REVIEW OF LITERATURE

Chapter II

20 Chapter I1

2.1 Introduction

The present study on the various species of Indigofera was designed with three

aims. 1. To achieve a standardized tissue culture technology for micropropagation of

the medicinally important species of Indigofera, 2. To develop a proliferating callus

from the cultured explants and conduct in vitro screening studies against drug

resistant pathogenic bacteria using crude callus extract for detecting antibacterial

activity, 3. To screen the crude extracts of the stem, leaves and roots of selected

species of Indigofera, in various solvents, for the presence of antibacterial activity

and to identify the bioactive compounds responsible for it through phytochemical

screening of the various fractions of the extract. In order to conduct the investigation,

a review of the literature regarding the micropropagation of the medicinal plants in

general, as well as that of Leguminosae family to which Indigofera belongs, was

done. Also a survey of the relevant studies regarding the antibacterial activity of the

crude extracts of the various plants and the callus obtained in in vitro culture was

undertaken. Above all, a thorough study of the available literature of the studies so

far reported in the various species of Indigofera was also done in order to design the

present set of experiments.

2.2 Micropropagation of Medicinal Plants:

The recent urge for plant based drugs has induced a great enthusiasm among

the tissue culture scientists to develop a standardized micropropagation technology in

medicinal plants of common use so that sufficient planting materials can be made

available to the interested cultivators. Unlike the seedlings, the micro propagated

planting materials have the advantages of being disease free, healthy ones and can be

produced in large numbers from limited number of explants. Generally there are two

methods of micropropagation (i) direct regeneration of shoots from the cultured

explants (ii) indirect regeneration of plants from the callus developed from the

explants in the culture medium.

The direct method of multiple shoot formation was reported from the cultured

shoot tip explants of Catharanthus roseus (Seth and Mathur, 2005), Aegle marmelos

(Das et al., 2008), Piper nigrum (Philip et al.,1992), Adhatoda vasica (Nath and

Buragohain, 2003; Khalekuzzman et al., 2008), Ocimum basilicum (Begum et al.,

Review of Literature 21

2002), Embelia ribes (Raghu et al., 2006), Heliotropium keralense (Sebastian et al.,

2006), Vitex negundo (Rani and Nair, 2006), Evolvulus alsinoides (Tejavathi and

Purushotham, 2004), Cayratia pedata (Anupama et al., 2005), Piper longum

(Soniya and Das, 2002), Rauwolfia tetraphylla (Gosh and Banerjee, 2003), Gloriosa

superba (Sayeed and Shyamal, 2005), Aloe vera (Hashemabadi and Kaviani, 2008;

Ahmed et al., 2007), Phyla nodiflora (Ali Ahamad et al., 2005) etc. The direct plant

regeneration is usually through in vitro axillary shoot proliferation from the axils of

the shoot tip / nodal explants as it was noticed in Clitoria ternatea (Barik et al.,

2007). The advantage of the multiple shoots obtained through direct method is that

they are more true to type compared to the callus regenerated ones.

Since the nodal explants are available more in comparison to shoot tip

explants, in several plants the nodal explants were used as the initial tissue for

multiple shoot induction in in vitro. The nodal explants responded well and initiated

direct multiple shoot formation successfully in Ocimum sanctum (Sharma and Devi,

2006), Nilgirianthus ciliatus (Devi and Kamalam, 2007), Centella asiatica (Kavindra

et al., 2000), Rauwolfia serpentina (Nandy et al., 2004), Terminalia bellerica

(Ramesh et al., 2005), Moringa oleifera, (Islam et al., 2005), Ptetrocarpus santalinus

(Prakash et al., 2006), Ruta graveolens (Bohindar et al., 2008), Adhatoda vasica

(Jaiswal et al., 1989), Boerhaavia diffusa L. (Roy, 2008), Phyllanthus amarus

(Pagare et al., 2008) and Asparagus adescendens (Mehta and Subramanian, 2005).

Thoyajaksha and Rai (2006) developed multiple shoots from the rhizome buds of

Ammomum microstephanum, an endangered medicinal plant. Not only from shoot tip

and nodal explants, direct plant regeneration from leaf explants was also reported in

some plants like Plumbago rosea and Plumbago zeylanica (Das and Rout, 2002).

Even though, the plants obtained through direct regeneration were reported to

have more clonal fidelity in comparison to the callus regenerated plants, the

regeneration through callus has got the advantage of requiring only few explants

from which callus can be induced and regeneration of large number of plants can be

obtained. For induction of callus, shoot tip, node, stem and leaf segments were used

as explants. Successful callus formation followed by regeneration of shoots was

observed in the cotyledon and hypocotyl explants of Eucalyptus tereticornis (Prakash

and Gurumurthi, 2005). Plant regeneration through somatic embryogenesis in leaf

22 Chapter I1

derived callus was reported in Hemidesmus indicus (Swaroop et al., 2008). Rahman

et al., (2004) was able to demonstrate shoot bud regeneration in the callus initiated

from internodal explants of Elaeocarpus robustus.

The development of micropropagation technique is all the more important in

endangered medicinal plants; for, with a few explants, a large number of plantlets

can be produced. For example, in Rauwolfia serpentina Linn., an endangered plant,

large number of plants can be produced from the callus initiated from leaf explants

(Singh et al., 2009) and shoot tip explants (Ahmed et al., 2002). Similar reports are

also available in Sarcostemma brevistigma (Thomas and Shankar, 2008), a rare plant,

and Boesenbergia pulcherrima, a threatened plant species (Anish et al., 2008).

The role of plant growth regulators and their interactions in micropropagation

technique is really significant. Usually the cytokinin BAP with or without a low

concentration auxin like NAA was found critical in callus regeneration as it was

found in Rauwolfia tetraphylla (Gosh and Banerjee, 2003), Echinacea purpurea

(Korch et al., 2001), Dahlia (Fatima et al., 2007), Pepper (Bhat et al., 1992),

Asparagus (Pontaroli and Camadro, 2005) etc. Kaladhar et al., (2010) reported callus

formation and organogenesis from shoot tip and flower explants of Merremia

tridentata L. in MS medium with IAA as auxin component instead of NAA and

BAP. In contrast there are reports on response of explants on media containing plant

growth regulators and showing direct organogenesis instead of callus formation in

Dioscorea (Chen et al., 2007), Perilla (Hou and Jia, 2005), Thapsia (Makunga et al.,

2005) and Chestnut rose (Wen and Deng, 2005), Gloriosa superba (Sayeed and

Shyamal, 2005) etc.

2. 3 In vitro Propagation of Leguminous Plants.

The Indigofera genus belongs to the Leguminosae family. In vitro

micropropagation studies were reported in several members of the family. Multiple

shoot formation has been reported from shoot tip and nodal explants of Acacia

chundra (Rout et al., 2008) and nodal explants of Entada pursaetha (Vidhya et al.,

2005) and Acacia catechu (Rohini and Gupta, 2002).

