INTRODUCTION AND REVIEW OF LITERATURE

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1 Chapter I INTRODUCTION AND REVIEW OF LITERATURE The Fabaceae or Leguminosae, commonly known as the legume, pea, or bean family, is a large and economically important of. The group is the third largest family, behind only the > and, with 730 genera and over 19,400 species (Stevens, 2001). The largest genera are (over 2,400 species), (over 950 species), (around 700 species), (around 700 species), and (around 500 species). Plants of this family are found throughout the world, growing in many different environments and climates. A numbers are important agricultural plants, including: (soybean), (beans), (pea), (chickpeas), (alfalfa), (peanut), and (licorice); which are among the best known members of Fabaceae. The name Fabaceae comes from the defunct genus Faba, now included in. The term "faba" comes from Latin, and appears to simply mean "bean". (Wiktionary, 2011) Leguminosae is an older name still considered valid, ( 4 & 3 ) Art (Vienna Code) and refers to the typical of these plants, which are called. Uraria is a genus belonging to tribe Desmodieae of the subfamily papilionoidae Leguminosae (Ohashi, 2005). It s basically characterized in having imparipinnately foliolate leaves, the leaflets of which have distinct principle lateral nerves extending to the margin 1

2 densely flowered long pseudoracemes with acuminate bracts longer than flowers, paired pedicels inflexed hooked at the apex with dense long hairs and jointed pods with folded articles. The genus comprises about 20 species distributed mainly in tropical and subtropical regions in south eastern Asia extending east Taiwan and Japan, south to Northern Australia and Africa.The center of diversity is considered to be India to Indochina (Ohashi 2005). The genus was first complied by (Yang and P.H. Huang, 1981) and there results were adopted in Flora Republicae popularis sinicae Vol.41 Leguminosae (3) in He recognized nine species of Urariopsis. (T.C. Huang and Ohashi,1993) compiled the genus in Taiwan and recognized four species. Aside from these species common in both works. Uraria aquilobata Hosokawa was recognized as a synonym of Uraria lagopodides (L) Desv. ex Dc. by Yang and P.H. Huang (1981 and 1995) but was treated as distinct species endemic to Taiwan and Hongkong by (Huang and Ohashi,1993). Uraria and Urariopsis were regarded as congeneric by (De Hass et. al.1980) and his broad circumscription of Uraria has been accepted by (Dy Phon,1887) and (Ohashi, 2005), Aurvedic formulations in the Indian system of medicine which contains powder of roots from ten different medicinal plant including Uraria picta. The preparation is used as source of hormone to cure post delivery problems, inflammations, detoxification of entire body, and an excellent sedative in balancing tri-doshas (Three imbalances):vata (regulation of body movement through the nervous system), PITTA (causes of all metabolic processes in the body), and KAPPHA (Principle of cohesion and function through the body fluids) (Kasar et.al. 2007), (Anonymous 2010). The root, leaf and pod extracts posses acaricidal, antiviral, and antimicrobial activities (Igboechi et. al.1989), (Rahman et.al. 2007), 2

3 Uraria picta shows antiulcerogenic property (S. Manonmani et.al. 1995). The tribes of Akole tahsil of Ahmadnagar districts pods of Uraria picta eat as a vegitable (Khyade et. al.2009), However the availability of Uraria picta root biomass is very less. Moreover, the unsustainable utilization from the wild habitat for the root source and limited success in propagation through seeds has led to its inclusion in the list of rare, endangered, and endemic plant species (Anand et. al.1998; Gurav et. al. 2008; Ahire et. al. 2009; Rai et. al. 2010). A bioprospecting study using medicinal plants is important both in terms of adding economic value for biological resources and also creating an economic stake in conservation (Feinsilver 1995). Plant tissue culture is an efficient technique for the conservation and large scale production of various rare, endangered, medicinal and aromatic plants (Anand et.al. 1998; Joshi and Dhar 2003; Zhang et.al.2004; Faisal et.al.2007 Gurav et.al. 2008; Srivastava et.al. 2009; Rai et.al. 2010; Ahire et.al.2011). Although in vitro propagation from nodal explant of Uraria picta has been reported (Anand et. al. 1998; Mukundan et. al. 2002; Gurav et. al. 2008; Rai et. al. 2010; and Ahire et. al In view of increasing demand of Uraria picta, Uraria rufescens and Uraria lagopus and its rare endangered and endemic status we describe here a reproducible and efficient propagation protocol to produce genetically uniform plants. We follow Uraria in this work. The study of gross internal structure of plant organs by the technique of section cutting is called plant anatomy. (Pandey, 2002). Various plant organ viz. root, stem, leaves, petiole show their typical anatomical structure after section cutting. The development and organization of tissue in plant organs is the result of their function, in words tissue in specific plant organ are developed to perform a specific function. Several anatomical features 3

