SAUSSAUREA LAPPA C.B. CLARKE (ASTERACEAE)

Introduction

Saussurea DC is a large genus of annual, biennial or perennial herbs

distributed in the temperate regions of Asia, Europe and North America. Out

of 125 species about 47 species are reported in India of which Saussurea

lappa is reputed for the medicinal properties of its roots.

Vernacular names

Sanskrit

Arabic and Persian

Hindi and BengaliGujaratil\/1arathiTeluguTamilMalayalamKannad

Kashmiri

Kushta

Kust

Kut, Kur, Pachak

Upaleta, Kur KushtaChangala, Kustam

Kostum, Putchuk, Goshtam

Sepuddy

KoshtaKuth, Chob-i-qut, post-khai

Habitat

it grows in alpine grasslands and rocky slopes: moist ravines in forests. It Is

distributed in Kashmir at altitudes from 2,700 - 4,000 m; and also cultivated in

Kashmir and neighbouring Himalayan regions. It is found growing wild only in

Jammu and Kashmir in the Kishenganga valley and the higher elevations of

the Chenab valley, Lahul in Himachal Pradesh and Garhwal in Uttaranchal.

Kuth can be propogated either by root cuttings or by seeds.

227

Morphology

A tall, robust, perennial herb. Stem erect, 1.2 - 2 m high, simple. Leaves

membranous, irregularly toothed; basal ones very large, 0.6 - 1.2 m long,

triangular, with a long lobately winged stalk, end-lobe often 30 cm diam.

Stem-leaves smaller, stalked or stalkless, with 2 half-stem-clasping lobes at

the base. Flower-heads stalkless, hard, rounded, 2.5 - 3.8 cm diam., 2.5

forming axillary and terminal clusters. Bracts surounding the heads many,

ovate-lanceolate, long-pointed, rigid, bent back, hairless. Corolla 2 cm long,

tubular, dark blue-purple or almost black. Stamens free. Anther-tails fringed.

Pappus-hairs 1.7 cm long, brown, all feathery. Fruit (achene) up to 8 mm

long, compressed, curved, tip nan^owed, with 1 rib on each face, top

contracted, cupped. (Kirtikarand Basu, 1993).

Plate 7.1: Dried Roots of Saussurea lappa B.C. Clarke

228

Fresh roots of Kuth are stout, up to 0.6 m long, 0.3 in girth and carrot like;

sometimes a number of roots are found joined together at the collar zone.

Root possesses a characteristic penetrating odour which can be smelt from a

distance and sticl<s to the soil and even the handling apparatus. The dried

roots of the kuth constitute the drug Saussurea which is official in India. They

have strong and sweet aromatic odour and a somewhat bitter taste. They are

greyish to dull brown, thick, light, stout, fusiform to cylindrical, 7-15 cm long

and 1-5 cm thick. Occassionally they are ridged and possess a short and

horny fracture. In powdered form the drug is deep brown or rust coloured and

contains not more than 2% percent foreign organic matter (Wealth of India,

1993).

Chemical Constituents

The roots of S. lappa contain reslnoids (6%), essential oil (1.5%), alkaloids

(0.05%), inulin (18%), fixed oil and other minor constituents like tannins and

sugars. The roots obtained from Kashmir are richer In essential oil contents

than root obtained from Garhwal and Nepal. The essential oil obtained from

roots of aged plant and samples collected during September-October is

higher percentage than those collected during earlier months (Rao, ef

a/.,1951).

The yield of essential oil obtained from Kashmir variety, range from 0.8 -

5.8%. The oil percentage Is totally depends upon the method by which it is

extracted (by steam distillation or by solvent extraction or by steam distillation

of resinoids obtained by alcohol or benzene extraction) (Handa ef a/.,1957;

Het Singh ef a/., 1957).

The constituents of the essential oil are mainly high boiling sesquiterpenes

and susquiterpenic alcohols. The oil, obtained by steam distillation of

powdered roots, has the following constants : sp. gr, 1.5045, + 12.3°;

acid value, 9.7; ester value, 58.1; ester value after acetylation, 161.0;

229

solubility, soluble in all volume of 90% alcohol. The following constituents

have been reported from the essential oil : constunolide, primary

sesquiterpene lactone, dehydro costus lactone, dihydro costus lactone,

dehydrocostuslactone, 12-methoxy-dihydrocostunolide, costol, p-sitosterol,

stigmasterol, betulin, aplotaxene, p-selinene, p-elemene, a-and p-ionones, a

bicyclic sesquiterpenic acid, a Ci3-ketone (Het Singh et a/.,1957; Saxena et

a/., 1993), a crystalline lactone-saussurea lactone which is isomeric with

costus lactone (Rao et a/.,1951; Kulkarni et a/.,1961), caryophyllene,

humulene, 1-pentadecene, dihydroapotaxene and 3,9,11-guaiatrlene - 12-

acid which was isolated as the methyl ester (Klein et a/., 1976), four

sesquiterpenes - (-) a - costol, {+) y- costol. (-) elema - 1,3,11(13)-trien -1 2 -

ol and (+) y - costal In addition to (-) a-selinene, (+) selina - 4 ,1 1-diene, (-) a-

trans-bergamotene, (-) a-costal, (+) p-costol, (-) elema -1,3,11 (13)- trien-12-

al, (-) E-trans-bergamota-2,12 - dien - 14-al, (-) curcumene and (-)

caryophyllene oxide, (E)-9-lsopropyl -6-methyl-5,9-decadiene-2-one, a

terpenoid C14- ketone isolated and its structure was established (Maurer and

Ohioff, 1977), (E) 6,10 - dimethyl - 9 - methyleneundec - 5-en - 2-one, acetic

acid, 3-methyl butyric acid, hexanolc acid, heptanoic acid, octanoic acid, 7-

octenoic acid, 3-isopropylpentanoic acid, 4-ethyloctanoic acid, a-amorphenic

acid were isolated as acidic components In the essential oil of Kuth (Rijke et

a/., 1978). A new guaianollde, 12-methoxy dihydrodehydro costus lactone, has

also been reported from the essential oil (Dhillon et al., 1987).

