Chapter 1 - Shodhganga : a reservoir of Indian theses ...shodhganga.inflibnet.ac.in/bitstream/10603/22728/3/ch-1.pdfdihydroxyphenyl) ethenyl ester and (Z, E) - 2 - (3, 5 - dihydroxyphenyl)

REVIEW OF LITERATURE

11

Chapter 1


12

Perilla is an aromatic herb belongs to fa mily Labiatae. Four to six species of

Perilla are found. Only one species is found in India. Commonly grows in

waste places, crop fields, road sides, occasionally cultivated to 3000m.

Leaves and flower tops of Perilla are used as flavouring. Seeds are edible or

used as condiments. Plant extract and powdered dried part of Perilla are

used for bronchitis and uterine ailments. Paste of Perilla leaf is used in

rheumatic arthritis1.The leaf of P. frutescens Britton (Japanese name shiso) is

one of the most popular garnishes in Japan 2 which is prescribed for cold,

cough and promoting digestion 3.The herb is reported to possess sedative,

antispasmodic and diaphoretic properties and prescribed for cephalic and

uterine troubles.

Seeds of Perilla contain 30-51% of a valuable drying oil known as Perilla

oil. Perilla oil finds extensive use in paints and varnishes, printing inks,

Japanese oil papers, waterproof clothes, artificial leather, cheap lacquers,

enamels and linoleum. In U. S.A., oil is mixed with soyabean oil for

protective coatings. The cake left after the expression of the oil is used in

Japan as a fertilizer for mulberry and rice. The essential oil possesses a

strong antiseptic action and is used as antimildew agent. The oil is used in

Japan chiefly for the preparation of the • -antialdoxime of perillaldehyde

which is 2,000 times as sweet as sugar and 4 to 8 times as sweet as

saccharin. The Japanese Government permits the use of these derivatives as

a substitute for maple sugar or liquorices in the sweetening of tobacco 4.

Spermadictyon is a small genus of shrub found in Indomalayan region and

China. It belongs to family Rubiaceae. One species of Spermadictyon occurs

in India. Spermadictyon suaveolens is a shrub upto 12 ft. high found almost

throughout India ascending upto an altitude of 6,500 ft. The plant is also

cultivated in gardens, flowers are blue or white.


13

Plant is used as tribal remedies against snake bites and scorpion stings in

Rajasthan5 and also used as a tribal remedy for diabetes 6. Hypoglycemic

activity of S. suaveolens was also observed7. The plant is used by Mundas of

Ranchi district (Bihar), along with mustard oil as an application for wounds.

The bark is ground and rubbed on the body in puerperal fever. The root is

given in diarrhea. The wood is dark grey and reported to be used for making

gunpowder charcoal8. Leaves are used as a insecticide for the stored grains 9.

Verbascum belongs to the family Scrophulariaceae is erect, pubescent herb.

About 360 species of Verbascum are found, only 6 species occur in India10.

The name ‘mullein’ has two possible derivations: It either comes from

mollis, which means soft in Latin or the Latin word mulandrum, which

comes from melanders and means leprosy - an illness this plant was used to

treat. Verbascum means ‘mullein’ in Latin. It is derived from the word

barbascum, which means ‘with beard’. The species name is thapsus because

mullein resembles the European genus Thapsia, named after an ancient town

in the present day Tunisia.

In Europe by settlers, it was used as medicinal herb as a remedy for cough

and diarrhoea and a respiratory stimulant for the lungs when smoked. A

methanol extract from common mullein has been used as an insecticide for

mosquito larvae. The American Indians dried the leaves, (especially the first

year, or new growth leaves) and smoked them to relieve asthma and other

respiratory problems. The leaf tea is also medicinal and is recognized by

herbalists as a traditional remedy for respiratory congestion and hemorrhage.

The flowers are said to have strong microbial qualities an d are used in oil

infusion for ear infections. The roots have been used for their tonic

properties, astringent to treat urinary incontinence. The seeds were also used

by American Indians as a paralytic fish p oison. The plant contains coumarin


14

and rotenone, especially in seeds. The leaves are a rubefacient which means

that if you rub them against your skin it will become red and irritated.

Mullein tea provides vitamins B -2, B-5, B-12, and D, choline, hesperidin ,

PABA, sulphur, magnesium, mucilage, saponins and other active

constituents.

People use the tea as beverage, but its best known as one of the safest, most

effective herbal cough remedies. Mullein is an expectorant and a tonic for

lungs, mucus membranes and glands. An infusion is good for colds,

emphysema, asthma, hay fever and whooping cough. Labo ratory tests have

shown that it is anti-inflammatory with antibiotic activity and that it inhibits

the tuberculosis bacillus. The Indians smoked dried mullein and coltsfoot

cigarettes for asthma and bronchitis. The tea is also a n astringent and

demulcent. It is good for diarrhoea and it is been used in compresses for

hemorrhoids since it was recommended b y Dioscorides centuries ago. It i s

also supposed to help other herbs get absorbed through the skin. Pliny of

ancient Rome, Gerard in sixteenth century England the Delaware Indians

and country folk in the south used the heated leaves in poultice for arthritis.

A tincture of the flowers is used for migraine headaches. An oil extract of

the flowers which contains a bactericide is used for ear infections 11.

The seeds are said to intoxicate fish when thrown into the water, and are

used by poachers for that purpose, being slightly narcotic. The seeds of

V. sinuatum, which are used in Greece as a fish poiso n, contain 6 to 13 per

cent of saponin. Traces of the same substance were found in the seeds of

V. phlomoides and V. thapsiforme, common in the south of Europe, which

have been used for the same purpose. V. pulverulentum of Madeira (also

used as a fish poison) and V. phlomoides are employed as taenicides

(expellers of tapeworm)12.


15

Great Mullein has been used as an alternative medicine for centuries and in

many countries throughout the world . The value of Great Mullein as a

proven medicinal herb is now backed by scientific evidence. Some valuable

constituents contained in Mullein are coumarin and h esperidin, they exhibit

many healing abilities. Researches indicate that some of the uses analgesic,

antihistaminic, antiinflamatory, anticancer, antioxidant, antiviral, bacteristat,

cardiodepressant, estrogenic, fungicide, hypnotic, sedative and pesticide are

valid. An infusion is taken internally in the treatment of a wide range of

chest complaints and also to treat d iarrhoea and bleeding of the lungs and

bowels. The leaves, roots and the flowers are anodyne, anti -inflammatory,

antiseptic, antispasmodic, astringent, demulcent, diuretic, emollient,

expectorant, nervine and vulnerary.

Mullein oil is very medicinal and va luable destroyer of disease germs. An

infusion of the flowers in olive oil is used as earache drops or as a local

application in the treatment of piles and other mucous membrane

inflammations. This infusion is a str ong antibacterial. The oil is used to treat

gums and mouth ulcers. A decoction of the roots is used to alleviate

toothache and also relieve cramps and convulsions. It is also used in

alternative medicine for the treatment of migraine headaches accompanied

with oppression of the ear. The whole plant possesses slightly sedative and

narcotic properties. The seeds are mostly used as narcotic and also contain

saponins. The dried leaves are sometimes smoked to relieve the irritation of

the respiratory mucus membranes. They can be employed with equal ben efit

when made into cigarettes for asthma and spasmodic coughs in general.

