STUDIES OF Wrightia tinctoria (Roxb) R.Br. LEAVES
A dissertation submitted to
THE TAMILNADU DR.M.G.R MEDICAL UNIVERSITY CHENNAI-600 032
In partial fulfillment of the requirements for the award of the degree of MASTER OF PHARMACY
IN
BRANCH – III PHARMACOGNOSY
Submitted by P.KOHILAVANI
261720705
Under the guidance of
Dr. A. KRISHNAVENI, M.Pharm., Ph.D., Department of Pharmacognosy
COLLEGE OF PHARMACY MADURAI MEDICAL COLLEGE
MADURAI – 625020
This is to certify that the dissertation entitled “PHARMACOGNOSTICAL, PHYTOCHEMICAL STUDIES, ANTI HYPERGLYCEMIC ACTIVITIY AND IN VITRO ANTI OXIDANT STUDIES OF Wrightia tinctoria (Roxb) R.Br. LEAVES” is a bonafide work done by Mrs. P.KOHILAVANI (261720705), DEPARTMENT OF PHARMACOGNOSY, COLLEGE OF PHARMACY, MADURAI MEDICAL COLLEGE, MADURAI-625020 in partial fulfillment of the The Tamilnadu Dr.M.G.R Medical university rules and regulation for award of MASTER OF PHARMACY IN PHARMACOGNOSY under my guidance and supervision during the academic year 2018-2019.
Name & Signature of the Guide:
Name & Signature of the Head of the Department:
I first and foremost express my revered regard and obeisance to the ALIMIGHTY GOD with my family members whose blessings I was able to complete my project work.
I am grateful to express my sincere thanks to Dr. K .Vanitha, MD., DCH., Dean, Madurai Medical College, Madurai, for giving an opportunity to carry out my project work.
I am thankful to Dr.Anitha Mohan, MD Physiology, Vice Principal, Madurai Medical College, Madurai for her support and encouragement to carry out the work.
I express my thanks to Prof. Dr. A. Abdul Hasan Sathali, M.Pharm., Ph.D., Principal and Head of Department of Pharmaceutics, College of Pharmacy, Madurai Medical College, Madurai for his support and valuable suggestions.
I express my sincere thanks to Dr. T.Venkat Rathna Kumar, M.Pharm., Ph.D., Professor and Head of Department of Pharmacognosy, College of Pharmacy, Madurai Medical College, Madurai for his support and valuable suggestions.
It is my privilege to express a deep and heartfelt sense of gratitude and my regards to our respected Mrs.Dr.A.Krishnaveni, M.Pharm., Ph.D., Assistant professor, Department of Pharmacognosy, College of Pharmacy, Madurai Medical College, Madurai for her active guidance, advice, help support and encouragement. I am very much obliged for her perseverance, without which my project work would not be completed.
I thank Mrs.Dr.A.Sethuramani M.Pharm., Mr.G. Sathya Balan M.Pharm.,
Madurai for their help.
I thank to Dr. D. Stephen, M.Sc., Ph.D., Department of Botany, American College, Madurai for the plant identification and to carry out this research work.
I special thanks to my classmates Mrs.P.Brindha, Miss.A.Deebika, Miss.R.Dhanalakshmi, Mr.K.Muthukrishnan, Mr.V.Rajamanickam, Mrs.J.Selvarani, Mr.M.Vijayaganesh for helping my project.
And I thanks to my Juniors Department of pharmacognosy Mr.M.Ahamedmueen, Miss.V.Dharani, Mrs.I.Jansyrani, Miss.T.Keerthana, Miss.R.Mohanapriya, Mr.M.Murugan, Miss.N.N.Nagaprashanthini, Mr.B.Naveenkumar, Miss.R. Soundariya
I extend my thanks to all the teaching and non-teaching staffs of other departments of College of Pharmacy, Madurai Medical College, Madurai who have rendered their help for the completion of the project.
S.NO TITLE PAGE NO
1 INTRODUCTION 1-8
2 LITERATURE REVIEW 9-35
3 AIM AND OBJECTIVE 36-37
4 MATERIALS AND METHODS 38-73
5 RESULTS AND DISCUSSION 74-115
6 SUMMARY 116-121
7 CONCLUSION 122-123
8 REFERENCES 124-136
CHAPTER-1
INTRODUCTION
INTRODUCTION
Human beings have depended on nature for their simple requirements as being the sources for medicines, shelters, food stuffs, fragrances, clothing, flavours, fertilizers and means of transportation throughout the ages. For the large proportions of world’s population medicinal plants continue to show a dominant role in the healthcare system and this is mainly true in developing countries, where herbal medicine has continuous history of long use. The development and recognition of medicinal and financial aids of these plants are on rise in both industrialized and developing nations.
Traditional system of plant medicines
The foundations of typical traditional systems of medicine for thousands of years that have been in existence have formed from plants. The plants remain to offer mankind with new medicines. Some of the beneficial properties ascribed to plants have recognised to be flawed and medicinal plant treatment is based on the experimental findings of hundreds to thousands of years. The earliest reports carved on clay tablets in cuneiform date from about 2600 BC are from Mesopotamia; among the materials that were used were oils of Commhiphora species (Myrrh), Cedrus species (Cedar), Glycyrrhiza glabra (Licorice), Papaver somniferum (Poppy juice) and Cupressus sempervirens (Cypress) are still used today for the cure of diseases extending from colds and coughs to inflammation and parasitic infections.
The traditional medicine practice is widespread in China, India, Japan, Pakistan, Sri Lanka and Thailand. About 40% of the total medicinal consumption is
and the Mimosaceae. It is estimated that in mid-90s, more than US$2.5 billion have resulted from the sales of herbal medicines. The herbal medicinal preparations are more in demand than mainstream pharmaceutical products in Japan. Testicular and neck cancer was isolated from the Catharanthus rosesus. Indian indigenous tree of Nothapodytes nimmoniana (Mappia foetida) are frequently used in Japan for the cure of cervical cancer.
Medicinal plants usage on modern medicine system
Even today, plants are not only indispensable in health care, but form the best hope of source for safe future medicines. In spite of the fact that now we have at our command a number of modern drugs, it is still genuinely urgent to discover and develop new therapeutic agents. It has been estimated that the acceptable therapy is available only for one third of the known human ailments. Therefore, the fight against diseases must be carried on relentlessly. Traditional plant medicines still enjoy significant position in the modern-day drug industries due to the minor side effects as well as the synergistic action of the combination of compounds.
Most of the important drugs of the past 50 years, which have revolutionized modern medicinal practice, have been isolated/derivatized from plants. These chemical ingredients exhibit therapeutic properties of plant and animal drugs. The WHO endorses and promotes the addition of herbal drugs in national health care programs because they are easily accessible at a price within the reach of a common man and are time tested and thus considered to be much safer than the modern synthetic drugs. Thus, the research of pharmacologically/ biologically active agents obtained by screening natural sources such as plant extracts had led to the detection of
diseases. The phytochemical-pharmacological research work has recently yielded effective solutions to certain diseases which synthetic drug industry has failed to afford. The most important among them are the research work on Artimisia annua, Cathranthus roseus, Taxus spp., Lantana camara and Baccopa spp. etc. Such plants were earlier considered as poisonous or useless, but now have been found to contain molecules of high drug values and are considered as medicinal herbs of great significance.
