GENETIC VARIABILITY AND PHYTOCHEMICAL STUDIES OF INDIGOFERA ASPALATHOIDES VAHL. EX.DC& I.TINCTORIA L., TWO ENDEMIC MEDICINAL PLANTS OF SOUTH TAMILNADU

Project Report Submitted to

Taminadu State Council for Higher Education(TANSCHE)

By

Dr. S. K. Sundar Assistant Professor, Research Department of Microbiology, M.R. Government Arts College, Mannargudi- 614 001.

INTRODUCTION

India is one of the mega biodiversity country in the world hosting 75,000 species of fauna and 45,000 species of flora. Of the 1.5 million species of identified organisms 0.2 million (13%) are known from India (Gadgil, 1996).Amongst the flora, the medicinal plants form an important component, as it is a very valuable heritage for humanity in the sense that its administration in various forms, relieves man from the burden of diseases to the improvement of the quality of life. Humans and other animals are almost totally depend on plants directly or indirectly, as a source of energy and the medicinal plants are potential renewable natural sources.

World wide, tens of thousands of species of higher plants and several hundred lower plants are currently used by human beings for a wide diversity of purposes such as food, fuel, fibre, oil herbs spices industrial crops and as forage and fodder for domesticated animals

(Hey wood et al., 1992).The wide spread use of herbal remedies of health care preparations, as those described in ancient texts such as the Vedas and the Bible, are obtained from commonly used traditional herbs and medicinal plants, India has a long historical use of large number of medicinal and aromatic plants. It is reported that almost every plant family in the world is represented in India‟s rich flora (Harsha et al., 2002).

A number of native plants have been used in various preparations of the traditional system of medicines such as Ayurveda and Siddha. India being an „Emporium of medicinal plants‟ the Ayurvedic and Siddha systems flourished well in the country The drugs used in

Ayurvedic and Siddha system are essentially of plant orgin. These systems incorporate nearly 700 plant drugs in several medicinal preparations for the management of human health care (Purohit et al., 2003).

The medicinal properties of plant species made an outstanding contribution in the origin and evolution of many traditional herbal therapies. These traditional knowledge systems have started to disappear with passage of time, due to scarcity of written documents and relatively low income in the traditions.Recently, considerable attention has been paid to utilize ecofriendly and biofriendly plant based products for the prevention and cure of different human diseases. It is documented that 80% of the World Population has faith in traditional medicine, particularly plant drugs for their primary healthy care (Singh, 2002)

Herbal medicine is a major component in all traditional medicine systems and common element in Ayurvedic, Chinese medicine and Native American Medicine (Lietava,

1992) It has been estimated that 25% of the prescribed medicines today are substance derived from plants (Rawat and Uniyal, 2003).

The practice of traditional medicine is wide spread in China, India, Japan, Pakistan,

Sri Lanka and Thailand. In China about 40% of the total medicinal consumption is attributed to traditionaltribalmedicines (Bensky et al., 2004)In Thailand, herbal medicines make use of legumes encountered in the Caesalpiniaceae, Fabaceae and Mimosaceae. In the mid – 90‟s it is estimated that receipts of more than US$ 2.5 billion have resulted from the sales of herbal medicines. And in Japan herbal medicinal prepartions are more in demand than main stream pharmaceutical products (Irwin et al., 2002).

In India, it is reported that traditional healers use 2500 plant species and 100 species of plants serve as regular source of medicine (Maruthi et al., 2000).Gorman (1992) drew attention to the power of Chinese folk medicinal portions in treating maladies from eczema and malaria to respiratory disorders. Examples Artemisia annua used as antimalarial drug.

Bupleurumchinese used as a popular remedy for hepatitis. More recently, the biochemistry of

Cucumber is being studied in USA to decipher the identity of compound Q an extract used in

China and credited with remedial and relief properties in AIDS sufferers.

In the last 40 years, many potent drugs have been derived from flowering plants, approximately half (125 000) of the world‟s flowering plant species are found in the tropical forests. Tropical rain forests continue to support a vast reservoir of potential drug species.

The potential for finding more compounds from these plants are enormous and as on date only about 1% of tropical species have been studied for their pharmaceutical potential. To date about 50 drugs have come from tropical plants. The probable undiscovered pharmaceuticals for modern medicine have often been cited as one of the most important reasons for protecting tropical forests. Therefore the high annual extinction rate is a matter for concern (Cragg and Newman, 2005).

Many of these indigenous medicinal plants are used as spices and food plants. They are also sometimes added to foods meant for pregnant and nursing mothers for medicinal purposes (Okwu, 1999, 2001). They have also provided an invaluable resource that has been used to find new drug molecules (Gurib-Fakim, 2006).

Medicinal plants are an integral component of ethno veterinary medicine and are widely used as a primary source of prevention and control of livestock diseases. In Mexico intestinal disorders of cows are treated with herbal extract of Polakowskiatacacco. Dietary supplement such as vitamin A in poultry feeds in Uganda are supplied through enrichments of Amaranthus species (Anjara et. al., 1996).

Around 70% of the India‟s medicinal plants are found in tropical area mostly in the various forest types spread across the Western and Eastern Ghats. Although less than 30% of medicinal plants are found in the temperate and alpine areas, India officially recognizes over

3000 plants for their medicinal values (Rajashekaran, 2002).