Externally, a medicinal poultice of the leaves is applied to sunburn, ulcers,

tumors and piles. A decoction of the seeds is used to soothe chilblains and

chapped skin.


16

It is also used as dye, insecticide, insulation, lighting, tinder and wick. A

yellow dye is made from the flowers by boiling them in water. When used

with dilute sulphuric acid they produce a rather permanent green dye, this

becomes brown with the addition of alkalis. An infusion of the flower is

sometimes used to dye the hair in golden colour. The leaves contain rotenone

which is used as an insecticide. The dried leaves are highly flammable and

can be used to ignite a fire quickly or as wick for candles13.

Isolated compounds and biological activity of Perilla frutescens,

Spermadictyon suaveolens and Verbascum thapsus are listed in Table 1.1,

1.2 and 1.3 respectively.

Rotenone (Fig.1.1) and its derivatives commonly referred to as rotenoids are

insecticidal compounds essentially extracted from seeds, roots and

sometimes leaves and stems of the tropical Leguminosae plants Derris,

Tephrosia and Lonchocarpous. Commercially important plants like Derris

elliptica and D. malaccensis contain 4-5% rotenone while Lonchocarpous

utilis and L. urucu contain 8-10% rotenone in dry roots. Rotenone comprises

of an isoflavone nucleus with an isoprene moiety.

Table 1.1. Isolated compounds/activity of different parts of Perilla species

S. No.

Plant Name

Plant Part

Isolated compounds/Activity

Ref.

1

Perilla nankinensis

Leaves

Apigenin glucuronide

14

Essential oil (leaves)

Shisofuran

15

2

Perilla setoyensis

Essential oil

4 - Terpenol and limonene

16


17

3

Perilla sikokiana

Leaves

Anti-inflammatory activity

17

Ethyl linolenate, linolenic acid and • -sitosterol

18

Aerial part

(3S,4R)3-hydroxy-4-(1-methyl-ethenyl 1-cyclohexene-1-carboxaldehyde

19

Perilloside A[ ( - ) perillyl 7 - O - • - D glucopyranoside eugenyl O - • - D - glucopyranoside]

20

Perilloside B [1- • - D -glucopyranosyl (-) perillate], Perilloside C [trans- dihydroperillyl 7 - O - • - D - glucopyranoside], Perilloside D [cis - dihydroperillyl 7 - O - • - D - glucopyranoside], • - sitosteryl O - • -D - glucopyranoside, protocatecheuic aldehyde methyl ferulate

21

7 - O - Diglucuronides of apigenin, luteolin and scutellar in

22

Benzyl • - D - glucopyranoside, ursolic acid, tormetic acid, methyl rosmarinate, Perilloside E [6 -methoxy-2, 3 - methylenedioxy - 5 - allylphenyl • - D - glucopyranoside]

23

Weak antidermatophytic activity

24

Sedative effect

25

Luteolin, rosmarinic acid and caffeic acid

4

Perilla frutescens

Leaves

Luteolin as a anti -inflammatory and antiallergic constituent

26


18

Rosmarinic acid

Tannic activity

27

Leaves

Caffeic esters, (Z, E) - 2 - (3, 4 -dihydroxyphenyl) ethenyl ester and (Z, E) - 2 - (3, 5 - dihydroxyphenyl) ethenyl ester of 3 - (3, 4 -dihydroxyphenyl) - 2 - propenoic acid

28

Novel antioxidants [5 - (3, 4 - dihydroxyphenyl mehyl oxazolidine 2, 4 - dione (1)] & [3 - (3, 4 - dihydroxyphenyl lactamide -2)]

29

Roasted seeds

Antioxidant activity

30

Stem

Perriloxin and dehydroperriloxin

31

Apigenin, 2,4,5 -trimethoxy cinnamic acid (TMCA)

Antidepressant effect

32

Cytotoxicity and antitumor activity

33

Antiallergic flavonoids

34

Vinyl caffeate, trans - p - menth - 8 -en - 7 - yl caffeate, 3, 4 -dihydroxybenzaldehyde, methyl caffeate, 3’, 4’, 5, 7 -tetrhydroxyflavone, caffeic acid, 6, 7 - dihydroxycoumarin and rosmarinic acid

35

Post coital antifertility effect

36

Glycoprotein

37

Neuropharmacological activity

38

Perilla frutescens

Whole Plant

Antioxidant vinyl caffeate

39


19

Limonene, piperitone, •-caryophyllene and germacrene

40

Essential oil (Aerial part)

Perillaketone, Isoegomaketone, egomaketone and perillene

41

Essential oil (leaves & racemes)

Perillaketone, 1 - (3 - furyl) - 4 -methyl - 2 - pentanone, 1 - (3 - furyl) - 4 - methyl - 2 - penten - 1 - one, 1 -(3 - furyl) - 4 - methyl - 3 - penten -1 - one

42

Essential oil (leaves & racemes)

40-55% Perillaldehyde (4 -isoprenyl-I-cyclohexen-7-al)

43

Essential oil (Green leaves)

Perillaldehyde, limonene, • - caryo - phyllene, • - bergomotene and linalool

44

Essential oil (Roasted seeds)

Luteolin, chrysoeriol and apigenin

45

Phenylpropanoid

46

Limonene, linalool, perillaketone and isoegomaketone

47

Limonene (9%) and small quantity of • - pinene

48

•-Caryophyllene, caryophyllene, thujopsene and • - farnescene

Perillaketone

49

Perilla frutescens

Essential oil

Perillaldehyde, elsholtziaketone, perillaketone, citral, perillene and phenylpropanoid

50


20

Table 1.2. Activity of different parts of Spermadictyon species

Table 1.3. Compound isolated/activity of different parts of Verbascum species

S. No.

Name of Plants

Plant Parts

Activity

Ref.

Roots

Hypoglycemic activity

7

1

Spermadictyon

suaveolens

Leaves

Insecticidal activity

9

S. No.

Name of Plants Plant Parts

Compound isolated/Activity Ref.

New sterol sigmasta - 5, 9 (11) - dien - 3β - ol, three new saponins: celsiogenin A [olean - 12, 17 (18) -dien - 3β, 23 - diol ], celsiogenin B [olean - 11, 13 (18) - dien - 3β, 23, 28 - triol ], celsiogenin C [olean - 11, 13 (18) - dien - 3β, 22β, 23, 28 - tetriol ].