Modern searches for bioactive molecules typically make use of sophisticated bioassays and bioassay-guided fractionation of medicinal plants used by traditional healers. This has led to the isolation of several new therapeutically important compounds. A good number of potent drugs and a large number of therapeutic leads and many new pharmacologically active constituents have been developed from herbal drugs due to the dedicated efforts of researchers. The manufacture of morphine on industrial scale by E. Merck, Germany in 1826 marks the beginning of commercialization of plant-derived drugs. Nearly half of the top selling pharmaceuticals in 1991 were either natural products or their derivatives.
Distribution of medicinal plants
The distribution analysis of the medicinal plants shows that they are distributed across diverse habitats and landscape elements. Nearly about 70% of the medicinal plants in India are found in tropical forests in Eastern and western Ghats, Chota Nagpur plateau, Aravalis, Vindhyas and the Himalayas. Among the Himalayas, Kashmir Himalayan region is nestled within the Among the Himalayas, Kashmir
part of main Himalayan range. Floristic wealth of this region includes a fairly good representation of medicinal plants. Kaul has listed 111 medicinal plants from Kashmir and Ladakh. He has also mentioned in his book about healing properties of 291 species of medicinal plants from these regions. The medicinal flora of Kashmir, however, has not been paid due attention and Kashmir alone may have at least two times this number. Some of the most important medicinal plants of Kashmir Himalaya include Dioscorea deltoidea, Rheum Emodi, Arnebia benthamii, Inula racemosa, Datura stramonium Aconitum heterophyllum,, Artemisia spp., Podophylum hexandrum, Juniperus macropoda, Hypercum perforatum, Hyoscyamus niger, Sassurea spp., and Picrorhiza kurroa etc., growing in abundance in areas like Yusmarg, PirPanjal, Sonamarg, Gurez, Lolab valley, Gulmarg, Khilanmarg, Pahalgam and Tilail valley. Besides there is a number of aromatic and medicinal plant species grown in different high-altitude regions of Kashmir Valley. The important aromatic plant species include Caraway (Carum cervi), Saffron (Crocus sativus), Siya zira (Bunium persicum), Garlic (Allium sativa), Coriander (Coriandrum sativum), Mint (Mentha spp.), Fennel (Foeniculum vulgare) and Hare’s foot (Trigonella foenum-graecum). Many of these plants are used in standardized plant extracts.
Future prospects of Medicinal Plants
There is a promising future of medicinal plants as there are about half million plants around the world, and most of them are not investigated yet for their medical activities and their hidden potential of medical activities could be decisive in the treatment of present and future studies.
In the development of human culture medicinal plants have played an essential
medicines, many of them are produced indirectly from medicinal plants, for example aspirin. Many food crops have medicinal effects, for example garlic. Studying medicinal plants helps to understand plant toxicity and protect human and animals from natural poisons. The medicinal effects of plants are due to secondary metabolite production of the plants. Keeping this in consideration there have been increased waves of interest in the field of research in natural product chemistry. This interest can be due to several factors, including therapeutic needs, the remarkable diversity of both chemical structure and biological activities of naturally occurring secondary metabolites, the utility of novel bioactive natural compounds as biochemical probes, the development of Among the variety of modern medicines, many of them are produced indirectly from medicinal plants, for example aspirin. Many food crops have medicinal effects, for example garlic. Studying medicinal plants helps to understand plant toxicity and protect human and animals from natural poisons. The medicinal effects of plants are due to secondary metabolite production of the plants. Keeping this in consideration there have been increased waves of interest in the field of research in natural product chemistry. This interest can be due to several factors, including therapeutic needs, the remarkable diversity of both chemical structure and biological activities of naturally occurring secondary metabolites, the utility of novel bioactive natural compounds as biochemical probes, the development of novel and sensitive techniques to detect biologically active natural products, improved techniques to isolate, purify, and structurally characterize these active constituents, and advances in solving the demand for supply of complex natural products . The importance of traditional medicine has also recognized by World Health Organization
medicines agro-industrial technologies need to be applied. Medicinal plants are resources of new drugs and many of the modern medicines are produced indirectly from plants.
In diversified industries the contribution of plants is remarkable such as fine chemicals, cosmetics, pharmaceuticals and drugs and industrial raw materials etc. For the development of new drug discovery medicinal plants perform a dynamic part.
Medicinal plants have proved their sole role in coping with a number of deadly diseases including cancer and the diseases associated with viral onslaught viz.
Hepatitis, AIDS etc. In the USA drug market approx. 100 plant made new drugs were presented during 1950-1970 which includes vincristine, reseinnamine, vinblastin, deseridine and reserpine which are from different plants. During 1971-1990 fresh medicines i.e., artmisinin, Zguggulsterone, ginkgolides, lectinam, E-guggulsterone, teniposide, ectoposide, plaunotol and nabilone appeared all around the world. The 2%
medicines which were presented during 1991-1995 include irinotecan, toptecan, paclitaxel and gomishin etc. The isolation of serpentine in 1953 from the Indian plant Rauwolfia serpentine root was an innovatory episode in treatment of hypertension and lowering of blood pressure. The Vinblastine used for the treatment of leukemia in children, Hodgkins choriocarinoma, non-Hodgkins lymphomas,
Herbalisam
The term of medicinal plants include a various types of plants used in herbalism and some of these plants have a medicinal activities. Medicinal plants are the “backbone” of traditional medicine, which means more than 3.3 billion people in the less developed countries utilize medicinal plants on a regular basis. These
development and synthesis. Besides that these plants play a critical role in the development of human cultures around the whole world .The Indian sub-continent has a very rich diversity of plant species in a wide range of ecosystems. There are about 17.000 species of higher plants, of which approximately8.000 species, are considered medicinal and used by village communities, particularly tribal communities, or in traditional medicinal systems, such as the Ayurveda.
The use of traditional medicine and medicinal plants in most developing countries, as a basis for the maintenance good health, has been widely observed by UNESCO, 1996 . Further more, an increasing reliance on the use of medicinal plants in the industrialized societies has been traced to the extraction and development of several drugs and chemotherapeutics from these plants as well as from traditionally used rural herbal remedies.
During the past decade, traditional systems of medicine have become a topic of global importance. Current estimates suggest that, in many developing countries, a large proportion of the population relies heavily on traditional practitioners and medicinal plants to meet primary health care needs. Although modern medicine may be available in these countries, herbal medicines (phytomedicines) have often maintained popularity for historical and cultural reasons.
Medicinal plants frequently used as raw materials for extraction of active ingredients which used in the synthesis of different drugs. Like in case of laxatives, blood thinners, antibiotics and anti-malarial medications, contain ingredients from plants. Moreover the active ingredients of Taxol, vincristine, and morphine isolated
Medicine, in several developing countries, using local traditions and beliefs, is still the mainstay of health care. As defined by WHO, health is a state of complete physical, mental, and social wellbeing and not merely the absence of disease or infirmity. Medicinal plants can make an important contribution to the WHO goal to ensure, by the year 2000, that all peoples, worldwide, will lead a sustainable socioeconomic productive life.
The practice of traditional medicine is widespread in China, India, Japan, Pakistan, Sri Lanka and Thailand. In China about 40% of the total medicinal consumption is attributed to traditional tribal medicines. In Thailand, herbal medicines make use of legumes encountered in the Caesalpiniaceae, the Fabaceae, and the Mimosaceae. In the mid-90s, it is estimated that receipts of more than US$2.5 billion have resulted from the sales of herbal medicines. And, in Japan, herbal medicinal preparations are more in demand than mainstream pharmaceutical products.
