1.08 2.81±8.83 1.32±0.19 100 mg/kg II 23.01±4.12 2.85±0.65 1.35±0.06 Chloroform 200 mg/kg III 20.08±1.35 2.52±0.30 1.18±0.83
300 mg/kg 1V 22.56±2.97 2.89±0.32 1.15±1.74 100 mg/kg V 21.87±3.19 2.79±0.48 1.31±0.22 Ethanol 200 mg/kg VI 18.67±1.94 2.84±0.05 1.34±0.26 300 mg/kg VII 21.69±1.32 2.87±0.58 1.34±0.14 100 mg/kg VIII 19.01±1.02 1.81±0.51 1.36±0.34 Aqueous 200 mg/kg IX 18.87±1.82 2.65±0.39 1.32±0.32 300 mg/kg X 20.43±1.29 2.97±0.33 1.35±0.18 100 mg/kg XI 21.95±1.25 1.18±0.47 1.02±0.12 Ethanol callus 200 mg/kg XII 21.43±1.28 2.10±1.21 1.06±0.07 300 mg/kg XIII 21.76±1.24 2.76±0.14 1.13±0.11 Diabetic (STZ) 40mg/kg XIV 61.13±1.18* 6.41±3.45* 2.52±0.54* 100 mg/kg XV 56.70±6.42# 4.92±1.53# 2.41±0.12# STZ + 200 mg/kg XVI 54.10±4.95# 4.69±0.98# 2.38±0.26# Chloroform 300 mg/kg XVII 53.40±3.58# 4.55±1.36# 2.37±0.30# 100 mg/kg XVIII 41.04±2.26# 3.18±0.90# 1.75±0.39# STZ + Ethanol 200 mg/kg XIX 40.01±5.45# 3.12±1.0# 1.72±0.22# 300 mg/kg XX 38.04±4.04# 3.11±0.04# 1.69±0.19# 100 mg/kg XXI 49.46±2.98# 4.18±1.55# 2.35±0.36# STZ + 200 mg/kg XXII 45.31±5.12# 3.67±1.20# 2.01±0.26# Aqueous 300 mg/kg XXIII 42.48±3.81# 3.19±1.34# 1.98±0.29# 100 mg/kg XXIV 36.14±1.89# 3.09±1.05# 1.64±0.91# STZ + Ethanol 200 mg/kg XXV 35.48±1.74# 3.05±1.12# 1.58±0.82# callus 300 mg/kg XXVI 34.05±1.76# 3.01±0.9# 1.51±0.21# STZ + 600 µg/kg XXVII 42.01±5.49# 4.38±0.22# 1.62±0.11# Glibenclamide Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 48. Changes in the level of urea, uric acid and creatinine in normal and experimental animals
70
60
50
40 Urea (mg/dl) 30
20 10 Uric acid (mg/dl) 0
Creatinine (mg/dl) Normal Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
animals treated with glibenclamide at the rate of 600 µg/kg the amount was 42.01 mg/dl.
The uric acid level in normal animals was 2.81 mg/dl. In the streptozotocin treated control it was 6.41 mg/dl on the 21stday. Diabetic animals treated with 300 mg/kg of the three extracts showed lesser uric acid content. Animals treated with chloroform leaf extract showed 4.55 mg/dl; animals treated with ethanol extract had
3.11 mg/dl; animals treated with aqueous extract had 3.19 mg/dl of uric acid, whereas those animals treated with ethanol extract of callus had 3.01 mg/dl. Animal treated with
600 µg/kg of glibenclamide had 4.38 mg/dl uric acid.
Similarly the creatinine level in normal animals was 1.32 mg/dl. In the streptozotocin treated control it was 2.52 mg/dl on the 21stday. Diabetic animals treated with 300 mg/kg of the three extracts showed varying levels of creatinine. It was (2.37 mg/dl) in animals treated with chloroform leaf extract. Those treated with ethanol extract recorded 1.69 mg/dl. Animal treated with aqueous extract showed (1.98 mg/dl) and those treated with ethanol extract of callus showed 1.51 mg/dl. Animals treated with 600 µg /kg of glibenclamide had 1.62 mg/dl of creatinine.
The glutathione peroxidase (GPx) levels were found significantly lower in the liver of diabetic animals. It was 3.49 (µmole of glutathione utilized/min). In normal animals it was 9.10 µmole. But the GPx level was higher in diabetic animals treated with 300 mg/kg of all the extracts. The amount of GPx varied from 7.89 µmole to 4.66
µmole. The highest figure was with the ethanol callus extract treated groups (7.89
µmole), with the ethanol extract treated group the figure was 6.05 µmole and 5.15
µmole with the aqueous extract treated groups and 5.57 µmole in the group treated with glibenclamide at the rate of 600 µg/kg. The GPx rate was lowest with the group treated with the chloroform leaf extract (4.66 µmole).
87 The GPx level was found significantly lower in kidney from diabetic animals. It was 2.89 (µmole of glutathione utilized/min). With normal animal it was 7.30 µmole.
But the GPx levels were higher in diabetic animals treated with all the three extracts at the rate of 300 mg/kg. The results were 3.72 µmole in the chloroform leaf extract treated groups, 5.01 µmole with the ethanol extract treated groups, 4.04 µmole in the aqueous extract treated groups, 5.13 µmole in the ethanol callus extract treated groups.
The value was 4.92 µmole in the group treated with glibenclamide at the rate of
600µg/kg (Table 23 & fig. 49).
The reduced glutathione (GSH), levels were found significantly lower in the liver of diabetic animals it was 25.12 mg/100mg tissue. With normal animal it was
67.79 mg/100mg tissue. But the GSH levels were higher in diabetic animals treated with 300 mg/kg of all the extracts. The levels were 50.10 mg/100 mg tissue in animals treated chloroform leaf extract, 58.25 mg/100 mg tissue in those treated with ethanol extract and 52.46 mg/100 mg tissue in the aqueous extract, 60.04 mg/100 mg tissue in the ethanol callus extract treated group. In the group treated with glibenclamide at the rate of 600 µg/kg it was 59.09 mg/100 mg tissue.
The GSH levels were found significantly lower in kidney from the diabetic animals. It was 12.12 μg/mg tissue. With normal animals it was 38.31 μg/mg tissue.
But the GSH levels were higher in diabetic animals treated with 300 mg/kg of all the extracts. With the chloroform leaf extract treated group it was 20.44 μg/mg tissue, with the ethanol extract treated group it was 28.76 μg/mg tissue, with the aqueous extract treated group it was 24.78 μg/mg tissue and with the ethanol callus extract treated group it was 34.65 μg/mg tissue. In the group treated with glibenclamide at the rate of
600 µg/kg it was 24.63 μg/mg tissue (Table 23 & fig. 49.1).
88 Table 23. Changes in the level of glutathione peroxidase (GPx) and reduced glutathione (GSH) in liver and kidney of normal and experimental animals
Glutathione peroxidase Reduced glutathione (µmole of glutathione (mg/100 mg tissue) Control & Treatment Groups utilized/min) Liver Kidney Liver Kidney Normal I 9.10±0.41 7.30±0.35 67.79±2.95 38.31±4.05 100 mg/kg II 7.05±0.31 6.35±0.39 67.45±1.48 37.21±3.85 Chloroform 200 mg/kg III 8.65±0.37 6.85±0.92 67.14±1.36 36.42±2.78
300 mg/kg 1V 7.30±0.28 6.46±0.75 67.79±1.59 44.53±2.16 100 mg/kg V 7.15±0.49 6.38±0.41 67. 79± 2.61 37.62±4.31 Ethanol 200 mg/kg VI 7.65±1.39 6.54±0.15 68.44±2.55 46.52±2.67 300 mg/kg VII 8.35±1.86 7.13±0.12 66.71±2.83 47.62±2.31 100 mg/kg VIII 7.12±0.12 6.31±0.37 68.01±2.42 34.5±2.83 Aqueous 200 mg/kg IX 7.94±0.95 6.56±0.13 67.24±1.86 46.79±2.34 300 mg/kg X 8.87±1.15 6.99±0.41 67.78±1.55 43.42±2.83 100 mg/kg XI 7.47±1.05 7.65±0.22 66.74±1.19 48.47±3.44 Ethanol callus 200 mg/kg XII 7.80±1.90 7.16±0.63 66.16±1.13 43.82±1.70 300 mg/kg XIII 7.16±1.41 7.73±0.14 67.21±1.65 46.12±2.40 Diabetic (STZ) 40mg/kg XIV 3.49±0.12* 2.89±0.29* 25.12±2.98* 12.12±1.42* 100 mg/kg XV 4.05±0.51# 3.55±0.32# 45.65±0.75# 15.60±1.20# STZ + 200 mg/kg XVI 4.52±0.56# 3.69±0.47# 48.91±0.25# 17.39±0.54# Chloroform 300 mg/kg XVII 4.66±0.59# 3.72±0.44# 50.10±0.13# 20.44±0.57# 100 mg/kg XVIII 5.21±0.24# 4.15±0.80# 57.32±1.35# 25.01±0.21# STZ + Ethanol 200 mg/kg XIX 5.51±0.65# 4.27±0.52# 57.88±1.41# 26.13±0.42# 300 mg/kg XX 6.05±0.21# 5.01±0.39# 58.25±3.14# 28.76±0.29# 100 mg/kg XXI 4.75±0.61# 3.91±0.41# 50.47±1.47# 21.09±2.92# STZ + Aqueous 200 mg/kg XXII 5.11±0.31# 3.95±0.59# 52.33±0.12# 21.42±2.84# 300 mg/kg XXIII 5.15±0.33# 4.04±0.46# 52.46±0.10# 24.78±1.39# 100 mg/kg XXIV 6.13±0.99# 5.09±0.45# 58.98±2.25# 30.05±0.34# STZ + Ethanol 200 mg/kg XXV 7.45±0.16# 5.10±0.46# 59.40±2.23# 33.80±0.21# callus 300 mg/kg XXVI 7.89±0.51# 5.13±0.25# 60.04±1.19# 34.65±2.55# STZ + 600 µg/kg XXVII 5.57±0.19# 4.92±0.52# 59.09±1.01# 24.63±0.68# Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control.