4 are specific-to-specific taxa. Hence these are used for delimitation of the species. These anatomical features having taxonomic values are used criteria for separating the species, genera and even families. The anatomy of plant parts gives the criteria of xylem, cortex, secondary phloem, modullary rays, fibers etc. which forms the important parameters. The word epidermis derived from two words of Greek origin. (Epi - upon and derma - skin). Parenchyma refers to a tissue composed of living cells variable in their morphology and physiology, but generally having thin walls and a polygonal shape. Scleroids are widely distributed in the plant body (De Bary, 1884). The xylem was introduced by (Nageli, 1858), and is derived from the Greek xylos, meaning wood. The term tracheary a name originally applied to certain primary xylem elements resembling insect tracheae (Esau, 1961). Phloem is principal food conducting tissue of the vascular plants. Sclerenchyma was originally proposed by (Mettenius, 1865). Many seeds fail to germinate after processing and placement in favorable growing condition such seeds are said to be dormant. In some dormant seeds morphological changes must takes place before germination can start. For others, parts of the seeds undergo physiological changes before germination can occur. Under natural conditions necessary changes takes place gradually under varying combinations of aeration, moisture, temperature and light. By duplicating key condition of the natural environment in the laboratory, dormant seeds can be induced to germinate with a reasonable length of time (Krugman et.al.1974). In general there are two types of seed dormancy: Seed coat dormancy and internal dormancy. Seeds with seed coat dormancy usually have a seed coat that is 4

5 impermeable to oxygen or water. Occasionally the dormancy is caused by an inhibiting chemical in the epidermis or adjacent interior membranes. Under natural conditions these seeds remain in the ground without germinating until they have weathered sufficiently, to allow penetration of water, exchange of gases or neutralization of inhibiting chemicals. Seeds of some species germinate only after being subjected to fire, the length of time involved can be several years or more depends upon the species and the environmental conditions. Internal dormancy is a general term encompassing a number of physiological conditions that delay germination. Not all of these conditions are fully understood or easy to counteract. The most common one is called after ripening. Seeds that require an after- ripening period, even though harvested when mature, germinate poorly or not at all until they have subjected to moisture and either high or low temperatures or both in sequence; sometimes a period of dry storage is sufficient to break dormancy. As might be expected, internal dormancy is most often found among species that grow in high mountains or deserts. The more common method for breaking internal dormancy is cold stratification. In some cases the use of chemicals can be substituted for part or the entire stratification requirement. (Emery, 1987). In modern era we are loosing many important plant species due to high pressure of urbanization, industrialization and deforestation. So the study of seedling morphology is very important to conserve these plants permanently and for easy identification. Several workers studied the seedling characters of different species of plants. (Hill,1982) illustrated and discussed briefly the variations in cotyledon morphology in Malvastrum. (Sampath Kumar, 1982) stressed the use of cotyledon morphology at the 5