MS medium with BAP and IAA effectively induced multiple shoots from

nodal explants of Prosopis cineraria (Kumar and Singh, 2009). Development of

Review of Literature 23

shoots had been reported in Cajanus cajan from callus initiated from intervenal leaf

lamina explants (Jain and Chaturvedi, 2004) as well as from hypocotyl, epicotyl and

cotyledon explants (Jeyachandran et al., 2004). Vasanth et al., (2004) reported shoot

formation through regeneration from the callus derived from cotyledon of Arachis

hypogea. Standardized technology for in vitro propagation of leguminous tree

species like Albizzia lebbeck (Gharyal and Maheswari, 1981), Dalbergia sissoo

(Mukhopadhyay and Chandra, 1983) Leucaena lucocephala (Dhawan and Bhojwani,

1983; Nangia and Singh, 1996) were also reported. Hamdy and Hattori, (2006)

successfully micropropagated two cultivars namely Wara Soramame and Cairo - 24

of Vicia faba L. by somatic embryogenesis and nodal explant culture.

An efficient protocol for in vitro propagation of Abrus laevigatus E. May by

callus culture and organogenesis was formulated by Pandhura et al., (2010). The

effect of high concentration of BAP in multiple shooting was well illustrated in this

study.

In most of the studies referred above, it is found that MS medium with critical

level of hormones is highly significant in micropropagation. Among the hormones,

the BAP was reported to be efficient in inducing multiple shoots as it was seen in

Terminalia bellerica (Ramesh et al., 2005), Moringa oleifera (Islam et al., 2005) and

Holostemma adakodien (Martin, 2002). Hence in the present study, experiments

were designed with MS medium supplemented with different combinations of

hormones to formulate a standardized technology for micropropagation of the three

selected species of Indigofera.

2.4 Antibacterial Activity of Medicinal Plants

During the last two decades a wide spectrum of research had been undertaken

regarding the antimicrobial property of several plants as well as to identify the

compounds responsible for the antibacterial activity. Several workers throughout the

world have carried out antibacterial studies on plants with known or unknown

medicinal properties. The Screening for antibacterial activity is done usually in three

steps.

24 Chapter I1

I. In vitro screening of the crude extracts in water and different organic

solvents.

II. Screening for identification of the fractions from the crude extracts that

demonstrated antibacterial activity.

III. Isolation and identification the compounds from the fractions that showed

antibacterial activity.

2.5 Screening for Antibacterial Activity of the Crude Extracts:

Earlier screening studies for antibacterial activity were conducted on plants

used in folk medicine / ethanomedicine. Anesini and Perez (1993) have detected

antimicrobial activity in the plants used in Argentine folk medicine. Similar studies

were reported in hundred Rwandese medicinal plants, which confirmed their

antibacterial property (Vlietinck et al., 1995). The evaluation of antibacterial activity

of 36 ethanol extracts from 24 plants, all of them currently used in Peruvian

traditional medicine for treatment of several infections and inflammatory disorders

against four pathogenic bacteria, showed that, twenty five extracts have some degree

of antibacterial activity, with Cestrum auriculatum demonstrating the greatest

antimicrobial activity (Rojas et al., 2003). Similar studies of antibacterial activity of

plants used in traditional medicine were also conducted by Mitscher et al., (1987),

Palombo et al., (2001), Portillo et al., (2001), Srinivasan et al., (2001), Matu and

Staden (2003), Camporese et al., (2003) and Babu et al., (2002). In the study to

assess the antibacterial potential of three medicinal plants namely, Strychnos nux-

vomica, Pergularia daemia and Toddalia asiatica used by the tribals in

Maruthamalai hills, Tamil Nadu against five strains of human pathogenic bacteria, all

the extracts were found to exhibit antibacterial activity at higher concentrations

(Senthil Kumar et al., 2005). Twelve different extracts of Avicennia marina,

Avicennia officinalis and Brugiviera sexangula having proved therapeutic value

against microbial infections (Bandaranayaka, 1998), prepared in petroleum ether,

chloroform, ethyl acetate and ethanol tested against five strains of antibiotic resistant

bacteria, collected from hospitals were found to exhibit different degree of growth

inhibition. The crude ethyl acetate leaf extracts of A.marina showed better inhibition

against all tested bacterial strains (Abeysinghe and Waxigalunge, 2006).

Review of Literature 25

Aqueous and alcoholic extracts of the leaves of Gloriosa superba subjected to

antimicrobial activity screening studies against five strains of pathogenic bacteria

were reported to show significant antibacterial activity against all the tested strains

(Subhashini et al., 2000). In vitro antibacterial assay study conducted in 12 medicinal

plants using various crude extracts revealed that the methanol extract of all the plants

were more active apart from all others (Parekh et al., 2005). Accroding to Mishra et

al., (2007) the ethanol extract of the dried stem bark of Neem is potent in inhibiting

the growth of gram negative bacterial strains. The crude organic extracts of the

leaves of Eupatorium triplinerve Vahl. were found to have good antibacterial activity

against human pathogenic bacteria (Rahman and Junaid, 2008). Evaluation of

twenty eight extracts prepared from the fruits of four species viz. Piper cubeba,

Piper retrofractens, Piper longum and Piper nigrun regarding antibacterial activity

by trials on four multidrug resistant bacterial strains gave the result that all the

extracts are having antibacterial activity (Khan and Siddiquie, 2007). The methanol

extract of Leucas aspera flowers and the flower juice was reported to exhibit very

good antibacterial activity (Mangathayaru et al., 2005). Studies using disc diffusion

method about antibacterial activity of the ethanolic extract of Cocculus hirsutus on

four strains of bacteria led to the conclusion that the extract was having good result

on the tried bacterial strains (Satish and Singhai, 2003). Inhibition of growth of gram

positive and gram negative bacteria was exhibited by benzene, alcoholic and aquatic

extracts of the root of Dalbergia spinosa Roxb. (Senthamarai et al., 2003). Crude

acetone extracts of Punica granatum, Garcinia gummygutta, Averrhoa carambola

and Spondias pinnata significantly inhibited the growth of selected pathogenic

bacteria (Babu et al., 2002).

Masika and Afolayan (2002) evaluated the effectiveness of antibacterial

activity of the water, methanol and acetone extracts of Combretum caffrum, Saliz

capensis and Schoria latifolia against the gram positive and gram negative bacteria

and found that the extracts were most active against gram positive bacteria and not

active against gram negative bacteria. Conversely, the aqueous extract, chloroform

extract, alcohol extract and oil obtained from the leaves of Ocimum sanctum

screened against gram positive and gram negative bacteria was observed to be

equally effective against the gram negative and positive bacteria (Mishra and Mishra,

2011) A slightly different response was obtained in the leaf extracts of Artemisia

nilagirica prepared in six organic solvents. All the extracts were found to exhibit

inhibitory activity for gram positive and gram negative bacteria except for Klebsiella

26 Chapter I1

pneumoniae, Enterococcus faecalis and Staphylococcus aureus (Ahameethunisa and

Hopper, 2010).