Fig. 49. Changes in the level of glutathione peroxidase (GPx) in liver and kidney of normal and experimental animals
10 9 8 7 Glutathione 6 peroxidase in liver 5 (µmole of 4 glutathione 3 utilized/min) 2 1 0 Glutathione peroxidase in
Normal kidney (µmole of glutathione utilized/min) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
Fig. 49.1. Changes in the level of reduced glutathione (GSH) in liver and kidney of normal and experimental animals
80 70 60 50 Reduced 40 glutathione in 30 liver (mg/ 100 mg tissue) 20 10 0 Reduced glutathione in kidney (mg/ 100 Normal mg tissue) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
The glutathione-S-transferase (GST), levels were found significantly lower in the liver from diabetic animals. It was 1.77 (µmole of CDNB-GSH conjugate formed/min/mg/protein). With normal animals it was 7.97 µmole on 21st day (Table 24
& fig. 50). The level of GST is increased in animals treated with all the three extracts.
With chloroform leaf extract treated animals it was 3.01 µmole. The corresponding figure with animals treated with the ethanol extract was 4.55 µmole, with the aqueous extract treated group 3.22 µmole, while with the ethanol callus extract treated groups it was 6.02 µmole. In animals treated with glibenclamide at the rate of 600 µg/kg the corresponding figure was 3.89 µmole of CDNB-GSH conjugate formed/min/mg/protein on the 21st day.
The GST levels decreased significantly in kidney from diabetic animals. It was
1.78 (µmole of CDNB-GSH conjugate formed/min/mg/protein). With the normal animals the figure was 6.25 µmole. The levels of GST increased in animals treated with all the three extracts. In the chloroform leaf extract treated groups it was 2.90 µmole. In the ethanol extract treated groups it was 3.40 µmole, whereas in the aqueous extract treated groups it was 2.95 µmole. In the group treated ethanol callus extract it was as high as for 4.04 µmole. In the group treated with glibenclamide at the rate of 600 µg/kg the score was 2.07 µmole of CDNB-GSH conjugate formed/min/mg/protein (Table 24
& fig. 50).
The superoxide dismutase (SOD), levels were found lower in liver from diabetic animals. It was 4.99 UA/mg protein, (A – The amount of enzyme required to inhibit 50%
NBT reduction). With normal animals it was14.84 UA/mg protein on 21st day (Table 24
& fig. 50.1). The levels of SOD increased in animals treated with all the extracts at the rate of 300 mg/kg. In the chloroform leaf extract treated animals it was 5.59 UA/mg protein; in the groups treated with ethanol extract animals it was 7.55 UA/mg protein; in the groups treated with aqueous extract it was 6.16 UA/mg protein. In groups treated
89 Table 24. Changes in the level of glutathione-S-transferase (GST), superoxide dismutase (SOD) in liver and kidney of normal and experimental animals
Superoxide dismutase Glutathione-S-transferase A (U /min/ mg of protein) (µmole of CDNB-GSH A Control & Treatment Groups conjugate formed/min/mg/ The amount of enzyme protein) required to inhibit 50% NBT reduction Liver Kidney Liver Kidney Normal I 7.97±0.86 6.25±0.29 14.84±0.12 15.70±0.98 100 mg/kg II 6.81±0.09 5.17±0.05 12.86±0.17 15.45±0.82 Chloroform 200 mg/kg III 6.95±0.44 5.64±0.78 11.42±0.04 15.65±0.54
300 mg/kg 1V 7.67±0.89 5.53±0.16 15.44±0.33 15.30±0.87 100 mg/kg V 7.78±0.07 6.18± 0.36 17.83±0.11 15.68±0.94 Ethanol 200 mg/kg VI 8.85±0.96 5.68±0.31 13.69±0.97 15.29±0.83 300 mg/kg VII 6.86±0.86 7.26±0.25 16.15±0.63 16.59±0.26 100 mg/kg VIII 7.82±0.08 5.15±0.31 15.88±0.21 16.71±1.10 Aqueous 200 mg/kg IX 7.87±0.26 7.44±0.83 12.46±0.39 16.57±0.88 300 mg/kg X 7.99±0.36 5.73±0.85 11.76±0.74 16.65±0.54 100 mg/kg XI 6.12±0.75 7.02±0.54 12.74±0.14 15.45±0.38 Ethanol callus 200 mg/kg XII 7.43±0.36 7.20±0.12 15.16±0.65 15.62±0.42 300 mg/kg XIII 7.85±0.87 7.71±0.31 12.21±0.13 16.24±0.19 Diabetic (STZ) 40mg/kg XIV 1.77±0.65* 1.78±0.12* 4.99±0.87* 4.35±0.06* 100 mg/kg XV 2.97±0.41# 2.81±0.73# 5.04±0.99# 7.08±3.25# STZ + 200 mg/kg XVI 2.99±0.15# 2.89±0.23# 5.35±0.48# 7.16±1.29# Chloroform 300 mg/kg XVII 3.01±0.16# 2.90±0.25# 5.59±0.62# 8.20±0.68# 100 mg/kg XVIII 3.52±0.29# 2.97±0.025# 6.78±0.44# 9.18±0.85# STZ + Ethanol 200 mg/kg XIX 4.45±0.39# 3.05±0.087# 7.13±0.52# 9.22±0.87# 300 mg/kg XX 4.55±0.32# 3.40±0.021# 7.55±0.03# 10.64±0.24# STZ + Aqueous 100 mg/kg XXI 3.12±0.87# 2.93±0.37# 5.87±0.55# 8.27±0.54# 200 mg/kg XXII 3.15±0.74# 2.94±0.41# 6.02±0.52# 8.43±0.75#
300 mg/kg XXIII 3.22±0.91# 2.95±0.35# 6.16±0.36# 9.01±0.50# 100 mg/kg XXIV 5.71±0.54# 3.72±0.24# 8.09±0.54# 11.21±0.25# STZ + Ethanol 200 mg/kg XXV 5.85±0.13# 3.99 ±0.41# 8.92±0.17# 11.63±0.39# callus 300 mg/kg XXVI 6.02±0.21# 4.04±0.91# 9.05±0.36# 12.09±0.87# STZ + 600 µg/kg XXVII 3.89±0.12# 2.07±0.19# 7.81±0.09# 9.32±0.92# Glibenclamide Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control.
Fig. 50. Changes in the level of glutathione-S-transferase (GST) in liver and kidney of normal and experimental animals
10 9 8 7 Glutathione-S- 6 transferase in liver 5 (µmole of CDNB-GSH 4 conjugate 3 formed/min/mg/protein) 2 1 0 Glutathione-S- transferase in kidney
Normal (µmole of CDNB-GSH conjugate formed/min/mg/protein) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
Fig. 50.1. Changes in the level of superoxide dismutase (SOD) in liver and kidney of normal and experimental animals
20 18 16 14 12 10 8 Superoxide dismutase in 6 liver (UA/min/ mg of 4 protein)A The amount of 2 enzyme required to inhibit 0 50% NBT reduction
Superoxide dismutase in Normal kidney (UA/min/ mg of protein)A The amount of enzyme required to inhibit 50% NBT reduction Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
with ethanol callus extract it was 9.05 UA/mg protein and 7.81 UA/mg protein, in the group treated with glibenclamide at the rate of 600 µg/kg.
The SOD level was found lower in kidney from diabetic animals. It was 4.35
UA/mg (A – The amount of enzyme required to inhibit 50% NBT reduction). With the normal animal it was 15.70 UA/mg protein on the 21st day (Table 24 and fig. 50.1). The levels of SOD increased in treated animals. It was 8.20 UA/mg protein in the chloroform leaf extract treated groups, in the ethanol extract groups the corresponding figure was 10.64 UA/mg protein. In the case of the aqueous extract treated group it was
9.01UA/mg protein and 12.09 UA/mg protein in the ethanol callus treated groups. In the group treated with glibenclamide at the rate of 600 µg/kg the SOD level was 9.32
UA/mg protein.
The catalase level was found lower in liver from diabetic animals. It was 42.12
B B - unit /mg protein ( µmoles of H2O2 utilized/min). In normal animals the rate was almost more than double 88.55 unitB/mg protein on 21st day (Table 25 & fig. 51). The levels of catalase increased in leaf extracts treated animals. The figure was 55.58 unitB/mg protein in the chloroform leaf extract treated groups. In the ethanol treated group it was 67.80 unitB/mg protein; in the aqueous extract treated group it was 61.02 unitB/mg protein; whereas in ethanol callus extract treated groups it was as high as
79.32 unitB/mg protein. In case of animals treated with glibenclamide 600 µg/kg the corresponding figure 74.11 unitB/mg protein.