Many plants synthesize substances that are useful to the maintenance of health in humans and other animals. These include aromatic substances most of which are phenols or their oxygen-substituted derivatives such as tannins. Many are secondary metabolites of which atleast 12,000 have been isolated. In many cases, these substances, particularly alkaloid serve as plant defense mechanism against micro organisms, insects and herbivores.

(Lai, 2004 ;Tapsell, 2006).Plants up regulate and down regulate their biochemical paths in to the local mix of herbivores, pollinators and microorganisms. Plants synthesize a bewildering variety of photochemical but most are derivatives of a few biochemical motifs.

Phytonutrients/phytochemicals are said to be incredibly beneficial to human health; however, what separates them from other nutrients is that they are not absolutely necessary for the normal functioning of the body i.e. our bodies will not shut down without them.

Rather they are the building blocks from which our bodies use to promote good health e.g. by improving the function of the immune system, reducing inflammation etc. (Ross, 2006).

Alkaloids contain a ring with nitrogen and many of these compounds have dramatic effects on the central nervous system. Phenolics contain phenol ring, whereas terpenoids are built up from terpene building blocks. Each terpene consists of two paired isoprenes. The names monoterpenes, sesquiterpenes, diterpenes and triterpenes are based on the number of isoprene units (Hayashi et al., 1993).Glycosides consists of a glucose moiety attached to an aglycone. The aglycone is a molecule that is bioactive in its free form but inert until the glycoside bond is broken by water or by enzymes. This mechanism allows the plants to defer the availability of the molecule to an appropriate time (Seigler, 1998). Flavonoids present in plants are responsible for the colour of flowers, fruits and sometimes leaves. Some may contribute to the colour by acting as a co-pigment. Biosynthetically they are derived from a combination of the Shikimic acid and the acetate pathways. Small differences in basic substitution patterns give rise to several sub-groups. In the plant, flavonoids can either occur as aglycones or as O- or C-glycosides (Gurib-Fakim, 2006). Some of the plant moieties used as drug in all forms of medicine and examples are inulin from the roots of dahlias, quinine from the Cinchona, morphine from the Poppy. The active ingredient in Willow bark, once prescribed Hippocrates is Salicin or Salicylic acid.

The discovery of Salicylic acid leads to the development of aspirin also known as acetylsalicylic acid. Aspirin was originally a brand name and is still a protected trademark in some countries. This medication was patented by Bayer AG (Hill, 1992).

Some animals tend to forage plants rich in secondary metabolite such as tannins and alkaloids, since these phytochemicals often have antiviral, antibacterial, antifungal and antihelminthic properties (Huffman, 2003).Molecular markers generally refer to biochemical constituents, including primary and secondary metabolites and other macromolecules such as nucleic acids. Secondary metabolites as markers have been extensively used in quality control and standardization of botanical drugs. (Liu et al., 1994).

Using chemical finger printing plants can be demarcated on the basis of their species, strain and geographical origin. Chromatographic techniques like Thin Layer

Chromatography, High Performance Liquid Chromatography can be used to obtain chemical constituents from plants and this technique is called chemoprofiling (Vieira et al., 2001).

A recent offshoot of this method is the use of biomarkers. The European Scientific Co- operative on Phytotherapy (ESCOP) has clearly specified the requirement of the standardization of phyto-pharmaceuticals on the basis of biomarkers that are unique to that species (Fico et al., 2003).Not all plants contain a unique chemical compound and even if there is a unique marker, it may not be biologically active. There is a significant overlap of many molecules, especially phenolics and sterols. The main cause of the failure of chemoprofiling is the presence of inprocess artifacts, which tend to confound the findings of chemical finger printing. Additional techniques are required to profile natural drugs particularly when profiling the genotypic differences (Vieira et al., 2001). Additional motivation for using DNA finger printing on commercial herbal drug is the availability of intact genomic DNA from plant samples after they are processed. DNA based molecular markers have utility in the fields like taxonomy, physiology, embryology, genetics etc. DNA based techniques have been widely used for authentication of plant species of medicinal importance (Mihalov et al., 2000).Study of inter and intraspecific variation at the molecular level provide a efficient tool for taxonomic and evolutionary studies and for devising strategies to protect genetic diversity of species. Genetic variability also can be exploited to select useful genotypes that could be utilized as cultivars to avoid batch – to – batch variation in extraction of standard drug. Despite the recent interest in molecular modelling, combinatorial chemistry, and other synthetic chemistry techniques by pharmaceutical companies and funding organizations, the natural products, and particularly that of medicinal plants, remain an important source of new drugs, drug leads, and chemical entities (Newman et al., 2000; Newman et al., 2003; Butler, 2004). In both 2001 and 2002, approximately one quarter of the best-selling drugs worldwide were natural products or were derived from natural products (Butler, 2004).

Despite the recent interest in molecular modelling, combinatorial chemistry, and other synthetic chemistry techniques by pharmaceutical companies and funding organizations, the natural products, and particularly that of medicinal plants, remain an important source of new drugs, drug leads, and chemical entities (Newman et al., 2000; Newman et al., 2003; Butler,

2004). In both 2001 and 2002, approximately one quarter of the best-selling drugs worldwide were natural products or were derived from natural products (Butler, 2004).

During the early period of research, classical strategies including comparative anatomy, physiology and embryology were employed in genetic analysis to determine inter- and intra-species variability. In the past decade, however, molecular markers have very rapidly complemented the classical strategies (Weising et al., 1995).

