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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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