51

1

Verbascum

chinensis

Whole plant

Fish poisoning

52

2

Verbascum georgicum

New iridoid 6 - alpha - L -rhamnopyranosyl descinnamoyl globularinin

53

Aerial part

New iridoid glycoside 6 - O - alpha - L (3” - O - n - coumaryl) rhamnopyranosylacubin

3

Verbascum laxum

Bark

Haparoside, 6 - O - alpha - L - (2”- O - 3”- O - acetyl, n - methoxy - trans-cinnamoyl) rhamnopyranosylacubin

54


21

Inflorescence

6 - O - p - coumaroylcatalpol

55

Verbascosaponin, luteolin 7 - O -glucoside, verbascoside and foraythoside B

4

Verbascum lychnitis

Antiproliferative effect

56

Aerial part

A new spermine alkaloid: verbascenine (C30H40N4O3)

57

5

Verbascum nigrum

Inflorescence

Verbascosaponin, luteolin 7 - O -glucoside, verbascoside, foraythoside B

58

Five flavones, one flavanone, four flavonols, two iridoids, two steroglycosides, four carbohydrate derivatives, two phenolic substances and one terpene derivative Antiproliferative effect

59

Flowers

A new iridoid ester glycoside acylated with p-coumaric acid, speciocide, caffeic esters, verbascoside, forsythoside B, desrhamnosylverbascosaponin

60

6

Verbascum phlomoids

Flowers (oil contents)

Fatty acids: Myristic, palmitic, stearic, oleic, linoleic, arachic and lignoceric Phenolics, p-hydroxy cinnamic acid, protocatechuic acid, p-hydroxybenzoic acid, p - coumaric acid, vanillic acid and ferulic acid

61


22

Aerial part

A new spermine alkaloid: verbascenine (C30H40N4O3)

58

Z isomer [13 - acetyl - 1 (Z) - cinnamoyl - 6 - oxo - 8 - phenyl - 1, 5, 9, 13 - tetraazacycloheptadecane, verballoscenine] of verbascenine and analogous N - 13 on acetyl alkaloids verbacine, verballocine and verballoscenine

62

7

Verbascum phoeniceum

17 - membered macrocyclic sperm ine lactam alkaloids ( -) protoverbine and its N, N’ - methylene - bridged natural analogue (+) - protomethine (-) -verbacine, (-) - verballocine, (+) - verbascenine, (+) - verballoscenine, (+) - verbamethine, (+) - incasine (B) and some of their isosteric analogues

63

Alkaloid verbaskine (C 29H36N4O3), (E) - cinnamamide

64

Lactam alkaloids: ( -) protoverbine and its N, N’ - methylene - bridged natural analogue (+) - protomethine (-) -verbacine, (-) - verballocine, (+) - verbascenine, (+) - verballoscenine, (+) - verbamethine, (+) - incasine (B) and some of their isosteric analogues

63

17 - membered macrocyclic spermine alkaloids (-) - (s) - verbasitrine (-) - (s) -isoverbasitrine, (+) - (s) verbametrine and (+) - (s) - isoverbametrine

65

8

Verbascum pseudonobile

Leaves

Novel 17 - membered lactam alkaloids: verbacine and verballocine containing spermine

66


23

9

Verbascum pulverulentum

Root

Two iridoids of 6 - O - alpha - L -rhamnopyranosyl - catalpol type

67

10

Verbascum pycnostachyum

Aerial part

sigmasterol, β-sitosterol and unidentified compound (C29H48O)

68

11

Verbascum saccatum

Aerial part

Aucubin, 6 - alpha - L -rhamnopyranosylcatalpol and new iridoid glycoside 6 - alpha - L - (2”- para - coumarin) rhamnopyranosylcatalpol (C39H38O16)

68

Orobol, orobol 7 - O -beta -D- glucoside, 5, 3’, 4’- trihydroxy-8-methyl isoflavone 7 - O - beta - D - glucoside, 5 - hydroxy, 3’- 4’-dimethoxyflavone 7 - O - alpha - L - rhamnoside and phenyl ethanoid glycoside acetoside

70

Aerial part

7 new saikosaponin homologues called mulleinsaponins having 13, 28 epoxyolean- 11 - ene skeleton isolated together with eight known saikosaponin homologues 3 -O-beta-D-fucopyranosyl saikogenin F, saikosaponin A, songarosaponins C, D, mimengoside A and buddlejasaponins I and IV

71

12

Verbascum sinaiticum

Leaves

Two flavonolignans hydrocarpin and novel sinaiticin and two flavones chrysoeriol and luteolin

72

Aerial part

New iridoid glycoside sacatoside 6 -alpha - L (3” - n - coumaroyl) rhamnopyranosylcatalpol and α - methyl rhamnopyranoside

73

Under- aerial part

New diacyliridoiddiglycoside 6 -O-(2”, 3” di - O -acyl) - α -L -rhamnopyranosyl catalpol

74

13

Verbascum sinuatum

Iridoid and phenylpropanoid glycoside

75


24

Aerial part

D-glucopyranosyl - (1 - 3) - beta - D -glucopyranosyl - (1 - 2) - beta - D -fucopyranosyl - 13 beta, 28 - epoxyolea - 11 - ene - 3 beta, 16 beta, 23 - triol

76

Aerial part

Two new triterpenoid saponins: songarosaponin E and F and known buddlejasaponin I

77

14

Verbascum songaricum

Root

Flavonoids: apigenin, quercetin, luteolin, cynaroside and daucosterol

78

15

Verbascum spinosum

Aerial part

A new iridoid glycoside verbaspinoside 6 - O - (2” - O - trans - cinnamoyl) -alpha - L - rhamnopyranosyl catalpol

79

Verbascosaponin, luteolin 7 - O -glucoside, Verbascoside and foraythoside B

Antiproliferative effect.

57

Amanthadine derivatives Flowers

Reduced the infectious and haemaglutination yields of influenza viruses

80

16

Verbascum thapsiforme

Aerial parts

7 new saikosaponin homologues called mulleinsaponins having 13, 28 epoxyolean - 11 - ene skeleton isolated together with eight known saikosaponin homologues 3 - O - beta - D -fucopyranosyl saikogenin F, saikosaponin A, songarosaponins C, D, mimengoside A and buddlejasaponins I and IV

71

Aerial parts

New iridoid glycoside unduloside 6 - O - [(2”- O - trans - feruloyl) - alpha - L -rhamnopyranosyl] aucubin

81

A novel macrocyclic dimer lactone: verbalactone,

17

Verbascum undulatum

Root Antibacterial activity

82


25

18

Verbascum fruticulosum

Aerial part

7 new saikosaponin homologues called mulleinsaponins having 13, 28 epoxyolean -11 - ene skeleton isolated together with eight known saikosaponin homologues 3 - O - beta-D - fucopyranosyl saikogenin F, saikosaponin A, songarosaponins C, D, mimengoside A and buddlejasaponins I and IV

71

Acetoside, a polyhydroxylated phenyl propanoid glycoside

19

Verbascum macrurum

Aerial part

Antioxidant activity

83

20

Verbascum wiedemannianum

Aerial part

Four new phenylethanoid glycoside (wiedemanniosides B - E) with wiedemannioside A, verbascoside, martynoside,echinacoside, leucosceptoside B

84

21

Verbascum gypsicola

Whole plant

Antimicrobial activity

85

Five novel iridoid glycosides which are classified into two types - one contain ajugol and others contain 6 - O - L - (alpha-L-rhamnopyranosyl) - catalpol

86

Five new phenylethanoid glycosides and one new lignan glycoside

87

22

Verbascum

thapsus

Whole plant

Verbacoside [Luteolin 5 - O - alpha (1 - 3) - beta - D - glucopyranosyl (1 - 6) - beta - D - glucopyranoside]

88


26

Sterones, iridoids and sesqueterpenes

89

Catalpol, harpagide, aucubin and ajugol

75

Mild astringent

90

Antiviral activity

91

Whole plant

In vitro - Antihepatoma activity

92

Leaves and Flowers

7, 4’ - dihydroxyflavone - 4’ -rhamnoside, 6 - hydroxyluteolin - 7 - glucoside - 3’-methylquercetin

93

Irridoid glycosides: La teroside, harpagoside, ajugol and aucubin

Verbascum thapsus

Roots

Phytotoxic

94

The genus Tephrosia, estimated to contain 300 species, is endowed with

insect controlling properties 95-98. Besides rotenone other insecticidal

principles include tephrosin (Fig.1.1), degu lin (Fig. 1.1) and isotephrosin 99.

