CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

Tephrosia is a genus of flowering plants in the pea

family, Fabaceae. The generic name is derived from

the Greek word τεφρος (tephros), meaning "ash-colored,"

referring to the greyish tint given to the leaves by

their dense trichomes. (Zafar et al,2000)

(Quattrocchi, 2000). They are distributed in warmer

regions of the world especially Africa and tropical

Australia.

Plant morphology describes the study of the

development, form, and structure of plants, and by

implication, this can be used to show the basis of

similarity or dissimilarity of plants and their

origin. (Harold, 1987). The word macro and micro

morphology in this context is comparative, meaning

that it involves examining the structure of the

leaves and other physical features of the same or

different species in order to draw comparisons about

their similarities. The morphology characters may be

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inadequate in thorough differentiation of plant when

there are tight or close specie of plant, then other

facet of science may be exploited to draw out

adequate differences. However very little cytological

work has been done so far (about 12%) in the genus

and previous studies mainly involved chromosome

counts. In view of this an attempt has been made to

study meiosis and root tip mitosis in available

species of this genus.

It is found that least work is reported for

phytochemical and for biological activities of this

genus. Hence, four species of this plant was selected

and carried for Phytochemical investigation. The

genus is well known for its richness in prenylated

flavonoids and is considered to possess insect

repellant, larvicidal, piscicidal, antimicrobial and

anticancer properties been the subject of human

curiosity and need. (Gomez-Garibay et al., 2001).

Different extracts and fractions of Tephrosia showed

the presence of flavonoids being the active

ingredient and other compounds such as alkaloids.

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GENERAL DESCRIPTION OF GENUS TEPHROSIA

Tephrosia is a genus of flowering plants in the peafamily, Fabaceae. The generic name is derived fromthe Greek word τεφρος (tephros), meaning "ash-colored,"referring to the greyish tint given to the leaves bytheir dense trichomes.(Zafar et al,2000)(Quattrocchi,2000).It is a genus of leguminousshrubby plants and herbs mostly found in tropicalcountries (Blommaert, 1950). It is a pantropicalgenus of more than 400 species of herbs and shrubs (Geesink, 1981). In Australia, the genus occurspredominantly in the tropics and subtropics,generally growing in well-drained sites in openhabitats. The most recent comprehensive treatment ofthe genus in Australia is that of Bentham (1864), whorecognized 23 species. Since then, additional specieshave been described by Mueller (1875, 1879, 1880,1883), Domin (1912, 1926), Fitzgerald (1918) and morerecently Pedley (1977), among others. Bosman & deHaas (1983) revised the genus for Malesia, withseveral species from that area also occurring innorthern Australia. The plant is established easilyfrom seeds (Barnes and Freyre, 1969). The foliage isoften ashy-pubescent, hence the name Tephrosia. Theherb comes in various species that include Tephrosiavirginiana (L.) pers. (Fabaceae), Tephrosia purpurea,Tephrosia candida, Tephrosia vogelii and Tephrosia toxicaria.

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Also, it is a short-lived, slow-growing, herbaceous,frost-susceptible perennial. Barnes et al (1967)suggested that for commercial production of rotenone,which is derived largely from the leaves, the plantsshould be grown at the rate of 30,000 to 37,000 perhectare. Barnes and Freyre (1969) reported that whenplants were spaced 1.0 to 8.9m apart, the seed yieldper plant ranged from 1.1 to 8.9g, with the highest-yielding line producing at the rate of only 70 kgseed/ha. The rotenone is the chemical responsible forthe pesticidal property of T. vogelii.It is aninteresting class of compounds showing mainlyactivity against insects which has an acute oraltoxicity of 132 -1500 mg/kg capable of killing fish.(Cesar et al 2002).

It is maintained as a semi-cultivated plant indooryards in some primitive areas where it is usedfor poisoning fish. Seeds are saved and planted, andthe plants are tended, yet the plant also occurs inthe wild state. It has not been grown commercially,although recent tests (Barnes et al. 1967, Gaskins et al.1972) indicate that production might be economicallyfeasible if the culture and handling of the cropcould be similar to that of some hay crops. Otherspecies of Tephrosia native to the United States havealso been tested and show some promise as sources ofrotenone (Sievers et al. 1938).

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Tephrosia is a postrate or erect perennial herbs orsoft or woody shrub; rarely annuals , the leaves arepinnate with opposite or slightly alternate leafletand a terminal leaflet, lacking stipels, rarely withthree leaflet or with a single leaflet. The leafletare opposite, often silky, entire, usually narrow atthe base and widest above the middle. Trichomes arefound almost all over the plant part except the root.It is found both on abaxial and adaxial part of theleaf in most Tephrosia species but found only at theabaxial part of T. bracteolata. The flowers are small orquite large, pink to purple, less commonly orange oryellow, lacking bracteoles, hairy on the back,stamens 10 or 9 filaments joined on a dorsally splittube, 1 or more or less free. Style is hairy orhairless, flattened. The pod is straight or slightlycurved and usually several seeded. The calyx isusually 5 sepals; gamosepalous, often imbricate butsometimes valvate, corolla is usually 5 petals, freeand unequal size, aestivation is vexillary.Androecium: stamen is 10, diadelphous and free,Gynoecium: one carpel, superior ovary, placentationis marginal.

HABIT

Most member of this genus are annuals while some areperennials. They may be herbs, shrubs or sub-shrubshaving a straight stem or twig; leaves arealternately compound, the leaves and stem are

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pubescent giving a silvery and hoary appearance. Mostspecies thrive well in the tropics. Although they canbe found in the sand savannas, open wood and rockysoils.

Many individual plant produce good seed yields,whereas other are poor, indicating that considerableimprovement through breeding for seed production ispossible. Based on variations among accessions inseed set and other agronomic traits, Martin andCabanillas (1970) suggested that cross breedingprocedures might result in improve seed production.Gaskins et al (1972) stated that seed production isimpeded by the flowering requirement of the species,by a naturally occurring system of sterility, and byfrequent shortages of pollinating insects.

INFLORESENCE

The flowers are typically papilionaceous about aninch across, and the purple with white markings. Theflowers are borne on compact racemes that blown overa 3-6 week period having flowers of differentcombination, flower colour ranging from white withpink to yellow, purple, red and so on depending onthe specie. There may be 20-30 flowers per racemewith upto 200 flowers per plant (Gaskins et al 1972).The flowers have a faint but definite pleasant aromaand bees visit them freely for both nectar andpollen. Flowering occurs on the decreasing daylenght.If the plant is grown in the United States, it is

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likely to be killed by frost before flowers appear;therefore the plant requires a tropical home for seedproduction. The flower may last about 2days duringcool or rainy weather but only 30hours during dryweather (Martin and Cabanillas 1970). The flowers arepolypetalous and zygomorphic.

POLLINATION

The plant is considered to be self pollinated. Thestigma appears to be receptive to pollination atanthesis, often it is in contact with dehiscedanthers, particularly if the stamens are long. Alsoflowers bagged before anthesis frequently producesome pods with seed; however when viewed from anagronomic standpoint, the seed-set is poor, and largedifferences in seed production occur in differentlocations.

Martin and Cabanillas (1970) concluded that “beesappear to have a role in pollinating Tephrosia. Honeybees particularly were the most frequent visitors,some were nectar seeking bees that visited onlyyounger flowers and usually did not touch a stigma,pollen seeking bees on the other hand reportedlyvisited older flowers. Though visiting bees,carpenter bees or largely honey bees caused scratcheson the stigma, possibly making them more receptive topollination. Although their conclusion is not clear,they further added that carpenter bees (Xylocopabrazilianorum) were the principal pollinators.

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Gaskins et al 1972 concluded that insects facilitateself-pollination but contribute little to crosspollination. Thus, they concluded, bees facilitateself pollination by changing the relative position ofkeel, stigma and pollen, without tripping, preparingthe stigma for pollination by breaking up a stigmaticsurface. Watson, 1981.