CHAPTER-2
LITERATURE REVIEW
LITERATURE REVIEW
PLANT DESCRIPTION
BOTANICAL CLASSIFICATION Kingdom : Plantae-plants
Subkingdom : Tracheobionta – vascular plants Phyllum / division : Angiospermae
Super division : Spermatophyta – seed plants Class : Magnoliopsida (Dicotyledonous) Sub Class : Rosidae
Order : Gentianales Family : Apocynaceae Sub family : Mimosoidae Genus : Wrightia Species : tinctoria
Synonym : Wrightia rothii, Nerium tinctorium Roxb. Dyers’s Oleander Common Name : Tontampalai, Veppalai, Vetpalai, Irumpalai
VERNACULAR NAMES
English : Pala indigo plant
Gujerati : Runchallaodudhlo, Dudhlo
Kannada : Kirkodasige, Bepalli, Kodamurki Malayalam : Kotakappla, Ayyappla, tinnampala Sanskrit : vetakutajah, Hyamaraka
Tamil : Vetpalai, Thontampalai, Kodisha
Telugu : Jaddapala, Tedlapala, Kala kuta, Jedda pala
Bengali : Indarajau
Oriya : Pita karumn, Dudhokriya ORIGIN
India and Burma
GEOGRAPHICAL DISTRIBUTION
It is distributed throughout India upto 1,200 m. Widely distributed in Westren Ghats of Madras state, Madhya Pradesh, Rajamuntry Hills, Carnatic, the Circars, and Rajasthan, and peninsular India.
HABIT AND HABITAT
The tree w.tinctoria is a small or middle sized tree generally up to 1.8m tall and often under 60cm, ascending to an altitude of 1200 in the hills. The tree is moderate light demander and is often as an under growth species in deciduous forest.
It requires a mixture of peat, learn, and sand and is propagated by seeds or cutting which readily root –suckers. The growth is slow to moderate.
The annual increase in girth being 1.16-2.3 cm. White latex is collected from
The tree sheds it’s leaves during the cold season. About the beginning of April fresh leaves are formed together with the flowers. The seeds ripen in the following January. It`s wood is remarkable white, closely grained and coming near to ivory than that of any other.
DESCRIPTION Leaves
Colour : Pale green
Odour : No characterstic odour.
Taste : Astringent Texture : Smooth
Eliptic ovate, elliptic lanceolate, obovate oblong, or oblong to lanceolate, short petiole, 3-6 inches long and 1-21/2 inches broad, acuminate or cordate apex, acute or rounded bass with 6-12 pairs of main nerves which are faint till the leaf matures. The leaves in this tree yields a blue dye namely Pala Indigo. Petiole 1/8 to 1/6 inches long.
Latex
When the leaves were picked white colored latex was oozing from the leaf and stem.
Flowers
Flowers are numerous, bisexual, actinomorphic, hypogynous white color fragrance flowers, five stamens which are connivent. From a distance, the white flowers may appear like snow flakes on a tree. During dry season from the second
Calyx
Glabrous, glandular inside, five green colored sepals.
Corolla
Gamopetallous, regular salvar shaped, with a short slightly gibbous tube, crowing of the mouth of the corolla tube is a well developed corona of numerous whitish ramous linear scales. Corolla is divided into five elaborated petals.
Filaments
Epipetalous, short, rigid, inserted with the mouth of the tube or within the corolla.
Anthers
Exserted, fairy large, arrow shaped.
Pollen grains
Creamy white and sticky throughout the flower life.
Pistil
Bicarpellary, ovaries seemingly united. Both ovaries have a common style and stigma which are situated slightly below the level of the anthers and completely concealed by the conical filaments.
Stigma
Bilobed stigma with transparent gluten by which the pistil adheres to the inside of the anthers.
Fruit
Fruits of two, initially green and becomes brown when mature, very long, follicles in pairs, pendulous, slender, cylindrical in nature, globrate or smooth, slightly tapering at both ends and co adhering at their tips, length 6-18 inches, diameters 1/4 to 1/3 inch
Seeds
Numerous, 1/2 – 3/4 inch long, linear, glabrous, slender, pointed at apex with a fine silky hairs often more than 11/2 inch long, non endospermous, embryo inverse.
Bark
Pale, grey, smooth thin bark abounding in yellow milky juice with opposite divaricated branches, reddish brown in color.
Wood
Uniformly white when first exposed, turning ivory colored with age, even grained, lustrous, smooth, straight, or some wavy or curly, even – textured.
Mean of propagation
Seedlings and by vegetable propagation.
ETHANOMEDICAL USES
Leaves
A.K.Natkarani, et al., 1954 had studied the leaves of wrigthia tinctoria (Apocynaceae) use for tooth ache treatment.
Vaidyaratnam et al., 1996 had reported the plant wrightia tinctoria (Apocynaceae) leaves are used for hypotension.
Kottai muthu.R et al., 2008 had identified and surveyed the ethnobotany of the valaiyans of sirumalai and karandamalai, Dindigul district, Tamil nadu. In India, the use of leaf paste Wrightia tinctoria applied on aching teeth to get relief from tooth ache.
Ganesen.S, et al., 2008 had evaluated ethnomedical survey of Algarkoil hills (reserved forest) T.N .India. They had reported the leaf paste of Wrightia tinctoria used with neem oil for eczema treatment.
Kinston.C, et al., 2009 had reported Indigenous knowledge of using medicinal plants in treating skin diseases in kanyakumari district southern India. They found the leaves of Wrightia tinctoria (Apocynaceae) mixed with coconut oil for the treatment of psoriasis.
Chatterjee, et al., 2011 had identified the leaves of Wrightia tinctoria (Apocynaceae) leaves traditionally used as febrifuge, stomahic and antidysentric
Vinoth, et al., 2014 had surveyed ethnomedical value of plants in Nagapattinam District of Tamil nadu, India. They identified the leaves of Wrightia tinctoria
Rajani srivastava and Alok mukerjee., et al 2017 had evaluated traditional use of Jharkhand region .The seeds of w.tinctoria used in the treatment of hyper glycemia and hyperlipidrmie.
Whole plant
Ignacimuthu.S. et al., 2006 had identified the plant leaves of wrightia tinctoria used for the treatment of gynecological disorders.
Kannan Ponnusamy, et al., 2010 had reported the plant Wrightia tinctoria used for various treatment of skin disorders
Khyde.S, et al., 2014 had reported the whole plant of Wrightia tinctoria used in various Indian system of medicine such as ayurvedha, siddha and unani for the treatment of jaundice,malaria, psoriasis and many other ailments.
Seed
A.K .Natkarani, et al., 1954 had evaluated the seeds of Wrightia tinctoria (Apocynaceae) used for seminal weakness.
R.N. chopra, et al., 1956 had studied the seeds of Wrightia tinctoria (Apocynaceae) traditionally used for aphrodiasis.
Kiritikar and Basu, et al., 1993 had reported the seeds of Wringtia tinctoria used for aphrodisiac effect.
Challaiah muthu, et al., 2006 had reported the seeds of Wrightia tinctoria taken orally for indigestion by the traditionally healers of kancheepuram.
Chatterjee, et al.,2011 had identified the seeds of Wringtia tinctoria (Apocynaceae)
Rajani srivastava and Alok mukerjee. et al 2017 had evaluated traditional use of Jharkhand region .The seeds of w.tinctoria used in the treatment of hyper glycemia and hyperlipidrmie.
Flower
Ramalakshmi.S et al., 2012 had evaluated the flowers of wrightia tinctoria combined with keezhanelli juice used in the treatment of Jaundice.
Bark
A.K.Natkarani, 1954 had submitted the bark of Wrightia tinctoria (Apocynaceae) use for stomachic tonic, febrifuge.