Molecular markers include biochemical constituents (e.g. secondary metabolites in plants) and macromolecules, viz.,., proteins and deoxyribonucleic acids (DNA). Amongst the molecular markers used, DNA markers are more suitable and ubiquitous to most of the living organisms (Swati et al., 1999).

Genetic diversity of medicinal plants have been studied by various means such as biometrical analysis, biochemical analysis (isozyme) and molecular markers (RAPD). The recently developed Random Amplified polymorphic DNA (Williams et al., 1990) based on polymerise chain reaction has been readily adopted in gene mapping and finger printing studies. RAPD technology was used for the identification of phylogenetic relationship among the varieties (Yu and Nguyen, 1994 and Henry, 2001).Electrophoretic analysis of plant protein or isozymes of several species have been investigated by many authors. Isozyme analyses are useful in determination of the genetic structure of a population and in comparison among the individuals of the population. This technique may also be used to examine the genetic structure of rare and endemic plant species (Crawford et al., 2001).

With the advent of molecular markers, a new generation of markers has been introduced over the last two decades, which has revolutionized the entire scenario of biological sciences. DNA-based molecular markers have acted as versatile tools and have found their own position in various fields like taxonomy, physiology, embryology, genetic engineering, etc. They are no longer looked upon as simple DNA fingerprinting markers in variability studies or as mere forensic tools. Ever since their development, they are constantly being modified to enhance their utility and to bring about automation in the process of genome analysis. The discovery of PCR (polymerase chain reaction) was a landmark in this effort and proved to be a unique process that brought about a new class of DNA profiling markers. These DNA markers offer several advantages over traditional phenotypic markers, as they provide data that can be analyzed objectively (Swati et al., 1999).

A number of PCR-based techniques can be used to detect polymorphisms in plants.

For their wide-scale usage in germplasm characterization and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn needs to be standardised to yield reproducible results (Jones et al., 1997).

Resistance to Citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. BLAST searches of the GenBank database revealed high sequence similarities between two markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata (Fang et al., 1998).

Considering the above the facts the present study will be carried out to collect two medicinally important Indigofera species from various places of Southern districts of

TamilNadu and to analyse the phytochemical constituents present in the two plants and to study their genetic variability pattern.

\ MATERIALS AND METHODS

Medicinally important I.aspalatoides plants collected from five different sites of

Kanyakumari District ,South TamilNadu were authenticated and subjected to qualitative and quantitative analysis of various biochemical constituents and genetic analysis using RAPD technique. The same study was conducted on natural dye yielding and medicinally important

I.tinctooria plants collected from four sites and in the fifth site Chitharal it was not present.

Plants in the study area was collected according to the Field and Herbarium techniques (Lawrence, 1951; Davis and Heywood, 1965 and Jain and Rao, 1977). Polythene bags were used to keep the collected materials in fresh condition. The morphological characters of the plants were recorded at the time of collection in the field note book using hand lens and the collected plants were brought to the laboratory for authentication. The plants were identified with the help of flora of Tamil Nadu and Carnatic by Mathew (1982) and flora of Tamil Nadu by Nair and Henry (1983).

The identified plants were verified and confirmed by the herbarium and voucher specimens maintained in the Department of Botany, M.R.Government Arts

College.Mannargudi. The photographs of selected plants were also taken during the field trips.

Description of the study site

The five study sites viz., Surulacode, Boothapandi, Aralvaimozhi, Thirunanthikarai,

Chitharal selected for the collection of medicinally important Indigofera sp. were located in

Kanyakumari District, Tamil Nadu. Kanyakumari District is located in the southern most tip of peninsular India where Indian Ocean, the Arabian Sea and the Bay of Bengal embrace one another. The district lies between 77o 15' and 77o 36' of the Eastern Longitudes and 8o 03‟ and 8o 35‟ of the Northern Latitudes. The study site- 1, Surulacode is a picturesque township located about 18 km north- west of Nagercoil in Kanyakumari District. With its small hills and paddy fields, the place is noted for its calm and serene atmosphere. The river Pazhayar originates near Surulacode and finally outfalls into Arabian Sea near Manakudy after traversing a distance of 35 Kms.

The study site- 3, Aralvaimozhi (or Aramboly) is a panchayat town in Kanniyakumari

District. It is a small village situated in southern India. Aralvaimozhi is famous for a Catholic church located in the hills of Kattadi malai where Saint Devasahayam Pillai was martyred.

The name "Aral" was derived as it was having a fort, and the remains of the fort can be seen near railway station.

The study site- 4, Thirunanthikarai is a village situated in Thirparappu panchayath, in the Kanyakumari district. Thirunanthikarai Nanthishwaran Temple and Thirunanthikarai

Cave Temple are the important places of worship.

The study site- 5, Chitharal is a small village situated at a distance of 7 km from

Marthandam and 55 km from Kanyakumari. Chitharal is historically known as

Thirucharanathupalli – the abode of Jain monks belonging to Digambara sect. It is famous for the hillock which has a cave containing rock-cut sculptures of Thirthankaras and attendant deities carved inside and outside the caves dates back to the 9th century A.D.

Phytochemical screening of various solvent extracts of I. aspalathoidesand I.tinctoria

Healthy plants of I. aspalathoides and I.tinctoria were cleaned and shade dried for 15 days and ground well into a fine powder. The powdered plant material was extracted with various solvents such as methanol, water and ethanol for the elucidation of secondary metabolites. All the solvents and aqueous extracts were subjected to phytochemical analysis as described by Harborne (1973), Trease and Evans (1989) and Sofowara (1993).Table – 1.