The leaves contain taraxasterol (C30H50O) (Namboodiripad era/.,1968) and

an alkaloid, saussurine, (Ghosh et a/., 1929); The same alkaloid is also

reported from the basic fraction (0.002%) of alcoholic extract of the root,

kushtin a non-nitrogeneous constituents probably an artefact resulting from

the interaction of ammonia with the non nitrogeneous constituents (Salooja. ef

a/.,1950; Dutta et a/.,1960; Sastry ef a/.,1961). a-amyrin stearate, p>amyrin

230

palmitate and lupeol palmitate have been also reported in hexane extract of

leaves (Pai and Kulkarni, 1977).

Other neutral products which have been reported from its roots are ; 22-

dihydrostigmasterol (Jain e? a/., 1968), costunolide, dehydrocostuslactone and

costic, palmitic and linoleic acids besides p-sitosterol and

a-cyclocostunolide from Punjab vareity and alantolactone, p-cyclocostunolide,

isoalantolactone and a-cyclocostunolide from Kashmir variety (Govindan et

a/.,1977). 4p-methoxy dehydrocostus lactone was isolated from the mother

liquor of a cooled petrol extract of its roots (Singh et a/.,1992) and a

sesquterpenoid-saussureal a new ring A -contracted aldehydrolactone, of the

modified eudesmanolide type, was isolated from the biologically active

fraction of the oil of roots from Kashmir variety (Talwar et al.,1992) were

isolated from roots and their structure established. Five amino acid-

sesquiterpene adducts-saussureamine A,B,C,D, and E and a lignan

glycoside- (-) massoniresinol - 4"-0-p-D-glucoside isolated from roots and

characterised (Yoshikawa et a/., 1993). Two guaianolids, namely

isodehydrocostus lactone and Isozaluzanin C. are isolated in minor quantity

from Kashmir variety (Kalsi et al..1963), two sesquiterpene lactones with the

same molecular formula but having different physical states, i.e., solid and

liquid forms and a-, p-, unsaturated aldehydic group present in their molecule

are isolated (Kalsi et a/., 1995). A sesquiterpene lactone, 15-Hydroxy

dehydrocostus lactone, was isolated and its stmcture has been assigned on

the basis of chemical degradation and spectral data (Saxena and Dixit,

1992).

The isolated compounds from this plant and their synthetic derivatives

prepared have been reported for plant growth regulating activities. When the

activity was compared between dehydrocostuslactone and two derived C-16

lactones from them in which a trisubstituted double bond and a cyclopnspane

ring are conjugated with the lactone carbonyl, then it has been observed that

231

the letter compounds are slightly more active then deliydrocostuslactone

(Kalsi et a/.,1977). Twenty derivatives have been prepared by synthesis from

isodehydrocostusiactone and isozaluzanin C, guaianolides isolated from S.

lappa and the conjugated lactones which have an exocyclic methylene group

at C-4, conjugated with a C-3 ketone showed distinct enhancement in their

root forming potential, as compared with their 3-deoxy derivatives. It has also

been seen that these ketones displayed maximum activity only at lower

concentrations (Kalsi et a/., 1984).

Dihydrocostus lactone, isolated from roots was tasted for the affect on

gastrointestinal motility, when its acetone extract given orally (25n-gm./kg)

then It enhanced the gartrointortinol motility (Yamahara et a/., 1990).

The major bfological parameters studied was rooting in stem cuttings of

Phaseolus aureus Roxb. In other case Vigna radiata is choosen to see plant

growth regulatory activity of saussurea, a sesquiterpenoid with a new type of

modified eudesmane skeleton having a five membered ring with the a-

methylene-7-lactone moiety, displayed significant biological activity potential

to generate adventitious root formation on hypocotyl cuttings of Vigna radiata

(Kalsi et a/.,1980; Singh et a/.,1992 and Talwar et a/,,1992).

Isodehydrocostus lactone Dehydrocostus lactone

232

Elema-1,3,11 (13)trien“ 12-ol

CH20H

®-CostolCH 2OH

-CostalR=CHO

^-Costol

R=CH20H

O

6,10 - Dimethyl-9- methylideneundec -5-ei>2-one

Isozaluzanin C 12-Methoxy-dihydrodehydrocostuslactone

233

HO

H

-■■k

\

HTaraxasterol

22- Dihydrostigmasterol

a-Cyciocostunolide

O

4 - p -methoxydehydrocostus lactone

CHO

New sesquiterpene lactone with an a . p - unsaturated aldehydic group

234

OHC

H

%O

New sesquiterpene lactone with an a - , p - unsaturated aldehydic group 15- Hydroxydehydro costus lactone

R= p -2Carboxy pyrralidinyl

Saussureamine C R = NHCH(C00H)CH2C0NH2

235

CHjOMe

12- Methoxydihydro costunolide

^cH L . u ,

Haa - Amorphenic acid

Chemical constituents reported from Saussurea lappa Clarke

Medicinal Use

The essential oil has a strong aromatic, penetrating and fragrant odour. It has

antiseptic and disinfectant properties. It relaxes the involuntary muscle

tissues. It is a cardiac stimulant, a camninative. an expectorant and a diuretic.

An alcoholic extract of the root containing both the essential oil and the

alkaloid has been found very useful in the treatment of bronchial asthma,

particularly of the vagotonic type (Kaul, 1997).

