2.0. Review of literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/20814/8/08_chapter...

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2.0. Review of literature

2.1. Secondary metabolites from plants

Secondary metabolites are generally derived through modifications

from primary metabolites, such as glycosylation, methylation and

hydroxylation. Secondary metabolites are structurally complex and are

classified on the basis of composition (presence or absence of nitrogen),

chemical structure (e. g., sugar, aromatic rings), the pathway by which

they are synthesized and their solubility in various solvents. They are

categorized into terpenes (composed of carbon and hydrogen), phenolics

(presence of benzene rings, oxygen and carbon, simple sugars) and sulfur

and/or nitrogen containing compounds (Chinou, 2008) (Table 2.1). Each

plant species, genus and family produce a mix of these metabolites.

Terpenes Phenols Sulfur and/or nitrogen

containing compounds

Monoterpenes: Limonene Phenolic acids: Caffei Alkaloids: nicotine

Sesquiterpene: Farnesol Coumarins: Umbelliferone Glucosinolates: sinigrin

Diterpenes: Taxol Lignans: podophyllin

Triterpenes, cardiac glycosides: Flavonoids: anthocyanin

Digitogenin

Tetraterpenoids: Carotene Tannins: gallotannin

Sterols: Spinasterol Lignin: lignin

Table 2.1. Classification of secondary metabolites

11

2.1.1. Terpenoids

Terpenoids, terpenes or Isoterpenes are polymers or trimers, dimers

of Isoprene units, which are usually joined by a head and tail fashion. In

plants building block of each type of terpenoid in the active form of the

Isoprene unit (Isopentanyl pyrophosphate) is synthesized either by the

methyl erythritol phosphate pathway (eg. mono and diterpenoids) or the

mevalonoic acid pathway (eg. sesquiterpenoids) (Taiz and zeiger, 2006).

Isoprene unit

Fig. 2.1. Main pathway leading to secondary metabolites

(Taiz and Zeiger, 2006)

12

The Isoprene units usually condense to form ring compounds or

linear ring commonly containing carbon atom numbers of 30 (the

triterpenoids), 20 (the diterpenoids), 15 (the sesquiterpenoids) or 10 (the

monoterpenoids).

Monoterpenoids

Essential oils commonly contain monoterpenoids e.g., Pyrethrins and

Iridoids. Pharmaceutical properties of monoterpenes range from

analgesic to anti-inflammatory and they are widely used as insecticides.

α-Pinene β-Pinene Linalool Menthol Borneol 1,8-cineol

Fig. 2.2. Monoterpenes commonly found in essential oils

Sesquiterpenes

Many plant essential oils contain sesquiterpenes e. g., caryophyllene,

bisabolol, humulene. Sesquiterpene lactones occur in families like

Asteraceae.

Sesquiterpene compounds possess epoxides and α-methylene-γ-

lactone moiety, having a broad range of acivities. Their pharmaceutical

activities include antifungal, antibacterial, antimalarial, molluscicidal

13

and anthelminitic, e.g., Santonin, which is used as antimalarial and

anthelmintic (Gurib Fakim, 2006).

CH3

CH3

O

O

CH3

O

Fig. 2.3. α-Santonin

Diterpenes

Diterpenes are present in plants and have therapeutic applications

e.g., anticancer drug taxol and its derivatives. Forskolin is another

example, which has antihypertensive activity, Steroside is a sweetening

agent while Zoapatanol is an abortifacient (Gurib Fakim, 2006).

Triterpenoids

C30 compounds are triterpenoids arising from the cyclization of

squalene. Steroids are included in triterpenoids and they are structurally

diverse compounds (Taiz and Zeiger, 2006).

Steroids contain a ring system of one five membered and three six

membered rings. Many natural steroids together with a considerable

number of semisynthetic and synthetic steroidal compounds and owing

to the profound biological activities are employed in medicine (e.g.,

mammalian sex hormones, corticosteroid hormones, steroidal saponins

14

and cardioactive glycosides). The pharmaceutical applications of steroids

and triterpenes are considerable (Gurib Fakim, 2006).

2.1.2. Phenolic compounds

All phenolic compounds contain various attached substituted groups

such as methoxy (-O-CH3) and hydroxyl groups, an aromatic ring and

often other non-aromatic ring structures.

Phenolic compounds are synthesized via the shikimic acetate or acid

pathway and subsequent reactions. Phenolic compounds have wide

range of pharmaceutical activities such as antitumor, anti HIV,

analgestic, anti-inflammatory, antihepatic, antioxidant, antilipopolytic,

antiulcerogenin, immunostimulant and vasodilatory. Phenolic

compounds serve as an effective defence against herbivores by plants

(Wink 1999 and Gurib Fakim, 2006).

2.1.3. Flavonoids

Flavonoids are generally distributed throughout the plant kingdom

with 15 carbon compounds. From plants, more than 2000 flavonoids

have been identified (Taiz and Zeiger, 2006). Flavonoids are responsible

for the colour of fruits, flowers and sometimes leaves. Acting as a co

15

pigment some may contribute to the colour. The Latin word ‘flavus’

meaning yellow refers the name flavonoid.

Chalcones Dihydroflavonols Flavonones

Flavones Flavonols Isoflavonoids

Fig. 2.4. Basic structures of some flavonoids

Flavonoids play a role in attracting animals with their colours in the

pollination and protect the plant from UV damaging effects (Gurib Fakim,

2006). The basic structure of flavonoids is 2-phenyl chromane skeleton.

Flavonoids are derived from a combination of shikimate acetate or acid

pathways biosynthetically. Flavonoids can either occur as -O-C-

glycosides or as aglycones (Gurib Fakim, 2006).

16

2.1.4. Nitrogen containing compounds

Plant secondary metabolites in a large variety, have nitrogen in their

structures, alkaloids and cyanogenic glycosides included in this category

contain well known antiherbivore compounds.

2.1.5. Alkaloids

Alkaloids are defined as cyclic organic compounds which have limited

distribution in living organisms. It contains nitrogen in a negative

oxidation state (Taiz and Zeiger, 2006). Alkaloids are divided into several

subgroups, according to their basic ring structure. The example of a

bisbenzylisoquinoline alkaloid is tetrandrin, solasodine is a triterpene

alkaloid while a non-heterocyclic or pseudoalkaloid is mescaline (Gurib

Fakim, 2006).

Mescaline Solasodine Tetrandrin

Fig. 2.5. Structure of some alkoloids

Alkaloids are pharmaceutically significant e.g., L - hyoscyamine as

antispasmodic and for pupil dilation, quinine as an antiarrythmic,

codeine in the treatment of cough and pain, colchicine in the treatment

of gout and morphine as a narcotic analgesic.

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2.1.6. Cyanogenic glycosides

Cyanogenic glycosides are defence-related secondary metabolites.

They are themselves toxic and they are voluntarily broken down to give

off volatile poisons when the plant is crushed. Hydrogen cyanide and

respiratory poisonous gases are released by the well known cyanogenic

glycosides.

All plants produce secondary metabolites, which are often specific to

an individual genus or species and environmental conditions. Compared

with primary metabolites, secondary metabolites (pesticides,

pharmaceuticals, fragrance and flavors) are considered high value and

they are fine chemicals with their cost ranging from $500 to $10000 per

kg (Ravishankar and Rao, 2000; Balandrin et al., 1985) (Table 2.2).