Similarly catalase level was found lower in kidney from diabetic animals. It was
21.59 unitB/mg protein. In normal animals it was almost thrice (59.38) on the 21st day
(Table 25 & fig. 51). In leaf extract treated animals the rate was 29.88 unitB/mg protein in the animals treated with chloroform leaf extract. In the ethanol extract treated group it was 43.21 unitB/mg protein, while in the aqueous extract treated group it was 34.54
90 Table 25. Changes in the level of catalase in liver and kidney of normal and experimental animals
Catalase B B Control & Treatment Groups (unit /mg protein) µmoles of H O 2 2 utilized/min Normal I 88.55±5.81 59.38±3.01 100 mg/kg II 91.22±4.46 50.31±2.12 Chloroform 200 mg/kg III 86.78±4.34 54.20±2.15
300 mg/kg 1V 88.55±5.81 59.38±3.01 100 mg/kg V 88.36±3.35 52.18±2.48 Ethanol 200 mg/kg VI 85.96±2.21 56.36±2.57 300 mg/kg VII 89.53±2.23 57.35±2.24 100 mg/kg VIII 89.40±2.35 59.12±2.31 Aqueous 200 mg/kg IX 88.61±2.67 58.46±2.33 300 mg/kg X 86.56±2.31 55.76±2.74 100 mg/kg XI 85.21±2.49 53.11±2.12 Ethanol callus 200 mg/kg XII 85.67±2.18 55.74±2.29 300 mg/kg XIII 83.21±2.85 56.47±2.21 Diabetic (STZ) 40mg/kg XIV 42.12±1.15* 21.59±1.86* 100 mg/kg XV 48.30±1.82# 23.66±1.92# STZ + Chloroform 200 mg/kg XVI 51.52±1.35# 25.47±1.07# 300 mg/kg XVII 55.58±1.21# 29.88±2.04# 100 mg/kg XVIII 64.3±3.71# 35.98±0.42# STZ + Ethanol 200 mg/kg XIX 65.47±2.23# 36.04±2.11#
300 mg/kg XX 67.80±2.31# 43.21±1.43# 100 mg/kg XXI 58.45±1.83# 31.01±2.84# STZ + Aqueous 200 mg/kg XXII 60.2±5.04# 33.44 ±2.29# 300 mg/kg XXIII 61.02±2.09# 34.54±2.60# 100 mg/kg XXIV 69.61±2.41# 44.65±1.28# STZ + Ethanol 200 mg/kg XXV 70.02±1.20# 45.89±1.96# callus 300 mg/kg XXVI 79.32±4.39# 45.91±2.19 # STZ + 600 µg/kg XXVII 74.11±0.46# 41.88±2.06# Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control.
Fig. 51. Changes in the level of catalase in liver and kidney of normal and experimental animals
100 90 80 70 60 Catalase in liver 50 (unitB/mg protein) 40 Bµmoles of H2O2 30 utilized/min 20 10 0 Catalase in kidney (unitB/mg protein) Bµmoles of H2O2
Normal utilized/min Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
unitB/mg protein. In the ethanol callus extract the figure was 45.91 unitB/mg protein. In the group treated with glibenclamide 600 µg/kg corresponding figure 41.88 unitB/mg protein.
The hydroperoxides (HPx) level were higher in liver from diabetic animals
90.94 µmole/mg of protein. In normal animal the figure was 66.11 µmole/mg of protein. But the hydroperoxides level decreased in diabetic animals treated with 300 mg/kg each of the three extract. It was 85.42 µmole/mg of protein of in the case of animals treated with chloroform leaf extract; in the ethanol extract treated groups it was
79.12; in the aqueous extract treated groups 81.01 and 73.28 in the ethanol callus extract treated groups. In the group treated with glibenclamide at the rate of 600 µg/kg it was 89.17 µmole/mg of protein (Table 26 & fig. 52).
The level of hydroperoxides (HPx) was found significantly higher in kidney from diabetic animals. The figure was 75.45 µmole/mg of protein. This was much lower than that in normal animals 34.12 µmole/mg of protein. But the hydroperoxides levels were lower in diabetic animal treated with 300 mg/kg of all the three extracts.
The figure was 65.10 µmole/mg of protein with chloroform leaf extract treated groups;
49.86 µmole/mg of protein with ethanol extract treated groups; 51.22 µmole/mg of protein with aqueous extract treated groups and 41.34 µmole/mg of protein in the case of ethanol callus treated groups. In the glibenclamide treated groups it was 69.26
µmole/mg of protein (Table 26 & fig. 52).
The tissue thiobarbituric acid reactive substances (TBARS), level was found higher in liver from diabetic animals. It was 4.25 nmol/mg of protein. With the normal animals it was 1.67 nmol/mg of protein on 21st day (Table 26 & fig. 52.1). The levels of
TBARS decreased in the experimental groups. The values were 4.15 nmol/mg of protein in the chloroform leaf extract treated groups; it was 3.28 nmol/mg of protein in
91 Table 26. Changes in the level of hydro peroxides (HPx) and tissue thiobarbituric acid reactive substances (TBARS) in liver and kidney of normal and experimental animals
Tissue thiobarbituric Hydro peroxides acid reactive substances (µmole/mg of protein) Control & Treatment Groups (nmole/mg of protein) Liver Kidney Liver Kidney Normal I 66.11±4.02 34.12±4.79 1.67±0.02 1.62±4.79 100 mg/kg II 55.18±3.54 34.02±3.81 1.34±0.05 1.65±0.78 Chloroform 200 mg/kg III 57.8±3.61 36.4±4.79 1.32±0.04 1.22±0.61
300 mg/kg 1V 52.54±3.59 34.53±2.16 1.62±1.02 1.73±0.81 100 mg/kg V 65.97±3.87 29.63±5.10 1.44±0.18 1.63±0.49 Ethanol 200 mg/kg VI 57.8±4.70 34.40±3.67 1.36±0.14 1.92±0.82 300 mg/kg VII 58.82±4.20 34.62±3.32 1.86±0.13 1.72±0.49 100 mg/kg VIII 66.3±4.05 35.18±4.90 1.56±0.10 1.69±0.52 Aqueous 200 mg/kg IX 64.76±3.99 34.65±3.39 1.75±0.04 1.39±0.44 300 mg/kg X 53.76±4.86 29.61±3.54 1.86±0.02 1.24±0.32 100 mg/kg XI 55.40±4.64 32.31±3.19 1.59±0.32 1.75±0.24 Ethanol callus 200 mg/kg XII 55.85±4.66 33.45±3.20 1.15±0.15 1.74±0.48 300 mg/kg XIII 55.37±4.39 31.42±3.06 1.18±0.26 1.45±0.77 Diabetic (STZ) 40mg/kg XIV 90.94±6.85* 75.45±5.57* 4.25±0.31* 4.97±0.32* 100 mg/kg XV 89.17±5.82# 73.26±5.81# 4.18±0.13# 4.86±0.58# STZ + 200 mg/kg XVI 87.29±5.79# 71.02±5.29# 4.16±0.08# 4.82±0.72# Chloroform 300 mg/kg XVII 85.42±4.32# 65.10±6.01# 4.15±0.25# 3.97±0.70# 100 mg/kg XVIII 80.40±5.97# 50.52±4.82# 3.75±0.12# 3.53±0.31# STZ + Ethanol 200 mg/kg XIX 79.96±5.86# 50.49±1.36# 3.62 ±0.37# 3.50±0.14# 300 mg/kg XX 79.12±5.75# 49.86±3.57# 3.28±0.21# 3.33±0.19# 100 mg/kg XXI 83.87±2.04# 62.87±3.03# 4.08±0.33# 3.86±0.11# STZ + 200 mg/kg XXII 82.05±4.12# 53.33±4.57# 4.01±0.15# 3.72±0.45# Aqueous 300 mg/kg XXIII 81.01±4.55# 51.22±4.51# 3.86±0.19# 3.64±0.23# 100 mg/kg XXIV 76.09±4.02# 47.57±2.81# 3.18±0.64# 3.26±0.18# STZ + Ethanol 200 mg/kg XXV 74.25±5.05# 45.39±2.64# 2.35±0.14# 3.14±0.19# callus 300 mg/kg XXVI 73.28±2.91# 41.34±3.12# 5.16±0.09# 4.32±0.73# STZ + 600 µg/kg XXVII 89.17±3.82# 69.26±4.14# 3.85±0.04# 5.18±0.09# Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 52. Changes in the level of hydro peroxides (HPx) in liver and kidney of normal and experimental animals
100 90 80 70 60 50 Hydro peroxides 40 in liver 30 (µmole/mg of 20 protein) 10 0
Hydro peroxides in kidney Normal (µmole/mg of protein) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
Fig. 52. Changes in the level of hydro peroxides (HPx) in liver and kidney of normal and experimental animals
100 90 80 70 60 50 Hydro peroxides 40 in liver 30 (µmole/mg of 20 protein) 10 0
Hydro peroxides in kidney Normal (µmole/mg of protein) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
Fig. 52.1. Changes in the level of tissue thiobarbituric acid reactive substances (TBARS) and in liver and kidney of normal and experimental animals
6
5
4
3 Tissue thiobarbituric 2 acid reactive substances in liver 1 (nmole/mg of protein)
0 Tissue thiobarbituric acid reactive
Normal substances in kidney (nmole/mg of protein) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ + Glibenclamide 600 µg/kg STZ
the ethanol extract treated groups; in the aqueous extract treated groups it was 3.86 nmol/mg of protein. Where as in the ethanol callus extract treated groups it was 5.16 nmol/mg of protein. In the glibenclamide treated groups the levels of TBARS was as low as 3.85 nmol/mg of protein.