Crude ethanol extracts from the stem and leaf of Momordica charantia

exhibited broad spectrum inhibitory activity against Bacillus subtilis, Staphylococcus

aureus, Lactococcus lactis, Listeria innocua and Lactobacillus plantarum (Areekul

and Chandrapatya, 2009). Sati and Joshi (2010) have reported significant sensitivity

of five pathogenic multidrug resistant bacteria against five crude organic extracts and

aqueous extracts of Juniperus communis. Mathur et al., (2007) reported that the

rotenoids and flavonoids isolated from stem, leaf and pods of Derris indica is having

high antimicrobial activity.

Evaluation of extracts of Polyalthia longifolia Benth & Hook. in five organic

solvents and water demonstrated that the petroleum ether extract was having the

highest activity against six tested bacteria. Further, the inhibitory effect of the extract

was found very identical in magnitude and comparable with that of standard

antibiotics used (Jain and Sharma, 2009). A lot of studies related to antimicrobial

activities of crude plant extracts of various species have been reported during the

last two decades like in Calendula officinalis (Chakraborthy, 2008); Bougainvillea

spectabilis (Umamaheswari et al., 2008), Raphanus sativus (Sultan Beevi et al.,

2009), Terminalia albida (Ayodele et al., 2010), Anethum graveolens (Jana and

Shekhawat, 2010), Passiflora edulis (Johnson et al., 2008), Nephelium longam (Ripa

et al., 2010) Rhinacanthus narsutus (Siripong et al., 2006), Cistus incanus and Cistus

monspeliensis (Bouamama et al., 1999) Rauwolfia tetraphylla and Physalis minima

(Shariff et al., 2006) Sida cordifolia, Tinospora cordifolia, Withania somnifera and

Ziziphus mauritiana (Mahesh and Satish, 2008), Thymus capitatus (Kandil et al.,

1994), Anacardium occidentale (Mustapha and Hafsat, 2007), Nerium oleander

(Hussain and Gorsi, 2004) Vernonia tenoreana (Ogundare et al., 2006) Plectranthus

amboinicus (Sunayana et al., 2003) Solanum incanum (Britto, 2001), Cassia alata

(Somchit et al., 2002), Eucalyptus canaldulensis (Abubakar, 2010), Senna auriculata

and Pongamia glabra (Selvakumar and Karunakaran, 2010), Croton zambericus

(Reuben et al., 2008), Gmelina arborea (Audipudi et al., 2010). Table-2.1 gives a list

of some prominent traditional medicinal plants and the antimicrobial compounds

identified from them.

Review of Literature 27

Table-2.1 Some of the traditional medicinal plants and the class of Anti-microbial compounds identified in them

Scientific names Parts/solvents Class Compounds Mechanism of traditional medicine References

Baccharis grisebachii

Hieron (Asteraceae) Resinous exudates

Diterpenes, P-coumaric acid derivatives, flavones

3-Prenyl-p-coumaric acid and 3,5-disprenyl-p-coumaric acid

Argentinian traditional medicine showed activity towards dermatophytes and bacteria (MICs

50, 100 and 125µg/ml)

Feresin et al.,2003

Cassia podocarpa

Guill et Perr. (Caesalpiniaceae)

Leaf and flower

Glycosides Anthraquinone glycosides, anthraquinones, free aglycone

Optimum laxative activity and reduced toxicity

Abo and Adeyemi, 2002

Chrysanthemum

morifolium Ramat. (Compositae)

Flowers Flavonoids Apigenin7-o-β-D glucurnoide Glucuronide showed strong HIV-1 integrase inhibitory activity in a cell culture assay using HIV- IIIIB- infected MT-4 cells.

Lee-Huang et al., 2003

Curcuma longa L.

Rhizome

(Zingiberaceae)

Rhizome Flavonoids Curcumin and curminoids A number of different molecules involved in inflammation that are inhibited by curcumin including lipo- oxygenase, phosphopolipase and elastase

Chainani-Wu, 2003

Acacia mellifera Stem bark Flavonoids -3-(z)-Ciscoumaroylbetulin, 30 hydroxylup-20-en-3 beta-ol

Used against pneumonia and malaria

Mutai et al., 2009

28 Chapter I1

Osmitopsis

asteriscoides L. (Asteraceae)

Aerial parts Essential Oil Cineole and camphor, Camphor and 1,8 – cineole

Anti- microbial activity (0.5-2% v/v) synergistic effect is presented as a possible explanation for the traditional use for chest pain in South Africa.

Viljoen et al.,2003

Santiria trimera (Oliv.) Aubrev. (Burseraceae)

Bark Essential Oil monoterpenes

β-Pinene (20.0%),- pinene (66.6%)

Plant widely used by the traditional healers, anti-microbial activity (MIC. 11-0.71 µg/ ml).

Martins et al., 2003

Stephania dinklagei Engl. (Menispermaceae)

Ethanol extract

Aporphine alkaloids

Liriodenine, corydine, isocorydine, atherospermidine, stephalagine and dehydrostephalagine

Liriodenine showed strong cytotoxic activity while corydine and atherospermidine showed DNA damaging activity

Gören et al., 2003

Warburgia

ugandensis Sprague (Cancellaceae) Zanthoxylum chalybeum Engl. (Rutaceae)

Seed Alkaloid Skimmianine Ugandan plants showed anti- microbial activity by agar well assay.

Olila et al., 2001

Hydrastis canadensis L. (Ranunculaceae)

Whole plant Alkaloid Berberine-etraphydroberberine and 8- oxoberberine

Chinese herb-exhibited vasodilator activity, that has been attributed to multiple cellular mechanisms. Its derivates are attributed to the blockade of K+ channels [delayed rectifier and K (ATP)] and stimulation of Na+-Ca (2+) exchanger.

Lau et al., 2001

Review of Literature 29

Boswellia serrata Roxb.

(Burseraceae)

Gum resin Pentacyclic triterpenes

Boswellic acids Boswellic acids inhibit the leukotriene biosynthesis in neutrophilic granulocytes by a non-redox, non-competitive inhibition of 5-lipoxygenase.

Ammon, 2001

Bougainvillea xbuttiana (Nyctaginaceae)

Leaf Proteins Lysine The inhibitor showed N- glycosidase activity on 25SrRNA of tobacco ribosomes, which interfered with virus multiplication through ribosome interaction

Narwal et al., 2001

Zingiber officinale Rosc. (Zingiberaceae)

Rhizome Polyphenol- ics 6-,8-,10-gingerol and 6-shogoal The gingerols inhibit the growth of H.pylori CagA+ strains in vitro at 6.25-50 µg/ml

Mahady et al., 2003

30 Chapter II

2.6 Antibacterial Activity of Leguminous Plants

Leguminosae, the third largest family in the plant kingdom comprises

approximately 19325 species in 727 genera (Caesalpineacae – 2250 sps, Mimosae –

3270 sps and Papilionaceae – 13800 sps) (Lewis et al., (2005). Several reports are

there regarding the antibacterial activity of the extracts from Leguminosae plants.

Since, Indigofera genus belongs to this family, those studies are of special

significance for us. Mutai et al., (2009) evaluated the antimicrobial effects of extracts

from the stem bark of Acacia mellifera against bacterial strains and the results

support the use of A.mellifera stem bark for the treatment of infectious diseases.