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Product Plant species Cost in US $ / Kg

Application

Ajmalicine Catharanthus roseus 37, 000 Antihypertensive

Berberine Coptis japonica 3250 Antihypertensive

Camptothecin Camptotheca

acuminate

432, 000 Antitumour

Colchicine Colchicum autumnale 35, 000 Antitumour

Digoxin Digitalis lanata 3000 Heart stimulant

Ellipticine Orchrosia elliptica 240, 000 Antitumour

Metformin Galega officinalis 1000 Antidiabetic

Morphine Papaver somniferum 340, 000 Sedative

Shikonin Lithospermum

erythrorhizon

4500 Antibacterial

Taxol Taxus brevifolia 600, 000 Anticancer

Vincristine Catharanthus roseus 2, 000, 000 Antileukemic

Vinblastine Catharanthus roseus 1, 000, 000 Antileukemic

(Ravishanker and Rao (2000)

Table 2.2. Cost estimation of plant derived secondary metabolites of

commercial importance in pharmaceutical industry

Many secondary metabolites have been characterized and their

mode of action has been determined. The mode of action of some of the

secondary metabolites has been depicted in table (Table. 2.3).

19

Class Example Compounds

Example sources

Some Effects and uses

Nitrogen- Containing

Alkaloids nicotine, cocaine, theobromine

tobacco, coca plant chocolate (cocao)

interferes with neurotransmission blocks enzyme action

Nitrogen & Sulfur-containing

TERPENOIDS Monoterpenes menthol,

linalool mint and relatives

interferes with neurotransmission, block ion transport

Sesquiterpenes Parthenolide Parthenium and relatives (Asteraceae)

contact dermatitis

Diterpenes Gossypol Cotton blocks phosphorylation; toxic

Triterpenes, cardiac glycosides

Digitogenin Digitalis (foxglove)

stimulate heart muscle, alter ion transport

Tetraterpenoids Carotene many plants antioxidant; orange coloring

Terpene polymers Rubber Hevea (rubber) trees, dandelion

gum up insects; airplane tires

Sterols Spinasterol Spinach interferes with animal hormone action

PHENOLICS Phenolic acids caffeic,

chlorogenic all plants causes oxidative damage

browning in fruits and wine

Coumarins Umbelliferone carrots, parsnip cross-link DNA, block cell division

Lignans podophyllin urushiol

mayapple, poison ivy

cathartic, vomiting, allergic dermatitis

Flavonoids anthocyanin, catechin

almost all plants flower, leaf color, inhibit enzymes, anti- and pro-oxidants, estrogenic

Tannins gallotannin, condensed tannin

oak, hemlock trees, birdsfoot trefoil, legumes

bind to proteins, enzymes, block digestion, antioxidants

Lignin Lignin all land plants structure, toughness,fiber (Perumal Samy and Gopalakrishnakone, 2007)

Table 2.3 : List of the important secondary metabolites and their mode of action

20

Table 2.4 represents the plant derived ethnotherapeutics and their

role in traditional modern medicine.

S. No. Drug Basic investigation

1. Codeine,

morphine

Opium the latex of Papaver somnifrum used by

ancient Sumarians. Egyptians and Greeks for the

treatment of headaches, anthritis and sleep.

2. Atropine,

hyoscyamine

Atropa belladonna, Hyoscyamus niger etc., were

important drugs in Babylonium folklore.

3. Ephedrine Crude drug (astringent yellow) derived from

Ephedra sinica had been used by Chinese for

respiratory ailments since 2700 BC

4. Quinine Cinchona sp were used by Peruvian Indians for the

treatment of fevers

5. Emetine Brazilians, Indians and several others South

American tribes used root and rhizomes of

Cephaelis sp to treat vomiting and crude dysentry.

6. Colchicine Use of Colchicum in the treatment of gout has been

known in Europe since 78 AD.

7. Digoxin Digitalis leaves were being used in heart therapy in

Europe during the 18th century.

(Perumal Samy and Gopalakrishnakone, 2007)

Table 2.4 : Plant derived ethnotherapeutics and their role in

traditional modern medicine

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The important medicinal plants used for major modern drugs for

cancer has been represented in Table 2.5.

(Perumal Samy and Gopalakrishnakone, 2007)

Table 2.5. Some of the important medicinal plants used for

chemotherapy for cancer

Drugs discovered for ethnobotanical leads have been characterized

and their mechanism of action or clinical use has been determined

(Table 2.6).

Plant

name/family

Drugs Treatment

Cathranthus

roseus L.

(Apocynaceae)

Vinblastine and vincristine Hodgkins,

Lymphosarcomas and

children leukemia.

Podophyllum

emodi Wall.

(Beriberidaceae)

Podophyllotoxin Testicular cancer, small

cell lung cancer and

lymphomas

Taxus brevifolia

(Taxaceae)

Paciltaxel, taxotere Ovarian cancer, lung

cancer and malignant

melanoma.

Mappia foetida

Miers.

Comptothecin, lrenoteccan

and topotecan

Lung, ovarian and

cervical cancer.

Comptotheca

acuminate

Quinoline and

comptothecin alkaloids

used in Japan for the

treatment of cervical

cancer

Juniperus

communis L.

(Cupressaceae)

Teniposide and etoposie Lung cancer

22

Plant source Drug Action or clinical use

Digitalis lanata Acetyldigoxin Cardiotonic

Rauwolfia serpentina

Ajmaline Circulatory disorders

Filipendula ulmaria Aspirin Analgesic, anti-inflammatory

Atropa belladonna Atropine Anticholinergic

Ardisia japonica Bergenin Antitussive

Ananas comosus Bromelain Anti-inflammatory, proteolytic agent

Camellia sinensis Caffeine Stimulant

Potentilla fragaroides

(+)-Catechin Haemostatic

Erythoxylum coca Cocaine Local anaesthetic

Papaver somniferum

Codeine Analgesic, antitussive

Colchicum autumnale

Colchicine Antitumor agent, antigout

Cassia spp. Danthron Laxative

Rawvolfia canescens

Deserpidine Antihypertensive, tranquilizer

Digitalis purpurea Digotoxin Cardiotonic

Digitalis purpurea Digoxin Cardiotonic

Cephaelis ipecacuanha

Emetine Amoebicide, emetic

Ephedra sinica Ephedrine sympathomimetic

Podophyllum peltatum

Etoposide Antitumor agent

Digitalis purpurea Gitalin Cardiotonic

Gossypium spp. Gossypol Male contraceptive

Hydrastis canadensis

Hydrastine Hemostatic, astringent

Hyoscyamus niger Hyoscyamine Anticholinergic

Piper methysicum Kawain Tranquilizer

Ammi visnaga Khellin Bronchodilator

Lobelia inflata Lobeline Smoking, deterrent, respiratory stimulant

Papaver somniferum

Morphine Analgesic

Papaver somniferum

Noscapine Antitussive

Sthrophanthus gratus

Ouabain Cardiotonic

Cont….