Among alkaloids, nicotine (Fig. 1.1) is probably the most well known and

widely used insecticide100. Nor-nicotine (Fig.1.1) and anabasine (Fig.1.1) are

other toxic constituents present in crude extractives of Nicotiana species.

Acylated nor-nicotinoids gave 100 % kill of I st instar larvae of tobacco

hornworm, Manduca sexta101. The powder of dried flowers of

Chrysanthemum cinerariaefolium or Tanacetum cinerariaefolium also

referred as pyrethrum is well known for insecticidal properties.


27

The pyrethrum powder is usually extracted with hexane / kerosene to obtain

viscous oleoresin concentrate containing about 30% pyrethrins. It contains

six closely related insecticidal esters namely pyrethrins I and I I, cinerins I

and II and jasmolins I and II102-103.

Members of family Zingiberaceae have attracted continuous phytochemical

interest due to their considerable importance as natural species or as

medicinal plants. Curcuma longa and Kaempferia pandurata are, on the

other hand, important medicinal plants of South Asia used as folk medicine

for the treatment of stomach discomfort as expectorants or as antiseptic for

wounds104-107. Taxa of Zingiberaceae have also been studied for insecticidal

activity. Dried and powdered rhizomes of C. longa, e.g. have been reported

to deter storage pest insects such as Tribolium castaneum 108.

In Columbia there is a long tradition of plants being used for their

insecticidal, repellent or antifeedant properties to protect crop fr om insect

attack (Perez Arbelaez, E., 1953). Ageratum conyzoides known to contain

precocene I and precocene II109-110 has been used not only in folk medicine,

as a cure for several diseases but also as an insecticide. n -Hexane fraction of

extract is active against Musca domestica larvae but the precocene

containing fraction is not. Some flavonoids are reported 111 to have toxic

effect on insects.

The essential oil of Artemesia monosperma obtained by steam distillation of

the aerial part of the plant was show n to have insecticidal activity against

housefly, cotton leafworm and rice weevil. The chemical structure of the

active ingredient from the steam distillate was shown to be 3 -methyl,

3-phenyl-1, 4-pentadiyne112 (Fig.1.2).


28

OO

O

O

O

MeOMe

MeOMe

H

H

H

O

MeO

MeO

O OO

O

OH

OO

O

O

MeOMe

O

N

Me

H

N

H

H

N

H

H

OO

O

O

O

Rotenone Tephrosin

Deguelin Amorpholone

Nicotine Nor-nicotine Anabasine

Fig. 1.1

Fig. 1.1


29

C CCC CH H

Me

Fig. 1.2

DIFFERENT CLASSES OF NATURAL COMPOUNDS AS

INSECTICIDES

MONOTERPENES

Monoterpenoids have profound effect on insects. They are widely distr ibuted

in the plant kingdom and are utilized as attractants, defensive and

allelopathic agents. When applied to flies, cockroaches and the western corn

rootworm, limonene, linalool and pulegone exhibited insecticidal and

antifeedant activity113. Monoterpenoids in the essential oils of Ocimum

basilicum have activity as deterrents and toxicants. The major active

constituents in the essential oils of Ocimum basilicum include linalool,

methyl chavicol, eugenol, methyl eugenol, geraniol, geranial and neral 114.

Zanthoxylum bungeanum, has been reported to possess insect repellent and

antifeedant action115. Among the principal constituents 1, 8-cineole, linalool,

4-terpineol, α-terpineol, piperitone, 4-terpinoyl acetate, α-terpene, α-

terpineyl acetate and caryophyllene exhibited the strongest feeding deterrent

activity comparable to commercial repellent N, N-diethyl-m-toluamide

(DEET)116. Piperitone, a major component of some oth er essential oils as

well117 acts as feeding deterrent to white pine weevil, Pissodes strobi.


30

Linalool is also repellent towards aphids 118 and mosquitoes. 4-Terpineol is

antifeedant against the locusts and repellent to mosquitoe s119.

SESQUITERPENES AND DITERPENES

Many plant sesquiterpenes and diterpenes exhibit biological activity against

insects ranging from insect feeding deterrence to toxocity 120-122. Several

insecticidal and antifeedant sesquiterpenoids 123-127 and diterpenes128 are

known as major deterrents in insect plant interactions. Floral chemicals,

besides being attractive to pollina tors are known for their ant iherbivore

action against insects. Several feeding deterrents have been isolated from the

inflorescence of cultivated sunflower such as sesquiterpene lactone angelate

argophyllin-A (Fig.1.3) and 3-O-methyl niveusin-A, which are most potent.

Such antifeedants produced symptoms in western corn rootworm 129-130.

A sesquiterpene 4,11-selinadien-3-one, also known as α-cyperone, isolated

from the nutgrass tubers showed insecticidal activity against diamondback

moth, Plutella xylostella131. Drimane group of sesquiterpenes, possess a

broad spectrum of activity including antibacterial, antifungal, antifeedant,

plant growth regulatory, cytotoxic, phytotoxic, piscicidal and molluscicidal

activities. Besides, occurring in plants of green Warburgia, Cinnamosina,

Winterana and Cinnamodendron (Cannellaceae) such compounds also occur

in the marsh pepper Polygonum hydropiper (Polygonaceae). They have also

been reported from some fungi and some molluscs. Polygodial , warburganal

and muzigadial are among some potential drimane sesquiterpenes having

anti-insect and antifungal properties 132.

The powdered bark of Chinese bittersweet, Celestrus angulatus , is

traditionally used in China to protect plants from insect damage. An insect

antifeedant celangulin with two possible structures has been reported from

this plant. It is a non-alkaloidal sesquiterpene polyol ester compound that has


31

a dihydroagarofuran skeleton with seven hydroxyl functions, five of which

are acetylated, one benzoylated and one free 133. Insecticidal alkaloids with a

β-dehydroagarofuran skeleton such as wilfordine from Tripterygium

wilfordii134 and wilforine alkaloid from Maytenus rigide as insect

antifeedant135 have also been reported.

ACETYLENES AND THIOPHENES

The oil of the desert plant, Artemisia monosperma has been reported to

contain 3-methyl 3-phenyl-1, 4-pentadiyne which is as active as DDT

against housefly and cotton leafworms, S. littolaris larvae. It is five fold

more active against the rice weevil, Sitophilus oryzae136.

PHENYLPROPENOIDS

Phenylpropenoids have some potential as evident fro m the bioactivity shown

by the essential oil of sweetflag, Acorus calamus. The oil has insecticidal,

ovicidal, antigonadal, antifeedant and insect growth inhibitory activities,

which have been attributed to the presence of asarones, the

phenylpropenoids occurring in very high percentage in plant 137. Asarone also

referred to cis asarone is a major constituent though other isomers such as

trans asarone and isoasarone has also been reported from this plant138

(Fig.1.3).

