QHAPrE - I

CHEMICAL COI'm'ITUEITS OF JIARSILEA IIIUTA LINI

1

CHEMICAL CONSTITUENTS OF MARSILEA M!NUTA LINI

Ma:rsilea min uta Linn 1-' (Ma:rsileaoeae > is a genus of aquati o

or aubaquatio ferne widely distributed in the tropical and temperate

regions of the world. About nine species are found in India. Many

of the Indian floras refer th1 s common Indian species to Marsilea

guadrifolia Linn4• The plant has long and thin stem and with

creeping rhizome and quadrifoliolate leaves. It grows by the side

of the ponds and irrigation ohanaele throughout the greater pa:rt of

India. It can be propagated by divisions of the rhizome or by spores.

!he stalks and leaves of the plant are eaten as pol-herb, especially

in times of scarcity.

It is a pOpular drug in folk medioiae. Leaves and whole plants

are used for sedation and in insomnia as it has been mentioned in

Ayurvedio medicine to induce sleep in insomic persona.

The hypnotic activity of Marsilia minuta was tirst studied

experimentally in 1961 by M.L. Chatterjee et al5 at the school of

Tropical Medicine, Calcutta. An alcoholic extract of the leaves was

given orally to white mice (20-30) through a syringe fitted with a

special type o:t needle. Doses varying trom 5 to 100 mg/kg contained

in a five volume o:t 0.2 ml of the suspension were used. Investigations

were made (a' on the action of Nare1lea minuta given alone and (b) on

its effec~ on the hypnosis induced by pentobarbital sodium (50 mg/k:g)

intraperitoneally administered 30 minutes later. Initially a control

study was also made With vehicle (gum acacia sol.) along with that o!

. 2

pentobarbital sodium and extract of Jfareilea llinuta. The tranquilising

effect with the extract of Marsilea minuta was found to be most

effective in doses between 5-10 mg/Kaorally in white mice. Lack of

significant differ enoe in the effect w1 th 5 ,1 0 and 20 mg/kc d osee

wae found which may probably be due to the crude preparation of the

extract.

Ohatter jee et a16 in 1963 however worked with Kareilea Jlinuta

Id.nn with the object of isolating the active principle present in

the plant. They isolated the only compound deeignated as 118Xsilin

c29Hs 40 (M = 418 from mass spectrometric analysis and by Raet method) 30.

m.p. 85-86•. (oe]]) l O• (CHC13) from the ~hloroform. extract of the

leaves. They observed the following che~cal 8l'ld physical properties

of the compound. It did not give any teet tor sterols or triterpenes '

and did not reduce Fehling' a solution or Toll en• s reagent a. Tetra-

ni tromethane had no action on this compound. The infrared epectrum

showed peak absorption fOr carbonyl at 5.76 p (in KBr diek) but

fails to .produce any oxime or semicarbazone •. Lithium aluminium

hydride reduction of the compound afforded a product, c291fs60, m.p.

80•, showed peak absorption for hydroxyl at ,.1 ;U. Nuclear JI.Bgnetio

resonance spectrum showed a sharp signal at 1 .26 which they accounted

for the pr esenoe of one -c-CH3 funct1 on. From analysis an4 the above

ohemioal and physical studies they designated marsilin as a

macr ocyolic ketone. They performed pharmacal ogioal test of mar silin

and :from their obs.ervati one they described marsilin as the sedative

and the anticonvulsant principle of Yarailea m1nuta Linn. It has been

said that it is specially effective against electro and ohemooonvuleive

agent and it may be a promising drug in the treatment of epilepsy and

.. a

in various mental disorders. The sedative action of marsilin was 7-10 subsequently described by other workers as well· • They olai11.ed

the confirmation of their views by clinical trial on some epileptic

patients.

While searching for saponin bearing plant the fern Marsilea

minuta :U.nn was brought to our notice as the aqueous solution of

the leaves of the plant ga'fe a stable froth indicating the presence

of saponin in it. As such a detailed chemical exam1nat1 on was

undertaken •12

The air dried powdered leaves and stems were extracted successively

with.petrolium ether (60-80°), chloroform and ethanol in a eoxblet.

Each fraction was studied separately and as critically as possible.

Treatment of Petroleum ether extract 1

The residue from petroleum ethe:r extract waa subjected to

chromatographic separation over Brockmann alumina (Table-5-experimental}.

156 (A1-A156 ' t:ractions were collected from this chromatography and

3 major components were obtained. Their purity was assayed by TJ.C. It

is worthwhile mentioning that while working with the higher aliphatic

hydrocarbon, ketone and alcohol the TLC often fails to give true

picture. Ther! are some inseparable mixtures whioh can only be detected

by GLC.