In the kidney from diabetic animals TBARS was found at a high level. It was
4.97 nmol/mg of protein. With the normal animals it was 1.62 nmol/mg of protein on the 21st day (Table 26 & fig. 52.1). The levels of TBARS decreased in the leaf extracts treated animals. The value was 3.97 nmol/mg of protein, in the case of animals treated with chloroform leaf extract. In the ethanol extract treated group it was 3.33 nmol/mg of protein. The figure was in 3.64 nmol/mg of protein in the aqueous extract treated groups and 4.32 nmol/mg of protein in the case of the ethanol callus extract treated group. In the group treated with glibenclamide at the rate of 600 µg/kg the corresponding figures 5.18 nmol/mg of protein.
The level of vitamin-C in plasma was found lower in diabetic animals. It was
1.02 mg/dl. In the normal group it was 6.46 mg/dl on 21st day (Table 27 & fig. 53). The
Vitamin-C plasma levels were higher in diabetic animals treated with 300 mg/kg of the extracts. The figure was the highest in the ethanol callus extract treated group (4.05 mg/dl); in the ethanol extract treated group it was 3.15 mg/dl; in the aqueous extract treated group it was 2.09 mg/dl; and in the group treated with chloroform leaf extract it was 1.68 mg/dl. In animals treated with glibenclamide (600 µg/kg) the figure was 2.64 mg/dl.
The levels of vitamin-C in liver were found lower in diabetic animals. It was
0.98 µg/mg protein. In normal groups the figure was 3.93 µg/mg protein. The Vitamin-
C level was higher in diabetic animals treated with 300 mg/kg of the extracts. In the chloroform leaf extract treated groups it was 1.55 µg/mg protein; in the ethanol extract
92 Table 27. Changes in the level of vitamin-C in plasma, liver and kidney of normal and experimental animals
Liver Kidney Plasma Vitamin-C Vitamin-C Control & Treatment Groups Vitamin-C (µg/mg (µg/mg (mg/dl) protein) protein) Normal I 6.46±0.211 3.93±0.16 0.095±0.041
100 mg/kg II 6.75±0.218 3.97±0.12 0.098±0.048 Chloroform 200 mg/kg III 6.43±0.354 3.78±0.56 0.095±0.060
300 mg/kg 1V 7.67±0.687 4.37±0.55 0.098±0.080 100 mg/kg V 6.55±0.215 4.97±0.15 0.095±0.031 Ethanol 200 mg/kg VI 7.66±0.155 4.31±0.30 0.091±0.033 300 mg/kg VII 7.86±0.084 4.81±0.28 0.096±0.040 100 mg/kg VIII 6.81±0.302 3.99±0.31 0.095±0.035 Aqueous 200 mg/kg IX 6.76±0.318 3.46±0.10 0.098±0.063 300 mg/kg X 6.77±0.036 4.37±0.04 0.097±0.076 100 mg/kg XI 6.86±0.013 3.87±0.45 0.095±0.015 Ethanol callus 200 mg/kg XII 6.95±0.015 3.71±0.90 0.096±0.023 300 mg/kg XIII 6.12±0.044 3.94±0.91 0.097±0.028 Diabetic (STZ) 40mg/kg XIV 1.02±0.035* 0.98±0.07* 0.029±0.101* 100 mg/kg XV 1.55±0.049# 1.01±0.24# 0.043±0.067# STZ + 200 mg/kg XVI 1.61±0.047# 1.25±0.58# 0.045±0.080# Chloroform 300 mg/kg XVII 1.68±0.089# 1.55±0.35# 0.049±0.072# 100 mg/kg XVIII 2.19±0.044# 1.65±0.82# 0.075±0.025# STZ + Ethanol 200 mg/kg XIX 2.55±0.071# 1.67±0.71# 0.078±0.071# 300 mg/kg XX 3.15±0.038# 1.94±0.045# 0.079±0.014# 100 mg/kg XXI 1.75±0.052# 1.57±0.89# 0.055±0.086# STZ + Aqueous 200 mg/kg XXII 1.77±0.50# 1.60±0.76# 0.063±0.055# 300 mg/kg XXIII 2.09±0.068# 1.61±0.85# 0.072±0.013# 100 mg/kg XXIV 3.19±0.025# 1.97±0.048# 0.081±0.074# STZ + Ethanol 200 mg/kg XXV 3.45±0.016# 2.01±0.020# 0.085±0.092# callus 300 mg/kg XXVI 4.05±0.069# 2.07±0.091# 0.089±0.031# STZ + 600 µg/kg XXVII 2.64±0.057# 1.75±0.44# 0.076±0.030 # Glibenclamide Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control.
Fig. 53. Changes in the level of vitamin-C in plasma, liver and kidney of normal and experimental animals
9 8 7 6 5 4 Plasma Vitamin-C 3 (mg/dl) 2 1 Liver Vitamin-C 0 (µg/mg protein)
Normal Kidney Vitamin-C (µg/mg protein) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ + Glibenclamide 600 µg/kg STZ
treated groups it was 1.94µg/mg protein; in the aqueous extract treated groups it was
1.61µg/mg protein and the ethanol callus extract treated groups it was 2.07µg/mg protein. In the glibenclamide groups (600 µg/kg) it was 1.75 µg/mg protein.
Similarly the level of vitamin-C in Kidney from diabetic animals was found considerably lower. It was 0.029 µg/mg protein. In normal groups it was 0.095 µg/mg protein. However the vitamin-C level rose in diabetic animals group treated with 300 mg/kg all three extracts prepared. The level was 0.049 µg/mg protein in the chloroform extracts treated groups; it was 0.079µg/mg protein in the case of ethanol extract treated groups; it was 0.072 µg/mg protein in the case of aqueous extract treated groups and
0.089µg/mg protein in ethanol callus extract treated groups. In the glibenclamide treated groups the corresponding figure was 0.076 µg/mg protein.
The level of vitamin-E in plasma was significantly lower in diabetic animals. It was 0.62 mg/dl. In normal animal it was 3.02 mg/dl. The Vitamin-E in plasma level was rose in diabetic animals separately treated with each of the three extracts at the rate of 300 mg/kg. The figure was 0.97 mg/dl in animals treated with chloroform leaf extract, 1.53 mg/dl in animals treated with ethanol extract, 1.48 mg/dl in animals treated with aqueous extract and 2.87 mg/dl in the case of animals treated with ethanol callus. In animals treated with glibenclamide at the rate of 600 µg/kg the levels of
Vitamin-E in plasma was 1.23 mg/dl.
The level of vitamin-E in liver was significantly lower in diabetic animals. It was 1.02 µg/mg protein. In normal animals it was 4.34 µg/mg protein. However the
Vitamin-E levels were higher in the liver of diabetic animals separately treated with
300 mg/kg of each of the extracts. It was 1.54 µg/mg protein in animals treated with chloroform leaf extract, 2.55 µg/mg protein in the group treated with ethanol extract and 1.87 µg/mg protein in the group treated with aqueous extract and 3.92 µg/mg
93 protein in the group treated with ethanol callus extract. In the glibenclamide treated
(600 µg/kg) the figure was 3.62 µg/mg protein.
Similarly the level of vitamin-E in the Kidney was significantly lower in diabetic animals. It was 0.14 µg/mg protein, whereas in normal animals it was almost seven fold that is 0.98 µg/mg protein. Vitamin-E levels were higher in diabetic animals treated separately with each of the extracts at the rate of 300 mg/kg. The levels were
0.40 µg/mg protein in the case of animals treated with chloroform leaf extract. In the ethanol extract treated groups it was 0.75µg/mg protein; in the aqueous extract treated groups it was 0.48 µg/mg protein and in the ethanol callus extract treated groups it was
0.92 µg/mg protein. In the glibenclamide treated groups it was 0.87 µg/mg protein
(Table 28 & fig. 54).
The concentration of liver marker enzymes of acid phosphatase (ACP level) was higher in diabetic animals. It was 45.6 IU/I. In normal animals it was 19.25 IU/I on the 21st day (Table 29 & fig.55). The levels of ACP decreased in leaf extracts treated animals. It was 34.26 IU/I in the chloroform leaf extract treated animals. In the ethanol extract treated group it was 29.47 IU/I; in the aqueous extract treated group it was
31.83 IU/I, where as in the ethanol callus extract treated group it was 23.09 IU/I. In the case of animals treated with 600 µg/kg of glibenclamide the corresponding figure was
39.08 IU/I.
The alkaline phosphatase (ALP), level was higher in diabetic animals. It was
354.91 IU/I. In normal animals it was 220.20 IU/I on the 21st day. The levels of ALP decreased in the experimental groups. The rates were 336.74 IU/I in the chloroform leaf extract treated groups, whereas in the ethanol extract treated group it was 278.66 IU/I.