ACETOGENINS

Acetogenins from Annona sp. are waxy substances consisting of C -32 or

C-34 long chain fatty acids combined with a propan-2-ol unit at C-2 to form

•-lactone. Over 220 annonaceous acetogenins have been reported from 26

species139. Entire group of annonaceous acetogenins was patented as

pesticide in which asimicin was claimed as structurally defined pesticidal

acetogenin140.


32

ISOBUTYLAMIDES

A large number of unsaturated isobutyl amides have been isolated from

various species of genus Piper141. These compounds have been isolated

mainly from fruits, stem and leaves of various Piper species such as

P. nigrum, P. longum, P. pedicellosum and P. thomsoni. Some of the active

compounds include piperlonguminine, pip erine (Fig.1.4), guineesine,

retrofractamide, pipericide, dihydropipericide, and pellitorine 142-145. Being

neurotoxic, these amides showed both knockdown and lethal action against

pyrethroid susceptible and resistant insects.

QUASSINOIDS

Quassinoids are well known for their anti-inflammatory, antimalarial,

amoebicidal, antifeedant, insecticidal and herbicidal properties. At least 31

quassinoids reported from Picrasama ailanthoides146-149 are potent

antifeedant and insecticidal compounds against 3 rd instar larvae of

diamondback moth, P. xylostella. Some of the bioactive quassinoids include

bruceantin isobrucein and bruceanol A (Fig.1.4) from Bruceae

antidysenterica and bruceoside A, brucein E, bruceoside B and yadanzioside

A, B, C, F G and L from Brucea javanica150.

LIMONIN AND RELATED COMPOUNDS

Limonoids are a group of chemically related bitter tetranortriterpenes found

predominantly in Rutaceae and Meliaceae. Limonin (Fig.1.4) is one of the

principal bitter components of citrus seeds having anti -insect properties. In

addition to having anti -feedant action against various insects, recently it

has been demonstrated that it induces anti -feedant action against the 5 th

instar larvae of P. xylostella which have developed resistance to

conventional synthetic insecticide150. Different parts of the neem,

Azadirachta indica, particularly the seeds, contain the array of biologically


33

active tetranortriterpenoids based on apo -euphol or apotirucallol skeleton 152-

153. Two limonoids have been commercialized, azadi rachtin in many parts of

the world and toosendanin in China. Azadirachtin, the main active

compound was identified as a potent anti -feedant against Schistocerca

gregaria, the desert locust. Its structure has been finally established by Kraus

and it induces toxicological, behavioural and physiological responses in over

four hundred insect species.

MELIACINS

Among the various groups of meliacins, which differ from each other in

basic nuclear structure pattern of oxygenation, C -seco meliacins are most

important such as azadirachtins (A & B) (Fg.1.5), salanin and nimbin.

Toosendanin is the major bioactive material in bark of Melia azedarach and

M. toosendan . It possesses strong anti -feedant properties and also inhibits

insect growth development154.

CARDENOLIDE GLYCOSIDES

Fractionation of the methanol extracts of stem and bark of Anodendron

affine has led to the isolation of three cardenolide glycosides, 4,5 -dehydro-

12-oxo-affinoside E, 12-oxo-affinoside E and 16 •-hydroxy affinoside A155

along with previously known carbenolides, affinoside A, E and M

which inhibit growth of silkworm larvae , Bombyx mori at 1 to 3 ppm.

concentration.

SUGAR ESTERS

Resistance to insect pests in Nicotiana sp., wild tomato, Lycopersicon

birsutum, Solanum sp.156 occurs due to glandular trichomes and the exudates

like sugar esters produced by them. Plant sucrose o r glucose esters composed

of the lower fatty acids (C-2 to C-10) possess very interesting biological

properties. Mixtures of sugar esters cibarian, coronarian and karakin


34

(Fig.1.4) reported from the forage legume, Lotus pedunculus exhibit

antifeedant activity against white grass grub, Costelytra zealandica157.

FUROCHROMENES AND COUMARINS

Furochromenes and coumarins have mostly anti -feedant action against

insects. The compounds like visnagin, khellin, from Pimpinella monoica and

khellinol ethyl ether are very active158.

NON-PROTEIN AMINO ACIDS

Among the several non-protein amino/imino acids of botanical origin,

azetidine-2-carboxylic acid, 2,4-diaminobutyric acid, mimosine,

3-hydroxyproline, •-cyanoalanine, pipecolic acid and canavinene are

significant in causing insect growth inhibition 159-161.

MOULTING HORMONES, JUVENILE HORMONE MIMICS AND ANTI

JUVENILE HORMONES

The endocrine system is critical for gr owth and survival of the insects. The

biosynthesis and release of brain, juvenile, moulting, eclosion and diapause

hormones generally govern insect growth and moulting. Of these, juvenile

hormones (JHs) and moulting hormones (MHs) are most significant as t heir

mimics and/ or antagonists are capable of disrupting insect growth and

moulting162. Phytoecdysteroids, the chemicals structurally similar to the

MHs, have been found in many plants especially ferns and yews.

The use of Juvenile Hormones (JHs) in insec t control was first demonstrated

by Wiggles-Worth in 1935. JH mimics which functionally resemble natural

JHs have been isolated from plants and shown to disturb normal

metamorphosis moulting and reproductive process of insects. Some of the

important JH mimics include farnesol from several plant oils, juvabiones

(Fig.1.5) from Abies balsamea and juvocimenes from Ocimum basilicum163.


35

The compounds like farnesol, farnesal , sesamin, sesamolin are also JH

mimics164.

O

O

O

O

HO

O

O

O

O

OCH O

O

OCH O

O

OCH

R2

R1

a. Pyrethrin I R 1 =CH3, R 2 =CH=CH2b. Pyrethrin II R 1 =COOCH3, R 2 =CH=CH2

c. Cinerin I R 1 = R2 = CH3d. Cinerin II R 1 =COOCH3, R 2 =CH3e. Jasmolin I R 1 =CH3, R 2 = CH2-CH3f. Jasmolin II R 1 =COOCH3, R 2 =CH2-CH3

Agrophyllin-A

3 3 3

-Asarone Isoasarone

Fig.1.3

-Asarone

β-Asarone α-Asarone Isoasarone

Fig. 1.3


36

O

O

N

H

O

O

O

N

O

O

O

O

O

O

O

O

O

O

O

H

HHOH

H

H H

Piperlonguminine Piperine

R1

COOCH3

OR2

Bruceantin R1=OH, R2=COCH=C(CH 3)C(CH3)2

Isobrucein-B R1=H, R2=COCH3

Bruceanol-A R1=H, R2=COC6H5

Limonin

Cibarian R1=R3=COCH2CH2NO2

Coronarian R 1=R2=COCH2CH2NO2

Karakin R1=R2=R3=COCH2CH2NO2

Fig.1.4

CH2-O-R3

OR1

R2O

Fig. 1.4


37

O

O

OH

O

O

OH

O

OOO

OHOH

OOH

OH

O

O

OO

OOH

OHO

OH

OH

O

OH

CO2Me

AcOMeO2C

Azadirachtin A

Juvabione Dehydrojuvabione

CO2Me

MeO2C

Azadirachtin B

Fig.1.5

Fig. 1.5


38

CHEMICAL CLASSES OF NATURAL COMPOUNDS ISOLATED

The continuous use of plants as biologically important source of chemicals

encouraged chemists and biochemists to isolate various types of natural

products from plants. The important classes of compounds that have been

isolated from the undertaken plant species are:

TERPENOIDS

Terpenoids comprise the largest and most widespread group of natural plant

products and over twenty thousand such structures have been described from

plant sources. They all are derived biogenetically from the 5 -carbon

precursor isoprene and, hence, are known as isoprenoids.They are classified

according to whether they contain two (C 10), three (C15), four (C20), six (C30)

such units. They range from essential oil components, the volatile mono or

sesquiterpene (C10 & C15) through less volatile diterpene (C 20) to involatile

triterpenoids (C30).