Each fraction from A1-A20 was examined separately by T U! when a

single strip was obtained. These fractions ehaNed no absorption at

1720-1740 om-1 or 3600-3400 cm-1 in the IR (Pig. 16> confirming the e

absence of carbonyl and hydroxy containing cOJtpounds. GLC indicated a

mixture of normal hydrooatbons ranging from o27 to c33

with o27 , c29

,

LL.J Cl) ~ a Q. (/')

lJJ 0::

0: a 1-u lJJ 1-lJJ t:::l

~solvent

II ii

II /I

I

L ..... ... -······· ................. .

10 20

TIME IN MINUTES

-Mixture of alkanols from C5 -CB

--···Hydroxy Ketone (!) from 886-8105

I

21 23 25 27 29

CARBON NUMBER--._

o Standard compounds 0 Hydroxy ketone (I)

D. Alkano!s from c5-c8 o Synthetic Hentriacontan-16- of

...

30

31

.· . . ·. .. . .

FIG. 1. GL C OF ALI<ANOLS AND HYDROXY KETONE (I)

40

o31 and o33 predominating.

Fractions A21 -A45 gave only a very small quantity of aaterial

and was found to be mixture by TLO.

Fractions A46-A110 gave one elongated diatinct strip in TLC.

GLC r ~vealed that it is a mixture of several component e. These

fractions were mixed together and chromatographed over Argentized

eilicage1 14• 15 .120 (B1-B120) fractions were collected troa this

chromatography (Table-6).

:Fr aoti on s B11 -B20 gave a solid, mp. 9'3•, and having a o abeorpti on

for =0=0 or -OH 1 n the IR and it was found to be a mixture of' .

hydr ocerbons by GIC.

Fractions B21 -B80 gave a solid, mp. 95° IR absorption at '3600--1 '

3400 em showed the presence of hydroxyl containing compound and

GLC revealed that it was a mixture. It was then rechromato1raphed

over A.rgentized silica gel (Table-7). 2'3 (o1-c23) fractions were

collected from t hie chr omat ogr aphy.

Fractions B86-B105 eluted with benzene and petroleum ether

(40-60°' was found to be a single component by TLC and GLC (Fig. 1)

after crystallisation from petroleum ether (40~0°) and be1zene

mixture. On elemental an.alysis and molecular weight deter111nati on

t~e oo~pound · ~· was found to have molecular tormla c3c)B60o2 (452,Jf"),

mp. 102°, [oe] ~5.-9° (cHol3).

Anal. Found I O, 79·7~ 1 H, 13.01~ I

Calc. for c3cJI60o2:o, 79.64"1 H, 13·2~ 1

i ...

WAVELENGTH (MICRONS) '3 7 B 9 10 12. 13 5 6 4 14 11 100--~--_J~ __ _L ____ ~ __ _j ____ ~ ____ L_ __ ~-----L----L---~----~--~ 15

so ,......_

~ 0

~ 60 ltJ l..> <: ~ ~ 40 ~ (/)

<: ~

~ 20

0 400~0~~~~----~~------~~------_L ________ _L ____ ~--------L---------~--~ '3000 BOO 700 12.00 1'500 1000 900 2000

CM -f

Fig 2. I R curve of Hydroxy-l<etone I .

5

It did not give any oolouration with tetranitr omethane. It

also did not correspond to the teet for sterols and triterpenes.

The functions of the two oxygen atoms in the molecule were

revealed from IR spectrum of the compound wbioh showed absorptions

at 3210 and 1700 cm-1 (Pig. 2) assignable to a hydroxyl and a

ketonic group respectively. The significant absorption discernible

in the IR spectra are at 1455 om""1 (-cH2 bending), 1'75 011-1

(-o-aH3 bending), 1066 om-1 (-c-o stretching of secondary alcohol), -1 \ doublet 727 and 718 om - (CH2,n bending(n >4) vibration due to

straight chain methylene gx oups. Moreover, it furniehed a mono­

acetyl derivative and a mono-oxime. consequently the presence of

a hydroxyl and a ketonic group in thi molecule was ascertained.

Oxime of the gy~r oxy Ketone 1

Oxime of the hydroxy ketone was prepared by hydroxyl aDd.ne

hydrochloride in the usual WfJN and the oxime thus formed had . .

Anal • Found 1 I, 3~ 1

Calc. fOJ! c,oH61N02 IN, :5·1,. I

Acetate of the Hydroxy Ketone 1

The hydrox.yketone was acetylated with acetic anhydride and

pyridine in the usual way and the acetate thus formed had mp .ga• [ oC):o; :5 • ( CH013)

Anal. Found a C, 79 ·7~ J H, 12 .51" J

Calc • for c32B620, 1 0, 79. 76~; H, 12 .55" 1

The structure of the hydroxyket one was determined mainly from

its mass speot:ral fragmentation pattern. The absolute identification

+

d m;e 200 b m;e 252

Scheme-l

c m;e 310

Scheme- II

----------- - - ---- .... , 423 \

I I

--- - - - --- - -- .. \ I

393 \ l I 1

CH3 (CH2)tB CO (CH2) 7-: CHOH ~C2H5

I I 29 1 \ I 1

l 59

.... ___ ------

. ..... _______________ _

Scheme-m:

Chert I- Mess Fragmentation Patrern of Hydroxy- Ketone (I)

..