The corresponding figure in the aqueous extract treated group was 296.41 IU/I. Where
94 Table 28. Changes in the level of vitamin-E in plasma, liver and kidney of normal and experimental animals
Liver Kidney Plasma Vitamin-E Vitamin-E Control & Treatment Groups Vitamin-E (µg/mg (µg/mg (mg/dl) protein) protein) Normal I 3.02±0.065 4.34±0.11 0.98±0.421 100 mg/kg II 3.05±0.071 4.12±0.15 0.99±0.415 Chloroform 200 mg/kg III 3.42±0.014 4.80±0.49 0.96±0.185
300 mg/kg 1V 3.86±0.019 5.86±0.53 0.97±0.365 100 mg/kg V 4.03±0.067 4.37±0.18 0.97±0.321 Ethanol 200 mg/kg VI 3.65±0.086 5.14±0.32 0.105±0.216 300 mg/kg VII 3.87±0.023 4.12±0.37 0.109±0.143 100 mg/kg VIII 4.08±0.043 4.65±0.41 0.96±0.318 Aqueous 200 mg/kg IX 3.43±0.067 6.34±0.48 0.105±0.326 300 mg/kg X 3.02±0.063 4.18±0.23 0.108±0.317 100 mg/kg XI 3.67±0.021 4.27±0.40 0.106±0.154 Ethanol callus 200 mg/kg XII 3.21±0.091 4.67±0.91 0.103±0.127 300 mg/kg XIII 3.20±0.031 4.30±0.37 0.105±0.186 Diabetic (STZ) 40mg/kg XIV 0.62±0.083* 1.02±0.08* 0.14±0.264* 100 mg/kg XV 0.95±0.184# 1.45±1.88# 0.32±0.118# STZ + 200 mg/kg XVI 0.96±0.323# 1.52±1.01# 0.39±0.126# Chloroform 300 mg/kg XVII 0.97±0.139# 1.54±1.61# 0.40±0.135# 100 mg/kg XVIII 1.50±0.41# 2.01±0.47# 0.59±0.166# STZ + Ethanol 200 mg/kg XIX 1.52±0.029# 2.07±1.01# 0.63±0.271# 300 mg/kg XX 1.53±0.032# 2.55±0.86# 0.75±0.48# 100 mg/kg XXI 0.99±0.086# 1.72±1.04# 0.44±0.323# STZ + Aqueous 200 mg/kg XXII 1.01±0.098# 1.79±0.97# 0.46±0.127# 300 mg/kg XXIII 1.48±0.036# 1.87±0.56# 0.48±0.144# 100 mg/kg XXIV 1.97±0.16# 2.85±0.04# 0.81±0.161# STZ + Ethanol 200 mg/kg XXV 2.04±0.054# 3.02±0.19# 0.86±0.20# callus 300 mg/kg XXVI 2.87±0.06# 3.92±0.25# 0.92±0.49# STZ + 600 µg/kg XXVII 1.23±0.057# 3.62±1.49# 0.87±0.154# Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 54. Changes in the level of vitamin-E in plasma, liver and kidney of normal and experimental animals
7 6 5 Plasma Vitamin-E 4 (mg/dl) 3 2 Liver Vitamin-E (µg/mg protein) 1 0 Kidney Vitamin-E (µg/mg protein) Normal Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Glibenclamide 600 µg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ
Table 29. Changes in the level of acid phosphatase (ACP), alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT) levels in normal and experimental animals
Acid Alkaline Aspartate Alanine Control & Treatment Groups phosphatase phosphatase transaminase transaminase (IU/I) (IU/l) (IU/l) (IU/l) Normal I 19.25 ±1.15 220.20±2.52 71.32±1.48 40.41±3.72 100 mg/kg II 19.01± 1.12 186.68±2.45 65.6±2.172 40.01±2.85 Chloroform 200 mg/kg III 18.49±1.83 206.37±1.18 70.05±1.60 35.98±1.04
300 mg/kg 1V 18.32±1.34 198.80±1.86 71.86±1.78 37.42±3.33 100 mg/kg V 19.28±1.17 191.07±1.55 64.86±2.47 39.30±2.68 Ethanol 200 mg/kg VI 19.01±1.36 190.48±1.47 64.33±1.04 38.26±3.48 300 mg/kg VII 17.63±1.97 194.36±1.84 71.82±1.86 33.89±2.52 100 mg/kg VIII 19.55± 1.33 189.11±0.32 66.1±2.12 40.25±3.81 Aqueous 200 mg/kg IX 16.79±1.34 183.97±1.29 68.23±1.42 31.29±2.88 300 mg/kg X 18.27±1.18 192.61±1.24 67.49±2.67 36.78±2.89 100 mg/kg XI 17.30±1.48 204.61±1.65 71.95±1.96 33.16±2.54 Ethanol callus 200 mg/kg XII 17.85±1.24 184.49±1.29 72.84±1.37 34.44±2.63 300 mg/kg XIII 17.94±1.40 189.62±1.33 65.62±1.48 39.23±2.17 Diabetic (STZ) 40mg/kg XIV 45.6 ±1.95* 354.91±0.23* 215.1±2.36* 103±6.52* 100 mg/kg XV 40.25±1.82# 345.14±1.80# 185.95±1.50# 95.11±1.16# STZ + 200 mg/kg XVI 38.62±1.75# 342.24±1.30# 178.13±1.35# 93.80±1.68# Chloroform 300 mg/kg XVII 34.26±1.09# 336.74±1.62# 176.49±1.04# 90.04±1.75# 100 mg/kg XVIII 31.08± 1.17# 287.56±0.58# 124.46±1.81# 75.25±1.98# STZ + Ethanol 200 mg/kg XIX 30.47±1.29# 283.08±0.31# 121.46±3.72# 72.42±5.48# 300 mg/kg XX 29.47±0.87# 278.66±1.34# 101.18±0.30# 69.91±1.36# 100 mg/kg XXI 33.44 ±1.78# 329.87±1.43# 160.93±2.24# 89.81±0.18# STZ + Aqueous 200 mg/kg XXII 31.99±0.47# 321.46±0.28# 155.6± 0.56# 83.41±2.42# 300 mg/kg XXIII 31.83±0.32# 296.41±0.26# 135.98±1.17# 81.92±2.26# 100 mg/kg XXIV 25.16±0.25# 245.36±1.58# 91.20±0.125# 55.97±2.14# STZ + Ethanol 200 mg/kg XXV 24.01±0.19# 244.10±0.33# 85.43±0.066# 48.3±2.32# callus 300 mg/kg XXVI 23.09±0.13# 234.32±0.39# 80.21±1.65# 45.96±1.46# STZ + 39.08±1.45# 335.18±2.45# 189.32±0.39# 96.21±3.52# 600 µg/kg XXVII Glibenclamide
Values are expressed as mean±SD (n=6). Diabetic control is compared with normal; *Values are statistically significant at P* <0.05 compared to normal. Treated groups are compared with diabetic control; # Values are statistically significant at P# <0.05 compared to diabetic control. Fig. 55. Changes in the level of acid phosphatase (ACP), alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT) levels in normal and experimental animals
400 350 300 Acid phosphatase 250 (IU/I) 200 150 Alkaline phosphatase (IU/l 100 50 Aspartate 0 transaminase (IU/l)
Alanine
Normal transaminase (IU/l) Ethanol 100 mg/kg Ethanol 200 mg/kg Ethanol 300 mg/kg Aqueous 100 mg/kg Aqueous 200 mg/kg Aqueous 300 mg/kg Chloroform 200 mg/kg Chloroform 300 mg/kg Chloroform 100 mg/kg Diabetic (STZ 40 mg/kg) Diabetic (STZ Ethanol callus 100 mg/kg Ethanol callus 200 mg/kg Ethanol callus 300 mg/kg STZ + Ethanol 100 mg/kg STZ + Ethanol 200 mg/kg STZ + Ethanol 300 mg/kg STZ STZ + Aqueous 100 mg/kg + STZ Aqueous 200 mg/kg + STZ Aqueous 300 mg/kg + STZ STZ + Chloroform 100 mg/kg STZ + Chloroform 200 mg/kg STZ + Chloroform 300 mg/kg STZ STZ + Ethanol callus 100 mg/kg STZ + Ethanol callus 200 mg/kg STZ + Ethanol callus 300 mg/kg STZ + Glibenclamide 600 µg/kg STZ
in the ethanol callus extract treated group it was 234.32IU/I. In the animals treated with glibenclamide (600 µg/kg) the figure was 335.18 IU/I (Table 29 & fig. 55).
The activity of aspartase transaminase (AST) was found higher in diabetic animals. It was 215.1 IU/I. In normal animal it was almost one third 71.32 IU/I on the
21st day (Table 29 & fig. 53). The levels of AST decreased in leaf extracts treated groups. It was 176.49 IU/I in animals treated with chloroform leaf extract at the rate of
300 mg/kg; in animals treated with a similar dose of ethanol extract it was 101.18 IU/I; in the case of aqueous extract treated animals it was 135.98 IU/I and 80.21 in the case of animals treated with ethanol callus extract. In the glibenclamide treated group (600
µg/kg) the figure was 189.32 IU/I
The activity of alanine transaminase (ALT) level was found higher in diabetic animals. It was 103 IU/I, where as in normal animals it was 40.41 IU/I on 21st day. The level of ALT decreased in leaf extracts treated animals. It was recorded as 90.04 IU/I in the case of animals treated with chloroform leaf extract at the rate of 300 mg/kg. A similar dose of ethanol extract produced 69.91 IU/I. In the aqueous extract treated groups the figure was 81.92 IU/I, while in the ethanol callus treated group it was 45.96.
In the group treated with glibenclamide at the rate of 600 µg/kg the corresponding figure was 96.21 IU/I (Table 29 & fig. 55).