ESSENTIAL OILS

Chemically the terpenes of essential oils can be divided into two classes, the

mono and sesquiterpenes, C10 and C15 isoprenoids, which differ in their

boiling point range (monoterpenes b.p. 140 - 180 oC, sesquiterpenes b.p. >

200 oC). In monoterpenes, these substances can be further divided into three

groups depending on whether they are acycli c (e.g. geraniol), monocyclic

(e.g. limonene) or bicyclic (e.g. • - and •-pinene) or irregular (γ-thujaplicin).

1. Monoterpenes

Monoterpenes may be simple unsaturated hydrocarbons (e.g. limonene) or

may have functional groups and be alcohols (e.g. menthol), aldehydes or

ketones (e.g. menthone, carvone). Also included with monoterpenes on

biosynthetic grounds are monoterpene lactones (better known as iridiods)

and tropolones.


39

A typical monoterpene lactone is nepetalactone, the principal odour

constituent of catmint, Nepeta cataria (Labiatae), a plant which has a

peculiar attraction for the domestic cat because of its odour. Other iridoids

such as loganin are of interest because they are intermediates in the

biosynthesis of the indole alkaloids. A typical tropo lone is •-thujaplicin;

these substances have a restricted distribution in certain fungi 165. The

structures of a range of typical monoterpenoids are collected in Fig.1.14

Simple monoterpenes are widespread and tend to occur as components of the

majority of essential oils. Some compounds are regularly found together in

leaf oils, especially • - and • - pinene, limonene, •3- carene, •-phellandrene

and myrcene. Flower and seed oils tend to have more specialized

monoterpenes.

2. Sesquiterpenes

Like the monoterpenes, the sesquiterpenes fall chemically into groups

according to the basic carbon skeleton; the common ones are either acyclic

(e.g. farnesol), monocyclic (e.g. • -bisabolene) or bicyclic (e.g. • -selinene,

carotol). However, within each group there are ma ny different compounds

known indeed, according to a recent estimate, there are several thousand

sesquiterpenoids with well -defined structures, belonging to some 200

skeletal types. The chemical formulae of the various sesquiterpenes

mentioned above are shown in Fig.1.15.

EXTRACTION

For their isolation from plant tissues, mono -and sesquiterpenes are

nowadays separated by extraction into ether, petroleum or acetone. The

classic procedure for essential oils is separation from fresh tissue by steam

distillation. This step is now often omitted, because of the danger of artifact

formation at the raised temperatures involved. Terpenes may either undergo


40

rearrangement (e.g. dehydration in the case of tertiary alcohols) or

polymerization. The volatility of the simpl e terpenes means that they are

ideal subjects for separation by GLC. Many have fragrant odours and indeed

can often be recognized in plant distillates directly, if present as the major

constituent.

The mono- and sesquiterpenes identified as volatile consti tuents in most

common fruits and vegetables have been exhaustively listed by Johnson

et al. (1971)166. The more recent general reviews are those of Charlwood and

Banthorpe (1978)167 and Loomis and Croteau (1980)168.

RECOMMENDED TECHNIQUES FOR MONO- AND SESQUITERPENES

1. Gas liquid chromatography

This is undoubtedly the most important technique for study of essential oils

since it yields in one operation both qualitative and quantitative analysis.

This is particularly important when a similar set of c ompounds occur

throughout a particular plant group, since it is the quantitative variations that

are most significant. Certainly, GLC is an indispensable tool for

chemotaxonomic studies of essential oils in leaf or bark, as in the

gymnosperms.

For the identification of volatile terpenes in any plant material, it is essential

to combine the use of GLC with other procedures, and especially with TLC.

TLC is useful, for example, for monitoring fractions separated by

preparative GLC; on the other hand, if a pre parative GLC apparatus is not

available, large-scale separations can be carried out on TLC, with the TLC

fractions subsequently being monitored by GLC. Some examples of relative

retention times are shown in Table. 1.4. These are given to illustrate the nee d

for using more than one column for purpose of terpene separation and

identification. In the detailed analysis of individual oil constituents of a


41

particular plant material, it is normal practice to use a range of GLC columns

for identification purposes.

Table 1.4. Relative retention times of terpenes in gas liquid chromatography

RRTs on column*

Terpene 10% Apiezon N

15% Polyethylene glycol

15% Polyethylene glycol bispropionitrile

•-Pinene

42

29

30

Camphene 50 41 44

•-Pinene 63 55 54

•3-Carene 82 73 67

Myreene 60 82 88

•-Phellandrene 82 82 86

Limonene 100 100 100

β-Phellandrene 97 106 116

p-cymene 100 175 232

* RRTs relative to limonene, from isothermal runs at 65 oC on a 300 cm column169

2. Thin layer chromatography

It is even possible, in the absence of a GLC apparatus to analyse essential

oils using TLC as the only separation technique 170. Even when GLC is

available, TLC is useful at all stages for separation and analysis of these

terpenes. Silica gel is the most widely adsorbent with solvents such as

benzene, chloroform, benzene-chloroform (1:1) and benzene -ethyl acetate

(19:1). For the analysis of oxygen-containing terpenes (e.g. carvone), silica

gel layers should not be activated prior to use since the moisture present aids


42

the separation. Terpene alcohols are best separated on paraffin -impregnated

plates in 70% methanol. Activated silica gel plates are first immersed in 5%

paraffin in petroleum for 1 min and then allowed to dry before use; the

chromatographic solvent, 70% methanol must also be saturated with paraffin

oil. Another modification to separate terpenes according to the number of

double bonds involves TLC on silica gel plates spread as a slurry with 2.5%

aqueous AgNO3 instead of with water. The solvent system to employ with

the AgNO3 treated plates is methylene dichloride -chloroform-ethyl acetate-

n-propanol (10:10:1:1).

General methods of detection include spraying with 0.2% aqueous KMnO 4,

5% antimony chloride in chloroform, conc. H 2SO4 or vanillin-H2SO4. The

latter reagent is prepared fresh by adding 8 ml ethanol with cooling to 0.5 g

vanillin in 2 ml conc. H 2SO4. The plates are heated after spraying at

100-105 oC until full development of colours has occurred. More selective

agents are available for de tecting terpenes with double bonds (bromine

vapour) and those with ketonic groupings (2, 4 -dinitrophenyl-hydrazine).