The alkaloid saussurine has a depressant action on the vagus centre in the

medulla as well as on the Involuntary muscle fibres of the bronchioles and

gastro-intestinal tract. It produces a slight but persistent rise of blood-pressure

and increases the force of contraction and amplitude of the ventricles. The

total alkaloidal preparation of the drug showed a depressant action both

subcutaneously and orally and the relief obtained was comparable to that of

conventional bronchodilators without any side effects like a rise in blood

236

pressure, tremor, sweating or headache, even on repeated administration. As

a useful drug for chronic bronchitis and asthma, Tinctura Saussurea', devoid

of the petroleum ether soluble fraction responsible for bronchoconstrictor

action, would be suitable (Sastry and Dutta, 1961; Gupta et a/.,1967; Kirtikar

and Basu, 1993).

The paroxysms are cut short by the combined action of the essential oil and

the alkaloid present in the root. The essential oil during its excretion in the

lungs not only relaxes the bronchial muscle but has a marked expectorant

action which relieves turgescene of the mucosa. It may be pointed out,

however, that although the drug relieves asthamatic attacks, it does not

produce permanent cure unless the casual factors are investigated and

removed. The drug is also useful in persistent high cough (Wealth of India,

1993).

The roots are carminative, analgesic, anthelmintic, depurative,

emmenagogue, aphrodisiac, stimulate the brain and cure diseases of the

blood, liver and kidney; prescribed in the advanced stage of typhous fever

and in rheumatism.

The root and stem are prescribed in snake-bite (Charaka, Sushruta,

Vagbhata, Bhavaprakasha, Yogaratnakara, Rasaratnakara,

Sharangdharasamhita) and scorpion-sting (Charaka, Sushruta, Vagbhata,

Brihannighantaratnakara, Rasaratnakara, Yogaratnakara, Nighantaratnakara,

Vaidyavinoda). In snake-bite, the root is given internally in powder form or in

the form of a decoction (Kirtikar and Basu, 1993).

It is also reported that the root and stem are not an effective antidote to either

snake-bite or scorpion-venom (Kirtikar and Basu , 1993).

237

The root improves the complexion and cures leucoderma, as an insect

repellent The root is used as hair wash to kill lice and also having properties

of turning grey hair to black. The root oil is v\/idely recommended in various

types of skin infections due to its antiseptic, disinfectant and antimicrobial

properties especially against Streptococcus and Staphylococcus micro­

organisms (Ray and Mujumdar, 1976). The roots are also used as insecticide

to protect shawls and woollon fabrics. Dried stems of the plant are given as

fodder in Lahul, Himachal Pradesh in winter (Wealth of India, 1993; Kaul,

1997). Alcoholic extracts of its roots have been found to contain a very potent

antifungal substance with a broad spectrum. It strongly inhibited the growth of

Trichophyton rubrum, T. mentagrophytes and Epidermophyton floccosum.

Other organisms inhibited to lesser extents included Candida albicans, C.

tropicalis, Madurella grisea, Microsporum gypseum, Monosporium

apiospermum [Petriellidium boydii], Sporotrichum [Sporothrix] schenckii, T.

tonsurans, T. simii, Aspergillus niger, A. favus Penicillium chrysogenum and

Cryptococcus neofomnans. It showed no activity against T. cutaneum (Ray

and Muzumdar, 1977). The antitubercular activity in vitro has also been

studied (Nath and Gupta, 1968), crude extract obtained from its roots, having

constunolide and dehydrocostunolide lactone, showed strong suppressive

effect on the expression of the hepatitits B surface antigen (HBs Ag) in human

hepatoma Hep 3B cells, but have little effect on the viability of the cells (Chen

et a/., 1995).

As a medicine the root is considered carminative and stimulant in China.

238

The root improves the complexion and cures leucoderma, as an insect

repellent. The root is used as hair wash to kill lice and also having properties

of turning grey hair to black. The root oil is v/idely recommended in various

types of skin infections due to its antiseptic, disinfectant and antimicrobial

properties especially against Streptococcus and Staphylococcus micro­

organisms (Ray and Mujumdar, 1976). The roots are also used as insecticide

to protect shavi/ls and woollon fabrics. Dried stems of the plant are given as

fodder in Lahul, Himachal Pradesh in winter (Wealth of India, 1993; Kaul,

1997). Alcoholic extracts of its roots have been found to contain a very potent

antifungal substance with a broad spectrum. It strongly inhibited the growth of

Trichophyton rubrum, T. mentagmphytes and Epidermophyton Hoccosum.

Other organisms inhibited to lesser extents included Candida albicans, C.

tropicalis, Madurella grisea, Microsponim gypseum, Monosporium

apiospetmum [Petriellidium boydii], Sporotrichum [Sporothrix] schenckii, T.

tonsurans, T. simil, Aspergillus niger, A. favus Penicillium chrysogenum and

Cryptococcus neoformans. It showed no activity against T. cutaneum (Ray

and Muzumdar, 1977). The antitubercular activity in vitro has also been

studied (Nath and Gupta, 1968), crude extract obtained from its roots, having

constunolide and dehydrocostunolide lactone, showed strong suppressive

effect on the expression of the hepatitits B surface antigen (HBs Ag) in human

hepatoma Hep 3B cells, but have little effect on the viability of the cells (Chen

efa/.,1995).

As a medicine the root is considered carminative and stimulant in China.

238

Chapter 7

SAUSSUREA LAPPA CB. CLARKE

EXPERIMENTAL, ISOLATION AND STRUCTURE ELUCIDATION

Extraction And isolation Of Chemical Constituents From The

Roots Of Saussaurea Lappa C.B. Clarke

Plant Material

The roots of S, lappa were purchased from the Khari Baoli Market, Delhi.

Extraction

The air dried and coarsely powdered material (1.950 kg) was exhaustively

extracted with EtOH (95%) in a Soxhlet apparatus for 72 hours. The

combined extracts were dried under reduced pressure to obtain a dark

brownish coloured residue (400 gm) (20.51%).

isolation of chemical constituents

The dried ethanollc extracts (260 gm) was dissolved in minimum quantity of

MeOH and absorbed on silica gel to form a slurry. The slurry was air dried

and subjected to silica gel column chromatography. The column was eluted

with petroleum ether, petroleum ether - chloroform (99:1, 98:2, 95:5, 9:1, 3:1,

1:1,1:3) chroloroform, chloroform - methanol (9:1,8:2, 3:1,1:1) to isolate the

following compounds.