6 generic, specific and varietal levels in the family. (Kamilya and Paria,1993), studied on seedling morphology of some members of polygonaceae and its taxonomic implications. The seedlings of the genus Uraria have been virtually neglected from the viewpoint of taxonomy. The seedling morphology of only a few species among the several hundred of the genus has been studied, invariably showing two epigeal cotyledons and elongated epicotyls. However i have found that the seedlings of Uraria are much diversified in morphology, and have come to realize the importance of seedling morphology for classification and delimit the species. Uraria picta (Jacq) Desv. Ex Dc. Belonging to family fabaceae is a native of tropical zone including Nepal, Srilanka, Northern Austrelia, China Burma (Anonymous 1976). This suffruticose herb which grows upto 1-2 m tall is found in dry grasslands, waste places open deciduous forests and in all planes of India extending from Himalayas to Ceylon, Malaysia and Phillippines (J.D.Hooker 1973). The plant is reported to be endangered in some parts of India (M.C.Joshi 1988).Leaf, roots and pods of the plants produce medicinal substances which are found to posses acaricidal, antiviral, antimicrobial activity ( Igboechi et. al.1989 ). Its use in Dashmula capsules, an aurvedic medicine has shown significant improvement in patients of primary neurological disorder (Gupta et. al.1984). In Ghana, the plant is used in heart troubles (Chauhan, 1999).The plant is supposed to be a good antidote to venom of Echis carinata (Vaidya, 1968), (Sharma, 1996). It is also used in the preparation of Abana, an Aurvedic drug remedy useful in the treatment of hypertension, tachycardia and angina (Pasani et.al. 1988).The whole plants is medicinally important and is used by certain Adivasi and native tribes ( Jain and Defilipps,1991). 6

7 Traditionally the plant is used as an antidote to the venome of a dangerous Indian snake, Echis carinata (Kirtikar and Basu 1993). The fruit and pods are effective against oral sores in children, and the roots are used against cough, chills and fever (Kirtikar and Basu 1993), and (Yusuf et. al. 1994).Two isoflavones isolated from Uraria picta have antimicrobial activity (Rahman et.al. 2007).The plants is also known for healing fractures, as it has the property of accumulation of phosphorous and deposition of calcium. The species is increasly becoming rare due to overexploitation by various pharmaceutical industries as well as local tribes for medicine and trade coupled with poor seed viability (Anand et.al.1998).the pulverized leaves of this plant are used medicinally in Southern Nigeria as a remedy for gonorrhea; and in India the plant is used as an antidote against the bites of certain vipers (Allein and Allein,1981), The root has aphrodiastic properties and the decoction is prescribed for cough, chills and fever (Anonymous, 1976). Bioprospecting studies using medicinal plants is increasingly important both in terms of adding economic value for biological resources and also creating and economic stake in conservation (Feinsilver, 1995).However with the growing need for medicinal plant those that are rare and endangered in nature, propagation of these species in large numbers is a necessity In vitro technique offer a rapid means for multiplication of such elite and rare germplasm (Bajaj,1986) and an alternative method for Ex situ conservation (Kartha, 1985). Hence there is an urgent need to develop in vitro methods for rapid production of plants for providing uniform raw material for medicinal purposes as well as for its rehabilitation in natural habitat for conservation, and sustainable utilization. 7

8 AREA OF STUDY: A. Physiography Maharashtra State (Plate I) was a part of Bombay Presidency during the British rule. Maharashtra was carved as new State on 1st May With present political boundaries the State comprises an area of 3, 07, 690 sq. km and is about 800 km east-west and north south, lying between to north and to east longitude. The State of Maharashtra constitutes 35 districts, which are physiographically divided into 5 division s viz. Konkan, Desh or Deccan, Khandesh, Marathwada and Vidarbha. 1) Konkan: Konkan is a narrow coastal strip. It is about 27 to 48 km in breadth and about 800 km in length, which starts from Goa to Tapi basin. Politically it consists of 5 districts viz. Bombay, Raigad, Ratnagiri, Sindhudurg and Thane. The region consists of highly undulating lateritic hilly tracks and the coastal sandy low land belts with series of backwaters and estuaries as intrusion from the sea. The region is mainly dominated by littoral or mangrove forests, open scrub forest, moist deciduous forest and herbaceous flora of lateritic plateaus. 2) Desh or Deccan: Deccan is lying to the east of Sahyadris, which approximately 565 km in length. Politically it consists of 7 districts viz. Ahmednagar, Kolhapur, Nasik, Pune, Sangli, Satara and Solapur. The average height of Sahyadri hills in Deccan is 1200 m. The highest peaks of Sahyadris are Kalsubai (1646 m), Harishchandragad (1424 m) and Mahabaleshwar (1438 m). The vegetation of this region is varied. It consists of moist and dry deciduous forest, semi evergreen forest. 8