Martin and Cabanillas (1970) recommended that plantings be made close to weedy areas and in abandoned fields where dead trees or legume plantingsoccur. They also recommended that methods of increasing population of carpenter bees must be conducted. Their discussion of the pollination of this crop is so reminiscent of the early history of alfalfa pollination that one is led to wonder if flooding the field of Tephrosia with honey bees might not have the same beneficial effect it has had on alfalfa seed production.

DISTRIBUTION AND PREVALENCE

According to literature, Tephrosia, is said to be nativeto Africa and it is widely distributed in thefollowing regions;

AFRICA

Northern Africa: Algeria, Egypt North East Tropical Africa: Chad, Ethiopia

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East Tropical Africa: Kenya, Tanzania, Uganda West Central Tropical Africa: Burundi, Cameroun West Tropical Africa: Cote D’Ivoire, Nigeria,

Benin South Tropical Africa: Angola, Mozambique Southern Africa: Botswana, Namibia, South Africa Western Indian Ocean: Madagascar, Mauritius

ASIA-TEMPERATE

Arabian Peninsula: Oman, Saudi Arabia, Yemen Western Asia: Iran China: Fujian, Guangdong, Guangxi, Yunnan Eastern Asia: Taiwan

ASIA-TROPICAL

Indian Subcontinent: India, Nepal, Pakistan, SriLanka

Malaysia: Indonesia

AUSTRALIA

Australia: Australian Northern and WesternTerritory, Queensland

NOMENCLATURE HISTORY

The taxonomy of Tephrosia was for many years underconsiderable confusion prior to Wood’s (1949)monograph (Bowles 1994). There are two tribes ofTephrosia, one group with glabrous styles (subgenusTephrosia), and the other, larger group, to which

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Virginia Goat’s-rue belongs, with bearded or barbatestyles (subgenus Barbistyla). Two varieties ofVirginia Goat’s-rue have been described previouslybased on variation in pubescence. Tephrosia virginianavar. glabra has been applied to plants with short andtightly appressed trichomes and to glabrous plants.Tephrosia virginiana var. holosericea has been applied toplants with the upper surfaces of the leaflets hairy,versus the typical form with upper leaf surfacesglabrous. However, these varieties are consideredtaxonomically insignificant by Wood (1949), thelatest monographer of the species, who concludedTephrosia virginiana is a single, widespread, geneticallydiverse species lacking distinct morphological andgeographical variations. No infra specific taxa arepresently recognized for the species.It is earlier been classified by many taxonomistseither into sections or subgenera, based mainly onthe morphological traits. De candolle (1825)classified Tephrosia into four sections namely:

Mundulea Brissonia Craccoides and Reineria

Bentham (1865) and Baker (1876) classified the genusinto three subgenera as:

Macronyx, which includes T. tenuis Brissonia that includes T. candida and

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Reineria which includes rest of the species ofTephrosia

Wood (1949) categorized the new world species ofTephrosia into two groups, those with

Glabrous style Pubescent style.

Subsequently Brummitt (1981) divide Tephrosia into twosubgenera:

Tephrosia with a glabrous style. Examples includeT. pumila, T. purpurea, T. spinosa, T. strigosa, T. villosa etcwere included and

Tephrosia with a trichiferous style, whichinclude T. candida, T. maxima, T. pulcherrima, T. tinctoriaetc.

It is worthy of note that the genus Tephrosia has beenfocused primarily from morphological data which isadequate for its classification but there arespecies, whose relationships are difficult to judge,hence phytochemical studies especially flavonoids areused.

Systematic classification of genus tephrosia

Kingdom: Plantae

Subkingdom: Tracheobionta12

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: Rosidae

Order: Fabales

Family: Fabaceae

Genus: Tephrosia pers- hoarypea

Floral formula and diagram

FORMULA K (5) C5 A(10) G1

KEY: Actinomorphic

Bisexual

K - Calyx (gamosepalous)

C - Corolla

A - Androecium (fused)

G - Gynoecium (superior ovary)

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CULTIVATION OF TEPHROSIA

HABITAT

Tephrosia is native to tropical Africa, and is foundgrowing naturally in widely varying habitats,including savannah-like vegetation, grassland, forestmargins and shrubland.

PREPARATION OF LAND

Tephrosia does not require a special land preparationbecause they can withstand wide range of soils andcan compete for nutrient and space.

PLANTING

Tephrosia can be planted in rows or stands. For agreen manure crop, the recommended spacing is 40cm ×40cm, with 2-3 seeds per hole; when planted forhedges the spacing should be 1.5m between the rows.For large plantations, sufficient seedlings should beavailable for replanting in case of a low survivalrate. When sown in rows, the recommended sowing rateis 5kg/ha and when broadcast 8-13kg/ha. An easy wayto plant Tephrosia for soil improvement is to sow itdirectly in the field between maize stations. Theseeds should be planted at a spacing of 90cm (3feet)with 2-3 seeds in each location in every other row ofmaize. The seeds can be planted on the ridgebetweeoaking n maize, in the furrow. It is important

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that the site is not waterlogged, as Tephrosia doesnot do well on such sites.

PLANTING TIME

Tephrosia should be sown at the beginning or middleof the rainy season. If planting with maize,Tephrosia can be sown at the same time or any timeafter maize planting until the middle of the rainyseason to make sure it gets sufficient rain forestablishment. However, germination increases if theseeds are pre-treated by soaking in sulphuric acidfor 10 to 12 minutes. Most seeds will have germinated8-10 days after sowing. Without treatment, thegermination percentage is 65%, and the seedlingsurvival rate about 60%. Soaking in warm water (450C)or sulphuric acid stimulates germination.

Tephrosia germinate to mature plant in about sixmonths, it is however a perennial shrub lasting for 3to 4 years.

CLIMATE

Tephrosia grows ataltitude up to 2100mm, requiresaverage annual temperature of 120C - 270C. It grows inareas with average annual rainfall of 850mm-2650mm,strives well on andosol soils not subject to floodingand on well drained loams with optimum pH 5-6.5. Theplant is also tolerant to poor soils with low pH(acid soils), although can be prone to diseases.

PESTS AND DISEASES15

Unfortunately, Tephrosia suffers from the same root-knot nematodes that attack tomato, tobacco, egg-plant, paprika and green peppers. This meansTephrosia plant should not be planted at a site whereany of these or related plants have been grownrecently, or where it is planted to grow them in thenext season. This roundworm causes severe damage whenit infects the roots of various plants (tobacco,tomato, beans, etc). For this reason, only plants notattacked by roundworms, such as maize, should beplanted in rotation after Tephrosia.

ACTIVE PRINCIPLES

In Tephrosia species, a wide variety of activepytochemicals including the flavonoids, terpenoids,ligans, rotenone, quercetin, deguelin, rutin,sulfides, polyphenolics, tephrosin, carotenoids,caumarins have been identified. The genus Tephrosia isknown to contain a wide variety of flavonoids(Dewick, 1993). As the Tephrosia genus representspotential source for flavonoids and variousbiological activities. According to Ayurvedha theplant is useful as an anthelmentic, anti-pyretic andas well as an alexiteric drug. It is also activeagainst leprosy, ulcers and used as alternative curesfor diseases of the liver, spleen, heart and blood.

SOME COMPOUNDS ISOLATED FROM TEPHROSIA16

Compounds isolated from Tephrosia include flavonoids,glycosides, steroids, tannins and reducing sugars(Ekpendu 1998). Rotenone is the compound of primaryinterest in Tephrosia (as far as its insecticidalproperty is concerned) but it is usually found incombination with several related rotenoids, theprincipal one being deguelin (Barnes 1967).

Analysis of the rotenoid content of leaf extracts ofTephrosia showed that rotenolone, tephrosin, rotenoneand deguelin are the main rotenoids produced (Lambert1993). Rotenolone and tephrosin are believed to bethe oxidation products of rotenone and deguelinrespectively. Although rotenone is considered themost active (insecticidal) ingredient in Tephrosia,the other extractives also possess appreciableactivity (Matsumura 1975).