Table: 1 Protocols for phytochemical analysis

Sl. No Compound Experimental detail Observation 1. Alkaloid Sample +2ml Wagners reagent Reddish brown precipitate 2. Flavanoids a) Sample + 5ml dilute Yellow colour ammonia + Conc. H2SO4 (disappears on standing) b) Sample – boil with 10ml of Yellow colour ethyl acetate + 1ml dilute ammonia 3. Tannins a) Sample +10% alcoholic ferric Dark blue or greenish grey chloride colour b) Sample – boiled in 20 ml Brownish green water + few drops 0.1% ferric or chloride Blue black colour 4. Saponins Sample – boiled in 10ml water + Formation of emulsion 3 drops of olive oil 5. Cardiac Sample + 2ml glacial acetic acid Brown ring of the interface glycosides + 1 drop ferric chloride + 1ml indicates a deoxy sugar (Keller – conc. H2SO4. characteris-tics of Killani test) cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout the thin layer 6. Steroids Sample + 2ml acetic anhydride + Colour changed from violet 2ml H2SO4. to blue or green 7. Terpenoids Sample + 2ml chloroform + 3ml Cherry red colour (Salkowski conc. H2SO4. test)

8. Libermann- Sample + 1ml chloroform + 2ml Dark green colour indicate Burchardt test acetic anhydride + 1-2 drops the presence of steroids. for steroids conc. H2SO4. Dark pink or red colour and indicate the presence of terpenoids terpenoids

Determination of Alkaloids (Harborne, 1973)

5g of dried sample was extracted using 10% acetic acid in ethanol and allowed to stand for 4 hr. This was filtered and the extract was concentrated on a water bath to one quarter of the original volume. Concentrated ammonium hydroxide was added dropwise to the extract until the precipitation gets completed. The whole solution was allowed to settle and the precipitate was collected and washed with dilute ammonium hydroxide (1%) and then filtered. The residue alkaloid thus obtained was expressed as mg/g dry weight of plant.

Determination of Flavanoids (Bohm and KocipaiAbyazan, 1994)

Five grams of the plant sample was extracted repeatedly with 50ml of 80% aqueous methanol at room temperature. The whole solution was filtered and the filtrate was transferred to a crucible and evaporated to dryness over a water bath and weighed.

Determination of Saponins (Obadoni and Ochuko, 2001)

10g of the plant sample was extracted using 20% aqueous ethanol. The sample was then heated over a hot water bath for 4 hr with continuous stirring at about 550C. The mixture was filtered and the residue was re-extracted with 20% ethanol. The combined extracts were reduced to 40ml over water bath at about 900C. The concentrate was then transferred to a separatory funnel and diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. To the aqueous layer n-butanol was added.

The combined n-butanol extracts were washed twice with 10ml of 5% aqueous sodium chloride and was heated in a water bath. After evaporation, the samples were dried in oven to a constant weight.

Conformation of Phytochemicals by Thin Layer Chromatography.

The aqueous and organic solvent extracts of the plants were subjected to Thin Layer

Chromatography to confirm the presence of various Phytochemicals. Glass plates coated with silica gel „G‟ dried at room temperature were charged separately with crude extracts of different solvents and aqueous extract. Each sample was loaded 1cm above the edge of the plate. The plates were developed by various solvent mixture and of spraying reagents (Table -

2) according to the method of Wagner and Bladt, 1996 for the detection of alkaloid, flavanoid and saponin and Harborne, 1998 for phenols.

Table : 2Mobile solvents and spraying reagents used for the identification of

phytochemicals

Sl. No Active Principle Mobile Solvent Spraying reagent 1. Alkaloid Choloroform : Methanol Wagners reagent (9:1)

2. Flavanoid Chloroform : Methanol 5% alcoholic (19:1) aluminium chloride

3. Saponins Ethyl acetate : Iodine Vapours hexane (1:9)

4. Phenols Chloroform : Folin – ciocalteau Methanol (9:1) reagent

Genomic DNA isolation

Genomic DNA from the plant samples were isolated by using the protocol described by Doyle and Doyle (1987). 3.5 ml of the extraction buffer was mixed with 3.5 ml of 8 M

LiCl and warmed for 65°C. Fresh leaf sample of 5 g was homogenized and 1ml of the sample was transferred to micro centrifuge tubes followed by incubation at 65°C for 20 minutes. The homogenate was extracted with chloroform: iso amyl alcohol (24:1) and centrifuged at 13000 rpm for 10 minute at 4°C. The upper phase was transferred to a new tube and extracted again with (24:1) chloroform: iso amyl alcohol and the contents were centrifuged. The upper aqueous layer was pipetted into a new tube. 0.5 vol. 3 M potassium acetate (pH 4.8) and equal volume of isopropanol was added and centrifuged at 13000 rpm for 10 minute at 4°C.

The supernatant was pipetted into a new tube. Double the volume of ice cold absolute ethanol was then added and the tube was then placed at 4°C in a refrigerator for overnight. The contents were again centrifuged at 13000 rpm for 10 minutes at 4°C and the supernatant was discarded. The pellet obtained was air dried and dissolved in 50 µl of 1X TE buffer.

Electrophoretic separation of DNA

Electrophoretic separation of the DNA of plant samples were performed in

Sangenomics Lab, Banglore. The DNA was checked for quality and integrity by agarose gel electrophoresis. The isolated DNA was elctrophoresed in 30 ml of 1 X TAE buffer for 45 minutes at 100 volts and gel was viewed in the alpha imager.