R.N.Chopra, et al., 1956 had studied the bark of wrightia tinctoria (Apocynaceae) traditionally used for tonic.
Kritikar, et al., 1993 had reported the bark of Wrightia tinctoria (Apocynaceae) used for skin diseases, diarrhoea and piles.
Vaidyaratnam, et al., 1996 had examined the bark of Wringtia tinctoria (Apocynaceae) used as carminative, digestive, stomachic, constipation, leprosy and psoriasis.
Chatterjee, et al., 2011 had found traditionally used the bark of Wrightia tinctoria (Apocynaceae) as tooth ache, stomachic, antidysentric.
PHARMACOLOGICAL REVIEW
Leaves
Anti microbial activity
Moorthy.K. et al., 2012, had evaluated W.tinctoria leaves showed significant antimicrobial activity against Cryptococcus neoformans (36.0mm), staphylococcus aureus(27.2mm), candida albicans(25.0mm), S.epidermidis(23.2mm) and bacillus subtilis(20.2) whereas petroleum ether leaves extract showed significant antimicrobial activity against s.aureus (25.0mm), C.neoformans(21.8mm) S.epidermidis(18.5mm) and c.albicans(16.0mm)were observed. According to broth dilution method, the methanolic extract of plant material showed the MIC values against c.neoformans(256µg/ml), and S.aureus and C.albicans(512µg/ml) respectively.
Whereas, the petroleum ether extract of wrightia tinctoria showed the MIC values against s.aureus and C.neoformans (512µg/ml) and S.epidermidis and C.albicans(1,024µg/ml) with a significant inhibitory activity.
Vijayaragavan, et al., 2013 had done anti microbial studies on the leaf of W.
tinctoria R.Br. It concludes there is little evidence of leaf on Anti-microbial studies.
Vedhanarayanan.P, et al., 2013, had evaluated the antibacterial activity of different extracts (chloroform, ethanol and methanol) of wrightia tinctoria has been studied against the human pathogenic bacterial strains, Escherichia coli, Bacillus subtilis, Staphylococcus aureus and pseudomonas aeruginosa by disc diffusion method on agar. Among the three solvents tested, ethanol extract of leaf showed higher inhibition zone. Ethanol extract of wrightia tinctoria exhibits maximum zone of inhibition
Hemalatha et al., 2017, had evaluated the crude hexane, ethyl acetate and aqueous leaf extracts of medicinal plant wrightia tinctoria R.Br were evaluated for in vitro antimicrobial activity against selected bacterial and fungal strains. The extracts were screened against three gram positive strains (Bacillus subtilis, Bacillus licheniformis and streptococcus pneumonia), two gram negative strains (Aspergillus flavus, Aspergillus niger and candida albicans). Antimicrobial activity of these crude extracts was determined using agar well diffusion method. Agar broth and potato dextrose broth dilution method was used to determine the minimum inhibitory concentration. Ampicillin and flucanazole were used as standards for bacteria and fungi respectively.
Lakshmi Manokari, et al., 2017 had done A study on the extraction process of Wrightia tinctoria and evaluation of its antimicrobial activity. Response Surface Methodology (RSM) with Central Composite Design (CCD) is performed in the present study under different experiment runs varying the weight of the Wrightia tinctoria leaf powder. Aqueous, ethanol and ethyl acetate extracts are derived from Wrightia tinctoria plant leaf powder using Soxhlet apparatus under various experimental conditions. Antimicrobial activity of the plant extracts are investigated using agar disc diffusion method.
Anti bacterial activity
Ravi Shankar, et al., 2010 had evaluated antibacterial activity of Wrightia tinctoria . Ethyl acetate and methanol extracts showed significant antibacterial activity against gram negative bacteria; the MIC was 50 g/ml for Xanthomonas campestris and
properties against plant pathogenic bacteria suggesting that the active principles may be useful in the control of plant disease.
Beena,et al., 2014 had observed The antibacterial activitiy of the leaf extracts of Wrightia tinctoria (Pala Indigo) were investigated. The antibacterial potential of the leaf coconut oil extracts, petroleum ether, chloroform, ethyl acetate and methanol extracts of the leaves of Wrightia tinctoria were studied against human pathogenic bacteria viz. Bacillus cereus, Enterobacter faecalis, Salmonella paratyphi, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens activity against ‘agar well diffusion’ method. Leaf methanol extracts of Wrightia tinctoria exhibited pronounced activity against Gram-positive and Gramnegative bacteria and their activity is quite comparable with the standard antibiotics such as tobramycin and gentamycin sulphate screened under similar conditions.
Shakthivel Muthu, et al., 2017 had experimented Antibacterial serine protease from Wrightia tinctoria. Serine protease had a potent antibacterial activity against both Gram-positive and Gramnegative bacteria. A 10 g/ml of serine protease was tested against S. aureus, M. luteus, R aeruginosa and K. pneumonia which had 21, 20, 18 and 17 mm of zone of inhibition respectively. Serine protease from W. tinctoria degrades the peptidoglycan layer of bacteria which was visualized by transmission electron microscopic analysis
Anti cancer activity
Jayesh, et al., 2015 had identified DW-F5: a novel formulation against malignant melanoma from Wrightia tinctoria. The major pathways in melanoma signaling
MITF-M, the master regulator of melanomagenesis, were inhibited by DW-F5, leading to complete abolition of MITF-M. Purification of DW-F5 led to the isolation of two cytotoxic components, one being tryptanthrin and the other being an unidentified aliphatic fraction.
Ashish dixit et al., 2017 had reported the methanolic extract of wrightia tinctoria leaves effective against the cancer cells. The potency of each plant extract was calculated in terms of percent decrease in viable HeLa cells as compared to the control values. The extract showed dose dependent anticancer activity
Antioxidant activity
Sawale, et al., 2014 had observed In vitro antioxidant activity of the methanolic extract of wrightia tinctoria leaves was determined by measuring its 1,1 diphenyl-2- pycryl hydrazyl(DPPH) radical scavenging activity, reducing power and ability to inhibit lipid peroxidation.
Beena, et al., 2014 had evaluated the DPPH free radical scavenging activity of Wrightia tinctoria R. Br. Leaf extract. DPPH radical could be scavenged most effectively by Wrightia tinctoria bark ethyl acetate extract with IC50 value 67.302.4 g/ml and was quite comparable with the standard antioxidant L-ascorbic acid (IC50 value 62.52.5 g/ml). The antioxidant activity of W. tinctoria leaf methanol extract (IC50: 72.93.5 g/ml) was not significantly different from that of L ascorbic acid (IC50: 62.52.5 g/ml). The remarkable antioxidant activity exhibited by the plant extracts can be attributed to the synergic effect of the active compounds present in it.
Sathiyanarayanan et al., 2016 had observed the ethanolic extract of w.tinctoria the
scavenging assay was observed to be 290µg/mL. The IC50 of hydrogen peroxide value was noted to be 410µg/mL.
Larvicidal activity
Murugesan, et al., 2014 had examined Larvicidal activity of Wrightia tinctoria R.
BR. (Apocynaceae) fruit and leaf extracts against the filarial vector Culex quinquefasciatus Say (Diptera: Culicidae). Among the plant parts tested, aqueous fruit extract exhibited highest larvicidal activity followed by aqueous leaf extract with LC50 values of 0.17% and 0.09%; 0.21% and 0.11% after 24 and 48 h respectively.