RotenoneThe chemical structures of rotenone and itsderivatives (collectively called rotenoids) wereindependently determined by Butenandt and McCartney(1932), Laforge and Haller (1932), and Takei et al(1932). The rotenoids are composed of an isoflavonenucleus (C6-C3-C6) with an isoprene moiety attachedto it. They are classified into the isoflavonoidfamily, the end product of the phenylpropanoidpathway (Hahlbrock 1975). Rotenoids are advancedisoflavonoids (Crombie 1998).

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Rotenone is colourless and odourless, and has anempirical formula of C23H22O6 and a molecular weightof 394.41. Its melting point is 165-166°C (Watt 1962,Ware 1983). It is very soluble in many organicsolvents, for example alcohol and acetone, but isalmost completely insoluble in water (Tomlin 2000).Rotenone is generally unstable and decomposes quicklyin water, sometimes as fast as two weeks after itsapplication. However it may persist for up to sixmonths depending on a variety of factors includinglight, temperature, depth, dose and presence oforganic debris (Copping 1998). Rotenone readilybreaks down in the presence of light into at least 20products, only one of which, 6ab, 12ab-rotenolone, istoxic. None of the other degradation products istoxic meaning it is considered safe for use on landand in water (Newsome 1980). The decompositionprocess occurs at a faster rate as the temperature ofthe water increases. The depth and the presence oforganic debris in the water affect the amount oflight and therefore the rate of rotenone degradation.Lack of light decreases the rate of degradation(Marking 1988). The two main commercial uses ofrotenone today are as a piscicide and as aninsecticide. Rotenone has acaricidal (lice, tick,mite and spider killing) properties (Menichini 1982,Hollingworth 1995). Although rotenone is consideredthe most insecticidal ingredient in T. vogelii, theother rotenoids (deguelin, rotenolone and tephrosin)

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also possess appreciable insecticidal activity(Hagermann 1972, Matsumura 1975, Uddin 1979).

Deguelin Deguelin is a dimethoxylactone [(7aS, BaS)--13, 13a-dihydro-9, 10-dimethoxy-3, 3-dimethyl-3H-bis [1]benzo-pyrano [3, 4-b: 6', 5'-e] pyran-7 (7aH)-one],pale green in colour and has a chemical formula of[C.sub.23][H.sub.22][O.sub.6]; its melting point is171°C. Deguelin mediates antiproliferative propertiesin a variety of cell types. It exerts its growthinhibitory effects via the induction of apoptosis. Itwas found to suppress the growth of HT-29 coloncancer cells, with an IC (50) of 4.32 x 10 (-8) M,through the induction of apoptosis and cell cyclearrest (Murillo 2002).

Tephrosin Tephrosin is a nearly colourless crystallinesubstance with a chemical formula of [C.sub.23][H.sub.22][O.sub.7] and melting point of 198°C, andis thought to be the oxidation product of deguelin(Watt 1962, Murillo 2002).

Quercetin  The chemical formula of quercetin is C15H10O7-3D. Itis a member of a group of naturally occurringcompounds, the flavonoids, which have a commonflavone nucleus composed of two benzene rings, linked

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through a heterocyclic pyrone ring. Quercetin is awater soluble plant pigment (Young 1999). Thesynonyms for quercetin are: CI natural yellow 10; CI75670; cyanidelonon 1522; flavin meletin; quercetine;quercitol; quertin; quertine; sophoretin;xanthaurine; 3,3',4',5,7-pentahydroxyflavone;3,5,7,3',4'-pentahydroxyflavone; 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one. Quercetin is a natural compound found in T.vogelii and it helps to reduce painful swellingbecause of its potent anti-inflammatory capabilities(Shoskes 1999). It is a water soluble plant pigmentthat may help prevent heart disease. It also blockssorbitol accumulation, which seems to bring aboutnervous conditions in diabetics. Quercetin is anantihistamine, anti-inflammatory and an antioxidantagent. It promotes proper circulation of blood in thebody (Stoewsand 1984, Ishikawa 1985, Castillo 1989,Hertog 1994, Miodini 1999). It is effective inreducing allergic reactions and may be beneficial intreating canker sores, hives, asthma and otherinflammatory conditions (Lieberman 2003). Otherconditions for which quercetin may be helpful includediabetes, dysentery, gout, cataracts, and atopicdermatitis (Duthie 1997, Hollman 1997, Young 1999,Damianaki 2000, Feng 2001, Begum 2002). Quercetin hasbeen shown to possess antiviral properties(Weisburger2000, Feng 2001).

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Rutin Rutin, which is a component of T. vogelii, has importantmedicinal application in blood circulation andcapillary fragility (Oliver-Bever 1982).

ECONOMIC IMPORTANCE AND USES OF TEPHROSIA PLANT

The genus Tephrosia is of value to the generalpopulation owing to its variety of uses in differentaspects of science. Some of which includes;

MEDICINAL USES

Tonic, laxative, diuretic and deobstruent; used inbronchitis and bilious febrile attacks, and also forboils, pimples and bleeding piles. Pharmacologicalstudies have shown that extract of the herb is usefulin insufficiency of liver, but is not effective ininfantile cirrhosis. Roots and seeds are insecticidaland piscicidal (reports conflicting). Decoction ofroots given in dyspepsia, diarrhoea, rheumatism,asthma and urinary disorders; roots given with blackpepper in colic. A liniment prepared from the rootsis used in elephantiasis. Pulverized roots smoked forrelief from asthma and cough, decoction of pods usedas a vermifuge and to stop vomiting. Seeds yields oilsaid to be specific against scabies, itch, eczema andother skin eruptions. Fresh root-bark, ground andmade into a pill, with a little blackpepper, is

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frequently given in cases of obstinate colic. Theplant is used internally as a purifier of the blood,and is considered a cordiac tonic. An infusion of theseeds is given as a cooling medicine. In Ceylon, itis employed as an anthelmintic for children. InFrench Guiana, the root is used to stupefy or fishpoison. This drug is said to be useful in cough andin derangement of the kidneys. A decoction of thedrug was administered in one ounce doses to cases ofBright’s disease with dropsy and found to possessdiuretic properties in a mild degree (Kritikar andBasu, 1980).

Juice of the I. tinctoria leaves used for hydrophobia.Leaf juice and paste apply on the wound, dogs biteand snake bite. 10ml leaf extract mixed with honeyand drink for curing jaundice and spleen swelling.Fruit used to cure constipation. Root is used ininflammation of the liver, nervine tonic and antidotefor poisoning. (Maheshwari, 1996) The tribals use thereadily available local plants to anual the snakepoison.The species including I. tinctoria often used.(Masilamani, 1997) The whole plant of I. tinctoria iscrushed into a paste, mixed with water and given tothe dog and to detect rabies. If the dog has rabies,infection will express the symptoms soon. (Mini andSivadasan, 2007) Khanna et al. (1994) collectedinformation on unreported medicinal uses of eighteenplant species belonging to sixteen families exploitedas aphrodisiac among the fork- lores of Uttar Pradesh

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plains. Ethnomedical investigation of traditionaluses of medicinal plants by the tribal inhabiting theforest areas of Uttar Pradesh have brought to lightsome plants which are utilized for treatment ofvarious skin diseases, boils and blisters (Singh andPrakash, 1996). Plant of T. purpurea is tonic, laxative,anthelmitic to children given to purify the blood andas cordial, decoction is tonic. Root is bitter chewedto cure colic pain, used in asthma. Juice is mixedwith molasses and given for stomach pain applied onskin eruptions. Powder is smoked for cough, asthmaand respiratory diseases, as paste applied on bellyto cure dyspepsia, powdered and boiled in milk isapplied on leprosy and wounds. (Maheswari, 2000)Hiremath et al. (2007) collected sixty indigenousmedicinal plants from Dharwad and its surroundings.The traditional uses of these plants were given afterdiscussion with local healers and experienced adults.For each medicinal plant including T. purpurea, thescientific name, its family, local name, medicinaluse and method of preparation or administration havebeen given. The same work had been done on 47different medicinal plants by Sudha Rani et al. (2007)Based on the ethno botanical studies T. purpurea rootsused as an ailment for fractures, gas troubles andgastritis. (Lancelot, 2007)