Lappasterol (1)

Elution of the column with petroleum ether-chloroform (1:1)

(fractions 17 to 20) afforded cream-coloured needles of 1, recrystallized from

chloroform -methanol (1:1), 170 mg (0.0087 % yield ), mp 130-132 Rf 0.70

(petroleum ether: chloroform: methanol: 5:3:0.5)

[a]o“ 0.00 (C=0.14, CHCI3)

UV Xmax (MeOH) 263 nm (log e 2.6)

239

IR 3450. 2965, 2870, 1640,1480, 1455, 1395, 1275, 1115, 1075, 1035,

810 cm '\

NMR - Table 7.1

C NMR - Table 7.1

EIMS miz (rel.int.) 410 [M l (C29H46O) (74.3), 396 (100), 395 (34.5), 392

(40.8), 380 (34.0), 379 (6 8 .8 ), 377 (32.5), 365 (38.5), 347 (24.9), 286 (36.2),

272 (33.5), 253 (46.2), 230 (19.3), 212 (33.8), 197 (14.2), 190 (8.2), 176

(11.0), 172 (10.8), 158 (28.8), 147 (28.9), 132 (33.1), 124 (11.8), 118 (27.6),

106 (43.1), 95 (44.7), 83 (43.5), 80 (56.2), 72 (2.3), 70 (61.8), 56 (93.5), 54

(83.2), 43(91.0).

3-ep/-Lappasterol (2)

Elution of the column with petroleum ether-chloroform (1:3)

(fractions 21-25) famished colourless crystals of 2, recrystallized from

chloroform ; methanol (1:1), 180 mg (0.0092 % yield ), mp 126-28°C, Rf 0.61

(petroleum ether; chloroform; methanol; 5:3:0.5),

[a]o ® (+) 345.2° (C:= 0.04, CHCI3)

UVXmax262nm(loge4.2)

IR Vmax 3450, 2915, 2850, 1640, 1465, 1375, 1265, 1105, 1045, 1015, 800

cm

’H NMR Table 7.2

NMR Table 7.2

EIMS m/z (rel. int.) 410 [M]" (C29H46O) (81.3), 395 (53.7), 392 (47.0), 380

(34.8), 377 (28.5), 365 (18.0), 347 (20.0), 286 (15.2), 272 (9.1), 270 (27.5),

240

253 (43.5), 230 (17.7), 212 (30.3), 197(12.1), 176 (9.4), 172 (14.5), 158

(35.8), 147 (16.4), 132 (35.6), 124 (6.3), 118 (31.6), 106 (63.8), 95 (41.3), 83

(68.0), 80 (58.1). 70 (21.5), 68 (62.8), 56 (53.4). 54 (100), 43 (71.8).

Jone's oxidation of 3-«p/>lappaasterol (2). Compound (50 mg) was

dissolved In acetone (60 ml) and treated with freshly prepared Jone's reagent

(5 ml). The reaction mixture was stirred at room temperature till the reaction

was completed (TLC monitoring ). It was diluted with water and extracted with

EtaO. Removal of the solvent from the extract yielded iappasterone (2a).

(32 mg), mp 111- 112° IR v ax 1705, 885 cm■ EIMS m/z 408 (C29H44O)

(3.6).

Reduction of Iappasterone (2a)

Lappasterone (15 mg) was dissolved in ethanol (10 ml) and NaBH (1 g) was

then added with stining for 1 hour. After dilution with water, the reaction

mixture was extracted with chloraform, the chloroform layer was washed with

water, dried and evaporated to obtain lappasterol (1) (12 mg), mp and mmp

131-132®, Co - TLC comparable.

Lappalanasterol (3)

Elution of the column with chloroform-methanol (99:1) (fractions

28-32) furnished pale yellow crystals of 3, recrystallized from chloroform -

methanol (1 ;3), 230 mg (0.011 % yield ), mp 280-282°, Rf 0.64 (petroleum

ether: chloroform; methanol; 5:3:1).

(-) 336.8 ° (C= 0.16, CHCI3)

UVXmax 228 nm (logs 6.2).

241

IR Vmax (KBr). 3460, 2990, 2930, 2875, 1710, 1680, 1630, 1465, 1420, 1370,

1310,1290,1275,1210, 1130, 1095,1075,1025,910, 790, 735 cm ’

’HNMR Table 7.3

NMR Table 7.3

EIMS m/z (rel. int.): 502 [M]* (C30H46O6) (1.0), 346 (6.3), 328 (33.0), 313

(100), 271(10.1), 268 (5.3), 238 (5.2), 224 (4.5), 222 (6.1), 206 (9.3), 205

(9.2), 191 (13.6), 186 (12.8), 182 (4.5), 168 (7.8), 167 (8.5), 154 (6.3), 150

(10.2), 145(11.5), 141 (7.9), 139 (9.8), 135(13.1), 129(12.0), 126 (23.7), 123

(10.9), 114 (8.3), 111 (18.5), 97 (18.2), 91 (22.1), 83 (22.0), 77 (31.2), 69

(41.3), 55 (43.0), 43 (89.4).

P-Sitosterol-3-P‘D'glucoside (4)

Elution of the column with chloroform - methanol (9:1) (fractions

33-50) furnished colourless amorphous powder of 4, recrystallized from

methanol, 245 mg (0.0125 % yield), mp 280-281°, Rf 0.53 (petroleum ether;

chloroform; methanol; 13:7; 4).