The responses of some of the common terpenes to a range of detection

agents are indicated in Table 1.5. TRITERPENOIDS

Plant triterpenoids have been classified into tetracyclic (Fig.1.6) and

pentacyclic triterpenes according to their skeletal type and are widespread in

nature (Fig.1.7). Squalene is universally present in green plants since it is the

basic precursor of all triterpenoids and tetraterpenoids171.


43

Table. 1.5. Detection of monoterpenes on thin layer chromatography

plates

Response to test

Terpene UV

Bromine

2,4-DNP

Conc. H2SO4

Limonene

-

+

-

Brown

•-Pinene - + - Brown

Pulegone + + + Yellow

Geraniol - + - Purple

Carvone + + + Pink

p-cymene + - - -

•-Terpineol - + - Green

1,8 Cineoic - - - Green

Key: UV = examine in short UV light; bromine = spray with 0.5% fluorescein in water,

expose plate to bromine vapour, yellow spots on a red background; 2,4 DNP = spray with

0.4g2.4-DNP in 100 ml 2 M HCl, yellow spots on white background, conc. H2SO4 =

spray with conc. H2SO4 and heat plate at 100 oC for 10 min.


44

Cucurbitane Drammarane

Euphane Halimone

Lanostane Trucalane

Basic skeleton of naturally occurring tetracyclic triterpenes

Fig. 1.6


45

Triterpenic Groups

Tetracyclic Pentacyclic

(A) Cucurbitane (a) Pleanane

(b) Dammarane (b) Ursane

(c) Lanostane (c) Friedelane

(d) Euphane (d) Serratane

(e) Halimone (e) Strictane

(f) Tirucalane (f) Taraxasterane

(g) Lupane

(h) Hopane

(i) Feranane

FATTY OILS OR FIXED OILS

Fatty oils or fixed oils are chemi cally triglyceraldehyde or simply

glyceraldehyde i.e., esters of glycerols with saturated or unsaturated fatty

acids containing minor portion of sterols (free or as esters), vitamins,

pigments, hydrocarbons and other substances. The natural stuff consistin g of

glycerol esters, which is solid at room temperature, is commonly called fat

(saturated) and one that is liquid at room temperature is called oil. The two

terms are purely conventional since the same substance may be a fat in one

climate and oil in another.


46

EXTRACTION

Fresh leaf tissue is extracted by maceration in 20 vols of cold isopropanol

(this alcohol de-activates hydrolytic enzymes) and this is followed by re -

extraction with chloroform-methanol (2:1). Seed tissue can be extracted

directly with the latter solvent mixture or with petroleum. For tissues in

which the lipids are very tightly bound such as cereals, extraction with

chloroform-ethanol-water (40:19:1) is advisable.

1. Thin layer chromatography

The total lipids of plant tissues can be a nalysed by two-dimensional TLC.

This was obtained from potato tuber 172 but most other plant tissues show a

range of similar components. The solvent in the first direction is chloroform -

methanol-acetic acid-water (170:25:25:4), and that in the second,

chloroform-methanol-7M NH4OH (65:30:4). In order to avoid

decomposition of lipids during TLC, Galliard (1968) recommends adding

BHT (5 mg) to the first solvent and drying the plate after the first run at

50 oC in an atmosphere of nitrogen.

Phospho-and glycolipids can be separated one-dimensionally on silica gel

plates using solvents such as chloroform -methanol-acetic acid-water

(170:30:20:7) and acetone-benzene-water (90:30:8). Phosphatidylglycerol is

better separated from phosphatidylethanolamine by running the second

solvent on plates previously impregnated with 0.15M ammonium

sulphate173.

2. Gas liquid chromatography

The fatty acids obtained after acid hydrolysis are converted to the methyl

esters with ethereal diazomethane and then analysed by GLC. Alte rnatively,

the fatty acid methyl esters can be obtained directly by transmethylation of


47

the parent lipids by refluxing them for 90 min with methanol -benzene-

H2SO4 (20:10:1). Some unsaturated acids cannot be identified by GLC

alone. In such cases, it is necessary to carry out argentation TLC in order to

determine the degree of unsaturation.

3. High performance liquid chromatography

Although HPLC has been applied to many lipid separations, there is still the

major difficulty of finding a suitable detection s ystem for a class of

compounds essentially lacking UV absorbance. In the case of the fatty acids

this can be overcome by derivatizing them and separating them as their

phenacyl or p-bromophenacyl esters. In the case of the bound lipids it is

possible to measure their end absorption at about 195 nm or use a refractive

index detector, but in general HPLC has not yet become a routine procedure

in plant lipid studies.

The common fatty acids are either saturated or simple unsaturated

compounds of C16 or C18 chain length (Fig. 1.6). Palmitic acid, a C 16 acid is

the major saturated acid in leaf lipids and also occur in some seed oils.

Stearic acid, C18 is less prominent in leaf lipids but is a major saturated acid

in seed fats in a number of plant families 174.

STEROIDS

Steroids are based on the 1,2-cyclopentenophenanthrene (Fig.1.7 a) skeleton,

and form a group of structurally related compounds, which are widely

distributed in animals and plants. Many natural steroids are unsaturated

(mostly at C-5) and are designed as ‘sterols’. On dehydrogenation with

selenium at 420 oC, all steroids give chrysene as the main product with small

amount of pinene. They all give D iel’s hydrocarbons among other products.

α-Spinasterol, ergosterol, campasterol, stigmasterol and β-sitosterol are

common plant steroids. These steroids are sometimes present in glycosidic


48

forms and as acetate derivatives. The aglycones of this group, possessing

spirostane nuclei having rings A B C D E and F were isolated first

(Fig.1.7 b). Some steroidal glycosides have open F rings (Fig.1.7 c) and

known as furostanol glycosides or bisdesmoside 175.

The most common steroid, β-sitosterol has been isolated invariably from

almost all plant species. Due to the different types of pharmacological

activities like antinflamatory, antifungal, antirheumatic etc., the vast majority

of steroids play an important role in field of medicines. They occur

invariably, where life exists and have profound importance in animal

metabolism. They are structurally related to hor mones (oestrogen group and

male sex hormones), co-enzymes, bile acids and provitamin-D.

FLAVONOIDS

The term flavonoid embraces a large group of naturally occurring

compounds such as anthocyanins, leucoanthocyanins, chalcones,

dihydrochalcones and aurones e tc. Flavonoids are benzo-γ-pyrone

derivatives, which resemble coumarine and are ubiquitous in

photosynthesizing cells. Their occurrence is therefore widespread in the

plant kingdom. A wide variety of flavonoids are known for centuries.

Preparations which contain flavonoids as principal physiologically active

constituents have been used by Laymen and Physicians in attempt to treat

human diseases176. Flavonoids occur as aglycones, glycosides and

methylated derivatives176-179. All the flavonoid aglycones consi st of a

benzene ring (A) condensed with a six membered heterocyclic ring (C),

which is either a γ-pyrone (Chromone) or its dehydro derivative (4 -

chromone). 4-Chromanone substituted by an aryl ring (B) at position -2 gives

rise to flavonones and dihydroflavo noids (Fig.1.8). The position of aryl ring


49

O

O

OH

H

H

H

H

H

(b)

(c)

Me

2720

(a)

Fig. 1.7


50

O

O

O

O

O

O

O

O

O

O

OH

O

O

OH

O

O

OH O

O

OH

Chromone 4-Chromanone

Flavone Flavanone

Flavonol Flavanol

Isoflavonol Isoflavanol

Fig. 1.8


51

divides the flavonoids class into flavonoids (2-position) and isoflavonoids

(3-position). Flavonoids differ from flavonoids by OH -group in 3-position

and C2-C3 double bond. Flavonoids are often hydroxylated at 3, 5, 7, 3’, 4’

and 5’-positions. Methyl ethers and acetyl ethers of the alcohol groups ar e

known to occur in nature. When glycosides are formed, the glycosidic

linkage is normally located at 3 - or 7-position and the carbohydrate can be

L-rhamnose, D-glucose, glucorhamnose, galactose or arabinose 176.