PHARMACOLOGY

Leaves23

Naraikulam et al. (2010) aimed at assessing theprotective effect of I. tinctoria on Carbon Tetrachloride(CCl4) induced hepatotoxicity. The leaf extract of I.tinctoria increased the antioxidant potential andhepatoprotective effect of the animal. Inhistopathological profile, CCl4 treated rat showshepatocellular necrosis, fatty degeneration andextensive vacuolation. The treatment with I. tinctoriaextract enhances the recovery from CCl4 inducedhepatic damage due to its antioxidant andhepatoprotective property. Jain et al. (2006) showedthat the ethanol extracts of leaves and flavonoid(isolated from leaf extract) from T. purpurea wereevaluated for hepatoprotective activity in rats byinducing hepatotoxicity with carbon tetrachloride.Serum level of transaminases, alkaline phosphate andtotal bilirubin and histopathological changes inliver were used as biochemical markers ofhepatotoxicity. Hence, these indicated that thehepatoprotective activity was more in ethanolicextract of leaves than that in isolated flavonoid.The maximum free radical scavenging activity ofethanolic extract T. purpurea was made the basis ofselection of in vivo study. The extract showedsignificant reduction in the elevated blood urea andserum creatinine. Histopathological changes and in vivoantioxidant activity were determined in line withbiochemical findings. The ethanol extract of T.purpurea leaves possesses marked nephroprotective andcurative activities without any toxicity. The

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proposed mechanisms of activities are antioxidantactivity and inhibition of overproduction of NO andCox-2 expression and it may be attributed to phenolicand flavonoidal compounds like quercetin. (Jain andSinghai, 2009)

SeedPavana et al. (2007) and Pamu Pavana et al. (2008)investigated that the beneficial role of T. purpureaethanolic seed extract on glycoprotein components instreptozotocin induced diabetic andantilipidperoxidative rats. Blood glucose and plasmainsulin were measured and glycoproteincomponents (protein bound hexose, protein boundhexosamine, fucose and sialic acid) in plasma,erythrocyte membrane, liver and kidney wereinvestigated and hyperglycemia associated withaltered hexokinase and glucose 6 phosphataseactivities, elevated lipid peroxidation, disturbedenzymatic and non-enzymatic antioxidants status wereobserved in streptozotocin induced diabetic rats.Oral administration indicates that TpEt has potentrole in modifying altered glycoprotein components instreptozotocin induced diabetic rats. Pavana et al.(2009) evaluated the effects of aqueous seed extractof T. purpurea (TpASet) on blood glucose andantioxidant status in streptozotocin induced diabeticrats. Hyperglycemia associated with an alteredhexokinase and glucose-6-phosphatase activities,elevated lipid peroxidation, disturbed enzymatic and

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non enzymatic antioxidant status 23 were observed.“TpASet” has potent antihyperglycemic and antioxidanteffects and therefore further studies are warrantedto isolate and characterize the bioactive principlesfrom “TpASet”.

StemA new butenylflavanone, a new rotenoid, three activeflavonoids and nine known inactive compounds wereisolated from an ethyl acetate-soluble extract of thestems of T. toxicaria, using a bioassay based on theinduction of quinone reductase (QR) in cultured HepaIcIc 7 mouseheptoma cells to moniter chromatographicfractionation. Selected compounds were tested in amouse mammary organ culture assay to evaluate theinhibition of 7, 12-di-methylben {a} anthracene(DMBA)- induced preneoplastic lesions. (Jang et al.,2003)

RootDeshpande et al. (2002) reported that the aqueousextracts of roots of T. purpurea was studied usingvarious ulcer models in albino rats. Results suggestthat AETP possesses significant antiulcer propertywhich could be either due to cytprotective action ofthe drug or inhibition of acid secretion. Kavitha andManoharan (2006) investigated that the

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chemopreventive potential and antilipidperoxidativeeffects of ethanolic root extract of T. purpurea (TpEt)on 7, 12 dimethylbenz (a)anthracene (DMBA) inducedhamster buccal pouch carcinoma. TpEt has potentchemopreventive efficacy and significantantilipidperoxidative effect, in DMBA induced oralcarcinogenesis. Further studies are needed to isolateand characterize the bioactive principle. Anti-hyperglycemic activity of roots of T. villosa wasevaluated by Balakrishnan et al. (2007). Aqueous extractof T. villosa were significantly superior in reducingblood sugar on long treatment. Anti-implantationactivity was lead out by Rajan et al. (2007) onmethanolic extract of Schleichera oleosa (stem bark) andT. purpurea (root) were screened and both the dosesreduced the number of implantation sitessignificantly and a dose-related response wasobserved. Roots of crude drug of T. purpurea extractedsuccessively using various solvents [Pet-Ether (60-800C), 95% ethanol and aqueous alcohol (60% water +40% ethanol). Single dose administration of all theextracts of T. purpurea did not showed any hypoglycemicor anti-diabetic activity. Repeated doseadministration of alcoholic and hydro-alcoholicextract showed significant hypoglycemic andantidiabetic activity at the end of 7th day. Theseresults suggest that aqueous and hydro-alcoholicextract possess antidiabetic activity. (Joshi et al.,2008) Aqueous extract of the roots of T. purpurea wasevaluated by Swathi et al. (2008) for its

27

antilithiatic activity, in two models ofurolithiasis. Gentamicin (s.c.) and 5% ammoniumoxalate mixed with rat feed was used to inducecalcium oxalate stones; the foreign body implantationmodel which makes use of zinc discs, was used toinduce magnesium ammonium phosphate stones. Theaqueous extract of T. purpurea was found to be effectivein reducing the formation of and dissolving existingcalcium oxalate and magnesium ammonium phosphatestones.

T. purpurea was evaluated for its efficacy in rats byinducing hepatotoxicity with D galactosamine HCl(acute) and carbon tetrachloride (chronic). T. purpurea(aerial parts) powder was administered orally andserum levels of transaminases (SGOT and SGPT)bilirubin and histopathological changes in the liverwere used as the biochemical markers ofhepatotoxicity. The administration of T. purpurea alongwith the hepatotoxins offered a protective action inboth acute (D-galactosamine) and chronic (CCl4)models. (Murthy and Srinivasan, 1993) Gokhale andSaraf (2000) showed the ethanolic extract of driedaerial parts of T. purpurea inhibited passive pawanaphylaxis in rats and also passive peritonealanaphylax is showing antiallergic activity.Significantly reduced an elevated WBC count inresponse to antigen challenge in sensitized mice. Theextract also significantly inhibited eosinophil

28

infiltration without any significant change in themononuclear cell population. The extract failed toalter neutrophil adhesion to nylon fibres. Itproduced a significant inhibitory activity on enzymelipoxygenase. The inhibitory effect of ethanolicextract of T. purpurea on late-phase allergy could beattributed to the inhibition of leukotrienesynthesis. Wound healing potential of T. purpurea L. wasexperimented by Lodhi et al. (2006) by using ethanolicextract of aerial parts in the form of simpleointment using three types of wound models in rats asincision wound, excision wound and dead space wound.Histopathological study showed significant (Plessthan 0.05) increase in fibroblast cells, collagenfibres and blood vessels formation.