[a]o^® 152.5° (C= 0.05, CHCI3)

UV (MeOH) 217 nm (log e 4.5)

IR Vmax 3450, 2955, 2910, 2845, 1610, 1460, 1375, 1255, 1155, 1 1 0 0 , 1080,

1020.795 c m \

NMR (DMSO-de) : 8 5.29 (1H, brs, H-6 ), 4.81 (1H, d, J = 9.61 Hz, H-1),

4.78 (1H. d, J = 6.0 Hz. H-5'), 4.30 (1H, m, H-4'), 4.23 (1H, dd, J = 7.63, 6.0

Hz, H-2’), 3.65 (1H, m. H-3'), 3.47 (1H, brm, Wy, = 16.50 Hz, H-3 a), 3.08 (2H,

242

brs, Hz - 6'), 0.95 (3H, brs, Me-19), 0.91 (3H, d, J = 6.5Hz, Me-21), 0.89 (3H,

d. J= 7.0 Hz, Me - 29), 0.81 (3H, d. J= 5.69 Hz, Me - 26), 0.77 (3H, d, J = 6.39

Hz, Me - 27), 0. 64 (3H, brs. M e-18).

NMR (DMSO-de): 6 36.10 (C-1), 31.29 (C-2), 79.07 (C-3), 41.72 (C-4),

140.90 (C-5), 121.01 (C-6), 33.27 (C-7), 33.03 (C-8), 50.51 (C-9), 35.38.

(C-10), 22.48 (C-11), 36.72 (C-12), 45.08 (C-13), 56.07 (C-14), 25.38 (C-15).

28.56 (C-16). 55.32 (C-17). 11.60 (C-18), 20.46 (C-19), 35.12 (C-20). 18.92

(C-21), 31.70 (C-22), 27. 62 (C-23), 49.51 (C-24), 29.14 (C-25), 19.48

(C-26), 19.83 (C-27), 23.72 (C-28), 11.47 (C-29), 100.76 (C-1'). 73.29 (C-2’),

78.63 (C-3'), 69.96 (C-4'), 78.19 (C-5'), 61.06 (C-6').

EIMS m/z (rel. int.), 576 [M]" (CasHeoOe) (N.O.), 413 (4.3), 398 (15.5), 396

(20.5), 381 (15.3), 272 (4.3), 255 (11.7), 240 (7.6), 213 (8.2), 186 (10.3), 159

(14.8), 145 (15.9), 138 (13.5), 133 (14.7), 131 (16.1), 121 (18.5), 105 (31.6),

91 (32.7), 81 (37.8), 77 (48.1), 69 (24.3), 57 (63.8), 43 (100).

Acid hydrolysis of compound (4) Compound 4. (15 mg) was refluxed with

2N HCl in 80% MeOH (1:1, 15 ml) for four liours. After cooling, the reaction

mixture was poured into crushed ice, and the hydrolysate was then extracted

wth EtOAc to give the aglycone, mp and mmp 138-140°, Co- TLC

comparable. The neutralized and concentrated aqueous hydrolysate showed

the presence of glucose on comparison with authentic sugar on silica gel

TLC. Rf 0.4 (EtO Ac: HOAc : H2O : MeOH; 6:1:1:2).

243

DISCUSSIONS

Compound 1, designated as lappasterol, was obtained as cream coloured

needles from petroleum ether - chloroform (1:1) eluents . It responded

positively to Liebermann- Burchard test and exhibited IR absorption bands for

hydroxyl group (3450 cm"’) and unsaturation (1640 c m I t s molecular weight

was established as 410 (C29H46O) on the basis of mass and NMR

spectra. It indicated seven double bond equivalents; four of them were

adjusted in tetracyclic carbon skeleton of a sterol, two in olefinic linkage and

the remaining one in cyciized side chain. The diagnostically important ion

peaks in the mass spectrum appearing at m/z 395 [M-Me] , 380 [M -2 x Me]" ,

365 [M-3 X Me]", 392 [M-H2O]". 377 (392-Me]^ 272 [M-Cio H19. side chain]",

253 [273-H20]", 230 [272-ring D]*, 212 [230- H2O Y and 197 [212-Me]"

suggested a steroidal carbon framework of the molecule with two vinylic

linkages, the hydroxyl group in rings A -D and a cyciized side chain at C-17.

The prominent ion peaks at m/z 83 [€2,3 - C5,10 -C7.8 fission]", 72 [ Ci,io-C4.5

fission]", 54 [72- H2O]", 124, 286 [C6.7-C9.10 fission] 106 (124- H2O]". 147

[286 - C10H19, side chain]" and 132[147-Me]" indicated the location of the

hydroxyl group in ring A, placed at C-3 on the basis of biogenetic

considerations, and one of the olefinic linkage in ring B at C- 5. The ion peaks

at m/z 176 [ Ca.14- Cn .12 fission]", 158 [176- H2O]". 95 [M-176-side chain]", 80

[95-Me]", 190 [C12.13 - Ce.i4 fission]" and 172 [190 - H2O]" supported the

existence of another vinylic linkage at C-9 (11) (Budzlkiewicz et a/., 1964;

Gupta ef a/.,1981; Das ef a/,,1992) (Scheme-7.1).

The NMR spectrum of 1 displayed two one-proton signals as a doublet at 5

5.28 (J = 4.6 Hz) and as a multiple! at 8 5.04 assigned to C-6 and C-11 vinylic

Compound 1

244

protons, respectively. A one-proton carbinol broad multiplet at 5 3.47

(wvi = 15.24 Hz) was ascribed to 3 a-mettiine proton. Three singlets at 5

0.61, 1.18 and 0.93, integrated for three protons each, were attributed

correspondingly to C-18, C-19 and C-21 tertiary methyls. Three doublets at 8

0.77 [J- 6.12 Hz), 0.73 (J = 4.23 Hz) and 0.86 (J = 6.32 Hz), integrated three

protons each, were associated with C-26, C-27 and C-29 secondary methyls,

respectively. The appearance of all the methyls in the region 1.18 - 0.61

suggested that these functionalities were attached to the saturated carbons.