Histological examination of the normal liver tissue (Group. I) section shows hepatic vein, normal hepatocyte congestion (fig. 56). The sections of liver tissues of healthy rats treated with P. daemia (Groups. II-XIII) did not show any significant changes in the liver architecture, and no pathological changes when compared to the group-I (fig. 56 - fig. 56B. a). The liver section of diabetic animal showed dilated vein, vascular inflammation, periductal inflammation, damaged bile duct, nuclear vacuolation, dissolution of hepatocyte cytoplasm (fig. 56B. b).
95
P.daemia leaves chloroform extract, ethanol extract, aqueous extract and ethanol leaves derived callus extract at the various concentration of (100 mg/kg, 200 mg/kg and 300 mg/kg) and 600 µg/kg of glibenclamide treatment brought back the cellular arrangement. The portal tracts shows vascular proliferation, mild dilation of sinusoids, venous congestion, sinusoidal congestion kuffer cell hyperplasia-lymphoid aggregates canalicular widening, dilation of hepatic vein, congestion, no persisting changes with focal necrosis of hepatocytes. It almost restored the cellular arrangement of hepatocyte around the central vein and reduced fibril. It also supports to bring the blood vessels to normal condition (fig. 56B - fig. 56D).
Kidney section of the normal animal (Group. I) showed the congestion, glomerular reaction, tubular epithelial changes (fig. 57). Kidney sections of healthy rats treated with P.daemia (Group. II-XIII) did not show any significant changes in the kidney architecture, and no pathological changes when compared to the group-I (fig.
57- fig. 57B). Diabetic rat kidney showed the thickening on the capillary loops with increases in the thickness of the wall with degenerated glomerulus with distorted proximal and distal convoluted tubules (fig. 57B. b).
In P.daemia leaf extract and leaf callus extract (100 mg/kg, 200 mg/kg and 300 mg/kg) and 600 µg/kg of glibenclamide treated animal shows the wall rejuvenated proximal and distal convoluted tubules, glomerulus with diffuse glomerulosclerosis, increased mesengial matrix and thickening of capillary wall, stromal congestion and normal architecturiure, tubular changes, vacuolation of tubular cells, normal proximal and distal convoluted tubules (fig. 57B - fig. 57D).
Many of the oral anti diabetic agents have a number of serious side effects; so managing diabetes without any side effects is still a challenge (Radermecker and
96
Scheen, 2007). Therefore a search for a more and safer hypoglycemic agent is still a challenge and a vital area of research.
The solvent extract at different doses in P.daemia have been studied extensively chloroform, ethanol, aqueous and ethanol callus extracts of P.daemia were tested on diabetic rats. They were proved to have significant hypoglycemic properties. These extracts improved the biochemical parameters assessed, also brought about regeneration of β-cells of the pancreas and increased insulin levels.
Streptozotocin at high doses selectively destroys the pancreatic insulin-secreting
β-cells, leaving less functional cells resulting in diabetes mellitus (Kamtchouing et al.,
1998; Sridhar et al, 2005; Pushparaj et al., 2006; Skyler, 2007). In most of the experimental studies hyperglycemia was induced by streptozotocin or alloxan
(Leatherdale et al., 1981; Hamada et al., 2006).
In the present investigation, daily administration of chloroform, ethanol, aqueous, ethanol callus extracts of P. daemia led to decrease in blood glucose levels in
STZ-induced diabetic rats. Though all the four extracts proved to be effective, the ethanol callus leaf extract and ethanol leaf extract of P. daemia had satisfactory capacity to restore glucose levels to near normal. P. daemia is claimed to be useful in diabetes in folklore medicine. The results of the present study indicate that the plant extract was found to reduce the blood glucose level in STZ-induced diabetic rats.
Increased levels of serum triglycerides and cholesterol observed in streptozotocin-induced diabetic rats were in more or less agreement with other studies
(Annida and Stanely Mainzen Prince, 2004; Tunali and Yanardag, 2006). The abnormally high concentration of serum lipids in diabetic animals is due mainly to an increase in the mobilization of free fatty acids from the peripheral fat depots, since insulin inhibits the hormone-sensitive lipase (Pushparaj, 2000). Excess fatty acid in the
97 serum of diabetic rats is converted into phospholipids and cholesterol in the liver. These two substances along with the excess triglycerides formed at the same time in the liver may be discharged into the blood in the form of lipoproteins (Bopanna et al., 1997).
Hypertriglyceridaemia is common among diabetic patients and is responsible for vascular complications (Kudchodkar et al., 1988). In the study of Bruan and
Severson (1992), it was concluded that deficiency of lipoprotein lipase activity may contribute significantly to the elevation of triglycerides in diabeties. Lopes Virella et al.
(1977) reported that treatment of diabetes with insulin served to lower plasma triglycerides levels by returning lipoprotein lipase levels to normal. In the present study the decreasing levels of plasma triglycerides and cholesterol levels following the treatment with P. daemia leaf extract and glibenclamide may be due to their stimulating effect on insulin secretion.
The observed increase in the levels of glycosylated haemoglobin (HbA1C) in the diabetic control group of rats is due to the presence of excessive amounts of blood glucose. During diabetes, the excess of glucose present in the blood reacts with haemoglobin (Alyassin and Ibrahim, 1981; Sheela and Augusti, 1992). The mechanism by which increased oxidative stress is involved in only diabetic complications like activation of transcription factors, advanced glycated end products and protein kinase-C are only partially known. Glycosylated haemoglobin was found to be increased over a long period of time in cases of diabetic mellitus (Bunn et al., 1978). There is evidence that glycation may itself induce the regeneration of oxygen derived free radical in diabetic conditions (Gupta et al., 1997). In the present study, the oral administration of
P. daemia leaf extract and glibenclamide normalized the insulin and glycosylated haemoglobin level.
98 In streptozotocin-induced diabetic rats, increased food consumption and decreased body weight were observed. This indicates polyphagic condition and loss of weight due to excessive break-down of tissue proteins (Chatterjea and Shinde, 2002).
Hakim et al. (1997) stated that decreased body weight in diabetic rats could be due to dehydration and catabolism of fats and proteins. Increased catabolic reactions leading to muscle wasting might also be the cause for the reduced weight gain by diabetic rats
(Raj Kumar et al., 1991). P.daemia leaf extracts administration to diabetic rats decreased food consumption and improved body weight and this could be due to a better control of the hyperglycemic state in the diabetic rats. Decreased levels of blood glucose could improve body weight in streptozotocin-diabetic rats (Kamalakkanan et al., 2003; Babu and Stanely Mainzen Prince, 2004).
One of the key enzymes in the catabolism of glucose is hexokinase, which phosphorylates glucose and converts it into glucose-6-phosphate (Weber et al., 1996).
The activity of hexokinase, the rate limiting enzyme of glycosis, was found to be significantly higher on P. daemia treatment. Insulin was shown to be a potentiator of hexokinase/glucokinase (Laakso et al., 1995). Hexokinase activity was restored on
P. daemia mediation. In the present study, the hexokinase activity was found lower in the liver and kidney of diabetic rats, which may be due to the diabetes induced deficiency of insulin. Partial or total deficiency of insulin in the activity of regulatory enzymes of glycosis and glycogen synthesis was reported by Huang et al. (1996) and
Anand et al. (2009). The key enzymes regulating the glycolytic metabolite pools and glycolytic pools are very important to maintain the normal blood glucose levels by keeping a balance between glucose production and its utilization in the body. The reduction of blood glucose in P. daemia treated diabetic rats may be due to the insulinomimetric action islet of pancreas and restoration of hexokinase activity.
99 Insulin decreases gluconeogenesis by decreasing the activities of the key enzymes, such as glucose-6-phosphatase and fructose-1,6-bisphosphatase. Glucose-6- phosphatase is an important enzyme in homeostasis of blood glucose as it catalyzes the terminal step both in gluconeogenesis and glycogenolysis (Heris et al., 1989). Fructose-
1,6-bisphosphatase is one of the key enzymes of gluconeogenic pathway. It is presented in the liver and kidney of diabetic rats may be due to insulin deficiency. Administration of P. daemia and glibenclamide decreased the gluconeogenic enzyme activities in diabetic rats enhanced glucose utilization. This increased utilization leads to decreased blood glucose level.
A decrease in serum protein content with concomitant increase in protein level of diabetic rats was observed in the present study. Advanced oxidative protein products
(AOPP), reactive oxygen species (ROS) and free radicals produce protein carbonyl products (PCO) and are considered as markers of oxygen-mediated protein damage.
They also indicate changes in glomerular filtration barrier that result in the increased permeability of the membrane (Reznick and Packer, 1994; Madianov et al., 2000).
Serum protein, albumin and haemoglobin levels were reduced in diabetic rats as reported by Prakasam et al. (2004) from their herbal drug antidiabetic study. Serum protein, albumin and haemoglobin level reduction may be due to increased protein catabolism caused by streptozotocin (Almdal and Vilstrup, 1988). Administration of
P. daemia and glibenclamide showing the improvement in renal function.
STZ administration elevated renal markers, i.e. serum urea, uric acid and creatinine, which are responsible for proper functioning of the kidney and changes in the glomerular filtration rate (Mauer et al., 1981). The plasma levels of urea, uric acid and creatinine levels were measured, as streptozoction causes renal damage in diabetic rats due to abnormal glucose regulation, including elevated glucose and glycosylated protein tissue levels, haemodynamic changes within the kidney tissue, and increased
100 oxidative stress. The STZ-induced diabetic rats exhibited significantly higher urea, uric acid and creatinine levels compared to the control group. However, the P. daemia extract supplement lowered these values to a control range. These results are in agreement with previous studies (Eidi et al., 2009; Ram Kumar et al., 2009).