23

Plant source Drug Action or clinical use

Carica papaya Papain Proteolytic, mucolytic

Physostigma venenosum

Physostigmine Cholinesterase inhibitor

Anamirta cocculus Picrotoxin Analeptic

Pilocarpus jaborandi

Pilocarpine parasympathomimetic

Veratrum album Protoveratrines A & B Antihypertensive

Ephedra sinica Pseudoephedrine Sympathomimetic

Cinchona ledgeriana

Quinine Antimalarial

Quisqualis indica Quisqualic acid Anthelmintic


Rescinnamine Antihypertensive, tranquilizer


Reserpine Antihypertensive, tranquilizer

Rorippa indica Rorifone Antitussive

Lonchocarpus nicou Rotenone Piscicide

Salix alba Salicin Analgesic

Stevia rebaudiana Stevioside Sweetener

Podophyllum peltatum

Teniposide Antitumor agent

Corydalis ambigua Tetrahydropalmatine Analgesic, sedative

Theobroma cacao Theobromine Diuretic, bronchodilator

Thymus vulgaris Trichosanthin Abortifacient

Chondodendron tomentosum

Tubocurarine Skeletal muscle relaxant

Vinca minor Vincamine Cerebral stimulant

Ammi majus Xanthotoxin Leukoderma, vitiligo

Pausinystalia yohimbe

Yohimbine Aphrodisiac

Daphne genkwa Yuanhuacine Abortifacient

(Fabricant and Farnsworth, 2001)

Table 2.6. Drugs discovery by ethnobotanical leads

24

2.2. Salacia species

Salacia species (Family: Celastraceae) are widely distributed in Sri

Lanka, India, China and other Southeast Asian countries, and many

plants from this genus (e.g., S. reticulata, S. oblonga, and S. prinoides)

have been used for thousands of years in traditional medicines. Salacia

species has been used for the treatment of diabetes, obesity,

rheumatism, gonorrhea and asthma (Jayaweera, 1981; Chandrasena,

1935; Vaidyaratnam, 1996). Several reports from studies in animals have

described Salacia species having hypoglycemic activity, including S.

oblonga (Krishnakumar et al., 1999, Augusti et al., 1995; 2000;

Matsuura et al., 2004), S. macrosperma (Venkateswarlu et al., 1993), S.

prinoides (syn. S. chinensis) (Pillai et al., 1979) and S. reticulata

(Karunanayake et al., 1984; Sirasinghe et al., 1990; Shimoda et al.,

1998; Kumara et al., 2005). Further in human studies, hypoglycemic

activity of herbal preparations containing Salacia species have also been

reported (Kajimoto et al., 2000; Heacock et al., 2005; Collene et al., 2005;

Jayawardena et al., 2005). Deepa et al., 2004 reported that ethyl acetate

extracts of stem and leaf of S. beddomei showed very good antimicrobial

activity against many microbes.

Recently, Salacia species have been extensively consumed in the

United States, Japan, and other countries as a food supplement for the

prevention of diabetes and obesity, as well as being the subject of broad

studies for diabetes management (He et al., 2009; Yuhao et al., 2008).

25

2.2.1. Salacia oblonga Wall.

For at least 4000 years, Salacia plants have been used in the

traditional Ayurvedic system of medicine for the treatment of several

common ailments, including diabetes mellitus (Kowsalya et al., 1995;

Krishnakumar et al., 1999). The root bark of salacia oblonga is used for

the treatment of rheumatism, gonorrhea and skin diseases (Chopra et

al., 1956; Nadkerni, 1976; Kirtikar et al., 1984).

Botanical classification of Salacia oblonga

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsidae

Order : Celastrales

Family : Celastraceae

Genus : Salacia

Species : oblonga

26

Fig. 2.6. Salacia oblonga plant growing in its natural habitat in

Western Ghats, India

Fig. 2.7. Ripe fruit of Salacia oblonga

27

Botanical Description

S. oblonga is a large woody climbing shrub with hairless cylindrical

branchlets, densely sprinkled with lenticels. The leaves are arranged on

the stem opposite (phyllotaxy) with a leaf stalk of about 5-10 mm in

length. The leaves are hairless, oblong in shape, 7-15 x 3-5 cm in size

and possess lateral nerves in 7-9 pairs. The base and apex of the leaf are

acute and the leaf margins are toothed (round or saw like). It produces

flowers and fruits from December to May. Flowers are bisexual, greenish

yellow in color and arranged in axillary clusters of 3-6 together with

small stalks. Its fruits are drupes and are sub-globose or pear shaped

and 5-6cm in diameter. When ripe, the fruit is orange red in color with 1-

6 angled seeds embedded in a fleshy pulp. Salacia oblonga is distributed

in South India and Sri Lanka, the Western Ghats in Maharastra, Tamil

Nadu, Kerala, Goa and Karnataka. It is rarely seen in the Eastern Ghats

of Andhra Pradesh. In Kerala fairly common in Thrissur, Kollam and

Idukki districts, in Karnataka in Kodagu district, in Tamil Nadu reported

only from Coimbatore, Tirunelveli and Nilgiri hills.

2.3. The medicinal Importance of Salacia oblonga Wall.

Matsuda et al., (1999), subjected the ethyl acetate-soluble portion of

S. oblonga to normal phase and reverse-phase silica gel column

chromatography, and finally HPLC, to get the following fractions.

Kotalagenin 16-acetate (1), 26-hydroxy-1,3-friedelanedione (2),

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maytenfolic acid (3), 3b, 22a -dihydroxyolean-12- en-29-oic acid (4), 19-

hydroxyferruginol (5), lambertic acid (6), and (2)-49-

Omethylepigallocatechin (7). The water-soluble portion was also

separated by normal-phase silica gel (SiO2 and NH Chromatorex) column

chromatography and HPLC to give salacinol (8), kotalanol (9), dulcitol

(10), galactinol (11), 3-O-a -D-galactopyranosyl(1→6)-O-b -D-

galactopyranosyl- sn-glycerol (12), raffinose (13) and stachyose (14).

The chemical constituents obtained from ethyl acetate & water

soluble fractions from the extract of Salacia oblonga have been tabulated

(Table. 2.6).

Compound number

Chemical constituents

(1) Kotalagenin 16-acetate

(2) 26-Hydroxy-1,3-friedelanedione

(3) Maytenfolic acid

(4) 3b ,22a -Dihydroxyolean-12-en-29-oic acid

(5) 19-Hydroxyferruginol

(6) Lambertic acid

(7) (2)-49-O-Methylepigallocatechin

(8) Salacinol

(9) Kotalanol

(11) Galactinol

(12) 3-O-a-D-Galactopyranosyl(1→6)-O-b-Dgalactopyranosyl-sn-glycerol

(13) Raffinose

(14) Stachyose

(Matsuda et al., 1999)

Table 2.7. Chemical Constituents of EtOAc extract of S. oblonga

29

O

O

O

OH

C CH3

O

O

O

OH

Kotalagenin 16-acetate (1) 26-Hydroxy-1,3-friedelanedione (2)

HO

OH

COOH

Maytenfolic acid (3) 3b ,22a -Dihydroxyolean-12- en-29-oic acid (4)

HO

OH

HOOC

OH

19-Hydroxyferruginol (5) Lambertic acid (6)

O

OH

OH

OH

OH

HO

OH

(2)-49-Omethylepigallocatechin (7)