9 3) Khandesh: Khandesh consists of 3 districts viz. Dhule, Jalgaon and Nandurbar. It lies in valley of the Tapi valley and the Godavari valley separating it from western Maharashtra. Khandesh is situated between various parallel ranges of Satpudas. The land is mostly flat and plain. The vegetation of Khandesh is dry deciduous forest and thorn forest. 4) Marathwada: Politically Marathwada region consists of 8 districts viz. Aurangabad, Beed, Hingoli, Jalna, Latur, Nanded, Osmanabad and Parbhani. The total area of the region is 64, 798 sq. km. The entire region is situated at an average height of about m above mean sea level. The highest peak, (960 m) is situated in Aurangabad district. Tropical dry deciduous forests, open scrub jungles and vast tracts of grasslands, mainly dominate the region. 5) Vidarbha: Vidarbha or the eastern Maharashtra consists of 11 districts viz. Akola, Amaravati, Bhandara, Buldhana, Chandrapur, Gadchiroli, Gondia, Nagpur, Wardha, Wasim and Yavatmal. It is a continuous stretch of land with black cotton soils derived from the Deccan Trap basalt. The region is situated at an average height of 457 to 549 m elevations above mean sea level. The forest of this region is tropical moist deciduous forest and dry tropical forest. B. Soil Soils of Maharashtra are mainly formed from the Deccan Trapps, generally from the augite or amygdaloidal basalt. These soils are black, dark brown or reddish in colour and hence called as Black cotton soil. Black Cotton soils have high percentage of clay and are heavy in texture. They are sticky and plastic in nature and swell on wetting and shrink on drying.calcium and magnesium carbonates and iron are found in appreciable proportions. However, these soils are poor in organic matter, 9

10 nitrogen and phosphorous. The black cotton soil with sufficient water and nitrogen are capable of high productivity, particularly cash crops like sugarcane, chillis, groundnut, jowar, wheat etc. the black cotton soils in rain scarcity zones of Ahmednagar, Solapur, Jalna, Beed and Aurangabad are saline and therefore infertile. Lateritic soils of Konkan are derived from Deccan trap which are leached from laterite. They contain dehydrated iron-oxides which impair red colour. These soils are poor in plant nutrients and organic matters. * In Desh, light brown coloured and deep coloured soil is found. Light brown coloured soil is found in Mavals while deep coloured soil occurs in Kolhapur, Satara, Pune and Nasik districts. In this region, below the black soil coarsely powdered sedimentary rock occurs, which is called as Murum. * Coastal alluvial are deep sandy soils of very little clay content. In this sandy belt there are saline soils which are formed due to ingress of sea water or by the low lying nature. * Soils of Khandesh are fertile due to river Tapi and provide ideal conditions for different crops. While Vidarbha soils are formed by the Archeans, the Vindhyans, the Gondwanas and the Traps. They are sandy or loamy soils found at Nagpur, Bhandara and Chandrapur districts. C. Climate The climate of Maharashtra is monsoonal. The State faces extremely hot summers from March till the starting of rainy season in early June. The lush greenery is the result of monsoon season, which persists during the following mild winters. The State enjoys mild winters, but the season changing month of October is very unpleasant. Average rainfall in Maharashtra is mm and average temperature is 260 o C. Rainfall 10

11 and humidity in Konkan and hill slopes are equable and suitable for plant growth. Western Ghats and the coastal regions receive heaviest rains exceeding 2000 mm rainfall per annum. Rainfall over the ghats may exceed 5,000 mm annually. The temperature is lowest due to Arabian Sea. Western parts of Maharashtra (Desh or Deccan) receive annual rainfall of about 600 to 635 mm except Ahmednagar, Solapur and eastern part of Poona district. Average rainfall of Marathwada is about 900 mm and the average temperature ranges from 270 ºC to 380 ºC. The western part of Vidarbha is little hotter, receives about mm rainfall per annum. The annual mean temperature is more than about 400C. The eastern part of Vidarbha comprising Chandrapur, Bhandara and Nagpur temperature is lower than the western Vidarbha and rain fall is about mm. In Khandesh the temperature is high and rainfall is about mm. Konkan receives 94% of the annual rain during monsoon, Madhya Maharashtra and Marathwada receives 83% of the annual rain and Vidarbha receives 87% of annual rainfall. 11