Structurally flavonoids resemble with nucleo sides, isoalloxazine. and folic

acid and this similarly is the basis of many of the current hypothesis o f their

physiological action180.

EXTRACTION AND ISOLATION OF FLAVONOIDS

Since the last five decades number of techniques were developed and used

for the isolation of flavonoids from the plant material including solvent

extraction181-182.

Flavonoids are found in almost all parts of the plants and methods of

extractuion used basically depend on the type of flvonoids to be isolated. In

general, freshly dried plant material provides the ideal material for a

flavonoid analysis. The flavonoids can be conveniently and sequentially

extracted with the solvents of increasing polarity. This method is used to

separate the less polar and highly methylated flavonoids from the

hydroxylated flavonoids and their glycosides. In general, the solvents used

for the extraction are chosen according to the nature of the compounds to be

studied. Higly methylated flavonoids are soluble in the less polar solvents

such as petroleum ether, dichloromethane and chloroform. Flavonoids

possessing a number of unsubstituted hydroxyl groups or a sugar are soluble

in polar solvents183-184.


52

The isolation and purification of the flavonoids mixture along with other

constituents can be achieved by chromatographic techniques such as column

chromatography over silica gel, Sephdex -20 and preparative TLC183, 185. The

various methods (GC, MPLC, HPLC, and DCCC) are well established for

the isolation of the flavonoids 186. The choice of isolation techniq ues depends

largely on the solubility and nature of the compounds to be isolated. PC is

particularly useful to isolate the water -soluble constituents185-186.

METHODS OF IDENTIFICATION

1. Chemical Methods

Once the flavonoids are purified, the following steps are used to identify the

pure compounds.

1.1. Hydrolysis

1.1.1. Acidic Hydrolysis

Acidic hydrolysis of flavonoid glycoside is commonly carried out by

dissolving the compound in 2 -5 ml of 2-6% aqueous HCl or H2SO4 and

refluxed for 45 min., on water bath. The cooled solutionis extracted with

diethyl ether or eyhyl acetate to remove the aglycone portion of the

flavonoid glycoside. The aqueous layer contains the sugar moiety and

used for the estimation of sugar. More dilute acid e.g. formic acid in

cyclohexane or 10% acetic acid at lower temperature is used for partial

hydrolysis of di and triglycosides187-188. Acidic hydrolysis can be also be

carried out by using the Kiliani mixture 189.

1.1.2. Enzymatic Hydrolysis

Compound (1 mg) is treated with 2 ml of 0.5 M acetic acid-sodium

acetate buffer (pH = 5.0) in presence of 1mg β-glycosidase. The mixture

was allowed to stand over night at 37 oC, which afforded aglycone and


53

sugar moiety190.The sugar(s) thus obtained was identified by paper

chromatography and gas chromatography by comparing with the

authentic samples. The sugars can also be identified by PC, developed by

using the developing solvent systems such as n -butanol-pyridine-water

(6:4:3), n-butanol-acetic acid-water (4:1:5) or ethyl acetate -pyridine-

water (12:5:4)191.

2. Spectroscopic Methods of Identification

2.1. UV-VIS Spectroscopy

Flavonoid contains conjugated aromatic system and thus showed intense

absorption bands in the UV -VIS regions of the spectrum. The substitution

patterns of flavonoid nuleus can be determined by adding shift reagents.

Sodium methoxide, sodium acetate, sodium acetate -boric acid, aluminium

chloride and aluminum chloride -hydrochloric acid are used as shift

reagents192.

UV spectrophotometry of flavonoids dissolved in MeOH or EtOH and with

the addition of the classical shift reagents is a method of choice in structure

determination. Flavonoids exhibit two characteristic UV bands depending

upon the hydroxylation patterns of the aromatic ring. Band I is usually

recorded in the region 300-380 nm for all flavonoes and flavonols and band

II in the region 240-280 nm191.

2.2. IR Spectroscopy

The IR spectrum of flavonoids displayed characteristic absorption in the

region 3200-3450 cm-1 of free hydroxyl and chelated hydroxyl groups, two

bands in the region 1600-1700 for •, •-unsaturated carbonyl function and

the aromatic absorption in the region 1500 -1600 cm-1 193.


54

2.3. NMR Spectroscopy

The application of NMR in structure elucidation of flavonoids is well

established. Many methylated and acet ate derivatives of flavonoids are

sufficiently soluble in the commonly used solvent deuterated chloroform

(CDCl3) for direct NMR analysis. However, most naturally occurring

flavonoids including flavonoid glycosides have less or no solubility in

CDCl3. Thus the NMR spectra of highly hydroxylated and flavonoid

glycosides are recorded in CD 3OD, DMSO-d6, or C5D5N and TMS an

internal standard191-192 .

2.4. Mass Spectrometry

The MS is very useful in the characterization and structure elucidation of

6- and 8-C-flavonoid glycosides related to apigenin and luteolin 196.

C-glycosyl flavones show initial fragmentation of the sugar moiety. Electron

impact mass spectrometry (EI-MS) serves as valuable aid in determining the

structure of flavonoids especially when very small quantities are available.

Most aglycones are sufficiently volatile at probe temperature 100 -300 oC to

allow successful MS without derivatization 192, 195.

The fragmentation of flavonoids takes place by Retro-Diels Alder (RDA)

cleavage. The RDA fragmentation leads to the clevage of the flavonoid

molecules by the double bond in ring -C giving fragments derived from ring-

A and B which show the substitution pattern on each ring (Fig.1.9).

Fragment A1 gives information on the substitution pattern of A-ring and

fragment B1 and B2 show the substitution pattern of B -ring196-198.

The initial products of fragmentation of flavonols are the ion A 1 and B1,

while flavones give ions A2 and B2 showing that main cleavage involves the

bonds between oxygen and C-2 and C-3, C-4 (Fig.1.9). Another important

fission product of flavones involves elimination of C -4 carbonyl group with


55

the formation of an M-28 ion. Isoflavones give a similar fragmentation

pattern to that for flavones. Flavanones give A 1, A2 and B3 ions while

dihydroflavonols give A2, B3 and B4 and chalcones produce A 3, B3 and B5

ions (Fig.1.9)199-200.

O

O

O

C

O

C

CH

O

O

O

CO

CO

H

O

C

O

C

CH

C

C

O

+

+

R1

R1O

R3

R3

R1O

R1

R1O

R1O

R1O

R1O

+

+

A

A

B+

+

X

H

R

H2+

+

A

R2

R2

A1

A2

B1

B2

A3 B3, X=H or OH

B4 B5

C

Fig.1.9


56

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