(Alam et a/.,1994; Sharma and Ali, 1996; Gupta and Ali, 1999). The remaining

methylene and methine protons appeared in the region 5 2.20 - 1.01. The H3-

29 resonance of 24R - configuration (60.83) was more upshielded as

compared to the 24 S- resonance (8 0.86) (Rubinstein et a/., 1976). The

assignment of the chemical shifts were made by comparison with 8 values of

the corresponding protons in the stnjcturally similar sterols (Greca et a/.,1990;

Gupta ef a/.,1992,1994; Saba efa/.,1999).

Further evidence for the structure 1 was provided by Its NMR spectral

data which showed the existence of 29 carbon atoms in the molecule. Four

downfield signals at 8 139.77, 120.71, 137.31 and 128.30 were assigned to

C-5, C-6 , C-9 and C-11 unsaturated carbons, respectively. The C-3 carbinol

proton appeared at 5 70.80. The assignments of the carbon chemical shifts

were made by comparison with the Sc values of the corresponding carbon

atoms of p-sitosterol (Greca et al.,1990) and lawsaritols (Gupta et a/., 1992;

1994) and pleuchinol (Alam et a/.,1994). On the basis of the spectral data and

chemical reactions, the stmcture of 1 was characterized as (24S)- stigmast -

5 ,9{11)-dien-20,28*endo - 3 p-ol. This is a new member of the phytosterols.

245

Table 7.1. and NMR spectral data of Lappasterol (1) and NMR

values of p> Sitosterol

Position ’HNMR1

NMR

Alpha Beta 1 P -sitosterol

1 1.37 dddd (16.12,9.78, 5.37,4.58)

2.20 dddd (12.10,4.58, 9.72, 11.31)

38.79 37.33

2 1.93 m 1.88 m 32.72 31.63

3 3.47 brm (Wy, 15.24)

- 70.80 71.73

4 2.16 d (11.32) 1.79d(5.34) 41.32 42.20

5 - - 139.77 140.71

6 5.28 d (4.6) - 120.71 121.63

7 1.75 m 1.75 m 30.67 31.96

8 - 2.75 m 30.92 31.81

9 - 137.31 51.13

10 - - 36.27 36.43

11 5.04 m - 128.30 21.09

12 1.89 d (5.21) 1.75 m 39.48 39.79

13 - - 44.86 42.37

14 1.50 m - 55.78 56.75

15 1.09 m 1.41 m 23.31 24.15

16 1.61 m 1.41 m 28.70 28.25

246

17 1.47 dd (8.52, 5.56)

- 55.08 56.02

18 0.61 s - 10.99 11.84

19 1.18s - 19.20 19.46

20 - - 35.15 36.07

21 0.93 s - 18.81 18.68

22 1.41 s 1.01 m 32.97 33.95

23 1.14 m 2.13m 27.24 26.10

24 1.05 m - 49.16 45.82

25 1.50 m - 29.30 29.15

26 0.77 d (6.12) - 20.09 19.77

27 0.73 d (4.23) - 17.26 19.21

28 - 1.14 m 25.12 23.13

29 0.86 d (6.32) - 10.87 11.04

Coupling constants In Hertz are given in parentheses.

Compound 2

Compound 2, named 3>ep/-iappasterol, was obtained as colourless

crystalline mass from petroleum ether-chloroform (1:3) eluents. Its spectral

data were almost identical to that of lappasterol. However, difference in

physical constants, eluent's polarity and crystal shape suggested It to be an

epimer of the previous compound.

247

' \ /,CHo

C5H7O

m/z 83 (43.3)

m/z54(100) m/z 197 (14.2)

- f Mem/z212(33,8)

H2O m/z 230 (19.3)

m/^72(^3) m/z272(33.5) -* 2° m/z253 (46.2)

m/z 347(24.9)

t H2O m/z 365 (38.5)

' - Me m/z 360 (34.0)

t Me ^m/z 395 (34.5)

^ m/z 392 (40.8)H2O

m/z 377 (32.5) |-2 x Me

m/z 347 (24.9)

,>-m/r 106 (43.1)

Ci3^18 m/z 190 (8.2)

j-HaO

m/z 172 (10.8)

m/z 158 (28.8)

17 30m/^234 (N.0)

I CioHig(side chain)

m/z 95 (44.7)

j-Me

m/z 80 (56.2)

Scheme 7.1. Mass fragmentation pattern of Lappasterol (1)

C21H34 m/z 286 (36.2)

^ C i o H i 9(side chain)

m/^ 147 (28.9)I Me

m/z 132 (33.1)

In the ’H NMR spectrum of 2, the C>3 carblnol proton appeared as a one- proton multiplet and its half -width of 10.92 Hz indicated its p orientation. John's oxidation of 2 yielded iappasterone (2a). The latter gave a positive Zimmern:^ann test Indicating the presence of the 3 - oxo group (Barton and de Mayo, 1954). The NaBH4 reduction of 2a regenerated lappasterol (1), confirming equatorial orientation of the hydroxyl group in 3-ep/- lappasterol. On the basis of these findings, compound 2, was identified as (24S)- stigmast - 5,9(11) - dien - 20,28 ~ endo- 3 a - cl. This is a new phytosterol.

Table 7.2. and NMR spectral data of 3-epi- Lappasterol (2)Position 'HNMR '^C NMR

Alpha Beta

1 1.36 dddd (11.4, 3.05,9.58, 5.38)

2.21 dddd (5.39, 7.00, 6.44, 4.43)

37.27

2 1.93 m 1.87 m 31.66

3 3.43 brm {w» 10.92)

7.81

4 2.12 d (11.35) 1.75 d (5.92) 42.30

5 140.76

6 5.27 d (4.71) 121.72

7 1.79 m 1.79 m 31.91

8 2.75 m 31.66

9 138.32

10 36.15

11 5.00 m 21.09

249

12 1.87 d (5.36) 1.80 m 129.29

13 45.86

14 1.55 m 56.78

15 1.11 m 1.48 m 23.08

16 1.58 m 1.48 m 28.24

17 1.44 dd (11.4, 3.05)

55.99

18 0.16s 12.03

19 1.18s 19.40

20 36.15

21 0.93 s 18.79

22 1.26 m 1.01 m 33.96

23 1.14 m 2.12 m 26.10

24 1.07 m 45.85

25 1.58 m 29.17

26 0.77 d (3.37) 20.21

27 0.73 d (6.12) 18.26

28 1.14m 25.41

29 0.85 d (3.27) 11.87

Coupling constants in Hertz are provided in parentheses.