12 REVIEW OF LITERATURE: Study of genus Uraria was neglected by different scientist from long ago, firstly chromosome number of Uraria hamosa was studied by Gajapathy and recorded as n = 11in 1962 (Gajapathy 1962), In Uraria picta paracytic stomata occur on both surfaces where as anisocytic stomata occur only at abaxial surfaces. Amonocytic stomata are present on abaxial surface of Uraria picta and Uraria rufescens. (Kothari and Shah, 1975).Uraria picta used as a medicinal, leaf juice of Uraria picta is antipyretic (S. J. Bedi 1978). Uraria has been studied in China (Yang and Huang 1981). Uraria has been studied in Indochina (Dy Phon 1987), Bhutan (Grierson and Long 1987), Uraria picta reported to be endangered in some parts of India (M.C. Joshi 1988). Leaves of Uraria picta used in snake bite, antidote repositioning foetus, intra-uterine, aphrodiastic in the region of Garo hills of Meghalaya (Neogi et. al.1989). Germination response of Uraria picta to five environmental factors was investigated (O.T. Okusanya et.al. 1991) in Southern Nigeria. Uraria picta is an important medicinal woody herb (Okusanya et.al.1991) commanly known as Pithvan. Recently Uraria has been studied in India by Sanjappa, (Sanjappa 1992). Dy Phon (1987) revised Uraria in Cambodia, Laos and Vietnam and recognized 13 species including Uraria campanulata (Benth) Thoth.Gagnep.which is recognized as Chiristia campanulata (Benth) Thoth.and is excluded from Uraria following the previous treatment in Floras of Taiwan (Huang and Ohashi 1977, 1993). Uraria has been studied in Indochina (Lock and Heald 1994). Kumar and Sane (2003) enumerated 14 species from South Asia consisting of Bangladesh, India, Myanmar, Nepal, Sikkim, and Srilanka. Except for Uraria campanulata, seven species of the 13 recorded in South Asia are 12

13 distributed also in China. Six of these are common with those in Indochina as listed above and another is Uraria sinensis Franch. Uraria lagopus local name Prishniparni whole plant is used (Tiwari, 2004).Uraria picta seed paste used in Haemorrhoides(Jadeja, et.al. 2006) Uraria picta commonly known as Devla used in general debility, nervinetonic, cardiac and blood disorder, antidote for snake venom (Milind Parle and Nitin Bansal 2006). Uraria picta commonly known as Mahadevjata, Ishwarjata, root paste with honey (4:2) is given once daily for five days as abortifacient, Leaf paste is given twice daily as antidote to snakebite (Chakraborty and Bhattacharjee,2006).Uraria picta is used in Chyawnprash traditional polyherbal formulation which widely used as tonic, rejuvenator, anabolic, immunomodulator and Memory enhancer (Kasar et.al. 2007), Two isoflavones 5-7, Dihydroxy-20-methoxy-30-40mythelenedioxyisoflavone and 40,5 dihydroxy 20,30 dimethoxy hydroxychromen 7 yl isoflavone, triterpenes and steroides were isolated from root of Uraria picta. The compound showing antimicrobial activities against bacteria (both gram positive and gram negative) and fungi. (Rahman, et. al.2007) Uraria picta when used in dashmoola shows anxiolytic effect (Sharma, et.al.2007) Uraria picta used in dashmoola which is used to investigate the effect of Abana an Indian Aurvedic polyherbal formulation on memory and brain cholinesterase activity in mice. (Milind parle and Mani Vasudevan 2007). Uraria picta commonly known as Chinva in Madhya Pradesh, leaf paste is applied on cut and wound twice a day (Sikarwar, Pathak and Jaiswal 2008). Uraria picta root and stem were selected for estimation of six heavy metals namely Arsenic (As), Muecury (Hg), Lead (Pb), cadmium (Cd), Chromium (Cr),and nickel (Ni) apart from these the organochlorine pesticide residues viz. different metabolites of DDT, 13