Anthelmintic activity
Rajalakshmi, G. R, et al., 2013 had evaluated In vitro anthelmintic activity of Wrightia tinctoria. The three concentration (10, 25, 50 mg/ml) of extract were studied in activity which involved the determination of time of paralysis and time of death of the worm. The extract exhibited significant dose dependent anthelmintic activitiy.
Anti-inflammatory activity
Ravichandran, et al., 2013 had investigated anti inflammatory activity was evaluated using carageenan induced paw edema, cotton pellet granuloma pouch method in rats, membrane stabilizing potential and cyclooxygenase inhibition assay.
The ethonolic extract of wrightia tinctoria with a concentration of 400 mg/kg b.w.
showed maximum (56.25%) inhibition on carrageenan induced rat paw edema, significantly inhibited granuloma formation (p<0.01) in cotton pellet granuloma model. The ethanol extract (at concentration range of 0.10-0.40 mg/ml) significantly protect the rat erythrocyte membrane against lysis induced by hypotonic solution. The
enzyme inhibition which may behave established a pharmacological evidence for the folklore claim of the drug to be used as an anti inflammatory agent.
Anti-diabetic activity
Ashok raj.R et al., 2009 has investigate the hypoglycemic and hypolipidemic activity of petroleum ether extract of wrightia tinctoria L(PWT) in alloxan induced diabetes in albino wistar rats. Alloxan was asministered as a single dose (120mg/kg, b.wt) to induced diabetes. Administration of pet ether extracts from leaves of wrightia tinctoria L.(200 & 400mg/kg body weight/day) for 14 days, to alloxan-induced diabetic rats. The anti-diabetic and hypoglycemic activities of the pet ether extract of wrightia tinctoria L.(PWT) were similar to those produced by glibenclamide at 2.5mg/kg(positive control, p<0.05).
Shruthi.A, et al., 2012 had studied Anti-diabetic activity of the leaves extracts of Wrightia tinctoria on alloxan induced diabetic rats. Chloroform extract showed a significant antidiabetic activity when compared to the standard drug glibenclamide.
Anti-ulcer activity
Divakar, M. C. et al., 2011 had examined Antiulcer activity of Wrightia tinctoria (Roxb.) R.Br. The purpose of the present study was aimed at evaluating the antiulcer activity on leaves of Wrightia tinctoria (Roxb) R.Br (Family Apocynaceae) on albino rat. The antiulcer activity of the Wrightia tinctoria methanolic extract (TM) and Wrightia tinctoria 70% ethanolic extract (T70E) were compared with carboxy methyl cellulose (CMC), pylorus control, Aspirin and standard famotidine which was evaluated by employing aspirin plus pylorus ligation induced ulcer model. The
mg/kg body weight. The plant methanolic extract showed significant gastro protective activity of 65.89% when compared with the standard drug famotidine (20 mg/ kg) which showed 75.34%. The result suggested that the methanolic extract of Wrightia tinctoria leaves possesses anti-ulcer effect.
Anti-fungal activity
Krishna moorthy, et al 2006 had evaluated the extracts of W. tinctoria are also known to have an effect on keratinocyte proliferation the antifungal activity coupled with keratinocyte proliferation inhibition activity of W. tinctoria in dano makes the oil very effective in the management of dandruff. The antifungal activity especially against P.ovale. similarly, the bitter fraction of neem is also known to possess antifungal activity.
Beena, et al., 2014 had evaluated the antifungal activities of the leaf extracts of Wrightia tinctoria R.Br. The results obtained showed that the leaf methanol extracts of W. tinctoria can be considered as good sources of antimicrobial compounds and can be incorporated into the drug formulations.
Anti-psoriatic activity
Dhanabal, S. P, et al., 2012 had screened Wrightia tinctoria leaves for anti psoriatic activity. The extract produced significant (p<0.01) degree of orthokeratosis compared to control and the drug activity was found to be 70.18%, which is more potent than the standard (57.43%).
Bark
Anti-bacterial activity
Beena, et al., 2014 had evaluated the antibacterial activity of the bark extracts of Wrightia tinctoria R.Br. The results obtained showed that the bark methanol extracts of W. tinctoria can be considered as good sources of antimicrobial compounds and can be incorporated into the drug formulations.
Anti-oxidant activity
Beena, et al., 2014 had evaluated the DPPH free radical scavenging activity of Wrightia tinctoria R. Br.bark extracts. DPPH radical could be scavenged most effectively by Wrightia tinctoria bark ethyl acetate extract with IC50 value 67.302.4 g/ml and was quite comparable with the standard antioxidant L-ascorbic acid (IC50 value 62.52.5 g/ml).
Fatima, et al., 2016 had studied antioxidant potential and profiling of polyphenolic compounds of Wrightia tinctoria Roxb. (R.Br.) bark. The total phenolic content of WTBM was found to be 30.3 gallic acid equivalent mg/g dry weight of bark extract while IC 50 value for DPPH and ABTS radical scavenging activity was 72.2 2.8 mg/ml and 45.16 1.95 mg/ml, respectively.
Anti-cancer activity
Fatima, et al., 2016 had studied Anticancer, antioxidant potential and profiling of polyphenolic compounds of Wrightia tinctoria Roxb. (R.Br.) bark. WTBM significantly suppresses colony formation and induces apoptosis in both MDA-MB-
Anti-tuberculosis activity
Rajendra, et al., 2015 had studied Anti tuberculosis activity ethyl acetate extracts of Wrightia tinctoria bark. Ethyl acetate extract showed MIC 6.25 g/ml against M.
tuberculosis which was significantly different from other extract of WT bark.
Wound healing activity
Veerapur, V. P., et al., 2004 had examined The effect of ethanol extract of Wrightia tinctoria bark on wound healing in rats. The results of the present study reveal that ethanol extract of bark of W. tinctoria have significant wound healing activity. The prohealing action seems to be due to the increased synthesis of collagen, it's cross- linking as well as better alignment.
Anti-fungal activity
Beena, et al., 2014 had evaluated the antifungal activities of the bark extract of Wrightia tinctoria R.Br. The results obtained showed that the leaf and bark methanol extracts of W. tinctoria can be considered as good sources of antimicrobial compounds and can be incorporated into the drug formulations.
Anti-inflammatory activity
Bigoniya. P, et al., 2008 had made Pharmacological screening of Wrightia tinctoria bark hydro-alcoholic extract. Carrageenan-induced paw edema and cotton pellet induced granuloma model were employed to test anti-inflammatory activity.
Diuretic
Bigoniya. P, et al., 2008 had made Pharmacological screening of Wrightia tinctoria bark hydro alcoholic extract. Extract is devoid of any sedative activity. W. tinctoria
Antipsoriatic activity
Khyade, et al., 2014, made a Wrightia tinctoria R. Br.(-)a review on its pharmacological profile. Clinical studies indicated a broad range of applications in the treatment of psoriasis and other skin diseases.
Seed
Antimicrobial activity
Nagalakshmi, et al., 2012 had evaluated In vitro Antimicrobial Properties of Mature Seed Extracts of Wrightia tinctoria R. Br. The methanolic seed extracts were largely inhibitory against pathogenic yeasts like Trichophyton rubrum, Candida albicans, C.
parapsilosis and Cryptococcus. The results indicated that the methanolic extract of the brown variety seeds is pharmacologically more active than that of the beige variety seeds. The aqueous extracts of both the seed varieties were moderately effective against S. aurcus ATCC 25923 and S. citreus, with no effect against the fungal strains.