Carbonylation of proteins is a feature of irreversible oxidative damage, often leading to a loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease-derived protein dysfunction. When moderately carbonylated proteins are degraded by the proteasomal system, heavily carbonylated proteins tend to form high molecular weight aggregates which are resistant to degradation and accumulates as damaged or unfolded proteins. STZ-induced oxidative damage in proteins was revealed by the increased content of carbonylated proteins in the tissue (Cumaoglu et al., 2007). The treatment of STZ-injected animals with P. daemia extract lowered the proteins oxidant damage in rat liver and kidney tissues.
Oxidative stress in diabetes coexists with a reduction in the antioxidant capacity, which can increase the deleterious effects of the free radicals. The endogenous antioxidant system may counteract the ROS and reduce the oxidative stress with the enzymatic antioxidants SOD, CAT and GPx. SOD protects tissues against oxygen free radicals by catalysing the removal of superoxide radical, converting it into
H2O2 and molecular oxygen, which damage both the cell membrane and other biological structures (Arivazhagan et al., 2000). Catalase is a haem-protein, which is responsible for the detoxification of significant amounts of H2O2 (Cheng et al., 1981).
The kidney of diabetic animals showed decrease in free radical and reactive oxygen scavenging activity of the key antioxidant enzymes GPx, GST and GSH.
⎯ Reduced antioxidant activity results in over accumulation of O2 and H2O2, which further generate OH⎯ diabetic kidney damage (Anuradha and Selvam, 1993). P. daemia
101 administration increased the activities of antioxidants in the kidney of diabetic rats.
This may be due to the excess production of antioxidants due to P. daemia administration and further protection from toxic effects of free radical intermediates, so it is concluded that the P. daemia extract is very effective in diabetes and that the effects could be mediated through the pancreatic antioxidant without side effects.
Lipid peroxidation is a characteristic of diabetes mellitus. Lipid peroxidation is a free radical induced process leading to oxidative deterioration of polyunsaturated fatty acids. Under physiological conditions, the concentrations of lipid peroxides in the tissues are low. Karpen et al. (1982) reported elevated levels of lipid peroxides in the plasma of diabetic rats. Lipid peroxide-mediated tissue damage resulted in the development of both type I and II diabetes.
Low levels of lipid peroxides stimulate the secretion of insulin, but when the concentration of endogenous peroxides increases, it may initiate uncontrolled lipid peroxidation, thus leading to cellular infiltration and islet cell damage in type I diabetes
(Metz et al., 1984). The most commonly used indicators of lipid peroxidation are
TBARS products. The increased lipid peroxidation in the tissues of diabetic animals may be due to the observed increase in the concentration of TBARS (Lyons, 1991). In the present study, there was an increase in the concentration of TBARS and hydroperoxides were observed in the liver and kidney of diabetic rats. The increase in the level of TBARS and hydroperoxides suggests enhanced LPO leading to tissue injury and failure of the antioxidant defence mechanism to prevent the formation of excess free radicals. The administration of P. daemia and glibenclamide resulted in significant reduction of TBARS and hydroperoxides levels. This reduction may be due to the availability of antioxidants. Thus P. daemia leaf extract offered protection against oxidative stress by scavenging the free radicals that cause injuries.
102 The non-enzymatic antioxidant defence systems are the second line of defence against free radical damage. Vitamin-C, a potent water soluble non-enzymic antioxidant effectively intercepts oxidants in the aqueous phase before they attack and cause detectable oxidative damage (Beter, 1994). Vitamin-C plays an important role in the detoxification of reactive intermediates produced by cyt-P450 which detoxifies xenobiotics. In the present investigation, the decrease in vitamin-C level may be due to increased utilization of vitamin-C as an antioxidant defence against reactive oxygen species or to a decrease in GSH level, since GSH is required for recycling of vitamin-C
(Inofers and Sies, 1988). The observed decrease in the levels of vitamin-C in the diabetic condition is consistent with previous reports (Stanely Mainzen Prince and
Menon, 1999).
Vitamin-E is important radical scavenging antioxidants that interrupt the chain reaction of LPO by reacting with the lipid proxy radical (Buethner, 1999). In the present experiment, a decreased level of vitamin-E in tissues of diabetic rats was observed. Reduced vitamin-E levels in tissues may be due to the decreased vitamin-C level, because there is well established synergism between vitamin-C and vitamin-E.
This is in accordance with earlier investigations (Asayama et al., 1994), which showed increased levels of plasma tocopherol in STZ-diabetic rats. Treatment with P. daemia and glibenclamide showed normalization of non-enzymic antioxidant levels.
The present study indicates significant increases in the activity of ACP, ALP,
AST and ALT of STZ-diabetic animals. This concurs with the findings of Ahmed
(2005) and Derosa et al. (2007). In the present investigation, AST activity appears to be more elevated than ALT. According to Tanaka et al. (1988), AST had more activity than ALT in the liver of diabetic rat. This corroborates with the present findings. The higher AST level in diabetic rats in the present study is due to their greater need for gluconeogenic substrate. The elevation of both enzymes may also reflect the damage of
103 the hepatic cells. Rawi (1995); Kim et al. (2006) concluded that the elevation in AST and ALT levels may be due to destructive changes in the hepatic cells as a result of toxemia. On the other hand, other investigators have postulated that diabetes could induce defects in sarcolemmal enzymatic activities which led finally to such effects
(Micheal et al., 1985). The present study reveals that treatment of STZ-diabetic rats with the tested plant extracts and glibenclamide causes a detectable decrease in transaminases activity. The data recorded in the present investigation show a marked decrease in ACP and ALP activity after treatment with P. daemia extract and as glibenclamide. The present finding are in agreement with (Rawi, 1995) who reported that glibenclamide caused a significant decrease in serum hepatic enzyme activity, and also showed that glibenclamide causes restoration of the elevated enzyme to normal levels (Saha et al., 2008).
The streptozotocin is a broad spectrum antibiotic extracted from Streptomyces acromogenes. STZ-induced diabetes causes partial destruction of β-cells of the islets, which leads to a reduction in insulin release (Bailey and Flatt, 1986). Streptozotocin, not only destroys the pancretic cell, but also damages the liver and kidney (Zafar and
Syed Naeem Ul Hassan Naqvi, 2010). In the present study, the degenerative changes in the histology of liver and kidney brought about by STZ administration are similar to earlier observation (Selvan et al., 2008).
The histological structure of liver of STZ induced diabetic rats showed marked structural alterations as a result of absence of insulin. The major alteration was periportal fatty infiltration, vascular and periductal inflammation, necrosis of hepatocytes. Histopathology of the liver in normal animals showed normal hepatic cell wall and normal hepatocyte congestion. In the diabetic control groups, liver section showed damaged bile duct, nuclear vacuolation, dissolution of hepatocyte cytoplasm.
In diabetic rats treated with various extracts of P. daemia leaf and leaf derived callus
104 extract, liver sections maintained vascular proliferation, hepatic vein with mild periportal inflammation. A similar result was reported with Gymnema sylvestre in diabetic rabbits by Shanmuga Sundaram et al. (1983). El Soud et al. (2007) reported periportal necrosis, congestion in the portal veins and mononuclear cell infiltration in the portal area, three weeks after STZ-administration. In normal animals treated with
P. daemia leaf and leaf derived callus extract showed normal hepatic cells with well preserved cytoplasm, nucleus, nucleolus and central vein in which normal hepatic structure was maintained.
During diabetes the liver shows decrease in weight due to enhanced catabolic process such as glycogenolysis, lipolysis and proteolysis which is the outcome of lack of insulin and /or cellular glucose in liver cells (Meyer et al., 1998). These changes may lead to serious micro vascular renal complications, which involve a series of metabolic changes in the pathogenic of diabetic nephropathy (Raju et al., 2001). In the present study, treatment with P. daemia leaf and leaf derived callus extract significantly prevented the pathogenic symptoms, almost too normal levels.
The main function of the kidney is to excrete the waste products of metabolism and to regulate the body concentration of water and salt. Renal diseases are most common and severe in diabetes (Rhoads et al., 2006). Indeed, relationships between treatment–related alterations in urea concentration and histopathology of the kidney have been reported in rats. Creatinine, a marker of renal function is significantly higher in diabetic control animals (Siperstein et al., 1973). The total protein was restored to the normal range in Vinca rosea flower and leaf treated diabetic rats (Ghosh and
Suryawanshi, 2001). Different extracts of Elephantopus scaber brought about a significant increase in serum protein, and a decrease in urea and creatinine levels of
STZ-induced diabetic rats. Decrease in protein and increase in serum urea and creatinine concentrations, which are considered as a marker of kidney dysfunction,
105 have been rectified by administration of these extracts in STZ-induced diabetic rats.
The diabetic factors associated with hyperglycemia were responsible for dilation of proximal and distal tubules in the cortex in diabetic animal. The kidney showed signs of degenerated glomerulus with distorted proximal and distal convoluted tubules.