Estimation of purity of DNA

Purity of the samples were analyzed in Nanodrop Spectrophotometry in Chromopark

Research Lab , Namakkal and the results were interpreted.

Table- 3. Primer details

Operon Sequence Annealing code temperature OPW 6 5¹ ACGCCCGATG 3¹ 34ºC

OPW 7 5¹ CTGGACGTCA 3¹ 32ºC

OPW 8 5¹ GACTGCCTCT 3¹ 32ºC

OPW 9 5¹ GTGACCGAGT 3¹ 32ºC

OPW 10 5¹ TCGCATCCCT 3¹ 32ºC

PCR Reaction Mixture

The components were taken to match the final concentration; 10x assay buffer, 100 ng

DNA, 800 µM dNTP‟s, Taq DNA Polymerase 3U/µl, 30 pM of each primer and the volume was made up to 25 µl using double distilled water. The reaction mixture was then loaded into a thermal cycler for amplification.

PCR Reaction Conditions

All the above mentioned components were placed in a thermocycler and the following conditions were set to run the PCR. The initial denaturation was carried out at 94°C for 5 minutes and final denaturation for 1 minute. Annealing was performed at 35°C for 1 minute and initial extension for 2 minutes at 72°C and final extension was carried out at 72°C for 7 minutes.

RAPD Analysis

The amplified products were resolved by electrophoresis in 1.5% of agarose gel using

1x TAE buffer at 50 volt for 2 ½ hours . 100 base pair ladder was included as molecular marker. Gels containing the DNA bands were visualized by staining with Ethidium bromide and banding patterns were photographed in a gel documenter.

Cluster analysis using UPGMA

Phylogenetic variations among the different populations of the plants were determined by converting RAPD data into frequency similarity. The resulting data was analyzed by

Unweighted Pair Group Method with Arithmetic mean (UPGMA) cluster analysis to produce a phylogenetic tree.

RESULTS

Botanical description and medicinal uses of Indigofera aspalathoides and I. tinctonia

PAPILONACEAE(FABACEAE)

Twining or straggling shrubs or herb or trees. Leaves alternate, generally compound,often odd-pinnate or 1- or 3 foliolate, rarely simple, usually stipulate; stipels sometimes present;oftenpulvinate. Inflorescence in racemes, panicles, umbels, spikes or in a few-flowered clusters, rarely flower solitary. Flowers zygomorphic, bisexual.Calyx- tube usually campanulate; lobes 5, unequal, imbricate/valvate. Corolla papilionaceous; petals 5, imbricate; the upper (adaxial) exterior and forming the standard (vexillum); the 2 laterals the wings (alae), parallel to each other; the lower 2 interior and connate by their lower margins into a keel (carina). Disc rare. Stamens 10, rarely 9, mono-/di-adelphous, rarely free; anthers uniform/dimorphic. Ovary 1-celled; ovules , sometimes 1 or 2; styles often abruptly/gradually curved, bearded/glabrous. Pod dehiscent by 2 or 1 suture(s) or indehiscent, sometimes joined and breaking into 1-seeded segments; seeds sometimes strophiolate

(arillate).

Tribe: GALEGEAE

Herrbs or shrubs.Leaves imparipinnate, leaflets entire.Stamens diadelphous.Pod usually dehiscent or if indehiscent usually small.

1. Anthers apiculate, hairs fixed by the center ...... Indigofera{ XE "Indigofera" }

INDIGOFERA{ XE "INDIGOFERA" }L.

Herrbs, undershrubs or shrubs, with appressed laterally attached hairs, sometimes mixed with basifixed hairs, frequently silvery canescent. Leaves simple, trifoliolate or imparipinnate, the side leaflets usually opposite, but sometimes alternate, entire; stipules usually small, shortly adnate to the petiole; stipels setaceous or 0. Flowers generally very small, usually reddish or purple, in axillary racemes or spikes, rarely solitary, rarely panicled, each flower pedicelled in the axil of a caducous bract; bracteoles 0. Calyx minute, campanulate, teeth subequal or the lowest longest. Corolla more or less caduceus; standard ovate or orbicular, sessile or slightly clawed; wings oblong, slightly adherent to the kell; keel petals erect, obtuse, with a downward spur on each side near the base. Stamens diadelphous, the vexillary stamen free, the others with connate filaments; anthers uniform, apiculate.

Ovary sessile or subsessile, 1-2-or many- ovulate; style glabrous; stigma capitate, sometimes pedicellate. Pod usually linear-cylindric, rarely oblong or globose, straight or curved, sometimes angled, sometimes muricate, often torulose, septate within between the seeds.Seeds globose or cylindric and truncate; strophiole 0.

1. Undershrub,Leaflets 2,3 or 5, Leaves sessile, Flowers solitary,

Calyx linear-subulate,Corolla dark pink, Pods straight ...... I.aspalathoides

2. Shrub, Leaflets 7-15,Leavespetiolate, Flowers axillary

Racemes, Calyx long and acute, Corolla pink, Pods

Slightly curved ...... I.tinctoria

I. ASPALATHOIDES, Vahl ex DC.

A low much-branched erect undershrub; branches rigid, terete, divaricately spreading, the young ones argenteo-canescent, the hairs soon falling off, the older ones purple and nearly glabrous. Leaves 1-5 (often 3) foliolate, digitate, sessile,crowded on the young branches, but soon deciduous; stipules minute, subulate. Leaflets 2.5 - 6 mm long, sessile, linear or oblanceolate, apiculate, rather fleshy, with a few white appressed hairs. Flowers solitary, axillary; pedicels filiform, longer than the leaves, but shorter than the pods.Calyx 1.5 mm long; teeth linear-subulate. Corolla dark pink, exerted. Pods 1.2-1.5 cm long, somewhat turgid, straight, glabrous or with a few scattered hairs.Seeds 6-8.