S

OH

O

SO3

OH

HO

HO

HO

S

OH

O

SO3

HO

HO

HO

HO

OH

OH

OH

Salacinol (8) Kotalanol (9)

30

H

CH2OH

OH

HO H

HO H

H OH

CH2OH

OH

O

OH

OH

CH2OH

OH

OH

OH

HO

HO

OH

OH

O

OH

OH

HOH2C

OO

OH

H2C

OH

H

H2C

H OH

CH2OH

OH

Dulcitol (10) Galactinol (11) 3-O-a -galactopyranosyl(1→6)-O- b –

D-galactopyranosyl- sn-glycerol (12)

OH

O

OH

OH

HOH2C

OH

OH

H2C

OH OH

O

HOH2C

HO

O

H

CH2OH

OH

OH

Raffinose (13)

OH

O

OH

OH

HOH2C

OH

OH

H2C

OH OH

OHO

OH

OH

H2C

OH

OHO

HO

HOH2C

O

H

CH2OH

OH

OH

Stachyose (14)

Fig. 2.8. Chemical constituents of Salacia oblonga (1-14)

2.3.1. Antidiabetic activity

Salacia oblonga extract, was seen to lower the postprandial

glycemia in rats fed either with sucrose or maltose, which is consistent

with its α-glucosidase inhibitory effect.

Salacinol contains a zwitterion consisting of a sulfonium ion with an

internal sulfate counterion. It is hypothesized that the permanent

positive charge on the sulfur atom in the 1, 4 – anhydro – 4 – thio – D -

arabinitol moiety binds to alpha-glucosidase through mimicry of the

31

shape and charge of the oxacarbenium-ion intermediate in the hydrolysis

reaction mediated by alpha-glucosidase. Kotalanol contains the same

1, 4 – anhydro – 4 – thio – D - arabinitol moiety and is believed to work

via the same mechanism as salacinol.

Heacock et al., and Collene et al., (2005) have demonstrated that S.

oblonga extract reduces postprandial glycemia and insulinemia when it is

fed in addition to a liquid nutritional supplement containing mainly

maltodextrin (61% of available carbohydrate) as the carbohydrate source.

Heacock et al., 2005 measured the effects of varying doses of S. oblonga

extract on postprandial glycemia in 39 healthy subjects.

2.3.2. Anti-inflammatory activity

The anti-inflammatory activity of Salacia oblonga root bark powder

was assayed in male albino rats for cotton pellet granuloma (chronic

inflammation) and carrageenan-induced rat paw oedema (acute

inflammation). Both the crude drugs were maximally active at a dose of

1000 mg/kg (Ismail et al., 1997).

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2.4. Antimicrobial activity

Plants are precious source of pharmaceutical products as they have

an almost infinite capability to produce compounds that expose the

unreliable degree of antimicrobial activity against a range of pathogenic

and opportunistic microorganisms (Cowan, 1999).

During the last decade, the rapidity of expansion of new

antimicrobial drugs has slowed down, while the occurrence of resistance,

particularly multiple resistances, has increased (Clark, 1996). Hence

there is a great requirement for successful antibacterial agents with new

modes of action. Medicinal plants have many traditional claims including

the treatment of infections source ailments, and to confirm this, scientific

research is very significant. The effects of medicinal plant extracts on

pathogenic bacteria have been considered by a large number of

researchers in diverse parts of the world (Chen et al., 2008; Nogueira et

al., 2008; Turner and Usta, 2008; Costa et al., 2008; Prachayasittikul et

al., 2008; Al-Bayati and Al-Mola, 2008; Pesewu et al., 2008). Screening

for active compounds from plants has shown the way to the detection of

many novel medicinal drugs which have competent protection and

treatment roles against a variety of diseases, including Alzheimer’s

disease (Mukherjee et al., 2007) and Cancer (Kumar et al., 2004; Aa and

Mhel, 2008).

33

2.5. The role of solvents in the extraction

Solvents are broadly classified into polar and non-polar solvents. The

solvent classification is based on the polarity in comparison to the

polarity of water (It is indicated that the strong polarity of water at 20oC,

is by a dielectric constant of 80.10) (Lowery, T.H. and Richardson, K.S.,

1987). The solvent dielectric constant provides a rough measure of a

solvent polarity.

These solvents are chemically active, with high dielectric constants,

and form coordinate covalent bonds. The solvent molecules with no

charge and less dielectric constant are generally considered to be non

polar. The non polar compounds form weak bonds and, are chemically

less active. A non polar solvent is normally miscible in organic solutions,

where as a polar solvent in water.

For preparation of plant extracts, two types of solvents polar and non

polar are used. Plant material contains organic compounds both polar

and non polar along with water and salts. Polar organic compounds are

soluble in polar solvents like ethyl acetate and ethanol, whereas non

polar compounds are soluble in chloroform, petroleum ether and hexane.

The best way is to extract the non polar compounds first by using a non

polar solvent and then extract the polar compounds by using the polar

solvent.

34

Solvent

formula

MWa BPb

(°C)

MPc

(°C)

Density

(g/mL)

Solubility

in water

(g/100g)

Dielectric

Constant

Flash

Point

(°C)

Chloroform CHCl3 119.38 61.7 -63.7 1.498 0.795 4.81 --

diethyl ether C4H10O 74.12 34.6 -116.3 0.713 7.5 4.34 -45

ethanol C2H6O 46.07 78.5 -114.1 0.789 Miscible 24.6 13

ethyl acetate C4H8O2 88.11 77 -83.6 0.895 8.7 6(25) -4

hexane C6H14 86.18 69 -95 0.659 0.014 1.89 -22

Methanol CH4O 32.04 64.6 -98 0.791 Miscible 32.6(25) 12

Water H2O 18.02 100 0.00 0.998 -- 78.54 --

aMolecular weight, bBoiling point, cMelting point

(Vogel's Practical Organic Chemistry, 5th ed.)

Table 2.8. Common Solvents: Table of Properties

2.6. Microorganisms used in the study

2.6.1. Bacillus subtilis

Plate 2.1: Bacillus subtilis

Scientific classification

Kingdom : Bacteria

Phylum : Firmicutes

Class : Bacilli

Order : Bacillales

Family : Bacillaceae

Genus : Bacillus

Species : subtilis

35

Morphology

Bacillus subtilis is also known as the grass bacillus or hay bacillus,

commonly found in soil as sporulating, rod-shaped, gram positive

bacteria (Madigan and Martinko, 2005). The colonies are irregular flat

and dry, with lobate margins.

Distribution

B. subtilis is commonly found in decomposing plant residue, soil, air

and water.

Pathogenesis

B. subtilis generally contaminates food and is responsible for causing

rapines, a stringy and sticky consistency in the spoiled bread dough. But

rarely causes food poisoning (Ryan and Ray, 2004).

2.6.2. Enterococcus faecalis

Plate 2.2: Enterococcus faecalis

36


Kingdom : Bacteria

Phylum : Firmicutes

Class : Bacilli

Order : Lactobacillales

Family : Enterococcaceae

Genus : Enterococcus

Species : faecalis

Morphology

E. faecalis a gram positive (Ryan and Ray, 2004), facultatively

anaerobic, coccus, non motile. It forms pinpoint colonies with convex

elevation.