Sampaio et al., (2009) examined the antimicrobial activity of Caesalpinia ferrea

Marticus fruit extract against oral pathogens and found that the fruit extract inhibited

the in vitro growth of oral pathogens in planktonic and biofilm models supporting its

use for oral infections. The flavonoid fraction isolated from ethyl acetate fraction of

Butea frondosa (L.) stem bark exhibited distinct antibacterial activity against 9

different genera of both gram positive and gram negative types (Mishra et al., 2009).

The in vitro and in vivo antimicrobial activities of seeds of Caesalpinia bonduc (L.)

Roxb. was evaluated by Arif et al., (2009). The potentiality of Tephrosia purpurea

was examined as an anti-Helicobacter pylori agent (Chinniah et al., 2009) and

obtained positive response. Tephrosia purpurea showed significant antibiotic activity

against Pseudomonas aeruginosa and two coliform strains (Rangama et al., 2007).

The aqueous, methanol and ethyl acetate extracts of Acacia salicina was

demonstrated for its antibacterial potential against bacterial reference strains,

Staphylococcus aureus, Enterobacter aerogens, E.faecalis, Salmonella enteritidis

and Salmonella typhi-murium (Chatti et al., 2009).

The alcoholic extracts of leaves, stem and flowers of Saraca asoca exhibited

antibacterial activity due to the presence of certain antibacterial compounds (Shahid

et al., 2007). Parekh et al., (2005) demonstrated the potential antibacterial activity of

Caesalpinia pulcherima against the human pathogenic bacteria. The antimicrobial

activity of Acacia nilotica was exhibited against a panel of human pathogenic

microorganisms. The inhibition activity was found to be due to inhibition of protease

and amylase activities of these microorganisms following treatment with the extract

Review of Literature 31

(Elizabeth et al., 2006). Arya et al., (2010) reported the antimicrobial activity of the

leaves of Cassia occidentalis against various human pathogenic microbes. The crude

aqueous and acetone extracts of Tamarindus indica was found to exhibit antibacterial

activity against eight clinically important bacteria (Babu et al., 2002). The tests for

antibacterial activity of organic and aqueous extracts of Acacia aroma against

Staphylococcus aureus and S.epidermidis led to the conclusion that the ethanolic and

ethyl acetate extracts have highest activities against the tested organisms (Mattana et

al., 2010). Ethanol extracts of stem barks of Dipterix and Pterocarpus rohrii

demonstrated strong antibacterial effect against gram positive bacteria (Kloucek et

al., 2007).

From the studies reported above, it is clear that a good number of leguminous

plants are having significant antibacterial activity.

2.7 Antibacterial activity of In vitro Induced Callus

Recently a lot of studies related to antimicrobial activities of callus extracts

have been reported in medicinal plants such as Phyllanthus amarus, Rauwolfia

tetraphylla, Physalis minima (Shariff et al., 2006) Passiflora mollusima,Passiflora

edulis (Johnson et al., 2008); Bixa orellana (Castello et al., 2002); Balliospermum

axillare (Singh and Sudharshana, 2003) etc. In these studies the callus was found to

have the phytochemicals demonstrating antibacterial activity and can be compared in

this respect with the original plant (Parsaeimehr et al., 2010). Hence, in vitro

produced calli have been screened for the presence and activity of antimicrobial

substances (Mathes, 1967; Khanna et al., 1971; Jit and Nag, 1985; Veliky and Lata,

1974).

The ethanolic extract of the callus tissue of Asparagus officinalis exhibited

antibacterial activity against Bacillus subtilis (Khorasani et al., 2010). Methanol and

ethanol extracts of leaf and leaf derived callus extracts of Cardiospermum

halicacabum L. showed significant activity against the gram positive bacterial strains

tested (Girish et al., 2008). Landa et al., (2006) successfully evaluated the

antimicrobial activity of crude methanol extracts from callus cultures of Nigella

arvensis, N.danascena, N.hispamica, N.integrifolia and N.sativa. All the callus

extracts exhibited significant antimicrobial activity against Bacillus cereus,

32 Chapter II

Staphylococcus aureus and Staphylococcus epidermidis. Jana and Shekhawat (2010)

evaluated the antimicrobial potential of the aqueous and ethanolic extracts of the

callus of Anethum graveolens against important bacterial strains and in comparison

to in vivo, in vitro plant extracts were found to exhibit reduced activity. The ethanolic

callus extracts of Jasminum grandiflorum and J.sambac exhibited antibacterial

activity against the selected infectious pathogens viz, Staphylococcus albus, Proteus

mirabilis and Salmonella typhi (Joy and Raja, 2008). Salvador et al., (2003) reported

that the crude extracts of callus culture and adult plants from Alternanthera tenella

Colla. exhibit antibacterial property against the tested microorganisms. The plant cell

callus culture extracts induced in two different hormonal combinations of

Alternanthera maritima were found bioactive against the same strains to which the

adult plant extracts showed antibacterial activity. Ethanolic extracts of different parts

and the calli of Cassia occidentalis have been tested against human pathogenic

bacteria. The anthraquinones were found to be more active against E.coli and

S.aureus (Sharma et al., 1998).

In Saraca asoca, the alcoholic extracts derived from the callus showed

comparable antibacterial activity to the extracts from explants (Shahid et al., 2007).

Comparison of the crude extracts of the leaf and callus of Alophyllus cobbe L. in

chloroform, acetone, ethanol and water, showed that the acetone extract showed

maximum activity in both. Further it is noted that acetone extract of the callus is

more inhibitory (Hegde et al., 2010). Antimicrobial substances in callus cultures of

Ruta graveolens were identified and isolated by Wolters and Eilert (1981). The

acridone alkaloids were the most active substances against the tested bacterial

strains.

Broad spectrum antibacterial activity was recorded from ethanol and ether

extracts of callus cultures of Ricinus communis L.var. major and var. minor against

gram negative and gram positive bacteria. Callus cultures subcultured for 3-6

subcultures diffused antibacterial compounds into media during growth development.

Phytochemical analysis revealed that the antibacterial activity of var. major was due

to quercetin and ricinine, while that of var. minor was due to quercetin, kaempferol

and ricinine (Khafagi, 2007).

Review of Literature 33

2.8 Phytocomponents of Medicinal Plants and their Antimicrobial Activity

A special feature of higher plants is their capacity to produce large number of

organic chemicals of high structural diversity, the so called secondary metabolites

(Evans et al., 1986). Most of these phytochemical compounds are showing properties

like antimicrobial, anticancer, antitumor and antioxidant activities. Such metabolites

are divided into three different categories based on their mechanism of function viz.,

chemotherapeutic, bacteriostatic and antimicrobial (Purohit and Mathur, 1999).