250

Compound 3, named iappalanasterol, was obtained as pale yellow crystals

from chloroform - methanol (99:1) eluents. It gave positive Liebermann -

Burchard test and had IR absorption bands at 3460 (hydroxyl), 1710, 1680

(carbonyls) and 1630 cm' (C=C). Its mass and ^ C NMR spectra established

the molecular weight 502 consistent with a tetracyclic triterpene C30H46O6 . It

had eight degrees of unsaturation, which were adjusted in the lanastane-type

carbon framework with two carbonyl groups and two olefinic linkages. The

mass spectrum exhibited significant ion peaks at m/z 346 [M-HaO-Me]^, 328

[346- H2 0 ]^ 313 [328-Mer, 271 [371-ring D fission] and 123 [CaHnO]^

reflecting the presence of Ce-side chain with two olefinic linkages and an

aldehyde group. The ion peaks at m/z 83 [C3. 4 - Cs.io - C7.8 fission ]*, 114

[C1.10 - C4.5 fissionr , 97 [ 114 - OH ] M 8 2 [ C 7.8 - C 9.10 fission]". 167 [182 -

Me 1*. 150 [ 167-OH]^ 168 [Cej - C9.10 fission/182-CH2]*. 154 [C5.6-C9.10

fission/168- CH2]* and 139 [154-Me]" indicated saturated nature of rings A

and B. One hydroxyl group in ring A, placed at C-3 on the basis of biogenetic

grounds and a keto group at C-2. The ion peaks at m/z 224[C9,n - C a.i4

fission]\ 206[224 - HaOf. 191[206-Me]^ 238 [Cn,i2 - Ca.i4 fission] ,

205[ 238 - Me- HaO]" and 268 [ Ci2,ia - Ce,14 fission]* supported the saturated

nature of the ring C and existence of hydroxyl groups at C-9 and C- 12

(Scheme 7.2).

The NMR spectmm of 3 was consistent with the proposed structure and

clearly showed a one-proton downfield multiplet at 5 6.58 and a one-proton

downfield doublet at 8 5.85 (J = 7.69 Hz) assigned to vinylic H-22 and H - 24,

respectively. A one proton broad signal at 6 3.31 was due to 3 a - carbinol

proton. A one-proton double doublet at 5 4.23 with coupling interactions of

3.51 and 3.48 Hz was associated with C -12 p-carblnol proton. A one-proton

broad signal at 5 3.11 and a one-proton doublet at 8 3.03 (J = 3.74 Hz) were

ascribed to C- 18 hydroxy methylene protons. The presence of one proton

Compound 3

251

downfield signal at 6 9.63 attested the existence of aldehydic group at C-21 in

the molecule. Two three - proton signals at 6 1.96 and 1.90 were attributed to

C-26 and C- 27 methyl protons attached at C- 25 unsaturated carbon. Four

tertiary methyls resonated as broad signals at 81.33 (Me-19, Me -28), 1.19

(Me-29) and 0. 98 (Me-30). The remaining methylene and methine protons

resonated between 5 2.65-1.04 (Table 7.3).

The NMR spectrum of 3 displayed signals for C-2 oxo carbon (5 210.11),

C-21 aldehydic carbon (6 205.20), carbinol carbons (6 75.12, 81.11, 78.21,

73.22) and unsaturated carbons (8 148.46, 122.22, 128.62, 139.36) (Table

7.3).

Based on these evidences, the structure of 3 was characterized as ianosta -

20 (22), 24 - dien - 2-on- 3p, 9 a, 12a-trioi - 21-al. This is a new member of

lanastane - type triterpenes.

Table 7.3. and NMR spectral data of iappalanasterol (3)

Position NMR NMR

Alpha Beta

1 2.6 brs 2.36 35.29

2 - - 210.11

3 3.31 brs - 75.12

4 - - 40.57

252

5 1.59 dd (11.32, 5.6)

- 54.73

6 2.10 m 1.62 m 20.38

7 2.04 m 1.49 m 29.66

8 - 2.40 brs 43.97

9 - - 81.11

10 - - 36.89

11 152 d (9.41) 1.44 d (5.4) 23.24

12 - 4.23 dd (3.51, 3.48)

78.21

13 - - 52.08

14 - - 57,21

15 1.04 m 1.40 m 31.76

16 1.06 m 175 m 35.75

17 2.53 dd (5.03, 5.15)

- 56.37

18 3.11 brs 3.03 d (3.74)

“ 73.22

19 1.33 s - 13.76

20 - - 148.46

21 9.63 s - 205.20

253

2 2 6.58 m 1 2 2 .2 2

23 2.74 m 2.47 m 36.22

24

25

5.85 d(7.69)

128.62

139.36

26 1.96 s 21.88

27 1.90 s 21.98

28 1.33 s 14.70

29 1.19s 21.11

30 0.98 s 12.37

Coupling constants in Hertz are provided in parentheses.