Kreethi priya Yadala, et al., 2017 had studied Antimicrobial Activity Studies on Seed Fibers of Wrightia tinctoria (Roxb.) R.Br. which shows The different extract samples of seed fibres of Wrighita sp. were selected for testing against to their broad spectrum of antimicrobial activity resistance of different microorganisms as per the standard literature sources [11,12,1 the seed fiber extracts of Wrightia tinctoria (Roxb.) for their antimicrobial activity against 8 selected human pathogenic bacterial strains viz., Pseudomonas aeriginosa (10636), Staphylococcus aureus (6908), Bacillus subtilis (1305), Pseudomonas putida (1194), Streptococcus pyogenes (1926),
against the seed fibres extract samples of methonal, ethanol, chloroform, Ampicilin and DMSO individual and their combinations.
Anti-bacterial activity
Anchal Sharma, et.al., 2017 had performed Purification and Characterization of 2S Albumin from Seeds of Wrightia tinctoria Exhibiting Antibacterial and DNase Activity. In the present work a small 2S albumin (WTA) protein of similar to 16 kDa has been purified from the seeds of Wrightia tinctoria. The WTA is a heterodimer protein with a small subunit of similar to 5 kDa and a larger subunit of similar to 11 kDa bridged together through disulphide bonds. The protein exhibits deoxyribonucleases activity against closed circular pBR322 plasmid DNA and linear BL21 genomic DNA. The protein also showed antibacterial activity against Morexalla catarrhalis.
Anti-oxidant activity
Beena, et al., 2014 had evaluated the DPPH free radical scavenging activity of Wrightia tinctoria R. Br. seed extracts. DPPH radical could be scavenged most effectively by Wrightia tinctoria bark ethyl acetate extract with IC50 value 67.302.4 g/ml and was quite comparable with the standard antioxidant L-ascorbic acid (IC50 value 62.52.5 g/ml).
Whole plant
Anti-diabetic activity
Sunil, et al., 2014 had observed the effect of Wrightia tinctoria and Parthenocissus quinquefolia on blood glucose and insulin levels in the Zucker diabetic rat Model. W.
any correlation between blood glucose and insulin levels. Based on these findings, it can be concluded that hypoglycemic effects of W. tinctoria are more complicated than P. quinquefolia, and may involve other possible mechanism of action.
Anti-viral activity
Selvam et al., 2009 investigated methanolic extract of wrightia tinctoria leaves shown HCV-1 Integrase Inhibitory activity.the 50% effective concentration for inhibition of HCV in RNA subgenomic replicon replication in 5-2 cells (luciferase assay) by CWT was found to be 15µgmL.
Anti-fungal activity
Ponnusamy et al., 2010, had evaluated In vitro antifungal activity of indirubin isolated from plant Wrightia tinctoria R.Br. Antifungal activity was studied by spore germination test using agar dilution method. The minimum inhibitory concentration (MIC) was determined using broth microdilution method. Wrightia tinctoria showed promising activity against dermatophytic and non-dermatophytic fungi. Leaf chloroform extract showed activity at 0.5 mg/ml against Trichophyton rubrum, Epidermophyton floccosum, Aspergillus niger and Scopulariopsis brevicaulis. The major compound, identified as indirubin, exhibited activity against dermatophytes such as Epidermophyton floccosum (MIC = 6.25 mu g/ml); Trichophyton rubrum and Trichophyton tonsurans (MIC = 25 mu g/ml); Trichophyton mentagrophytes and Trichophyton simii (MIC = 50 mu g/ml). It was also active against non-dermatophytes (Aspergillus niger, Candida albicans and Cryptococcus sp.) with in a MIC range of 0.75-25 mu g/ml.
Root
Deshmukh, O.S. et al., 2017, had formed the various solvents ethanol, benzene, chloroform, acetone, petroleum ether and distilled water. The Gram-positive and Gram-negative bacteria yeast candida, Aspergillus niger, staphylococcus aureus, Eschericha coli, salmonella typhi, Bacillus subtilis, Pseudomonas fluorescence, kelbsiella pneumonia and Streptococus pyogenes.
Flower
Antimicrobial activity
Ramalakshini, et.al., 2012 had made an Investigation on antimicrobial of Wrightia tinctoria (Roxb.) R. Br. flower used in Indian medicine. The antibacterial studies of the ethanolic flower extract tested at different concentration of extracts, where 250 mg/mL concentration of extract showed good inhibitory activity against all the test pathogens compared with standard antibiotics like streptomycin and penicillin. The cytotoxic activity of flower extract was evaluated by brine shrimp lethality bioassay method and the LC50 value found to be 3.544 g/mL.
Anti oxidant activity
Ramalakshini, et.al., 2012 had made an Investigation on antioxidant of Wrightia tinctoria (Roxb.) R. Br. flower used in Indian medicine. Total phenols, flavonoids, carotenoids in the extract was found to be 55.290.45 mg GAE, 370.531.213 mg QE and 1.8250.321 mg/g respectively, where the reducing power, phosphomolybednum activity and metal chelating activity were increasing with increasing concentration of the flower extract
Anti-cancer activity
Ramalakshini, et.al., 2012 had made an Investigation on cytotoxic activity of Wrightia tinctoria (Roxb.) R. Br. flower used in Indian medicine. The cytotoxic activity of flower extract was evaluated by brine shrimp lethality bioassay method and the LC50 value found to be 3.544 g/Ml.
Stem
Anti-inflammatory activity
Tharkar, P. R., et al., 2010 had experimented Anti-inflammatory study of Wrightia tinctoria R.Br stem bark in experimental animal models. The various extracts showed inhibition of rat paw oedema and percent granuloma changes at dose of 200 mg/kg when compared to control group.
Fruit
Larvicidal activity
Murugesan sakthivadivel, et al., 2014, had determined the larvicidal activity of crude aqueous and petroleum ether extracts of Wrightia tinctoria fruits and leaves against the filarial vector, culex quinquefasciatus. It was evaluated at concentrations of 0.06%, 0.12%, 0.25%, 0.50%, and 1.00%. larval mortality was observed for 24 and 48 h. Aqueous fruit extract exhibited highest larvicidal activity followed by aqueous leaf extract with LC50 values 0f 0.17% and 0.09%; 0.21% and 0.11% after 24 and 48h respectively.
PHYTOCHEMICAL REVIEW
Whole plant
A.V.Muruganandam, S.Ghosal, et al., 2000 had investigated the members of the genus viz. W. tinctoria, W.tomentosa and W.coccinea, have been investigated for their chemical constituents with a view to locating their bioactive principles. Indigotin 3, indirubin6, tryptanthrin 8, istatin 5, anthranillate 7 and rutin 9 are major constituents of W.coccinea. The identities of these compounds have been established by comprehensive chromatrographic (HPTLC,HPLC) and spectroscopic (UV-VIS, IR, EI_MS) techniques, using markets and by synthesis, where possible.
T.Jeyamathi, et al., 2012, had studied GC-MS analysis of W.tinctoria ethanolic extract revealed the existence of the GC-MS chromatogram of the twenty two peaks presented. The major chemical constituents are 3-O-methyl-d-glucose (51.44%), squalene (16.52%), n-hexadecanoic acid (6.17%), phytol(4.47%) and 9,12- octadecadienoyl chloride (Z,Z)-(4.31%)
Sunayana Nath, et al., 2014, had studied medicinal plants have gained tremendous attention in recent years. The species Wrightia tinctoria (family Apocyancea) is a wild medicinal tree contains many active phtoconstituents which are of aphrodisiac, anthelminithic, anti-inflammatroy, analgesic as well as antipyretic importance and used to cure many human disease.