Many of these compounds such as ajoene, allicine, gallic acid, thymol and eugenol

have been isolated from different plants and are found effective against

microorganisms (Naganawa et al., 1996). Kandil et al., (1994) studied Thymus

capitatus for its antimicrobial activity. Preliminary phytochemical screening of this

plant exhibited the presence of saponins, resins, flavonoids, essential oils and fixed

oils. Aqueous and ethanolic extracts as well as saponins, resins and essential oil of

the plant were reported to inhibit the growth of several bacteria and fungi. The

essential oil was fractionated into two, namely, carvacrol and thymol. These two

were then subfractionated into 2-methyl-5-isopropyl phenol, 5-methyl-5-isopropyl-

phenol, 3-methyl-2,6-di-isopropylphenol,3-methyl-2,6-di-isopropylphenol, 5-methyl-

2,3-di-isopropylphenol,3-methyl-2,5-di-isopropylphenol,2,5-di-isopropylphenol, 4-

methyl-2,5-di-isopropylphenol and 5-methyl-2, 4-di-isopropylphenol. The phyto-

compound 2-Hydroxyheptane-3, 5-dione was obtained from the petroleum ether

soluble fraction of the ethanol extract of Potamogeton nodosus. Treatment with the

compound showed antibacterial activity against most tested microorganisms (Alam

et al., 1999). Biological screening of petroleum extract exhibited antibacterial

activity and cytotoxicity. Arambewela et al., (1999) reported that essential oils of

Kaempheria galanga root and rhizome showed antibacterial activity against E.coli

and S.aureus. The inhibitory effects of flavonoids and phenolic acids, which could be

transformed from flavonoids by human intestinal microflora on the growth of

Helicobacter pylori was investigated by Bae et al., (1999). In this study ponciretin,

hesperetin, naringenin and diosmetin were found active.

An activity guided fractionation of methanol- dichloromethane extract obtained

from the aerial parts of Eysenhardtia texana led to the isolation of two novel

antibacterial and antifungal flavonones together with a known flavonone. Their

34 Chapter II

structures were established as 4’ , 5,7-trihdroxy-8-methyl-6-(3-methyl- {2-butenyl })

– (2s)-flavonone, 4’ , 5,7-trihdroxy -6-methyl-8-(3-methyl-{2-butenyl }) – (2s)-

flavonone and 4’, 5-dihdroxy-7-methoxyl-6-(3-methyl-{2-butenyl }) – (2s)-flavonone

on the basis of their UV, 1D and 2D-NMR spectra (Watcher et al., 1999). The

following flavonoids–5, 7, 4’-trihydroxy-6-l-hydroxy-2-methylbuten-2-yl isoflavonone

(isogancaonin C), 7.2’-dihydroxy-4’--methoxy-l isoflav-3-ene (bolusanthin III), 6,6’-

dihydroxy-4’- methoxy -2-arylbenzofuran (bolusanthin IV), in addition to eight

known flavonoids were isolated from the root wood of Bolusanthus speciosus. The

compounds showed antimicrobial activity against E.coli, Bacillus subtilis,

Staphylococcus aureus and Candida mycoderma (Erasto et al., 2004). The flavonoids

- kumatakenin, pachypodol, 5-hydroxy-7,3’, 4’-trimethoxy flavones, velutin,

salvigenin, retusin and corymbosin have been isolated from the aerial parts of Ballota

glandulosissima. The antibacterial activity of kumatakenin, pachypodol, 5-hydroxy-

7,3’,4’-trimethoxy flavones, velutin and retusin were exhibited against Bacillus

subtilis, Staphylococcus aureus, Staphylococcus faecalis, E.coli and Pseudomonas

aeruginosa. The mono and sesquiterpene hydrocarbons: terpinolene, limonene and

alpha-humulene containg in the volatile oil isolated from the leaves of Psidia lucida

exhibited interesting antibacterial activity (Andriamanantoanina et al., 2004). The

essential oil of Achillea millefolium subsp. millifolium Afan, (Asteraceae) showed

antimicrobial activity against Streptococcus pneumoniae, Clostridium perfringens,

Candida albicans, Mycobacterium smegmatis, Acenetobacter lwoffii and Candida

krusei (Candan et al., 2003). A crude methanol extract of Syzygium aromaticum

(Clove) exhibited preferential growth inhibitory activity against gram negative

periodontal oral pathogen, including Porophyromonas gingivalis and Prevotella

intermedia. Eight active compounds were isolated from this extract and were

identified as 5,7-dihydroxy-2- methyl chromone 8-c-beta-D-glucopyranoside,

biflorine, kaempferol, rhamnocitrin, myricetin, gallic acid, ellagic acid and oleanolic

acid based on spectroscopic evidence. These pure compounds demonstrated

antibacterial activity against Streptococcus mutans, Actinomyces viscosus,

Pseudomonas gingivalis and Pseudomonas intermedia. Kaempferol and myricetin

demonstrated growth inhibitory activity against pathogens (Cai and Wu, 1996). Do et

al., (1996) isolated two constituents as the antibacterial principles from the

Review of Literature 35

methanolic extract of rhizomes of Dryopteris crassirhizoma against Streptococcus

mutans. They were identified as flavaspidic acid PB and flavaspidic acid AB and

exhibited strong antibacterial activity.

Isoflavonoids were isolated from the roots of Flemingia strobilifera (L.) and

were identified as 5,7,4- trihydroxy 8, 2’,5’-tri (3-methylbut-2-enyl) isoflavone along

with the known phytoconstituents ie., 5,7,2, 4’,-tetrahydroxy isoflavone, 5,7, 4’,-

trihydroxy isoflavone and beta-sitosterol. Structure assignments were performed on

the basis of spectroscopic data including Homo and Hetero Nuclear 1D, 2DNMR and

MS studies. The compounds were tested for in vitro antimicrobial activity and these

compounds except beta-sitosterol proved to be moderately active (Madan et al.,

2009). Three new isoflavonones, 5,7,3’-trihydroxy-4’- methoxy-6,5’-di (gamma,

gamma-dimethylallyl) - isoflavonone, 5, 3’ –dihydroxy-4’-methoxy-5’-gamma, gamma-

dimethylallyl- 2’’, 2’’-dimethylpyrano (5,6: 6,7 ) isoflavonone and 5,3’-dihydroxy 2’’,

2’’,- dimethyl pyrano (5,6 :6,7)- 2’’’, 2’’’ – dimethylpyrano (5,6:5,4) isoflavonone

along with two known isoflavonoids, cristacarpin and euchrenoneb were isolated

from the stem of Erythrina costaricensis. Their structure was established on the

basis of spectroscopic evidence (Tanaka et al., 2009). Extracts of Inga fendleriana

and the isolated flavonoids from it were studied for the antibacterial activity.

Quercetin 3-methyl ether, myricetin 3-o- rhamnoside and tricetin were found to show

antibacterial activity against the bacterial strains (Pistelli et al., 2009).

Various solvent fractions of the root extracts of Abrus precatorius exhibited

inhibitory activity against 13 gram negative and positive bacteria. The antibacterial

activity was localized to specific chromatophores in the chloroform fraction. Among

the four active principles isolated, 3 exhibited maximum activity (Zore et al., 2007).