Ten medicinal plants including alcohol dried extractof T. uniflora were pharmacologically screened for theircardiac activity on isolated rabbit heart and showedsignificant negative inotropic activity with negativechronotropic effects. In all cases alcohol driedextracts were used and reconstituted in either wateror ethyl acetate for tests. The plant extracts whichproduced cardiac depression (Sajeed et al., 1996). Duttaet al. (1997) worked on “Yakrifit" (a polyherbal productin which T. purpurea is a part) that was administered toall the animals either as liquid 25-30 ml orallytwice daily for 3-6 days or as one bolus twice dailyfor 5 days. All the animals recovered in 3 to 7 days,regained appetite for food and water and their

29

general condition had improved. The therapeuticefficacy of an herbal hepatoprotectivehepatostimulant AV/LTP/15 (a polyherbal productconsisting of T. purpurea) oral in removing anorexiasyndrome in dogs is reported by Singh (1997) andAgarwal (1999). AV/LTP/15 gave well to very goodresponse in 82.1 percent of the dogs. The formulationwas quite palatable and well-tolerated by dogs of allages. The protective effect of HD-03 was observed inall three types of intoxication, which are differentin their primary mechanism of inducinghepatotoxicity, a protective mode of action of HD-03,not specific to the hepatotoxic has been suggested byMitra et al. (1998). Anticholestatic acitivity wasinvestigated by Mitra et al. (1999) that HD- 03, aherbal formulation containing T. purpurea as one of theingredients in thioacetamide (TAA)- inducedexperimental cholestasis in anaesthetized guinea pigswhich significantly prevented thioacetamide inducedchanges in bile flow, bile acids and bile saltsexcretion. HD-03 has been suggested to serve as apotent choleretic and anticholestatic agent.

Therapeutic uses of T. purpurea as mentioned inAyurvedic texts, pharmacological activity andchemical constituents isolated from the plant havebeen reviewed by Patel et al. (1999). Lucas and AnanadaRajasekhar (2000) reviewed experimental studies onanti-hepatotoxic activity of certain medicinal plantsincluding T. purpurea used in Ayurveda. Out of those

30

medicinal plants, Boerhaavia diffusa exhibited cellregeneration effect and has a better anti-hepatotoxiceffect in comparison with other medicinal plants.Saleem et al. (2000) assessed the topical application ofT. purpurea 1h prior to each application of croton oil(phorbol ester) resulted in a significant protectionagainst cutaneous carcinogenesis in a dose-dependentmanner. The animals pre-treated with T. purpurea showeda decrease in both tumor incidence and tumor yield. Asignificant reduction in TPA (12-O-tetradecanoylphorbal-13-acetate) mediated induction in cutaneousornithine decarboxylase (ODC) activity and [H3]thymidine incorporation was also observed. Thetopical application of T. purpurea prior to TPA resultedin the significant recovery of TPA-mediateddepletion in the level of glutathione, glutathione S-transferase, glutathione reductase and catalase. T.purpurea can abrogate the tumor-promoting effect ofcroton oil (phorbol ester) in murine skin. Theprotective role of T. purpurea has been investigated byKumar et al. (2001) the alcohol extract of T. purpureashowed a significant hydroxyl radical scavengingactivity in vitro. Using a Trypan blue exclusion assay,it was found that the extract markedly increased thepercentage viability of the isolated rat kidneycortical cells in gentamicin-induced cell damage. Bythe evaluation of LDH activity and acid phosphatasecontent, it was established that the cell damage wasminimized in the case of cells treated with theextract of T. purpurea. The hydroxyl radical scavenging

31

effect of the extract was enhanced with increases inthe concentration of drug, suggesting the role offree radical scavengers in minimizing kidney celldamage. Taraphdar et al. (2002) examined the activitiesof T. purpurea in radiation induced haemopoietic injuryto total body irradiation. It induced significantincrease in haemoglobin and total RBC count. Afterirradiation there was no fall in RBC count andhaemoglobin and Tephrosia has a selectively effectiveon erythroid compartment. A fraction (F062) obtainedfrom n-butanol extract of T. purpurea showed consistentantileishmanial activity against Leishmania donovaniinfection in hamsters. Activity was further confirmedin a secondary model, i.e., Indian languor monkeys(Presbytis entellus). The fraction F062 from this plantpossesses potential to produce significantantileishmanial activity by oral route withoutproducing any toxic side effects. (Sharma et al., 2003)Soni et al. (2003) stated that, the dried alcoholicextract of T. purpurea was investigated for its freeradical scavenging activity, antilipid peroxidationpotential and hydroxyl radical scavenging activityand significant inhibition of lipid peroxidation.However, it failed to show any significant scavengingeffect on hydroxyl radicles.

The flavonoid fraction of T. purpurea (FFTP) was studiedfor its effect on cellular and humoral functions andon macrophage phagocytosis in mice. Oraladministration of FFTP (10–40 mg/Kg) significantly

32

inhibited sheep red blood cells (SRBC)-induceddelayed-type hypersensitivity reactions. It alsoproduced a significant, dose-related decrease insheep erythrocyte-specific haemagglutination antibodytitre. (Damre, 2003) Cao et al. (2004) reported that thebioassay guided fractionation of methanolic extractsof Mundulea chapelieri resulted in the isolation of twonew flavonoids and eight known flavonoids whichcontaining rotenolone, retoenone and tephrosin. Allthe isolated compounds were tested cytotoxicityagainst the A 2780 human ovarian cancer cell line,rotenolone and rotenone were the most potentcompounds isolated, with IC50 values of 0.5 and 0.7micr/mL, respectively. Anti inflammatory activity ofa flavonol glycoside from T. spinosa was investigatedby Chakradhar et al. (2005) and its activity wasevaluated against carrageenin induced paw edema. Thecompound exhibited significant activity. Prabhu Nair(2006) showed protective effect of Tefroli tonic (apolyherbal mixture containing T. purpurea) againstcadmium induced hepatotoxicity in experimental rats.Subcutaneous injection of cadmium chloride to ratscaused liver damage and was observed by analysis ofserum bilirubin and assay of marker enzymes such astrnsaminase and phosphates of body serum and liver.The administration of Tefroli tonic has maximumprotective effect against cadmium chloride inducedhepatotoxicity in rats. The ethanol extract of T.purpurea Linn. was found to significantly inhibit thecarbon tetrachloride-induced lipid peroxidation in vivo

33

and superoxide generation in vivo. The ethyl acetatefraction of the same extract was studied for freeradical scavenging and antilipid peroxidationactivity. The IC50 values in both of these in vitroassays were found to be significantly reduced forethyl acetate fraction compared with the ethanolicextract of the plant. The observation was furthersupported by comparing the in vivo antioxidant activityfor both the ethanolic extract and its ethyl acetatefraction. The ethanolic extract of T. purpurea exhibitsantioxidant activity in vivo and the ethyl acetatesoluble fraction has improved antioxidant potentialthan the extract. (Soni et al., 2006) Kishore kumar etal. (2007) concluded that significant hepatoprotectiveeffect was obtained against CCl4 induced liverdamage, by oral administration of methanolic extractof T. falciformis as evident from decreased levels ofserum enzymes in the treated groups, compared to thecontrols. Oral administration of T. purpurea andTecomella undulate resulted in a significant reductionin serum aspartate aminotransaminase, alanineaminotransaminase, gamma glutamyl transpeptidase,alkaline phosphatas, total bilirubin and liver MDAlevels and significant improvement in liverglutathione when compared with thioacetamide damagedrats. Histology of the liver sections of the animalstreated with the extracts also showed dose-dependentreduction of necrosis. (Khatri et al., 2008) Khan et al.(2008) investigate a chemopreventive efficacy of T.purpurea against N-diethylnitrosamine-initiated and

34

potassium bromate-mediated oxidative stress andtoxicity in rat kidney. The susceptibility of renalmicrosomal membrane for iron ascorbate-induced lipidperoxidation and xanthine oxidase activities weresignificantlyreduced. The depleted levels of glutathione, theinhibited activities of antioxidant enzymes, phase IImetabolizing enzymes and the enhanced levels of serumcreatinine and blood urea nitrogen were recovered toa significant level. All the antioxidant enzymes wererecovered dependently.

T. purpurea besides a skin antioxidant can be a potentchemopreventive agent. Polyherbal formulationavailable with a wide range of indications likeprotective to liver, appetite and growth promoters,gastrointestinal and hepatic regulator, as treatmentfor hepatic dysfunction, for hepatic regenerations aswell as liver stimulant and tonic. To evaluate thehepatoprotective activity of six commerciallyavailable formulations, namely Liv52, Livergen,Livokin, Octogen, Stimuliv and Tefroli in acutetoxicity in mice model induced by Paracetamol (PCM).(Girish et al.,2009).