14 Isomers of HCH and endosulfon in samples of crude drugs (Rai, et.al. 2008). Uraria picta used in quick healing of bone fracture, it is used as cardio and nervine tonic and has anti-inflammatory, expectorant and diuretic properties. The root of the Uraria picta plant is one of the ingredient of Dashmoola (NMPB 2008), Vol.I. In vitro propagation of the medicinal plant Uraria Picta from cotyledonary node and nodal explants (Gurav, et.al.2008). Uraria picta Chakama community commonly known as Bilai-langur and whole plant is used against paralysis (Roy, et. al. 2008). Uraria picta pod is generally eaten as raw and sometime salad and prickles are prepared from them (Khyade, et.al.2009). Liquid chromatographic method devoloped for the quqntitation of rhoifolin in Uraria picta a plant of high usage frequency in all traditional system of medicine (Yadav, et.al.2009). Uraria picta (Jacq) Dc. is an important medicinal woody herb commanly known as Pithvan or Prishnaparni (Ahire, et. al. 2009) it is widely distributed through India, China, Burma,Srilanka, Africa and Northern Austrelia (Yadav et.al.2009). Uraria picta used in fever (Jvaracikitsa) commonly known as Prishniparni (Mishra, 2009), Uraria picta used against pulmonary tuberculosis (Raghuvanshi, Pandya, Joshi 2009). Uraria picta commonly known as krushnaparni, in Deogarh Districts of Orissa the flowers of Uraria Picta used against sterility, 3 gm flower is made into paste and taken once a day in empty stomach to sterile women for one month for pregnancy (Sahu et. al. 2010). Rapid in vitro production of cloned plant of Uraria picta: A rare medicinal herb in long term culture (Rai, et.al.2010).the teaspoon is taken orally to treat sore mouth and half teaspoon of root decoction is taken orally for seven days in snake bite (Jain and Singh 2010)., A report on generic male sterility in Uraria picta (Bhattacharya and Datta 2011). Micropropagation of Uraria Picta 14

15 through adventitious bud regeneration and antimicrobial activity of callus (Ahire, et.al.2011).the preliminary study on the germination of Uraria picta were carried by (Waghire, et.al.2011).antioxidant activity of whole plant of Uraria picta studied by (Bharatkumar, et.al.2011).the antioxidant activity of root, stem and leaf studied by (Waghire, et.al.2012). Protective effect of aqueous extract of Uraria picta on acetaminophen induced nephrotoxicity in rat was studied by (Kale, et.al. 2012) he concluded that the protection offered by the extract due to presence of polyphenolic and carbohydrate compounds. OBJECTIVES 1. Field survey of genus Uraria in Maharashtra. 2. Detailed taxonomical studies of collected species. 3. Comparative studies of floral morphology including inflorescence flower Structure, calyx vexillum keel and carina supported by photographs. 4. Photographic documentation of taxonomically significant features of genus Uraria. 5. Anatomical studies of species of Uraria. 6. Seed germination pattern of the genus. 7. Seedling Morphology of the genus Uraria. 8. Scanning electron microscopic study of the seed, testa topography and leaf surfaces (Upper and Lower) of three species of genus Uraria 9. Biochemical study of the plant with GC-MS. 10. Thin layer chromatography of leaf stem and root powder. 11. Antioxidant activity of leaf stems and root powder and its scavenging property. 15

16 12. Gel electrophoresis and protein band pattern. 13. Ex-situ and in situ conservation of genus Uraria. The thesis embodies the results of five years field survey and observations on distribution, morphology, status, floral characters, biochemical investigation, Detection of chemicals by GCMS, protein band pattern by gel electrophoresis, Seed scanning electron microscopic studies and testa topography of the three species, antioxidant activity, seed germination, seedling morphology and tissue culture technique for conservation and rehabilitation of the three species i.e. Uraria lagopus, Uraria picta and Uraria rufescens. 16