Oviya, et al., 2015 had analysed the Phytochemical studies of Wrightia tinctoria have shown the presence of alkaloids, triterpenoids, steroids, flavonoids, lipids and carbohydrates.
Leaf
MadhuC. Divakar et al., 2011 had examined the HPTLC finger printing studies on two ethnomedicinally important wrightia species, viz., Wrightia tinctoria and Wrightia arborea. The high performance thin layer chromatographic finger print parameters have been developed for methanolic lead extracts to fix standards. At shorter (254nm) and longer (366 nm) wavelength, the resolution was better for these extracts and hence, these wavelengths can be taken for obtaining optimum HPTLC finger printing for this medicinal plant.
Beena, et al., 2014 had Analysed the essential oil from the leaves of Wrightia tinctoria R.Br. from south India. Thirty seven known compounds have been identified and quantified from the leaf essential oil of Wrightia tinctoria by GC-MS analysis.
The major compounds present in the leaf essential oil are urs-12-en-24-oic acid-3- oxo-methyl ester (34.28%), hydroquinone (13.24%), 1, 6-cyclodecadiene,1-methyl-5- methylene-8-(1-methylethyl) (9.70%), 3-methyl-2-(2-pentenyl)-2-cyclopentene-1-one (6.76%) and 9, 12, 15-octadecatrienoic acid (4.52%).
Ashish dixit, et al., 2014 has found the wrightia tinctoria is used for the treatment of several diseases including very severe disease like aids and cancer. In this review we want to highlight on different parameters of plant like general description and distribution, microcopy, chemical constituents and traditional uses with special emphasis of pharmacological action like analgesic, anti-inflammatory, anthelmintic, antiulcer, antidiabetic, anticancer, and antipyretic and antidysentric activity. This plant is also used in the treatment of skin disease like psoriasis and as indigo dyes.
extracts such as petroleum ether, chloroform, ethanol and methanol were prepared and phytochemical screening was made using standard procedures. It was apparent that the Rf values were mostly different irrespective of solvents used for extraction. The similarity in few Rf values of 0.21, 0.24 and 0.27 at 254nm and 0.21 and 0.44 at 366nm evidences the presence of specific components among solvent extracts of this plant. The difference in Rf values in most of the appeared peaks reflected qualitative variation in the phytocompounds. It can be concluded that HPTLC fingerprinting of Wrightia tinctoria may be useful in differentiating the species from the adulterant and act as a biochemical marker for authentication of this medicinally important plant in the pharmaceutical industry and plant systematic studies.
Sajitha.M and Subramani.K et al., 2017 had investigated the systematic chemical analysis of leaves of Wrightia tinctoria were found to contain two flavonoid glycosides
Kaempferol 3-O-rhamnoside and Quercetin 3-O-sophoroside and two flavonoid aglycone Kaempferol and Quercetin.
The detailed UV 1H 13C NMR and Mass spectral data confirm the characterization of the above compounds. All these compounds are reported first time from the leaves of Wrightia tinctoria.
Bark
Fatima, et al., 2016 had profiled polyphenolic compounds of Wrightia tinctoria Roxb. (R.Br.) bark. The total phenolic content of WTBM was found to be 30.3 gallic acid equivalent mg/g dry weight of bark extract.
Seeds
Nagalakshmi, et al., 2012 had evaluated phytochemical contituents of Mature Seed Extracts of Wrightia tinctoria R. Br. The phytochemical screening revealed the presence of carbohydrates, reducing sugars, alkaloids, sterols, glycosides, phenolics, tannins, flavonoids and amino acids
Rajani srivastava, et al., 2014 had studied the HPTLC fingerprinting profile developed for different extracts of w.tinctoria will help in proper identification and quantification of the marker compound. The satisfactory resolution was obtained in the solvent system tolune: ethyl acetate v/v (7:3:0.1) for alcoholic extract.
Rajani, et al., 2016 had experimented GC-MS analysis of phytocomponents in, pet ether fraction of Wrightia tinctoria seed. It could be concluded that, Wrightia tinctoria contains various bioactive compounds.
Anchal Sharma, et al., 2017 had Characterized of 2S Albumin from Seeds of Wrightia tinctoria. The conserved disulphide bonds in the protein suggest that the WTA is highly stable under high pH and temperature like other 2S albumin.
PHARMACOGNOSTICAL REVIEW
Leaf
Mahendra khydae ., 2009 had studied the total morphological and anatomical aspects of as well as different microscopical response of leaf.Physical constant values involving moisture content, ash value,extractive values as well as estimation of phytoconstituents of Wrightia tinctoria (Roxb) R.Br.leaves.
Lakhmi devi.S et al., 2012 had observed various pharmacognostical standard such as ashvalues extractive values, microsopical characters of leaf power and Various leaf extracts.
Vijaya ragavan, et al., 2013 had studied the pharmacognostical evalution of leaf powder of the plant.They had observed the various features of macroscopic, microscopic characters of the powder.
Beena, et al., 2014 had analysed the essential oil frm the leaves of Wrightia tinctoria R.Br.
Eswaran, et al., 2015 had observed the HPTLC finger print analysis of the Wrightia tinctoria leaf powder.
Seed
Rajani, et al.,2014 had reported the high performance thin layer chromatography finger printing important of seeds of w.tinctoria.
CHAPTER-3
AIM AND OBJECTIVE
AIM AND OBJECTIVE
AIM
The aim of the present research is to study the pharmacognostical, phytochemical evaluation and pharmacological activities of Wrightia tinctoria (Roxb.) leaf.
OBJECTIVE
The present research work has been planned to carry out the
PART-A
PHARMACOGNOSTICAL STUDIES- implicates
• Authentication and collection of plant
• Macroscopy of the leaf.
• Microscopy of the leaf
• Powder microscopy includes its identification of characters
• Behavioiral characters with different reagents.
PHYSICO-CHEMICAL PARAMETERS
• Foreign matter
• Loss on drying
• Extractive values with various solvents
• Ash value
PART-B PHYTOCHEMICAL STUDIES-involves the
• Preparation of hydro alcoholic extract
• Qualitative analysis
• TLC Profile
QUANTITATIVE ESTIMATION OF PHYTOCONSTITUENTS
• Gallic acid
• Tannic acid
• Rutin in terms of its equivalents
PART-C
PHARMACOLOGICAL ACTIVITIES ANTI HYPERGLYCEMIC ACTIVITY
IN VIVO STUDIES
• Alloxan induced diabetic rats with lipid profile
IN VITRO STUDIES
• In vitro alpha amylase inhibitory assay
IN VITRO ANTIOXIDANT ACTIVITY
• Hydrogen peroxide scavenging assay
• Reducing power assay
CHAPTER-4
MATERIALS AND METHODS
MATERIALS AND METHODS
PLANT COLLECTION & AUTHENTIFICATION
Fresh leaf of Wrightia tinctoria (Roxb).were collected from Kasampatti, Natham (Tk) Dindigul (Dt), Madurai (DT), during the month of August- 2018 and was authenticated by Dr. D. Stephen, M.Sc., Ph.D., Assistant professor, Department of Botany, American College, Madurai-20. The herbarium of this specimen was kept in the department for further reference.
PREPARATION OF LEAF POWDER
The leaves were collected and shade dried. It was powdered in a mixer,sieved and kept in a well closed container in a dry place.