PHYSIOLOGY AND BIOCHEMISTRYPlant tissue concentrations of various elements weretypical of species growing on ultramatic substrates.A usual finding was the discovery of T. villosa is one ofthe five hyper-accumulators of copper (more than 1000

35

36 micro g/g dry tissue) Copper hyper accumulation inultramatic substrate is a little known phenomenon andrequires further study (Rajakaruna and Bohm, 2002).Bhaskar Rao et al. (2007) evaluated in vitro antioxidantproperties of aqueous extracts of Alternanthera sessilis andT. purpurea. Both the plants are beneficial as anantioxidant sources and the plants possess significantlevels of enzymatic antioxidants, non-enzymaticantioxidants and also exhibits antioxidant capacity.

LeavesLambert et al. (1993) investigated that the rotenoidcompounds provided from the phenylproponoid pathwayare largely accumulated in the leaves of T. vogelii. Thephotomixotrophic cell line accumulated rotenone anddeguelin in Tephrosia leaves and the heterotrophic cellline produced essentially deguelin and tephrosin. Thein vitro antioxidant activity of ethanolic extract ofleaves of T. purpurea was investigated by DPPH freeradical scavenging and nitric oxide scavengingmethods. The ethanol extract showed good antioxidantactivity in these above methods. This activity may bedue to the presence of flavonoids (Jain et al., 2007).

SeedNagarajan and Merita (2001) stated that the twentyfive seeds of T. falciformis were given various treatmentsto establish the germination requirements. Seedtreatment significantly influenced germination ranged

36

from 1.7-25.0 and lowest value was recorded in seedssoaked in cold water treated for 12 hours and thehighest in mechanically scarified seeds followed byseeds soaked in cold water for 6 hours.Zafar et al. (2002) studied germination of T. purpureaseeds. The mechanical treatment i.e., removal of seedcoat at one end of the seed and its subsequenttreatment with Ethrel (1000ppm) gave 100 percentgermination within 5 days while the seeds withoutthis treatment took 12-20 days to germinate and therate of germination observed was 10 percent.

ETHNOMEDICINAL AND VETERINARY USE OF Tephrosia spp.

The plant genus Tephrosia is a legume with about 300species found in the tropical and subtropical regionsof the world, some of which have been used for manybeneficial purposes (Barnes 1967, Gaskins 1972).Tephrosia bracteolata is widespread in tropical Africa andprovides grazing for horses and other livestock(Dalziel 1937). It is cultivated on the Ivory Coastas a fish poison. In Tanzania the pounded root istaken twice daily as a therapeutic agent by pregnantSukuma women who are infected with syphilis (Burkill1995). The leaf and root are used for charms inNorthern Nigeria against injury by hunters andwarriors (Dalziel 1937). Tephrosia candida has been usedas fish poison in Bangladesh, Burma and Sierra Leone(Chadha 1976).

37

Tephrosia densiflora Hook. F. is very closely related toT. vogelii, and some authorities have taken it to be avariety. It is grown specifically for use as fishpoison in West Africa. The leaves and sometimes thefruit pods are crushed and thrown into streams forfishing (Kerharo 1950). The plant has abortifacientproperties and it is used against parasitic skindiseases (Oliver 1960). The leaf of Tephrosia elegans is occasionally cultivatedfor use as a fish poison. Where it is not cultivatedthe leaf is collected from wild plants, but is not afish-poison of importance as foliage is generallysparse (Kerharo 1950). In South Africa an arrowpoison is obtained from the root (Watt 1962). Tephrosiaflexuosa provides some grazing to cattle and otherlivestock. It is a fish poison, but not aparticularly good one (Dalziel 1937). Tephrosia linearis(Willd.) is grazed by all livestock in Senegal,pulped up leaves are used by the Fulanis to add tomilk, millet or guinea corn pap as a seasoning. InNorth western Nigeria the plant is given as apostnatal medicine, although the purpose is notdisclosed (Dalziel 1937). Tephrosia lupinifolia providessome grazing in Senegal for livestock. InBarotseland, West Zambia, a decoction obtained fromthe root is drunk as an abortifacient, acting to killthe fetus which is expelled in about 10 hours. Theroot is also used by women in suicide, the root beingpounded into a bolus which is inserted into the

38

vagina. This results into considerable local andabdominal swelling in about one hour and deathensues in 12-24 hours (Watt 1962).

Tephrosia nana is grazed by livestock in EasternCameroon. It serves as a fish poison on the IvoryCoast and Democratic Republic of Congo and is deemedto be as good as T. vogelii, with which it is sometimesmixed (Kerharo 1950). In the Congo (Brazzaville) theseed is used as bait in snares to catch rodentscalled nsibilikis, and in Nigeria Igbo people grindup the parched leaf for treating the sores of yaws(Burkill 1995). Tephrosia noctiflora is cultivated in Eastand West Africa as a source of fish poison, but thereis no report on its toxicity (Gillett 1971). Tephrosianubica (Boiss) provides grazing for livestock, andbirds take the flowers and seeds in Turkana Kenya.The whole plant is infused to produce a drink takenby women after childbirth (Burkill 1995). Tephrosiapedicellata Bak is grown on the Ivory Coast to producefish poison. In Sudan the Dinkas chew the root forthroat and lung complaints (Burkill 1985). Tephrosiaplatycarpa furnishes grazing for cattle, sheep andhorses. The seed is oil bearing, yielding a cookingoil used in the Kordofan of Sudan (Burkill 1995). The dried herb of Tephrosia purpurea (Linn.) is used inmedicine in India for its tonic, laxative, diureticand deobostruant properties, for bronchitis andbilious febrile attacks, the treatment of boils,pimples and bleeding piles, and for cough and kidney

39

disorders. The seed is said to be edible. The seedpod is used by extraction and administered for painand inflammation, to stop vomiting and as avermifuge. The seed oil is applied to scabies, itch,eczema and other eruptions of the skin (Chadha 1976,Rastogi 1990). In Tanzania the root slightly burnt ischewed for stomach pains. The root and leaf arerecognised as purgative and emetic (Burkill 1995). Itis used as a fish poison in Senegal and Gambia butits insecticidal action is low (Watt 1962). The planthas many medicinal uses in Senegal: for treatingdiarrhoea and whooping cough in children; vaginaldouches; diarrhoea in adults, spasmodic coughing,fevers, sterility, rickets and syphilis (Kerharo1964). In Nigeria the plant is used as a diuretic,blood purifier, a gargle and internally for coughs,colds etc. (Ainslie 1937).

Tephrosia subtriflora is often confused with T. uniflora andthe latter's usages may apply (Gillett 1958). Tephrosiauniflora provides grazing for cattle and elephants;stems are used as tooth brushes in Ethiopia. Stemsand roots of the plant have been shown to be toxic toBulinus globulus, a fresh water snail vector ofschistosomosis (Adewunmi 1980).

Biological activities for extracts of Tephrosia vogelii

Extracts of the leaf, bark, root, seed and/or flower

40

of Tephrosia vogelii possess numerous biological activities when tested in the laboratory.

* Molluscicidal activity

The water extract of the dried flowers, stem andleaves showed molluscicidal activity against Physopsisglobosa (Cowper 1948). The water extract of the driedleaf possessed molluscicidal activity against Bulinusglobosus (Chiotha 1986). The flower and flower bud ofTephrosia vogelii have been reported to be toxic toBulinus (Physopsis) globosus (Adewunmi 1980). Thepetroleum ether extract of the plant demonstratedmolluscicidal activity against Biomphalaria glabrata(Marston 1984). Ethanol (80%) extract of the driedleaf showed weak molluscicidal activity againstBiomphalaria pfeifferi and Bulinus truncatus (Abdel-Aziz 1990).Water extract of oven dried stem, leaf and seedshowed weak molluscicidal activity against Biomphalaria pfeifferi (Kloos 1987).