Distribution

It occurs as commensal in the gastrointestinal tract of human and

other mammals (Ryan and Ray, 2004).

Pathogenesis

E. faecalis which is life-threatening in humans is listed as the first to

the third leading cause of nosocomial infections. From the patient's

intestinal flora, many infecting strains originate. E. faecalis commonly

causes urinary tract infections, endocarditis, bacteremia, wound

infections, catheter-related infections and intra-abdominal and pelvic

infections. Pleural space infections, meningitis, and skin and soft-tissue

infections have also been reported, as well as bladder, epididymal and

37

prostate infections (Ryan and Ray, 2004; Pelletier, 1996). Nervous system

infections are less common.

2.6.3. Staphylococcus aureus

Plate 2.3: Staphylococcus aureus


Domain : Bacteria

Kingdom : Eubacteria

Phylum : Firmicutes

Class : Bacilli

Order : Bacillales

Family : Staphylococcaceae

Genus : Staphylococcus

Species : aureus

Morphology

Staphylococcus aureus is a gram positive coccus, spherical,

facultatively anaerobic bacterium, which appears as grape-like clusters

when viewed through a microscope. Staphylococcus aureus culture

shows circular, pinhead colonies which are convex with entire margins.

38

Often produces colonies which have a golden-brown color (Kluytmans et

al., 1997; Ryan and Ray, 2004).

Distribution

As a part of the skin flora found on the skin and the nose of a human

(Whitt et al., 2002), it is commonly found in soil.

Pathogenesis

It causes most common infections like staph infections, skin

infections such as folliculitis, pimples, boils (furuncles), cellulitis,

impetigo, carbuncles, abscesses and scalded skin syndrome. Also causes

life-threatening diseases such as bacteremia, pneumonia, meningitis,

endocarditis, osteomyelitis, toxic shock syndrome (TSS), nosocomial

infections, sepsis, post surgical wound infections and pneumonia. Its

incidence is from soft tissue, skin, respiratory, joint, bone, endovascular

to wound infections. In infants S. aureus infection can cause a severe

disease Staphylococcal scalded skin syndrome (SSSS) (Curran and Al-

Salihi, 1980).

39

2.6.4. Staphylococcus epidermidis

Plate 2.4: Staphylococcus epidermidis


Kingdom : Bacteria

Phylum : Firmicutes

Class : Cocci

Order : Bacillales

Family : Staphylococcaceae

Genus : Staphylococcus

Species : epidermidis

Morphology

S. epidermidis is gram-positive cocci, non-motile arranged in grape-

like clusters. It forms white, circular, pinhead colonies which are convex

with entire margins raised.

Distribution

S. epidermidis occurs as a part of the human skin flora, mucous

membrane and in animals (Queck and Otto, 2008).

40

Pathogenesis

S. epidermidis may cause both nosocomial or community acquired

infections. In patients with defective heart valves and sepsis in hospital

patients it also causes endocarditis. With implanted plastic device when

contaminated with this organism, infection can also occur in dialysis

patients (Otto M, 2009).

2.6.5. Listeria monocytogenes

Plate 2.5: Listeria monocytogenes


Domain : Bacteria

Kingdom : Eubacteria

Phylum : Firmicutes

Class : Bacilli

Order : Bacillalles

Family : Listeriaceae

Genus : Listeria

Species : monocytogenes

41

Morphology

L. monocytogenes is a gram positive, motile, non-sporing, rod shaped,

facultatively anaerobic bacterium (Ramaswamy, 2007; Grundling , 2004).

Distribution

L. monocytogenes are generally found in soil and other environmental

sources. It has been associated with foods such as cheeses, raw

vegetables, pasteurized fluid milk (Fleming, 1985), fermented raw-meat

sausages, raw milk, raw and cooked poultry, ice cream, raw and smoke

fish and raw meats. Human gastrointestinal tracts may be colonized by

L. monocytogenes.

Pathogenesis

Listeria monocytogenes is one of the most virulent food borne

pathogens and a facultative intracellular bacterium, leading to death. It

commonly causes gastrointestinal infection symptoms including

diarrhea, nausea and vomiting. It is the main causative agent of

Listeriosis in adult patients resulting in symptoms of infections including

pneumonia (Whitelock-Jones et al., 1989), encephalitis (Armstrong and

Fung, 1993), Meningitis (or Meningo encephalitis), septicemia (Gray and

Killinger, 1966), corneal ulcer (Holland et al., 1987) and intrauterine or

cervical infections in pregnant women.

42

2.6.6. Enterobacter aerogenes

Plate 2.6 : Enterobacter aerogenes


Kingdom : Bacteria

Phylum : Proteobacteria

Class : Gamma proteobacteria

Order : Enterobacteriales

Family : Enterobacteriaceae

Genus : Enterobacter

Species : aerogenes

Morphology

This is a gram negative, rod shaped bacteria and forms shiny colonies

with convex elevation in entire margins.

Distribution

E. aerogenes is a common contaminant of vegetable matter. It is

generally found in the human gastrointestinal tract. It has been found to

live in various hygienic chemicals, wastes and soil.

43

Pathogenesis

E. aerogenes is a pathogenic bacterium and a nosocomial that causes

opportunistic infections including lower respiratory tract infections,

urinary tract infections (UTIs), endocarditis, skin and soft-tissue

infections, intra-abdominal infections, osteomyelitis, septic arthritis and

ophthalmic infections and bacteremia (Sanders and Sanders, 1997).

2.6.7. Enterobacter cloacae

Plate 2.7. Enterobacter cloacae


Kingdom : Bacteria


Class : Gammaproteobacteria



Genus : Enterobacter

Species : cloacae

Morphology

Enterobacter cloacae is a gram-negative, rod-shaped facultative-

anaerobic bacterium.

44

Distribution

E. cloacae is found to live in soil and wastes.

Pathogenesis

Enterobacter species, particularly Enterobacter cloacae are responsible

for various infections and important nosocomial pathogens, including

bacteremia, endocarditis, lower respiratory tract infections, respiratory

tract infections, skin and soft-tissue infections, urinary tract

infections (UTIs), osteomyelitis, intra - abdominal infections, septic and

ophthalmic infections (Sanders and Sanders, 1997).

2.6.8. Escherichia coli

Plate 2.8. Escherichia coli


Kingdom : Bacteria





Genus : Escherichia

Species : coli

45

Morphology

E. coli is a rod shaped gram-negative, non-sporulating, facultatively

anaerobic bacteria that live in a wide variety of substrates (Vogt and

Dippold, 2005; Kubitschek, 1990).

Distribution

It is the primary facultative anaerobe of the human gastrointestinal

tract (Todar Kenneth, 2008). (Facultative anaerobes are organisms that

can grow in either the absence or presence of oxygen). It is also

commonly found in water, air and soil.

Pathogenesis

Virulent strains of E. coli can cause urinary tract infections,

gastroenteritis and neonatal meningitis. In rare cases, virulent strains

are also responsible for mastitis, peritonitis, haemolytic-uremic

syndrome (HUS), septicemia and gram-negative pneumonia (Todar

Kenneth, 2008). In traveler and infants especially in regions of poor

sanitation, it causes watery diarrhea.