2.9 Phytocompounds from Leguminous Plants and their Antibacterial Study

The nor-halimane diterpinoid tessmanic acid and its methyl, 2- methyl

isopropyl and 1- methyl butyl esters, the unusual isocoumarins 8-hydroxy-6-

methoxy-3- pentyl isocoumarin and 7-chloro-8-hydroxy-6-methoxy butyl aniline

isolated from the stem and root bark extracts of Tessmannia densiflora Harms

(Caesalpiniaceae) showed mosquito larvicidal activity. The tessmanic acid and its

36 Chapter II

methyl ester exhibited antibacterial activity, indicating Tessmannia species is a

potential source of bioactive natural products (Kihampa et al., 2009).

A new quinone methide diterpene with a cassane skeleton was isolated from

the root bark of Bobgunnia madagascariensis of the Leguminosae family. The

structure of the compounds was established as (4bS, 8aS)- 4b,5,6,7,8,8a- hexa hydro

-4- hydroxy- 2- (2- hydroxyethyl)-1,4b, 8,8-tetramethyl phenanthrene -3,9-dione by

spectroscopic methods including single crystal X-ray analysis (Schaller et al., 2000).

A protein designated Hypotin, with antibacterial activity was isolated from pea nut

(Arachis hypogea) seeds. This novel protein also exhibited antiproliferative activity

against tumour cells (Wang, et al., 2007). A Novel protease inhibitor, designated

mungoin, with antibacterial activity was isolated from the mung bean (Phaseolus

mungo) seeds (Wang et al., 2006). A non-specific lipid transfer peptide (nSLTP)

isolated from mung bean seeds exhibited antibacterial action against Staphylococcus

aureus but not against Salmonella typhi-murium (Wang et al., 2004). Chokchaisiri et

al., (2009) isolated one new dihydrochalcone, dihydro monospermoside from the

flowers of Butea monosperma together with three known chalcones, butein,

monospermoside and isoliquiritigenin, one flavones,7,3,4-tryhydroxyflavone, four

flavonones, (-)- butin, (-) – butrin, (+) – isomonospermoside and (-)- liquiritigenin

and three isoflavones, formononetin, afrormosin and formononetin -7-o-beta- D-

glucopyranoside. The flavonoid fraction isolated from the ethyl acetate fraction of

Butea frondosa (L.) stem bark exhibited distinct antimicrobial activity when tested

against both gram positive and gram negative bacteria (Mishra et al., 2009). Analysis

of the essential oil of the leaves of Tetrapleura tetraptera (Schum & Thonn.) Taubert

by GC-MS showed the presence of forty one compounds and all of them were

characterized. The essential oil was dominated by 1, 8- cineole, 6,10,14- trimethyl –

2- pentadecanone, phytol, alpha-pinene and geranyl acetone. The oil displayed weak

antibacterial activity against Bacillus subtilis (Aboaba et al., 2009). Mutai et al.,

(2009) examined the extracts from stem bark of Acacia mellifera for antibacterial

activity against different strains of bacteria and the extracts were further fractionated

to give 12 pure compounds. The activity guided fractionation led to isolation of

active compounds from the extracts : 3- (Z)–cis coumaryl betulin and 30 –

hydroxylup-20 (29)-en-3 beta-ol all of which showed antibacterial activity against

Review of Literature 37

Staphylococcus aureus, Microsporum gypseum, Trichophyton mentagrophytes and

Pseudomonas aeruginosa. Chatti et al., (2009) investigated the aqueous, methanol

and ethyl acetate extracts of Acacia salicina for their polyphenolic compound

content, antioxidative activity and antibacterial activity against gram positive and

gram negative bacterial reference strains and found that the antioxidant,

antimicrobial, antigenotoxic and cytotoxic activities exhibited by A.salicina

depended on the chemical composition of the tested extracts. Yenesev et al., (2005)

reported that the chloroform extract of Erythrina burtii have antibacterial activity and

flavonoids are responsible for it. The brief account given above makes clear that the

members of Leguminosae are rich sources of phytocompounds.

2.10 Secondary metabolites isolated from In vitro cultured cells

In view of the crucial importance gained by the secondary metabolites in recent

times, efficient production of bioactive compounds by tissue culture technology has

become a necessity (Vaniserce et al., 2004). Since the secondary metabolites often

have complex stereo- structures with many chiral centres, which may be essential for

biological activity, many of them cannot be synthezised economically on a

commercial basis. At the same time the continuous and non organized exploitation of

natural resources has resulted in many plants becoming rare and even extinct. To

overcome this limitations biotechnologists suggested “the use of cell and tissue

culture technology rather than to use the whole plant” for extraction of certain

secondary metabolites (Rajendra and D’ Souza, 2000). Plant cells are totipotent and

all the necessary genetic and physiological potentials for the secondary metabolite

production are expected to be present in isolated cells. According to this, the cultured

cells obtained from any part of the plant are expected to yield secondary metabolites

similar to those of the plant grown in vivo under suitable culture conditions (Brown

and Charlswood, 1986). Callus induction and in vitro production of anthocyanin

from callus cultures of Oxalis and Haplopappus gracilis was reported by Meyer and

Staden (1995) and Constable et al., (1971). Callus cultures derived from internodes

and leaf explants of the medicinally important plant, Cullen corylifolium, was

reported to produce Psoralen, a furano- coumarin and the product was isolated from

the callus. The content of the psoralen was found several folds higher in callus

cultures compared to the intact plant tissues (Sreelakshmi et al., 2007).

38 Chapter II

β-Sitosterol and Stigmasterol from tissue samples of Cissus quadrangularis

was isolated and both the sterols were found to be higher in concentration in 6 weeks

old callus tissue of the plant (Sharma and Patni, 2007). The callus cultures developed

from root explants of Centella asiatica were found to have biosynthetic potential to

produce asiaticoside (Sholapur et al., 2007). The production of the anticancer

alkaloid camptothecin and other secondary metabolites in the in vitro transformed

roots of Camptotheca acuminata was well achieved by Pasqua et al., (2005).

Many workers have reported the capability of in vitro induced callus to

produce phytochemicals similar to the parent plant. Table-2.2 presents the list of

several medicinal plants, callus / suspension of which contain secondary metabolites,

identified and isolated by suitable methods. Bahorun et al., (2005) reported abundant

occurrence of polyphenolics in both in vivo and in vitro extracts of Cassia fistula.

According to them these compounds may prove to be very important non-toxic,

antioxidative, chemopreventive agents against various oxidative stressess. Further,

they have isolated and characterized the chemical structures of 12 main

anthraquinone derivatives identified from C.fistula.