IndigoferaaspalathoidesVahl ex DC. Prodr.2: 231.1825; Wight, Ic.t.332.1840; Baker in Hook.f.Fl.Brit.India 2: 94. 1876; Gamble,Fl.Pres.Madras 1: 218. 1879 (repr.ed.).

Erect much branched undershrubs. Flowers red.SKS& BPN 262 (Plate-3 and Fig.

3).Found in and around the hills.

Medicinal properties and uses

The leaves, flowers and tender shoots are said to possess cooling and demulcent properties, and are employed in decoction, to treat leprosy and malignant tumors. The root is chewed as a remedy for tooth - ache and apathy. The whole plant, rubbed with butter, is applied to reduce oedematous tumours. A preparation is made from the ashes of the plant to remove dandruff from the hair. The leaves are applied to abscesses; and oil obtained from the root is used to anoint the head in erysipeals.

This is one of the important ingredients of the specific oil for syphilitic and other skin diseases. A decoction of the entire plant is given as an alternative in secondary syphilis, psoriasis, etc.

Common Names: Sanskrit-Sivanimba; Malayalam- Manali; Tamil- Shivanarvembu,

Iraivanvembu.

I. TINCTORIA, L.

A shrub 1.2-1.8m high; branches terete or more or less angular, slightly silvery from fine appressed hairs.Leaves 2.5-7.5 cm long; petioles 1.2-2.5 cm long; stipules small, subulate. Leaflets 9-13,opposite, membranous, green but drying a greayish black, 1.2-2.5 by

0.6-1.2 cm, oblong or oblanceolate, rounded, apiculate, glabrous above or nearly so, thinly clothed with appressed hairs beneath, base acute; petiolules of lateral leaflets 1.25-2 mm, those of the terminal reaching 6 mm long. Flowers numerous, in nearly sessile lax spicate racemes 5-10 cm long.Calyx 1.25-1.5 mm long, hairy outside; teeth triangular, acute, as long as the tube.Corolla pink, 4 mm long; standard pubescent at the back. Pods 2-3.2 cm long, linear, straight or slightly curved, apiculate, thickened at the sutures, glabrous, not torulose.

Seeds 8-12.

Indigofera { XE "Indigofera" }tinctoria L. Sp. Pl. 751.1753; Wight, Ic. t. 365. 1840;

Baker in Hook. f. FBI 2: 99.1876; Gamble, FPM 1: 220. 1957 (repr.ed.).FTNA 1: 113. 1983.

I.SumatranaGaertn. Fruct.2: 317. t. 148. f. 4. 1791; Gamble, FPM 1: 220. 1957

(repr.ed.).Branching shrub. Flowers pink found in and around the hills.

Medicinal properties and uses

The root and stem are hot with a sharp, bitter taste. In Ayurveda, it is used as laxative, expectorant, anthelmintic; promote the growth of hair; used in abdominal complaints, heart diseases, insanity; cure “vata” tumours, fever, leucoderma, enlarged spleen, cephalagia, injuries. The whole plant is used to treat intoxication, giddiness, fainting, constipation, regurgitation, ascites, hepatomegaly, splenomegaly, blood disorders, oedema, urinary calculi.

The root is also useful in difficult micturition, snake-bite, caries of the teeth, consumption.

In Siddha the root and leaf of the plant is used to cure poisoning, fever,jaundice, anaemia, arthitits, leucorrhoea, guinea-worm diseases. In Yunani the plant is used to cure inflammation; chronic bronchitis and asthma, especially of children; cures piles, leucoderma, bites of insects and reptiles, burns, scalds, ulcers, and skin diseases; good in lumbago, enlargement of the spleen and liver, flatulence; applied to the navel it acts as a diuretic and cathartic.

The juice of the leaves has great repute as a cure for hydrophobia, being administered both internally and externally. The root is used in hepatitis. An extract of the plant is given in epilepsy and nervous disorders. It is also used in bronchitis and as an ointment in sores, old ulcers, hemorrhoids.

Common Names: Sanskrit- Nilini; Malayalam- Amari; Tamil- Avuri, Nili

Preliminary phytochemical analysis of I.aspalathoides

Phytochemical analysis of the I. aspalathoidesplants revealed the presence of seven compounds namely alkaloids, flavonoids, saponins, terpenoids, steroids, tannins and cardiac glycosides in the aqueous extract of the plant.Acetone extract of I. aspalathoides was positive to alkaloids, flavonoids, terpenoids, steroids and tannins whereas, saponins and cardiac glycosides were absent.

Preliminary phytochemical analysis of I.tinctoria

Of the seven phytochemical constituents screened in I. tinctoria, aqueous extract revealed the presence ofalkaloids, flavonoids, saponins, terpenoids, tannins and cardiac glycosides whereas Steroids were absent in the plant. Acetone extract of the plant also did not elute steroids, saponins and cardiac glycosides, while the other four secondary metabolites were detected.