* Antimicrobial activity

Ethanol (80%) extract of the dried fruit showed weakantibacterial activity against Staphylococcus aureuson agar plate, weak antiviral activity againstmeasles virus on cell culture, strong antifungal

41

activity against Microsporum canis; and weak antifungalactivity against Trichophyton mentagrophytes on agar plate(Vlietinck 1995).

* Piscicidal activity

The hot water extract of the leaf showed piscicidal activity against goldfish Carassius auratus (Gaudin 1938).

* Insecticidal activity

Acetone extract of the leaf showed feeding deterrent activity against the insect Pieris rapae (Shin 1989), and the acetone extract of the seed showed larvicidalactivity against Aedes aegypti (Manson 1939).

* Anthelminthic activity

The methanolic leaf extract showed anthelminthic activity against Nippostrongylus braziliensis (Edeki 1997).

42

AIMS AND OBJECTIVES OF THE STUDY

The essence of the study is to focus on the following

aspect of the genus:

To study the morphology of selected species of Tephrosia and to deduce similarities or differences existing.

To illustrate the stages of mitotic cell divisionbetween the selected species.

To deduce the chromosome count in the genus Tephrosia.

Identification and separation of phytochemical compounds from selected species of the genus Tephrosia.

43

CHAPTER TWO

MATERIALS AND METHODS

Collection of Plant Materials

Different species of Tephrosia plant were carefully

observed at the Herbarium in Lagos state University

(LASU), for proper identification of the biotype in

the wild. Five selected specie of the Nigerian

representatives of the genus Tephrosia were collected

from different locations in Lagos and Ogun state of

Nigeria, and further preserved with various reagents

and herbarium.

Location of Collected Species are:

44

Tephrosia candida - Akason, Ogun state

Tephrosia purpurea - Erekiti, Ogun state

Tephrosia mossiensis - Apa kingdom, Owode Road. Ogun

state

Tephrosia linearis - Iragon, Lagos state.

Tephrosia bracteolata - Iragon, Lagos state.

Tephrosia lupinifolia - Erekiti, Ogun state

Since the selected species from the genus Tephrosia

is subjected to several treatments in order to

qualify and quantify their characters, the following

materials and methods are required in each of the

treatment.

Morphological studies

The morphology of a plant is the study of its

external features. The gross morphological characters

45

of each of the species collected was observed and

recorded.

These includes

• Phyllotaxy

• Color of flower

• Habit

LEAF MORPHOLOGICAL STUDIES

The materials used in this study are: Specimen

samples, Polythene bag, Camera, Meter rule, measuring

tape, White cardboard and White gum.

The images of the plant within the selected species

of Tephrosia were made taken to indicate their

morphological characters. However the morphology of

the leaf is of more priority and could be divided

into two types;

• The quantitative characters of the leaves

• The qualitative characters of the leaves

46

The quantitative characters examined includes the

following

a) Leaf length

b) Leaf width at the widest point

c) Leaf length from the widest point to the apex

d) Petiole length

e) Total leaf length

The qualitative characters examined includes the

following

a) Leaf shape

b) Leaf type

c) Leaf apex

d) Leaf blade

e) Leaf margin

f) Leaf texture

47

Plate 1: Tephrosia linearis

Plate 2: Tephrosia candida

Photographs of the collected species were taken forreference purposes.

s

48

Plate 3: Tephrosia purpurea

Plate 4: Tephrosia mossiensis

49

CYTOLOGICAL STUDIES

Materials

Solutions preparations

Preparation of Carnoy’s solution:

1:3 acetic acid and alcohol

1molarmass glacial acetic acid + 3

molar mass alcohol

And kept at a temperature of 40C.

Preparation of 45% acetic acid:

45ml of acetic acid + 55ml of

distilled water.

Preparation of acetocarmine:

Boil 2–4 gram of carmine dye in 100

cubic centimeters (cc) of 45% of

acetic acid for several minutes and

then cooled and filter. The

50

resulting solution is diluted with

45% acetic acid to obtain the

required strength.

Preparation of Normal HCL:

1molar mass HCL weighted on a

weighing balance (36.5g) +1 liter of

distilled water.

Collection of materials:

Two species were collected at different locations in

Nigeria between the hours of 11:00am – 1:00pm and

were carefully observed at the Herbarium in

University of Lagos for proper identification.

T. mossiensis - Apa kingdom, Owode road, Ogun state and T.

Purpurea - Ereketi, Ogun state.

Methodology

Culturing

Usually actively growing root tips are used for

cytological studies where cells are in continuous

51

process of division, which may be vegetative

(mitosis) or formation of gametes or spores

(Meiosis). In the genus Tephrosia, division occur in

meristem regions such as lateral roots, shoots, main

roots, leaf petiole expanding young leaf e.t.c.

After collection, viable seeds were collected from

each species pods daily and where sown in a petri

dish, placed with filter paper and soaked in

distilled water to develop the root tip.

Fixative

Fixative helps to stabilize cellular structure and

helped to preserve until time of usage, immediately

the root grow to 5 – 10cm tall, about 5mm of the root

tip was cut and fixed in a solution called Carnoy’s

solution. The time of harvest is recorded and is done

between the hours of 10:00am – 12:30pm and place in a

test tube.

Staining

Staining help to create optimal contrast between

cellular structure. The root tip was kept in a wash 52

glass containing ratio 9 : 1 drop of Acetocarmin

stain and normal HCL. The root tip in the mixture was

heated gently over a burner until steam is produced

from the stain. The preparation is covered for about

5minutes so that the stain penetrates into root tip.

Squashing

1mm of the stained root tip was cut and placed on a

clean slide containing drops of 45% of acetic acid

and covered with a cover slip. The slide was then

squashed gently.

Documentation of slide/cytological material

The prepared slides were then labeled

The preparation was observed via microscope or slide

projectors

Squashing of the root tips is prepared as follows:

• The initiation of the growth of the roots of the

plant by culturing the seeds

53

• When the roots grew up to 3-4cm long, then

harvested about 5mm from the root tips and placed

in wash glass and rinsed in water.

• Covered the root tips with ratio 9:1 drops of

Aceto-carmine (stain) and normal HCl

• Root tips were then heated over a gentle flame

until the steam is produced in the stir.

• The preparation was covered for 5minutes, so that

the steam penetrates into the root tip

• The root tip was then placed on a clean slide and

covered with 45% Acetic acid

• 1mm of the root tip was cut and the rest was

discarded.

• Covered the root tip with a clean cover slip and

made a squash of the root tip

• Observation was done under the microscope to

identify the different process of cell division.

54

Plate 7 : Pods of T.mossiensis containing seeds

55

Plate 8 : Pods of T.purpurea containing seeds

Plate 10: Growing root tips after 2daysPlate 9: Growing root tips after 2days

Germination of Tephrosia seeds in Petri dishes

56

Plate 11: Day 3, root tips reaching 1mmPlate 12: Day 3, root tips reaching 1mm

57

Plate 13: Root tips reaching

PHYTOCHEMISTRY

Collection of materials

Four species were collected for phytochemical identification of compounds. Parts such as the seeds,leaves and roots were used for the extraction.

Methodology

Steps involved: i) Preparation of the extracts

ii) Qualitative chemical analysis

iii) Chromatographic studies

iv) Isolation of phytochemical compounds

58

Plate 14: Root tips suitable for harvest

v) Characterization of isolated compounds.

Test for Alkaloids

1.0ml amount of the extract was dissolved in 2ml of diluted hydrochloric acid solution (HCL) and a few drops of solution of iodine in potassium iodide were added to the solution.

Test for Flavonoids

1.0g of each of the dried samples was boiled with 5.0ml of distilled water for ten minutes and filteredwhile hot. The filterates were allowed to cool. One drop of 10% Ferric chloride solution was added to thefilterate and shaken thoroughly well and observed; also diluted sodium hydroxide (1.0ml) NaOH was added to each of the filterates and observed the colour produced.