2.6.9. Klebsiella pneumonia

Plate 2.9 : Klebsiella pneumoniae

46


Kingdom : Bacteria





Genus : Klebsiella

Species : pneumoniae

Morphology

It is a gram-negative, encapsulated, non-motile, rod shaped,

facultatively anaerobic bacterium (Ryan and Ray, 2004).

Distribution

K. pneumoniae naturally occurs in the soil. It is also found in the

normal flora of skin, the mouth and intestine of humans (Ryan and Ray,

2004).

Pathogenesis

It causes pneumonia in humans. Characteristic sputum that is said

to resemble "red - currant jelly" in patients with this disease tends to

cough up. Klebsiella also causes urinary tract infections (Postgate, 1998).

47

2.6.10. Pseudomonas aeruginosa

Plate 2.10 : Pseudomonas aeruginosa


Kingdom : Bacteria



Order : Pseudomonadales

Family : Pseudomonadaceae

Genus : Pseudomonas

Species : aeruginosa

Morphology

This is a gram negative, rod shaped, aerobic bacteria which forms

mucoid colonies with umbonate elevation (Iglewski, 1996). Some strains

produce a distinctive fruity odor and a diffusible green pigment (Balcht et

al., 1994).

Distribution

P. aeruginosa is an opportunistic contaminant of wounds, burn

injuries such as cuts and gunshot. It is found in water, soil, most man-

made environments and skin flora throughout the world (Balcht et al.,

1994).

48

Pathogenesis

Pseudomonas aeruginosa is a common and an opportunistic

pathogen, which causes disease in humans, animals and also in the

plants (Anzai et al., 2002). It causes gastrointestinal tract, septicemia,

nosocomial, pneumonia, urinary tract, and skin and soft tissue

infections in immune compromised persons (Todar Kenneth, 2008).

2.6.11. Salmonella typhimurium

Plate 2.11 : Salmonella typhimurium


Kingdom : Bacteria





Genus : Salmonella

Species : typhimurium

49

Morphology

Salmonella is a gram-negative, rod-shaped, nonsporing, facultatively

anaerobic bacterium trivially known as "enteric" bacteria (Giannella,

1996; Murray et al., 2009).

Distribution

Salmonellae live in the intestinal tracts of cold and warm blooded

animals. Other species specifically adapt to a particular host (Todar

Kenneth, 2008).

Pathogenesis

In human beings, two types of infections are caused by Salmonella.

They are

Acute gastroenteritis: This result from food borne infection/intoxication

by salmonella.

Salmonellosis: Caused by the bacterial invasion of the bloodstream it

leading to enteric fever. All sorts of enteric fevers were characterized as

"typhoid” (Todar Kenneth, 2008).

2.7. The antimicrobial activity of plant extracts

The antimicrobial activity of medicinal plants against various human

pathogens has been represented in Table 2.8. As depicted in Table 2.8

many plant species have been tested for their antimicrobial propertities.

Studies were conducted with different plant parts viz., aerial (stem &

leaves), root, rhizome and root bark. The majority of the medicinal plants

50

have shown good antimicrobial activity with the extracts from aerial

parts. Plant extracts of Byrsonima crassifolia (Martinez et al., 1999),

Securidaca longipedunculata (Ajali et al., 2005), Solanum aculeastrum

(Wanyonyi et al., 2003), Rubia peregrine (Ozgena et al., 2003),

Aristolochia mollissima (Yu et al., 2007) and Alpinia galanga (Kiranmayee

Rao et al., 2010) root parts displayed antimicrobial activity.

The solvents used for the extraction of plant parts were ethyl acetate,

methanol, ethanol, water, petroleum ether, chloroform, dichloroethane,

butanol, ethylene glycol, hexane and acetone. Most of the medicinal

plants showed excellent antimicrobial activity in methanol, ethanol or

water extracts. For few plants two or three solvents were used for

extraction purpose. More than five solvents were used for the extraction

of phytochemicals from Raphanus sativus (Beevi et al., 2009).

The microorganisms used for the evaluation of antimicrobial activity,

were B. subtilis, E. aerogene, E. cloacae, E. faecalis, E. coli, K.

pneumoniae, P. aeruginosa, S. typhimurium, S. aureus, S. epidermidies

and L. monocytogenes. In some other studies different microorganisms

viz., Micrococcus luteus, S. pneumoniae, Shigella flexneri, B. anthracis, B.

pumilis, P. vulgaris, Salmonella newport, Salmonella pullorum, Salmonella

stanley, S. albus, S. agalactiae and Vibrio cholera were also studied. All

the medicinal plants have shown good to moderate antimicrobial activity

towards the tested microorganisms used in the study.

51

Scientific name Solvent used Parts used Organisms Authors Mutisia acuminate Water and

methanol Aerial P. aeruginosa, S. aureus and

L. monocytogenes Catalano et al., 1998

Saraca asoca Methanol and water

Leaf B. subtilis, P. aeruginosa and S. typhimurium

Annapurna et al., 1999

Byrsonima crassifolia (L.) H.B.K.

Ethyl acetate Roots M. luteus, S. pneumonia, S. aureus, S. epidermisdis, S.flexneri, K. pneumoniae, S. typhi and P. aeruginosa

Martinez et al., 1999

Hyptis suaveolens Water Leaves S. aureus, B. cereus, E. coli and P. aeruginosa

Asekun et al., 1999

Toddalia asiatica Ethylene glycol Leaves B. anthracis, B. pumilis, B. subtilis, E. coli, K. pneumoni, P. vulgaris, P. aeruginosa, S. newport, S.pullorum, S. stanley, S. albus, S. aureus, S. agalactiae, and V. cholera

Saxena et al., 1999a

Lantana aculeate Ethylene glycol Leaves B. anthracis, B. pumilis, B. subtilis, E. coli, K. pneumoniae, P. vulgaris, P. aeruginosa, S. newport,S. pullorum, S. stanley, S. albus, S. aureus,S. agalactiae and V. cholera

Saxena et al., 1999b

Alstonia macrophylla

Methanol Leaf E. coli, S. faecalis, S. aureus,

Chattopadhyay et al., 2001

Adesmia aegiceras Ethanol Aerial E. coli, S. aureus, S. saprophyticus, P. aeruginosa, and P. mirabilis

Agnese et al., 2001

Rhynchosia beddomei

Petroleum ether & EtOAc

Leaf B. subtilis, S. aureus, E. coli, P. aeruginosa Bakshu et al., 2001

Lepista nuda Methanol Aerial S. thyphimurium and K. pneumonia Dulgera et al., 2002

Solidago virgaurea

L. Ethanol and methanol

Aerial and root

S. epidermidis, S. aureus, B. subtilis, K. pneumonia, E. faecalis, P. aeruginosa and E. coli

Thiem et al., 2002

Strobilanthes callosus Nees.

Ethanol Aerial E. coli, S. aureus, E. cloacae,K. pneumonia and B. thuringiensis

Singh et al.,2002a

Cont….