Quantitative estimation of secondary metabolites

The results of quantitative estimation of secondary metabolites of

I. aspalathoides and I.tnctoria are presented in Table-3

Detection of Bioactive compounds in I.aspalathoides and I.tnctoria using TLC.

The presence of fluoresing spots confirm the presence of bioactive compounds in I. aspalathoides and I.tinctoria.

The presences of alkaloid in both the plants were confirmed by the development of orange fluoresing spot after spraying with Wagners reagent. Presence of flavanoids in the medicinal plants were confirmed by the development of yellow fluoresing spot on TLC plates after spraying with 5% alcoholic aluminium chloride.

Saponin in both the plants were confirmed by the presence of pink colour spots after incubating the plates in glass chamber saturated with iodine vapours, whereas presence of phenol was confirmed by the development of blue colourfluoresing spot after spraying with

Folinciocalteau reagent.

The preliminary analysis of secondary metabolites revealed the presence of various secondary metabolites. It has been confirmed through Thin layer chromatography. However, the various functional groups present in the two medicinal plants through IR spectroscopy and molecular docking of specific medical constituent present in the medicinal plants with anticancer (or) other disease targets using bioinformatics tools will be carried out in the second phase.

This docking study in the final phase may help to design new drugs from the bio active constituents of these medicinally important plants against dreadful diseases.

Table : 4 Preliminary phytochemical screening of Indigoferaaspalathoides

Aqueous Acetone Sl. No. Phytochemicals extract extract

1 Alkaloids + +

2 Flavonoids + +

3 Saponins + -

4 Terpenoids + +

5 Steroids + +

6 Tannins + +

7 Cardiac glycosides + -

+ Present , - Absent

Table : 5 Preliminary phytochemical screening of Indigoferatinctoria

Aqueous Acetone Sl. No. Phytochemicals extract extract

1 Alkaloids + +

2 Flavonoids + +

3 Saponins + -

4 Terpenoids + +

5 Steroids - -

6 Tannins + +

Cardiac 7 + - glycosides

+ Present , - Absent

Table :6 Quantitative analysis of phytochemical constituents of Indigofera aspalatheoides and Indigofera tinctoria.

S.No Secondary I. aspalatheoides I. tinctoria. metabolites 1. Alkaloid (%) 0.8 1.3

2. Flavonoid (%) 0.9 1.8

3. Saponine (%) 2.6 3.1

Preliminary phytochemical analysis of I.aspalathoides

Phytochemical analysis of the I. aspalathoides plants revealed the presence of seven compounds namely alkaloids, flavonoids, saponins, terpenoids, steroids, tannins and cardiac glycosides in the aqueous extract of the plant. Acetone extract of I. aspalathoides was positive to alkaloids, flavonoids, terpenoids, steroids and tannins whereas, saponins and cardiac glycosides were absent (Table 1).

Preliminary phytochemical analysis of I.tinctoria

Of the seven phytochemical constituents screened in I. tinctoria, aqueous extract revealed the presence of alkaloids, flavonoids, saponins, terpenoids, tannins and cardiac glycosides whereas Steroids were absent in the plant. Acetone extract of the plant also did not elute steroids, saponins and cardiac glycosides (Table1). Phytochemical studies were carried out in the ethanolic extracts of 45 Indian medicinal plants by Iqbal and Arina . They reported that qualitative phytochemical tests and thin layer chromatography of plant extracts demonstrated the presence of common phytocompounds, in that tannins and flavonoids are the major active constituents. The presence of flavonoids glycosides and steroids has been reported in

Tephrosia purea. The presences of alkaloid in both the plants were confirmed by the development of orange fluoresing spot after spraying with Wagners reagent in thin layer chromatography.

Presence of flavanoids in the medicinal plants were confirmed by the development of yellow fluoresing spot on TLC plates after spraying with 5% alcoholic aluminium chloride. Saponin in both the plants were confirmed by the presence of pink colour spots after incubating the plates in glass chamber saturated with iodine vapours(Plate-3), whereas presence of phenol was confirmed by the development of blue colourfluoresing spot after spraying with Folin ciocalteau reagent in the TLC plate.. The presence of the phytochemmical compounds alkaloids, flavonoids, coumarins and steroids were confirmed in Indigofera suffruticosa through chromatographic analysis [13].

Plate - 3

DNA isolation from plant samples

DNA of the plant samples were isolated using standard protocols and their separation was carried out using agarose gel electrophoresis. The bands were observed under UV light and the integrity of the isolated DNA was checked.

Estimation of DNA concentration and purity of I.aspalathoides and I.tinctoria

DNA concentration and purity of the marker DNA and the genomic DNA of the plant samples I.aspalathoides (IA1, IA2, IA 3, IA 44, IA 5) and I.tinctoria (it-1-IT-2,IT-3,IT-4) were analyzed using Nanodrop Spectrophotometer . The expected O. D value for A260/A280 ratio for pure DNA was in the range of 1.4 – 1.8. In the present study A260/A280 ratio for genomic DNA samples of the five populations of I. aspalathoides and four populations of

I.tinctoria were in the expected range and found to be pure in nature.

Plate-4 . Agarose gel electrophotogram of Genomic DNA isolated from five different populations of I.asalathoides

1 2 3 4 5

Lane1:IA1, Lane2: IA2, Lane3: IA3, Lane4: IA4, Lane5: IA5 Plate- 5 Agarose Gel Electrophotogram of Genomic DNA of four different poulations of I.tinctoria

1 2 3 4 L

Lane description:

Lane 1: IT1, Lane 2: IT2, Lane 3: IT3, Lane 4: I4, Lane L: Ladder.