CHAPTER THREE

59

RESULTS

QUANTITATIVE CHARACTERS

TABLE 1: MEAN AND STANDARD DEVIATION OF THE LEAVES OFFOUR SPECIES

SPECIENAME

LEAFLENGTH

LEAFWIDTHAT THEWIDESTPOINT

LEAFLENGTHFROMTHE

WIDESTPOINTTO THEAPEX

PETIOLELENGTH

TOTALLEAFLENGTH

Tephrosia purpurea

2.78 ± 0.19

0.98 ± 0.52

0.82 ± 0.51

0.24 ± 0.11

3.02 ± 0.24

Tephrosia mossiensis

1.97 ± 0.27

0.61 ± 0.16

0.52 ± 0.18

0.28 ± 0.06

2.25 ± 0.25

Tephrosia linearis

5.66 ± 1.90

0.38 ± 0.04

2.33 ± 0.93

0.35 ± 0.06

6.01 ± 1.90

Tephrosia candida

5.34 ± 0.49

0.43 ± 0.11

2.47 ± 0.21

0.23 ± 0.14

5.57 ± 0.44

60

T. purpurea T. mossiensis

T. linearis T.candida0

1

2

3

4

5

6

7

Leaf lengthLeaf wdth at the widest pointLeaf length from the widest pointPetioleTotal leaf length

61

Figure 1: A chart showing the mean and standard deviation of the leaves of four species.

QUALITATIVE CHARACTERS

TABLE 2: COMPARISON OF QUALITATIVE CHARACTERS IN SIX

SPECIES OF Tephrosia

SPECIE

NAME

SHAPE TYPE APEX MARGIN PETIOLE

Tephrosia

candida

Lanceola

te

Compound

Pinnate

Mucronat

e

Entire Petiola

teTephrosia

linearis

Linear Trifolia

te

Acuminat

e

Entire Petiola

teTephrosia

mossiensis

Elliptic Compound

Pinnate

Rounded Entire Petiola

te

Tephrosia

purpurea

Obovate Compound

Pinnate

Retuse Entire Petiola

te

Tephrosia

bracteolata

Oblong Compound

pinnate

Retuse Entire Petiola

teTephrosia

lupinifolia

Oblong Trifolia

te

Obtuse Entire Petiola

te

62

TABLE 3: TEXTURE OF LEAF SURFACES IN SIX SPECIES OF

Tephrosia

SPECIE NAME ADAXIAL PART ABAXIAL PARTTephrosia candida Hairy HairyTephrosia linearis Smooth SmoothTephrosia mossiensis Hairy HairyTephrosia purpurea Hairy HairyTephrosia bracteolata Smooth HairyTephrosia lupinifolia Hairy Hairy

63

Plate 15: Photomicrograph showing chromosome in T. mossiensis

Plate 14: Photomicrograph showing chromosome in T. purpurea

PLATES SHOWING STAGES OF MITOTIC CELL DIVISION

64

Plate 13: Photomicrograph showing chromosome

65

Plate 16: Photomicrograph showing chromosome in

TABLE 4: PHYTOCHEMICAL SCREENING OF COMPOUNDS IN Tephrosia

Test Observation InferenceAlkaloids

a) T.mossiensis

b) T. linearis

c) T. purpurea

d)T. candida

A brown precipitate was obtained.A brown precipitate was obtained.A brown precipitate was obtained.A brown precipitate was obtained.

Alkaloids present.

Alkaloids present.

Alkaloids present.

Alkaloids present.

Flavonoids

a) T.mossiensis

b) T. linearis

c) T. purpurea

d) T. candida

Greenish brown colour obtained.Greenish brown colour obtained.Greenish brown colour obtained.Greenish brown colour obtained.

Flavonoids present.

Flavonoids present.

Flavonoids present.

Flavonoids present.

1ml of filterates + 1ml of dilutedsodium

66

hydroxide solution (NaOH)

a) T.mossiensis

b) T. linearis

c) T. purpurea

d) T. candida

Greenish brown colour obtained.Greenish brown colour obtained.Greenish brown colour obtained.Greenish brown colour obtained.

Flavonoids confirmed.

Flavonoids confirmed.

Flavonoids confirmed.

Flavonoids confirmed.

CHAPTER FOUR

DISCUSSION

MORPHOLOGY

On investigation of the gross morphology of the four

selected species, considerable amount of variation

exist amongst the species. Deductions from the tables

as seen in the results shows the comparison of

morphological characters. The mean and standard

deviation were therefore computed in table1 to show

the quantitative characters of Tephrosia species.

From the table, it shows T.linearis has the highest leaf

67

length of 5.66cm and T.mossiensis has the lowest leaf

length of 1.97cm. It was also observed that the

T.purpurea has the highest leaf width of 0.98cm and

T.linearis has the lowest leaf width of 0.38cm. Regarding

the petiole length, ther is little variation in T.

purpurea, T. mossiensis and T. candida except for T. linearis

which has the highest petiole length of 0.35cm.

Certain degree of dissimilarity is also observe in

their leaf shapes such that T. candida is lanceolate,

T.linearis is linear T. mossiensis is elliptic and T. purpurea

is obovate. Their leaf type is majorly compound

pinnate except for T. linearis which is trifoliate.

The leaf texture among the species is mostly hairy

except for the adaxial surface of T. linearis which is

smooth. They have their margin being entire and are

all petiolated leaves. The seed show high

morphological similarities except for T. mossiensis seed

which are smaller.

There are variations and yet similarities between the

morphological attribute of the species due to the

fact that the plant were collected from different

ecological locations this characters are considered68

as unsuitable because of their likelihood to vary

with changes in ecological conditions implying that

there cannot be absolute determinant of the

similarities or dissimilarities of the species in the

genus. Stance, (1992) however pointed that there have

been relatively few studies of the effect of the

environment on the leaf morphology.

CYTOLOGY

Mitotic study

Two species of Tephrosia namely T. purpurea, and T.

mossiensis were analysed for mitotic cell division.

Eleven pairs of chromosomes where recorded in each

species. Photomicrographs of somatic metaphase plate

are presented in plates 13, 14, 15 and16

respectively. In this study more than one specimen of

every specie were analyzed to reveal the stages of

mitotic cell division and to show constancy of the

chromosome number in these species.

The stages of cell division show their distinct

characteristics.

69

The chromosomes were mainly metacentric and did

not vary greatly so that the level of asymmetry

was low.

We can deduce that no there is no variation in the

chromosome number recorded in any specie. They

possess a constant chromosome number 2n= 22; n=11

(Achison’s, 1951; Gupta et al., 1994). A haploid count

of n=11 was observed at metaphase 1. This confirms

the previous reports of 2n=22 by Gupta et al. (1994) and

n=11 by Jahan et al. (1994)

Achison’s, (1951); Gupta et al., (1994) reported that

Tephrosia is the only genus where 2n= 22 is almost

the exclusive chromosome number and therefore has a

unique position in this respect. Evoloutionary

significance of this situation is not clearly known

so far except that the member of this genus may be

related to members of tribe Dalbergieae more closely

than to members of Galegeae.

PHYTOCHEMISTRY

The chemical investigation were carried out to check

the presence of various phytoconstituents. From the70

table of results, a brown precipitate was obtained

from the pet ether and ethanol extract of the seeds,

roots and leaves of the plant inferring the presence

of alkaloids. The greenish brown precipitate obtained

from the ethanol and aqueous extracts indicates the

presence of flavonoids.

The isolation of the above bioactive chemical

constituents renders the medicinal significance of

the species which are presently been the subject of

human curiosity and need.

71

CONCLUSION

This project work show there is considerable

amount of variation among the six Tephrosia species

under study. They vary in shape, type, size, texture,

just to mention a few. We could also confer that

these species retain some ancestral characters. From

this study, Generally the chromosome numbers have

been found to be constant in this genus Tephrosieae

(2n=22). In conjunction with data from other sources

as in phytochemical investigation revealing

flavonoids as the active ingredient; these findings

could provide independent evidence of tribal as well

as generic relationships. The genus Tephrosia has

shown to be diverse prior to information regarding

morphology, cytology and phytochemistry. However

further work needs to be done in order to reveal

adequate information on the systematic of this genus.

72

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