52

Scientific name Solvent used Parts used Organisms Authors Heliotropium subulatum

Ethanol Aerial E. coli, S. aureus, S. pneumoniae B. subtilis and B. anthracis

Singh et al.,2002b

Ixora coccinea Ether Leaves E. coli, P. aeruginosa, S. typhimurium, Sarcina lutea, S. aureus and B. subtilis

Annapurna et al., 2003

Juniperus oxycedrus

Methanol Leaf Bacterial genera Bacillus, Escherichia, Staphylococcus, Enterobacter, Micrococcus, Brevundimonas, Brucella, Acinetobacter, Xanthomonas and Pseudomonas.

Karamana et al., 2003

Rubia peregrine Methanol Roots and

rhizomes

B. subtilis, S. aureus and E. coli Ozgena et al., 2003

Cassia alata Ethanol Leaves and barks

E. coli and S.aureus Somchit et al., 2003

Solanum aculeastrum

Methanol Root bark S. epidermidis, S. aureus, B. subtilis, K. pneumonia, P. aeruginosa, E. coli and E. faecalis

Wanyonyi et al., 2003

Satureja khuzistanica

Methanol Aerial S. aureus Amanloua et al., 2004

Artemisia species viz., A. scoparia, A. turanica and A. oliveriana

Methanol Aerial and root

B. subtilis, S. aureus, P. aeruginosa and E. coli

Ramezania et al., 2004

Artemisia douglasiana

Dichloro Methane

Leaves B. cereus, S. aureus P. aeruginosa and E.coli

Setzer et al., 2004

Anthemis xylopoda Water Leaves S. aureus, E. faecalis, B. cereus, B. subtilis, E. coli, K. pneumoniae , P. vulgaris, P. aeruginosa, P. fluorescens and S. typhimurium

Uzel et al., 2004

Scutellaria barbata Water Aerial S. aureus, E. coli, P. aeruginosa, S. epidermidis,S. heamolyticus, S. simulans, E. faecalis, C. freundii, K. pneumoniae, S. flexneri, S.typhi, S. paratyphi-A, S. liquefaciens, S. marcescens, S. maltophila

Yu et al., 2004

Cont…..

53

Scientific name Solvent used Parts used Organisms Authors

Salacia beddomei Petroleum ether, ethyl acetate and chloroform

Leaves and stem

S. aureus, B. subtilis, E. coli, K.pneumoniae , Pseudomonas phosphorescence and Aeromonas hydrophylla

Deepa and Narmada 2004

Datura innoxia Methanol Aerial S. aureus, E. faecalis and B. subtilis

Eftekhar et al., 2005

Viola tricolor Ethanol Aerial S. epidermidis, S. aureus, Bacillus cereus, K. pneumoniae, P. aeruginosa, E. coli and E. faecalis

Witkowska et al., 2005

Securidaca longipedunculata

Ethanol Roots S. aureus, B. subtilis, K. pneumoniae, P. aeruginosa and E. coli

Ajali et al., 2005

Hypericum species., H. hyssopifolium and H. lysimachioides

Water Leaves E. coli, B.brevis, B. cereus, Streptococcus pyogenes, P. aeruginosa and S. aureus

Toker et al., 2006

Loranthus micranthus

Methanol, etha-nol petroleum ether & CHCl3

Aerial and root

B.subtilis and E.coli Osadebe et al., 2006

Satureja subspicata

Water Aerial B. cereus, B. subtilis, E. faecium, E. faecalis, L. monocytogenes, S. aureus, Aeromonas hydrophila, E. coli, K. pneumoniae, P. aeruginosa, P. mirabilis, and S.typhimurium

Skocibusic et al., 2006

Euphorbia hirta Ethanol Aerial P. aeruginosa, E. coli, S. aureus and Proteus vulgaris

Sudhakar et al., 2006

. Cont ….

54

Scientific name Solvent used Parts used Organisms Authors Hypericum species H. alpinum, H. barbatum, H. rumeliacum, H. hirsutum, H. maculatum and H. perforatum

Water Aerial B. cereus, S. aureus, E. coli, P. mirabilis, P. aeruginosa, P. tolaasii and S. enteritidis

Saroglou et al., 2007

Rosmarinus officinalis

Methanol Aerial E. feacalis, K. pneumoniae, P. vulgaris, P. aeruginosa, S. aureus, B. subtilis, E. coli and S. epidermidis.

Celiktas et al., 2007

Berberis species viz. Berberis aristata, Berberis asiatica, Berberis chitria and Berberis lyceum

Hydro Alcoholic

Root, stem S. aureus, B. subtilis, B. cereus, Micrococcus luteus, E. aerogenes, S. pneumoniae, Proteus mirabilis, K. pneumoniae, S. typhimurium, P. aeruginosa and E. coli

Singh et al., 2007

Satureja species S. biflora,S. masukensis,S.ps-eudosimensis

Water Aerial and leaves

S. aureus, S. epidermidis E. coli, E. cloacae, K. pneumoniae and P. aeruginosa

Vagionas et al., 2007

Aristolochia mollissima

Water Rhizo-me and Aerial

S. aureus, E. coli, P. aeruginosa, S. saprophyticus, S. simulans, S. heamolyticus, E. faecalis, C.freundii, K. pneumoniae, S. flexneri, S. typhi, S. liquefaciens, S. marcescens, S. maltophilia, E. cloacae, E. aerogenes and Proteus mirabilis

Yu et al., 2007

Cont…..

55


Cynara cardunculus

Ethanol Aerial S. aureus, E. coli, S. typhimurium, B. subtilis and S. epidermidis

Kukic et al., 2008

Potentilla species: P. anserina, P. argentea, P. erecta, P.fruticosa, P. grandiflora, P. nepalensis P. recta, P. rupestris and P. thuringiaca

Aqueous extracts

Aerial S. epidermidis, S. aureus, B. subtilis, K. pneumoniae, P. aeruginosa and E. coli

Tomczyk et al., 2008

Andrographis paniculata

Chloroform Aerial B. subtilis, E. aerogenes, E. cloacae, E. faecalis, E. coli, K. pneumoniae, P. aeruginosa, S. typhimurium, S. aureus and S. epidermidis

Soma Roy et al., 2009

Raphanus sativus Water, methanol, acetone, ethyl acetate, chloroform and hexane

Root, stem and leaves

B. subtilis; S. aureus, S.epidermidis, E. faecalis,E. coli, S. typhimurium, E. cloacae, E. aerogenes, K. pneumoniae and P. aeruginosa.

Beevi et al., 2009

Craniotome furcata Ethyl acetate & n-butanol

Aerial S. faecalis, K. pneumoniae, and E. coli

Joshi et al., 2010

Clausena anisata Water Leaves E. coli, S. aureus, S. typhi, Shigella sp.,Proteus sp. and P. aeruginosa

Osei-Safo et al., 2010

Cont…..

56


Ocimum basilicum, O.kilimandscharic, O.lamiifolium, O. suave

Water Leaves S. aureus, S.epidermidis, S. mutans, S. viridians,E. coli, E. cloacae, K. pneumoniae and P.aeruginosa

Runyoro et al., 2010

Alpinia galanga (L) Willd

Methanol, acetone and diethyl ether extracts

Rhizome, leaves stem

B. subtilis, E.aerogene, E.cloacae, E.faecalis, E. coli, K. pneumoniae, P. aeruginosa, S. typhimurium, S. aureus and S.epidermidis

Kiranmayee Rao et al., 2010

Salacia oblonga Wall.