Table -2.2 Bioactive secondary metabolites from plant tissue cultures,

reported from 1990 onwards

No. Plant name Active ingredient Culture type Reference

1 Agave amanuensis Saponins Callus Andrijany et al., 1999

2 Ambrosia tenuifolia Altamisine Callus Goleniowski and Trippi, 1999

3 Brucea javanica (L.) Merr

Canthinone alkaloids Suspension Liu et al., 1990

4 Bupleurum falcatum L. Saikosaponins Root Kusakari et al., 2000

5 Camellia sinensis Theamine, -glutamyl derivatives

Suspension Orihara and Furuya, 1990

6 Canavalia ensiformis L Canavanine Callus Ramirez et al., 1992

7 Capsicum annuum L . Capsaicin Suspension Johnson et al., 1990

8 Cassia acutifolia Anthraquinones Suspension Nazif et al., 2000

Review of Literature 39

9

Catharanthus roseus Indole alkaloids Suspension Decendit,et al., 1992;

10 Catharanthus roseus Catharanthine Suspension Zhao et al., 2001b

11 Chrysanthemum

cinerariaefolium

Pyrethrins Callus Rajasekaran et al., 1991

12 Cinchona robusta Robustaquinones Suspension Schripsema et al., 1999

13 Cruciata glabra Anthraquinones Suspension Dornenburg and Knorr, 1996

14 Dioscorea doryophora Hance

Diosgenin Suspension Huang et al., 1993

15 Ephedra spp. L- Ephedrine Suspension O’Dowd et al., 1993

16 Eriobotrya japonica Triterpenes Callus Taniguchi et al., 2002

17 Gentiana sp Secoiridoid glucosides Callus Skrzypczak et al., 1993

18 Ginkgo biloba Ginkgolide A Suspension Carrier et al., 1991

19 Glehnia littoralis Furanocoumarin Suspension Kitamura et al., 1998

20 Glycyrrhiza glabra var. glandulifera

Triterpenes Callus Ayabe et al., 1990

21 Isoplexis isabellina Anthraquinones Suspension Arrebola et al., 1999

22 Linum flavum L 5-Methoxy podophyllotoxin

Suspension Uden et al., 1990

23 Lithospermum

erythrorhizon

Shikonin derivatives Suspension Fukui et al., 1990

24 Mentha arvensis Terpenoid Shoot Phatak and Heble, 2002

25 Morinda citrifolia Anthraquinones Suspension Bassetti et al., 1995

26 Mucuna pruriens L-DOPA Suspension Wichers et al., 1993

27 Ophiorrhiza pumila Camptothecin related alkaloids

Callus Kitajima et al., 1998

28 Panax notoginseng Ginsenosides Suspension Zhong and Zhu, 1995

40 Chapter II

29 Papaver somniferum Morphine, Codeine Suspension Siah and Doran, 1991

30 Polygala amarella Saponins Callus Desbene et al., 1999

31 Polygonum hydropiper Flavonoids Suspension Nakao et al., 1999

32 Rauwolfia sellowii Alkaloids Suspension Rech et al., 1998

33 Scutellaria columnae Phenolics Callus Stojakowska and Kisiel, 1999

34 Solanum chrysotrichum (Schldl.)

Spirostanol saponin Suspension Villarreal et al., 1997

35 Silybum marianum Flavonolignan Root Alikaridis et al., 2000

36 Solanum paludosum Solamargine Suspension Badaoui et al., 1996

37 Tabernaemontana

divaricata

Alkaloids Suspension Sierra et al., 1992

38 Taxus spp. Taxol Suspension Wu et al., 2001

39 Taxus baccata Taxol baccatin III Suspension Cusido et al., 1999

40 Rauwolfia serpentina x Rhazya stricta Hybrid plant

3-Oxo-rhazinilam Callus Gerasimenko et al., 2001

41 Rhus javanica Gallotannins Root Taniguchi et al., 2000

42 Ruta sp. Acridone and Furoquinoline alkaloids and coumarins

Callus Baumert et al., 1992

43 Salvia miltiorrhiza Lithospermic acid, Rosmarinic acid

Callus Morimoto et al., 1994

44 Torreya nucifera var. radicans

Diterpenoids Suspension Orihara et al., 2002

45 Withaina somnifera Withaferin A Shoot Ray and Jha, 2001

46 Gossypium arboreum

and G.hirssutum

Flavonoids Callus Chaturvedi et al., 2010

Review of Literature 41

47 Juniperus chinensis Podophyllotoxin Immobilized cell cultures and suspension cultures

Premjet et al., 2002;

Premjet and Tachibana, 2004

48 Datura metel Alkaloids Hairy root, callus

George et al., 2007

49 Datura stramonium Tropane alkaloids, Scopolamine, Hyoscyamine

Hairy root cultures

Maldonado-Mendoza

et al., 1992,1993,1995.

50 Ricinus communis Quercetin, ricinine Callus Khafagi, 2007

51 Azadirachta indica Azadirachtin Callus W’ewetzer,1998

2.11 Studies on Different Species of Indigofera

The genus Indigofera is having most of the species medicinally important. In

recent years a lot of studies had been undertaken to give a scientific basis to the use

of various species of Indigofera in traditional medicine.

Studies conducted with the ethanol extract of I.aspalathoides showed a

significant antiarthritic effect against complete Freaud’s adjuvant induced arthritis

(Rajkapoor, 2009). Christina et al., (2003) on testing the ethanolic extract of

I.aspalathoides in Swiss albino mice concluded that the plant is having protective

effect against Dalton’s ascitic lymphoma. Similarly, Rajkapoor et al., (2004, 2005)

reported that ethanol extract of I.aspalathoides possesses antitumor activity against

transplantable tumors and carcinogen. They have identified that the alcoholic extract

of I.aspalathoides is highly effective against Mycobacterium tuberculosis. By their

experimental studies they have confirmed that the ethanolic extract was cytotoxic to

HEP-2, HBL-100 and He La cancer lines (Rajkapoor et al., 2007). The methanolic

extract of I.tinctoria was reported to show significant decrease in blood glucose level

of rabbits in which diabetes was introduced artificially (Verma et al., 2009).

A new compound, flavone glycoside (5,4- Dihydroxy 6,8-dimethoxy 7-0

rhamnosyl flavonol isolated from the stem of I.aspalathoides and its structure was

identified by colour reactions and spectral analysis. Testing the compound against 56

42 Chapter II

human tumour cell lines showed that it is cytotoxic against human tumour cell lines

(Balasubramanian et al., 2007).

Acetone extracts of five Indigofera species of Burkina Faso, namely, I.colutea

(Burm.) Murril, I.macrocalyx Guilld et Perr, I.nigritana Hook F., I.pulchra Guilld.

and I.tinctoria L. evaluated for their antioxidant potentials using ferric reducing

antioxidant power, 2-2-diphenyl-1-picrylhydrazyl and 2, 2-azinobis assays demonstrated

that flavonoids, saponins, quinones sterols / triterpenes and tannins were present in

all these species except I.pulchra where quinones were not found. Gallic acid,

Caffeic acid, rutin and myricetin in I.colutea; Gallic acid, quercitrin, myricetin in

I.tinctoria, galangin and myricetin in I.macrocalyx were identified. Further, the

I.colutea, I.tinctoria, I.nigritana and I.macrocalyx were reported to have the highest

phenolic content and found to possess the best antioxidant activities (Bakasso et al.,

2009).