RAPD analysis of I.aspalathoides

The PCR amplified products of the DNA of the five populations of A. indica were subjected to RAPD analysis using five primers viz., OPW6, OPW7, OPW8, OPW9 and OPW10. The analysis resulted in 22 polymorphic bands of DNA of the five plant samples of

I.aspalathoides (Plate- 4), (Table- 8 ).

Table- 8. Percentage of polymorphic DNA bands of different populations of I.aspalathoides

Primer Total Bands Polymorphic Bands % of Polymorphism

OPW 6 17 3 17.64

OPW 7 14 6 42.85

OPW 8 12 3 25.00

OPW 9 17 4 23.52

OPW 10 19 6 31.57

The same type of bands occurred at different frequencies in all samples. The additional irreproducible DNA bands observed in the gel for the different populations of

I.aspalathoides were neglected. The genetic distance between the samples ranged from 15608 to 169.956 and genetic identity ranged from 0.250 to 0.844 (Table- 9 ).

Table - 9. Computation of genetic distance between the different populations of I.aspalathoides

IA1 IA2 IA3 IA4 IA5 IA1 0 25.000 75.000 165.938 63.832 IA2 0 100.000 165.938 15.608 IA3 0 67.031 136.168 IA4 0 169.956 IA5 0

Cluster analysis of I.aspalathoides

The DNA bands obtained from RAPD were analyzed using UPGMA software. The dendrogram obtained clearly indicates 2 clusters. IA2 and IA 5 in cluster one, IA 3 and IA4 in cluster three and IA1 in a separate clade in cluster one. Thus it was evident that there was a clear variability within the populations of I.aspalathoides collected from five different sites of Kanyakumari District. The phenogram (Fig. 2) based on genetic distance (UPGMA) reveals that the populations of I,aspalathoides from five different sites were highly differentiated by their own genetic distance.

Fig.2. Phylogenetic Tree of different cultivars of Indigofera aspalathoides

In the present study despite the morphological similarity, a great deal of polymorphism was observed in the different populations of I.aspalathoides. This variation at the genetic level may be due to environmental factors as reported by many authors.

I.aspalathoides is an important medicinal plant used against various disorders including alone and in combination with other plants. Hence this study will definitely help to evolve sound strategies to conserve the various phenotypes of this medicinally important plant.

RAPD Analysis of I.tinctoria

Genomic DNA was isolated from the four different populations of I.tinctoria and the

DNA was separated in Gel electrophoresis to assess their purity. The isolated DNA samples of the four bacterial isolates of I.tinctoria were amplified in PCR and the PCR amplified products of the DNA of the four isolates were subjected to RAPD analysis using a standard primer. The analysis revealed more polymorphic DNA bands for the four clinical isolates of

I.tinctoria. More bands were visualized for the isolate IT 2 followed by IT 1. The clinical isolates IT3 and IT4 revealed similar number of bands.

4.5.1 Distance matrix and Construction of Phylogenetic tree of I.tinctoria

Phylogenetic diversity between the four clinical isolates of I.tinctoria were determined by converting RAPD data into similarity matrix and analyzed by Neighbour– joining method to produce a phylogenetic tree. There were number of bands common to the four isolates. However, differences evident through the visual examination of the polymorphic bands of the DNA were further clarified by cluster analysis of the RAPD data.

The distance matrix similarity ranged from 0.35-0.62 (Table- 10). Phylogenetic tree constructed by Neighbour-Joining method revealed that IT3 and IT4 were closely related followed by IT1 with IT3 and IT4. However, the isolate IT2 appeared as a separate clade

(Fig. 3). I.tinctoria IT2 was totally different in its sensitivity pattern and polymorphic from the plants of other localities whereas IT3 was distantly related to the other locality plants.

These findings suggest the possibility of genetic polymorphism among the plants.

Table- 10. Computation of Distant Matrix using RAPD data

IT-4 IT-3 IT-1 IT-2

IT-4 - 0.53846 0.51111 0.55102

IT-3 0.53846 - 0.62162 0.46341

IT-1 0.51111 0.62162 - 0.35294

IT-2 0.55102 0.46341 0.35294 -

Fig.3. Phylogenetic Tree of different cultivars of Indigofera tinctoria The presence of various bioactive compounds and the confirmation of therapeutic properties justifies the use of whole plant for various ailments by traditional practitioners

.Separation of the compounds which are present in significant quantities in pure forms from the crude extracts may help us to discover new ligand molecules for therapeutic use.

Further, insilico docking of bioactive constituents (ligands) of the two medicinally important plants with bacterial, viral target proteins and cancer targets using bioinformatics tools such as AutoDock, PatchDock and also ascertaining the ADME properties of the compounds shall help to design new drugs against dreadful diseases.

Conclusion:

I.aspalathoides with potential anticancer activity is indigenous to south TamilNadu and

SriLanka and I.tinctoria possess dye yielding properties apart from its pharmaceutical applications. The secondary metabolites of the plants have potential therapeutic applications.

Hence conservation and mass cultivation of the plants and purification of bioactive constituents and subjecting them for further clinical trials may be a big boon for the health sector. Further, insilico docking of bioactive constituents (ligands) of the two medicinally important plants with bacterial, viral target proteins and cancer targets using bioinformatics tools such as AutoDock, PatchDock and also ascertaining the ADME properties of the compounds shall help to design new drugs against dreadful diseases.

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