PART A
PHARMACOGNOSTICAL STUDIES
Morphological and micro morphological examination and characterization of medicinal plants have always been accorded due credentials in the pharmacognostical studies. Botanical identity of the plants is an essential prerequisite for undertaking the analysis of medicinal properties of any plant. A researcher may succeed in getting a new compound or may find many useful pharmacological active properties in the plant. If the botanical identity of the plant happens to be dubious or erratic, the entire work on the plant becomes invalid. Thus it is needless to stress the botanical identity of the crude drug is the threshold in the processes of pharmacological investigations.
The researchers should be equipped with all possible diagnostic parameters of the
MORPHOLOGICAL STUDIES OF Wrightia tinctoria (Roxb)
Leaves were studied seperately for its morphological characters by organoleptic test.
MICROSCOPICAL STUDIES OF Wrightia tinctoria (Roxb)
Fresh leaves were selected for the microscopical parameters by using microscope
COLLECTION OF SPECIMEN
Care was taken to select healthy plants and for normal organs. Leaf, Petiole specimens were collected from a healthy plant by making a cut with petioles. The materials were cut into pieces and immediately immersed in fixative fluid FAA (Formalin – 5ml + Acetic acid – 5ml +70% Ethyl alcohol – 90ml).
DEHYDRATION
After 24 hours of fixing, the specimens were dehydrated with graded series of ethyl alcohol and tertiary-butyl alcohol (Sass, 1940). The specimen is kept is in each grade of the fluid for about 6 hrs. Every time the fluid is decanted and immediately the specimen were flooded with next grade of fluid.
INFILTRATION WITH PARAFFIN WAX
After dehydration, the shavings of paraffin wax were added to the vial containing the plant material with pure TBA. The paraffin shavings are added every 30mts at about 40-45ºC four or five times. Then the vials were filled with wax without damaging the tissues. The vial filled with wax is kept open in warm condition
CASTING TO MOLD
A boat made out of chart board, by folding the margin, is used to prepare a mold of wax containing specimens. The paraffin along with the leaf and petiole specimen was poured into the boat. With the help of heated needles, the specimen were arranged in parallel rows with enough space in between the specimens. The block was then immersed in chilled water and allowed to cool for few hours.
SECTIONING
The paraffin embedded specimens were sectioned with the help of microtome.
The thickness of the sections was 10-12μm. Dewaxing of the sections was by customary procedure. The sections were stained with Toluidine blue as per the method published by O’Brien et al (1964). Since toluidine blue is a poly chromatic strain, the straining results were remarkably good and some cytochemical reactions were also obtained. The dye rendered pink colour to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein bodies etc. Where ever necessary sections were also stained with safranin and fast- green and potassium iodide (for starch). For studying the stomatal morphology, venation patttern and trichome distribution, paradermal sections (sections taken parallel to the surface of leaf as well as clearing of leaf with 5% sodium hydroxide or epidermal peeling by partial maceration employing Jeffrey’s maceration fluid (Sass, 1940) were prepared. Glycerin mounted temporary preparations were made for macerated/cleared materials. Powdered materials of different parts were cleared with sodium hydroxide and mounted in glycerin medium after staining. Different cell
PHOTOMICROGRAPHS
Microscopic descriptions of tissues are supplemented with micrographs wherever necessary. Photographs of different magnifications were taken with Nikon labphot 2 Microscopic unit. For normal observations bright field was used. For the study of crystals, starch grains and lignified cells, polarized light were employed.
Since these structures have birefringent property, under polarized light they appear bright against dark background. Magnifigations of the figures are indicated by the scalebars. (Johansen DA, 1940).
PREPARATION OF LEAF POWDER
The leaves were collected and shade dried. It was powdered in a mixer. The coarse powder was sieved and was stored in a well closed container.
QUANTITATIVE MICROSCOPY OF Wrightia tinctoria (Roxb).
Fresh leaves of Wrightia tinctoria (Roxb). was subjected to microscopical study includes in stomatal number, stomatal index were determined on fresh leaves using standard procedure. (Wallis TE. 1953, Wallis TE, 1965).
VEIN ISLET NUMBER AND VEIN TERMINATION NUMBER
The term vein islet is used to denote the minute area of photosynthetic tissue encircled by the ultimate division of the conducting strands. The number of vein islets per sq.mm. Area is called vein islet number.
Vein terminal number may be defined as the number of vein terminals present in one sq.mm area of the photosynthetic tissue.
DETRMINATION OF VEIN ISLET NUMBER AND VEIN TERMINATION NUMBER
Small square portion from the lamina region of the leaf was cleared in chloral hydrates, stained and mounted on a slide. A camera lucida is set up and by means of a stage micrometer the paper is divided into squares of 1mm2 using a 16mm objective.
The stage micro meter is then replaced by the cleared preparation and the veins are raced in four continuous squares, either in a square 2mm x 2mm (or) rectangle 1mm x 4mm. When counting, it is convenient to number each vein-islet on the tracing. Each numbered area must be completely enclosed by veins, and those which are incomplete are excluded for the count if cut by top and left-hand sides of the square (or) rectangle but included if cut by the other two sides. Ten readings for vein islet and vein termination number were recorded.
STOMATAL INDEX
It is the percentage, which the numbers of stomata from the total number of epidermal cells, each stoma being counted as one cell.
I. Stomatal index = S/S+E x 100 Where, S = Number of stomata per unit area
E = Number of epidermal cells in the same unit area DETERMINATION OF STOMATAL INDEX
The procedure adopted in the determinations of stomatal number was observed under high power (45 X).The epidermal cells and the stomata were counted. From
POWDER MICROSCOPY
The coarse powder was treated with routine reagents to identify the diagnostic features of the plant.
PHYSIO-CHEMICAL PARAMETERS
The powder was subjected to physiochemical parameters such as foreign organic matter, loss on drying, ash values and extractive values with different solvents in increasing order of polarity, volatile oil, and total solids. The procedure was adapted as per WHO guidelines 1998, and James 1995.
DETERMINATION OF FOREIGN ORGANIC MATTER PROCEDURE
An accurately weighed 100g of air dried coarse drug and spread out in a thin layer. The sample drug was inspected with the unaided eye or with the use of 6x lens and the foreign organic matter was separated manually as completely as possible and weighed. The percentage of foreign organic matter was calculated with reference to the weight of the drug taken.
DETERMINATION OF MOISTURE CONTENT (LOSS ON DRYING) PROCEDURE
An accurately weighed 10 g of coarsely powdered drug was placed in a tarred evaporating dish. Then the dish was dried at 105oC for 5 h and weighed. The drying and weighing was continued at one hour intervals until the difference between the two successive weighing is not more than 0.25 %. The loss on drying was calculated with
DETERMINATION OF EXTRACTIVE VALUES PROCEDURE
An accurately weighed 5 g of the air dried coarsely powdered drug was macerated with 100mL of various solvents of increasing order of polarity (petroleum ether, benzene, chloroform, ethyl acetate, ethanol, methanol and water) in a closed flask for 24 h, shaking frequently during the first 6 h and allowed to stand for 18 h.
Thereafter filtered rapidly, taking precautions against loss of ethanol. Then evaporate 25 mL of the filtrate to dryness in a tarred flat bottomed shallow dish dry at 105o C and weighed.
DETERMINATION OF ASH VALUES ASH CONTENT
The residue remaining after incineration is the ash content of crude drug, which simply represents inorganic salts naturally occurring in the drug or adhering to it or deliberately added to it as a form of adulteration.
DETERMINATION OF TOTAL ASH PROCEDURE
An accurately weighed 3 g of air dried coarsely powdered drug was taken in a tarred silica crucible and incinerated at a temperature not exceeding 450o C, until free from carbon then allowed to cool and weighed. The percentage of ash was calculated with reference to the air dried drug.