EtOAc, Chloroform, methanol, ethanol, Petroleum ether, hexane, water

Aerial and root

B. subtilis, E.aerogene, E.cloacae, E.faecalis, E. coli, K. pneumoniae, P. aeruginosa, S. typhimurium, S. aureus , S. epidermidis and L. monocytogenes

Jayaram prakash rao et al., 2011

Table 2.8. Antimicrobial activity of medicinal plant species against various pathogens

57

2.8. GC-MS analysis

GC-MS, which is a method developed from the combination of GC and

MS, was the first of its kind to become helpful for research and

development purposes. Mass spectra attained by this technique offers

more structural information based on the explanation of fragmentations.

The fragment ions with diverse relative abundances can be compared

with library spectra. Compounds that are sufficiently small, volatile, and

constant in high temperature in GC conditions can be simply analyzed

by GC-MS. Sometimes, polar compounds, particularly those with a

number of hydroxyl groups, require to be derivatized for GC - MS

analysis. The most common derivatization procedure is the translation of

the analyte to its trimethylsilyl derivative.

The fatty acids from Ilex dipyrena Wallich leaves were subjected to

GC – MS analysis. The major constituents were methyl commate B

(7.45%), methyl cembratriene (7.69%), methyl ester (7.15%),

2 - hexadecen, 3, 7, 11, 15 tetramethyl (7.31%) and mangiferonic acid-

methyl cenbrenene (6.54%) (Sudhir kumar et al., 2010).

The essential oil from flowers of Nerium oleander their chemical

composition was determined by GC – MS. The major constituents were

digitoxigenine (11.25%), 1, 8 - cineole (6.58%), amorphane (8.11%),

58

α-pinene (5.54%), Limonene (5.01%), calarene (5.12%), β-phellandrene

(4.84%) (Elhoussine Derwich et al., 2010a).

The essential oil isolated from leaf of Pistcia lentiscus growing in

Moracco, GC – MS analysis twenty compounds were identified in the leaf

oil. The major compounds in aerial parts were pinene (24.25%) followed

by limonene (7.56%), α-pinene (12.58%), terpineol (4.89%) and terpinen-

4-ol (6.98%) (Elhoussine Derwich et al., 2010b).

The volatile constituents of Achillea clavennae L. (Asteraceae) rare

plant of Europe, have been analyzed using GC – MS. Twenty five

compounds with myrcene (5.5%), camphor (29.5%), 1, 8 cineole (5.3%),

linolool (4.9%) and β - caryophyllene (5.1%) being the major constituents

(Nada Bezic et al., 2003).

In a study the most important components identified in the Cordia

verbenacea extract were β - sitosterol, artemetin, β - caryophyllene and

α - hunulene among others (Eliane et al., 2009).

In earlier reports, for the stem of Mallotus philippensis (Lam) Muell

Arg. Var Philippensis (Euphorbiaceae) GC – MS analysis showed the

presence of ten compounds in ethanol extract (Jayaraman Velanganni et

al., 2011).

The essential oil of the leaves of Mentha rotundifolia and Mentha

pulegium grown on Morocco was determined by GC – MS. The identified

59

main constituents were menthol (40.50%), menthofuran (4.20%),

methone (5.0%) and menthyl acetate (4.50%). Twenty eight compounds

were identified in leaves oil of Mentha pulegium and the major component

was piperitone (35.56%) the other predominant constituents are menthol

(3.28%) and piperitone oxide (4.02%) (Derwich et al., 2010).

The aerial parts of Heliotropium indicum Linn. four components

were identified by GC – MS, Indicam 1,2,3 and 4 (Shoge Mansurat et al.,

2011).

The chemical composition of the essential oil obtained from the fruits

of Pimpinella affinis Lebeb (Apiaceae) was analyzed in GC – MS

technique. Twenty four compounds were identified in the essential oil of

P. affinis Lebeb whose major constituents were geijerene (17.68%),

limonene (12.86%), germacrene D (8.54%), pregeijerene (9.92%) and

trans – β - ocimen (4.94%) (Verdian-rizi Mohammadreza, 2008).

2.9. Silica gel chromatography

Column chromatography is a conservative technique, adopted

universally due to ease of its operation. It is carried out in glass columns

packed through a stationary phase like the Silica gel. The packing is

carried out by two methods- dry packing and the slurry packing (Salituro

and Dufresne, 2000).

60

Column chromatography remains a process of choice among the

phytochemists or the natural product chemists. A variety of natural

products like phenols, terpenoids, courmarins, neolignans, etc. have

been isolated using open column chromatography (Cowan, 1999).

The turmeric oil was extracted using hexane and the extract was

divided into three fractions using silica gel chromatography. The

turmeric oil fraction 1 and fraction 2 were analyzed by GC – MS,

ar - Turmerone, curlons and turmerone were found to be the major

compounds present in these fractions (Nagi et al., 1999).

The best antimicrobial activity results were obtained when methanol

aerial extract from Xanthium brasilicum was fractionated by silica gel

chromatography. The results showed the occurrence of two substances, a

Xanthanolide and a flavonoid (Fereshteh et al., 2007).

Phytochemical screening of methanolic extract and active principles of

hepatoprotective herb Eclipta alba revealed the presence of tannins,

flavonoids, coumestans, saponins and alkaloids etc., It’s EtOAc fraction

and when further isolated showed the presence of welelolactone as a

promising antimicrobial agent (Sunitha Dalal et al., 2010).

Phytochemical screening of Caesalpinia coriaria (Jacq) Willd pod and

leaf material revealed the presence of phenolics in acidic fraction.

Further separation of active fraction resulted in loss of antimicrobial

61

activity indicating a synergestic effect of the isolated active fraction

(Mohana and Reveesha, 2006).

2.10. LC - MS analysis

LC - MS qualitative analysis makes it possible to reconstruct an

unknown compound from MS data. The ionization techniques used in LC

- MS are usually soft ionization techniques that mainly exhibit the

molecular ion species with only a few fragment ions (Herderich et al.,

2007).

In GC – MS analysis of Mirabilis jalapa tubers dichloromethane and

methanol extracts showed that β - sitosterol and oleic acid were the

major compounds. LC – MS analysis of aqueous extract showed high

content of flavonol and flavanol derivatives (Mohammad et al., 2010).

Analysis of the extract of black cumin and garlic by GC – MS as well

as LC – MS confirmed that the main components of garlic were

γ –glutamyl – S - allylcysteine, allicin and allicin. Components of black

cumin were P - cymene, thymoquinone, pipine and P – tert - butyl

catechol (Jiben Roy et al., 2010).

Major compounds in cinnamon stick were tentatively identified by

GC – MS and LC – MS as a predominant volatile oil component [(E)-

Cinnamaldehyde] and several polyphenols (mainly proanthocyanidins

62

and (epi) catechins). This study suggests that cinnamon stick and its

bioactive components have potential for applications as natural food

preservatives (Bin shan et al., 2007).

The ethanol and acetone extracts obtained from Coleonema album

(Rutaceae) were analyzed by LC – MS. Identification and structural

information of the bioactive compounds were obtained by LC – MS. It

revealed the presence of coumarin aglycones which were responsible for

the observed antimicrobial activities.

2.0. Review of literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/20814/8/08_chapter...

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