Compound 4

Compound 4 gave a positive Liebennann - Burchard test and a negati

Ehrlich reaction. Its IR spectrum exhibited strong bands at 3450 and 10

cm’ characteristic of a glycoside. The El mass spectrum of 4 gave a fragmt

ion at m/z 413 [M-glucose]*, 398 [413-Me]*. 396 [576 - CgHiz Oe]\ 381 [3£

Me]^ 273 [4 1 3 -C10H21. side chain]*, 255 [273-H20]\ 240 [255-Me]" and 2

[255-ring D fission]"" which were characteristic for p-sitosterol 3-p-D-glycosid

254

CH:

CeHn m/zbZ (22.0)

m/z97(18.2)VOH m/z 150 (10.2)

t-OH m/z 167 (8.5)

t Me ...HO'-

CH

CHoy 'm /z 114 (8.3) ^

4

3 / C i -i H-,802

182 (4.5)

I<fflHliO__^^/2379(M.O) side chain

(7.8) CH2

m/z 154 (6.3)I Me

m/z 139 (9.8)

-H ,0

30 46 6 [M]+m/z 502(1)

OHHO'

C13H20O3

m/z 224 (4.5),, H2O

206 (9.3) j Me

m/z 191 (13.6)

OH

14 22 3 15* 24 4m/?268(5.2) m/z268(5.3)

Me H2O

m/z 205 (9.2)

m/z 346 (6.3)

, "H2O

m/z 328 (33.0) [-Me

m/z 313 (100) j-Ring D

m/^271 (10.1)

CgHiiO m/z 123 (10.9)

Scheme 7.2. Mass fragmentation pattern of Lappalanasteroi (3)

255

The C-18 and C-19 tertiary methyl protons appeared as broad signals at 5

0.64 and 0.95, respectively. Four three-proton doublets at 6 0.91 (J = 6.5 Hz),

0.89 (J = 7.0 Hz), 0.81 (J = 5.69 Hz) and 0.77 (J = 6.39 Hz) were due to C-21

(secondary), C-29 (primary) C-26 (secondary) and C-27 (secondary ) methyl

protons, respectively.

The NMR spectrum of 4 displayed 29 carbon signals for steroidal nucleus

including two vinylic carbons at 6 140.90 (C-5) and 121.01 (C-6) and one

carbinol carbon at 5 79.07 as well as six glucose carbons at 5 100.76 (C-1'),

73.29 (C-2'), 78.63 (C-3'), 69.96 (C-4‘), 78.19 (C-5*) and 61.06 (C-6'). Acid

hydrolysis of 4 yielded D-glucose and an aglycone which was identified as p-

sitosterol by spectral data and by direct comparison with the authentic

samples (Co - TLC, mmp). On the basis of these finding the structure of 4 w

as established as stigmast - 5-en - 3-0- p-0-g(ucopyranoside.

256

T able 7.4 Compounds isolated from the roots of Saussurea lappa Clarke

Code Name Columneluent

Rf.

mobilephase

Yield (%w/w)Solventsused forrecrysta*tllzatlon

m.p“ C

Value(CHCb)

Mol. Wt. [M]* m/z [Mol. For].

Nomenclature

1 Lappasterol A:B

1:1

0.70

A:B:C

5;3:0.5

0,0087

B-C(1:1)

130-132®

0.00

(C= 0.14)

410

C29H«0

(24S) - Stigmast-

5,9(11)-dien-20,

28- end0-3P'0l

(Now)

2 3-eph

lappasterol

A;B

1:3

0.61

A:B:C

5:3:0.5

0.0092

B-C(l.l)

126-128“

(+) 345.2

(0=0.04)

410

C29H46O

(24S) - Stigmast-

5,9(11}. dien-20,

28- endo-3a-ol

(New)

3 Lappalanas-

terol

A;B

(99:1)

0.64

A:B:C

5:3:1

.011

B-C(1:3)

280-282“

(-) 336.8

(0=0.16)

502

C3oH4eOa

Lanosta -20(22),

24 - dien-2-on-

3P- 9a-12a-tri ol

-21-at (New)

4 P-sitosterol -

3p-D-

glucoside

A:B

9:1

0.53

A:B:C

13:7:4

0.0125

0

280-281“

152.5

(0=0.05)

576

CssHeoOe

Stigma-5-en-3-o-

[i-D-gluco-

pyranoside

A =: Petroleum ether; B = Chloroform ; C= Methanol

257

Chemical Constituents isolated from the roots of Saussurea lappa

2 7

2 529 t* M' - , X ................... .......................- 2 6

2 3 2 4 ' - . . ,Z1 > 2 3

11:'r

17 ■ 18

2?'

HOH

2. 3-ep/-Lappasterol

1?

27

iZ8 21, i

'-V 20..., ..

f / .u M 1’'

- - / . I - ..'=1 0 fa ::

H H .25' / 26

2 4 ■,> 2 3

. ??'' ■

1119

HO

H

21CHO

HO- / ;2512 18/ / !

26

27

19 :

'■'12 lOi |8 :I OHi 30

OH. 14

.J-.3

HO- ,4 „r-'

29 2 8

OH CH2OH.A-., y

yfC..\ T H }

\ H OH

3. Lappalansterol

2 12 8 . ' 2 9

./ ,27\25

2 6

10 8 ;

- ^4

H4. -Sitosterol - 3 - -D- glucoside

HOH

258

2r

9S

1211

1 19

2 3

-2 5

H17 16

15

i ii 7': :l I

37S 410

l;j5157 7"

113

f i iMi' 'i i'l 4; !ii 'j'

364

rJi3^0 4<)0 ' ' 4 '5 0 ' 501

EIMS of Lappasterol (1)

NMR of Lappasterol (1)

259

27

EIMS of 3-ep/-Lappasterol (2)

260

”C NMR of 3-ep/-Lappasterol (2)

NMR of 3-ep/-Lappasterol (2)

261

’H NMR of Lappalanasterol (3)

NMR of LaoDalanasterol (3)

262

NMR of p-Silosterol-3-p-O-giucoside (4)

21C H O

\2 0 ^H C k .12 t

J O H 14

>27 . .

Q ' 233i 34S20C 215 305

E lM S o f L a p p a la n a s te ro l (3 )

555

263

382

45c

EIMS of p-S(tosterol-3-(3-D-glucoside (4)

’V . N M R of R-SitnRtfirol-3-n-D-alucoside (4)

,264

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