Diabetic animals treated with P. daemia leaf and leaf derived callus extract, the kidney showed normal histology. The recovery of renal function occurred on treatment with P. daemia leaf and leaf derived callus extract can be explained by the regenerative capability of the renal tubules. Similar results have been observed with the treatment in
Elephantopus scaber by Daisy and Edel Priya (2010). The role of P. daemia leaf and leaf derived callus extract in reversing the diabetic state at the cellular level and regulating the metabolic normalization further proves its potential as an antidiabetic drug. Histopathological studies of liver and kidney of diabetic control and P. daemia leaf and leaf derived callus extract treated groups indicate that the plant drug has cytoprotective properties.
The present studies indicate that P. daemia leaf and callus extract and glibenclamide led to improvement of histological factors in the liver and kidney of diabetic rats. This may be due to the presence of antidiabetic compound in the extract, which acts synergistically as antioxidants. Histological examination of liver and kidney showed the recovery of damaged tissues in the treated groups.
106 Chapter 5 SUMMARY
Medicinal plants have been in use in traditional health care systems since ancient times and are still the most important health care source for the vast majority of the population around the world. At present, 80% of the populations in developing countries rely largely on plant based drugs for their primary health care needs, and the world market for herbal products based on traditional knowledge was now estimated to be worth US $ 60 million. Among the 4,00,000 plant species on the earth, only a small percentage have been phytochemically investigated and the fraction submitted to biological or pharmacological screening is even smaller. Only 1,00,000 secondary plant products have so far been characterized. Thirty percent of the drugs sold worldwide contain compounds derived from plant material. The plant kingdom thus represents an enormous reservoir of pharmacologically valuable materials to be discovered.
Pergularia daemia – (Veliparuthi in Tamil). The plant is pungent, cooling, anthelmintic, laxative and antipyretic. It cures biliousness, asthma, ulcers. It is useful in eye complaints, urinary discharges, leucoderma, uterine complaints, inflammation, facilitates parturition, urinary disorders and cardiac problems. Decoction and juice of the leaves are reputed to be a cure for snakebite. The plant has a general reputation as an expectorant, emetic and also it is used in infantile diarrhea. It is commonly used among the folks of Sherveroy hills of Tamilnadu as a substitute for Gymnema sylvestre for the treatment of diabetes.
Pergularia daemia is an important plant described in the Ayurveda. More quantity of the plant is needed for the medicinal requirement and also need to genetic
107 stability. So, the standard protocol is needed for in vitro propagation of the plant and that will help to conservation.
In order to establish an efficient in vitro propagation system of P. daemia, the leaf explants were inoculated on MS solid medium supplemented with varying concentrations of either BAP/KIN alone and in combination with NAA respectively.
Leaf segments cultured on MS basal medium (without growth regulators) did not show any growth response. The combination of BAP (0.5 mg/l) and NAA (1.0mg/l) positively affected the multiplication rate of the P. daemia compared with BAP alone, successful results of shoot multiplication from leaf segments explants cultured on MS medium were obtained. The multiplication at the rate of 95% explants produced shoot with an average of 7.3 explants of P. daemia compared with BAP and Kinetin alone and kinetin combination with NAA. The optimum rooting efficiency for shoots (95%) as well as the best root number shoot-1 was obtained on MS media supplemented with
0.5 mg/l and 1.0 mg/l IAA respectively. IBA is clearly more effective in promoting root induction than NAA and IAA. The optimum rooting efficiency for shoots (95%) as well as the best root number per shoot was obtained on MS media supplemented with 0.5 mg/l and 1.0 mg/l IAA respectively. The hardening process of P. daemia was carried out by transferring 9-10 cm length rooted plantlets to 8.0 cm diameter plastic pot containing mixture of soil and vermicompost mixture 3:1 ratio. The in vitro grown potted plants that were to be hardened were incubated with growth chamber for 2 weeks.
Callus was initiated from stem and leaf explants of P. daemia on basal MS medium supplemented with 2,4-D, α-NAA, BAP, KIN and IBA at different concentrations (1.0 - 3.0 mg/l). The maximum induction rate was recorded as 94.2% in leaf explants of 2,4-D 2.0 mg/l combination with 0.5mg/l BAP and 1.0 mg/l α-NAA and 87.1% in stem explants on MS medium with 2.0 mg/l 2,4-D and 0.5 mg/l BAP and
108 1.0 mg/l α-NAA. The callus nature was delicate with pale green in colour. The yield of fresh biomass was high on medium with 2,4-D 141.25 gfw/l; 8.02 gdw/l followed by α-
NAA 114.64 gfw/l; 7.00 gdw/l and IBA 85.5 gfw/l; 5.21 gdw/l and BAP 136.10 gfw/l;
7.5 gdw/l and KIN 94.21 gfw/l; 6.21 gdw/l. The result of biomass yield from combination effect of 2,4-D and α-NAA with BAP. About 2.0 g of actively growing calli were inoculated in conical flasks each containing 30 ml of solid medium. The combination of 2,4-D, α-NAA and BAP showed more callus biomass yield than the combinations of auxin with Kinetin. These calli were used for phytochemical screening.
The results of the preliminary phytochemical investigation revealed the presence of alkaloids, flavonoids and steroids in all the solvent extracts, and glycosides and tannins in chloroform and ethanol extracts, carbohydrates in chloroform and ethanol extracts and terpenoids in ethanol extracts.
Five compounds were identified in chloroform extract of P. daemia by GC-MS
analysis such as benzeneethanamine,2,5-difluoro-β,3,4-trihydroxy-N-methyl; 2-
Naphthalenecarboxylic acid, 1,2,3,4-tetrahydro-3-hydroxy-8-methoxy-ethyl ester ;
l-Gala-l-ido-octose; 17-(1,5-Dimethylhexyl)-10,13-dimethyl-3-styrylhexadeca
hydrocyclopenta[a] phenanthren-2-one; 7,8-Epoxylanostan-11-ol,3-acetoxy.
Six compounds were identified in the ethanol extract of field grown plant by using GC-MS analysis, such as phenol,2,6-bis(1,1-dimethylethyl)-4-methyl- methylcarbamate; 1-Octanol, 2,7-dimethyl-; 3,7,11,15-Tetramethyl-2-hexadecen-1-ol;
1,10-Decanediol; 1-hexadecyne ; phytol.
Six compounds were identified in the aqueous extract of field grown plant GC-
MS analysis suc as methyl salicylate; diethyl phthalate; n-Hexadecanoic acid;
109 hexadecanoic acid ethyl ester; phytol; 9,12,15-Octadecatrienoic acid, ethyl ester,
(Z,Z,Z)-.
Ten compounds were identified in the ethanol extract of callus by GC-MS analysis, such as Propane, 1,1,3-triethoxy-; phytol; hexadecanoic acid, ethyl ester;
9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-; octadecanoic acid, ethyl ester;
1,2-Benzenedicarboxylic acid, diisooctyl ester; 5α-Androstan-16-one, cyclic ethylene mercaptole; methyl 3-oxours-12-en-23-oate; 2(1H)Naphthalenone, 3,5,6,7,8,8a- hexahydro-4,8a-dimethyl-6-(1-methylethenyl)-; decanoic acid, ethyl ester.
In the Pharmacology a total of 162 rats were used. Animals were randomly divided into 27 groups of 6 animals each and treatments continued in aqueous solution daily using an intragastric tube for 21 days.
After the termination of the experiment, all the animals were anaesthetized using ketamine chloride (24 mg/kg body weight) and sacrificed by cervical dislocation after an overnight fast. Blood was collected and tissues (liver and kidney) were immediately removed, blotted and kept at -20°C until use. Plasma, serum and tissue homogenates were separated after centrifugation and used for various biochemical estimations.
A portion of liver and kidney were removed after sacrifice and rapidly placed in
10% phosphate buffered-formalin for histological examination. Tissues were dehydrated in alcohol, embedded in paraffin wax, sectioned in 5 µm and stained with haematoxylin and eosin for Light Microscopy. The sections were examined at 40x.
The pharmocological studies indicate that P. daemia leaf and callus extract
(100 mg/kg, 200 mg/kg, 300 mg/kg) and glibenclamide (600 µg/kg) led to improvement of biochemical parameters and histological factors in the liver and kidney
110 of diabetes rats. The acute toxicity study revealed the non-toxic nature of P. daemia from chloroform, ethanol, aqueous and ethanol leaf callus extract at the tested concentrations. There were no lethal toxic reactions were observed until the end of the experiment. The morphological behavior, feed and fluid intake and body weight of the control and experimental animals were observed at regular interval.
The biochemical parameters were recorded from experimental and control animals such as blood glucose; total cholesterol; triglycerides; phospholipids; plasma insulin; glycosylated haemoglobin; hexokinase; glucose 6-phosphatase; fructose 1, 6- bisphosphatase; protein; haemoglobin; albumin; urea; uric acid; creatinine; glutathione peroxidise; reduced glutathione; glutathione-S-transferase; superoxide dismutase; catalase; hydroperoxides; tissue thiobarbituric acid; ascorbic acid; α–tocopherol; acid phosphatase; alkaline phospatase; aspartase transaminase and alanine transaminase are non significant difference between the normal and diabetes animal treated with alcohol extract of callus except diabetic control animal.
It may be concluded that the phytol and also other chemicals in the callus of
P. daemia responsible for antidiabetic activity. Based on the results, it is concluded that
P. daemia used as substitute for Gymnema sylvestre from Sherveroy hills of Tamilnadu proves scientifically its potential. Howevere, it is necessary and mandutory to isolate the pure bioactive compounds responsible for antidiabetic activity from the leaf callus extract/field grown plant.
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