Muthulingam et al., (2008) reported that oral administration of Indigofera

tinctoria (250-500 mg/Kg body weight) and silimarin to paracetamol treated rats

brought back the liver cells to near normal conditions, showing hepatoprotective

activity. The liver cells of the paracetamol treated rats showed fatty changes,

necrosis, vacuoles, space formation and loss of boundaries. Application of the

extracts of I.tinctoria was found to minimize the deleterious effects generated by the

hepatotoxin paracetamol. Indirubin, an isolated active compound from I.tinctoria

was found to be active against chronic myelocytic Leukemia (Han, 1991). Treatment

of HCT-116 cell lines with various concentrations of the methanol extract of

I.tinctoria were found to show a dose dependant rate of inhibition of growth and

induction of apoptosis. The result indicated that I.tinctoria may be a potential

candidate in the field of anticancer drug discovery (Magesh et al., 2009).

Analysis of the various plant parts of I.tinctoria at different growth stages have

shown that the total rotenoid contents decreased with age, maximum content was in

the leaves and minimum in the stem. Six rotenoids (deguelin, dehydrodeguelin,

rotenol, rotenone, tephrosin and sumatrol) were isolated, identified and qualified. Of

the six, only four were identified from eight week old callus cultures; sumatrol and

tephrosin were absent. The toxicological studies of the in vitro and in vivo extracts

Review of Literature 43

against the pulse beetle and mosquito showed that the in vivo rotenoid were more

effective against mosquito larvae, while the callus extract was found more effective

against both the animals tested. It is assumed that the higher bioefficacy of callus

extract may be due to various combinations of ratios of active rotenoids (Kamal and

Mangla, 1993).

Phytochemical screening of the ethanolic extract of I.tinctoria revealed the

presence of alkaloids, flavonoids, carbohydrates, glycosides, tannins, terpenoids,

phenols and the absence of saponins and steroids. Studies conducted in male albino

mice proved that, the flavonoids and tannins present in the extracts were responsible

for the analgesic activity of I.tinctoria. The study confirmed the analgesic effect

attributed to the plant in folklore medicine (Saravanakumar et al., 2009).

Purified indirubin obtained from methanol extract of I.tinctoria exhibited

inhibitory effect on MCF-7 breast cancer cells. Further, the concentration of

indirubin and the incubation time were reported to be the essential factors on the

cytotoxicity of MCF-7 cells (Aobechey et al., 2007).The antimicrobial activity of

I.tinctoria was found to be high against P.aeruginosa, Staphylococcus aureus and

B.subtilis as per the study reported by Selvakumar and Karunakaran (2010).

Fractionation of the ethyl acetate extract of Indigofera daleoides used for

treatment of diarrhoea by the traditional healers, led to the isolation of two pure

compounds identified as compound-1(6,2-0-glucopyranose) and compound-2 with a

novel skeleton (6,3’,4’- trihydroxyflavonoyl - β- D- O- glucopyranosides). Testing of

these two flavonoid compounds for antibacterial activity demonstrated that both have

bioactivity with compound -2 showing more activity (Mathabe et al., 2009).

Chatterji and Dutt (1937) had identified two compounds, namely, indigoferin

and enneaphyllin- two unsaturated hydrocarbons from the alcoholic and water extract

of I.enneaphylla. Majak et al., (1992) isolated, identified and characterized the

compounds, three 3- nitro propanoyl esters of glucose. The alcoholic extracts of

aerial parts of I.enneaphylla Linn. formed into an ointment that applied on two types

of wound models in rats, namely, excision wound model, and incision wound model

showed significant response in both types of wound models comparable to those of a

44 Chapter II

standard drug, nitrofurazene in terms of wound contracting ability, wound closure

time and tensile strength (Hemalatha et al., 2001).

Bharal and Rashid (1984) reported regeneration of complete plants from free

cell derived colonies of I.enneaphylla. According to them the factors such as plating

density, composition of the nutrient medium and the concentration of CO2 were

found essential for the growth of free cells.The methanolic extract of I.truxillensis

exhibited mutagenic activity in some strains of Salmonella typhi-murium. The

flavonoid and alkolid fractions of I.truxillensis and I.suffruticosa showed

mutagenesity. Chemical analysis of flavanoid fractions of both species resulted in

identification of kaempferol, quercetin and their derivates. The alkaloid fractions of

both species contained indigo and indirubin. Indigo was found to be the main

component responsible for the mutagenic effect (Calvo et al., 2009).

The flavonoids present in the methanol extract of the aerial parts of

I.truxillensis was found to be inducing a gastro protective action in several models of

gastric lesions in mice and rats. It is also reported that the methanol extract is not

toxic and protects, in a dose dependent way, the gastric mucosa from ethanol induced

ulcers at all doses. As a result the methanol extracts is found to have antiulcerogenic

activity (Cola- Miranda et al., 2006).

Oral administration of the ethanol extract of I.trita was found effective in

reducing solid tumour mass development induced by Ehrlich Ascites carcinoma

(EAC) cells in mice (Senthil Kumar et al., 2007).

Studies made on the effects of normal butanol portion of I.pulchra leaf extracts

of blood glucose levels of alloxan- induced diabetic and normo glycemic winstar rats

showed that the dosage of the extracts had significant hypoglycemic and

antihyperglycemic effects (Tanko et al., 2008). In vitro screening tests conducted

with crude methanol as well as ethyl acetate and - n-butanol fractions from the leaves

of I.pulchra against S.aureus, Bacillus subtilis, P.aeruginosa and E.coli

demonstrated that the crude methanol extract and n- butanol fraction has antibacterial

activity against all the organisms except E.coli, while the ethyl acetate fraction have

activity against P.aeruginosa only. Phytochemical screening revealed that the crude

Review of Literature 45

extracts and n-butanol fraction contains tannins, flavonoids and saponins, while the

ethyl acetate fraction contains tannins and flavonoids only (Musa et al., 2007).

Arriaga et al., (2008) identified nine chemical components from the essential

oil of I.microcarpa. The ethanolic extract of I.oblongifolia was reported to show

anticancer activity against FL-cells (Ali et al., 2001). High intake of the creeping

indigo, Indigofera spicata, by animals was found to be toxic to animals like rabbits.

Its leaves and seeds contain the toxin β-nitropropionic acid (Collectanea, 1989).

From the literature survey given above, it is seen that only one report is there

regarding the antibacterial activity of I.tinctoria; while no study was reported

regarding the antibacterial activity of I.enneaphylla and I.aspalathoides. Further even

in the case of I.tinctoria only a preliminary study alone was done. Hence, in the

present study experiments are designed to assess the antibacterial activity of

I.tinctoria I.enneaphylla and I.aspalathoides using in vitro screening tests. With

regard to tissue culture studies only one report is there about the regeneration of

plants from suspension in I.enneaphylla. No reports are there regarding the

antibacterial activity of in vitro induced callus in these three species. Hence in the

present study in vitro culture studies are conducted to develop a standardized

technology for production of callus/ suspension, from which antibacterial compounds

can be isolated. Identification of the antibacterial compounds from these species will

enhance the utilization of plant resources, which in turn, will boost the interest of

agriculturists to cultivate the plants. In such a situation, sufficient number of planting

materials can be made available only through micropropagation. With this view,

experiments were also devised to develop a technology for micropropagation of the

three species.