100

80

60

40

20

1 ·~

60

40

20 244

I I I

240

(I)

59

29

2.95

i· !

310 c

127

393

260 280 300 320 '340 390 410

Fig. 3. Mass spectrum of Hydroxy-l<etone (I)

200 d

IB6

220

430 450

6

of higher aliphatic ketone has given great trouble on acco•nt of •

very similar physical properties, IR and !MR spectra. The fragmenta­

tions obtained in mass spectra have been of immenee help in

elucidating the structure of higher aliphatic ketone, both symmetri­

cal and unsymmetrical19-23. The moet intense peaks are oi'tained by

-ex:: -fission and by (.3 -fission which is accompanied by Me-Lafferty

rearrangement with the addition of plus one (Chart-!). In the mase

spectrum of the compound, no significant (M-15'+ peak was observed

and the ratio of {M-15)+ /M+ was close to zero. This indicated that

the compound is a straight chain24 • Ma_ss spectrum showed peak at

0~1 )+ which is characteristic of unsymmetrical ketone25 •

Besides the molecular ion peak at e/m 452 (M+, 25~) the other

significant peaks desoernible were the following (Fig. 3)

42:5 ( oC -fission to the -OH group, 10)

393 { oC -fission to the -OH group, 12)

337 ( 6 -fission to the •C•O group, 5) -

323 ( 'l -fission to the =0=0 group, 9)

310 (fragment c, (? -fission to the •0•0 group - .

Mc-Lafferty plus one ion, 100)

296 ( .£-0~, 14)

295 (.Q. -oH, or oC -fission to the cC:zO group, 66)

292 (.Q.-H20, 17)

250 (.Q.-Hz0-3CH2,14)

244 (.!!-H2o, _20 ' 238 (~CH2 , 17,

237 (~C~ , 6 )

227 ( o -fission to the •0=0 group, 7)

CH3 (CH2>18 .co(CH2)7

. CHOH CzHs

1 1 Huar:g- Minion r~duction CH 3 (CH2) 26 . CHOH . CzHs

IT l Cr03/AcOH

][

+/H 0 I

C2.H 5 . c II CH2

n rnje 364 m m;e· 72

Chart II- Synthesis of compound ill and its Mass Fragmentation Pattern

.:

~ Q

t::::: -;::..., ......

'!I)

t::::: (l) ..... -S

IU -:::... ·--en -~

Q:

+o/H I

c2 H 5 - c II CH7.

m m;e 72

54

57

2.9

n m/e 364

72 m

364 !1

Fig. 4. Mass spectrum of the Ketone (m)

..

407

213 { Y-fission to the =0=0 group, 9)

200 ( fragment ~. @-fission to the =C=O group, Mc-

Ie.fferty plus one ion, 100)

186 {.!-CH2, 33)

185 (,!-ca, or oe-:f'ission to the =0=0 group, 33)

18~ (~-H20, 22)

167 (A-H20~0H3 , 18,

127 (!-OH3 ~ 25)

126 (!-CH2, 18,

59 ( oC - fission to the -OH group, 44)

29 ( oC -fission to the -QH group, 23)

From the above mass fragmentation data coupled with other

physical and chemical evidences the structure of the hydroxy ketone

may be represented by (I) 0 II

Me.(CH2)17 .cH2.o(cH2)7.cHOH.cH2Me

I

In order to ascertain further the position of the -OH group

in (I) it was reduced (Chart-II) to (II) by Huang-Minloa reduction26

having mp .gao, molecular formula, c30u62o. This on oxidation with

chl:omium trioxide gave a monoketo compound (III) having mp.92•,

molecular formula, c30H60~. The mass spectrum (Fig. 4) o:f' (III)

showed the mol~cular ion peak at m/e 436 (II'", 15~). The other

significant peaks appeared were the following•

422 (M--CH!,10)

408 ( M-2CH2

, 12)

,....... ~ 0 ....__

lU \..)

<: ~

lOO

80

60

:: 40 ~ U)

<: "<:(

n: 1-- 20

3 4 WAVELENGTH (MICRONS)

5 6 7 8 9 10 11 12 13 14 15

~

0----~----------~----------~--------~----------~------~--------~----------~--~ 4000 3000 2000 1500 1200 1000 9000

CM-t 800 700

Fig. 5. IR curve of the hydroxy compound (II}

WAVELENGTH (MICRONS) 3 4 5 6 7 8 9 10 11 12 13 14 15

100 .-~----~------~----~----~------~----~------L-----~----~------~-----L----~

80

'""" ~ 0 '-......

60 ltj (J

2: ;: !::: 40 ~ (J)

<::. "'(

0::. 1-- 20

0~--~----------~--------~----------~--------~------~--------~----------~--~ 4000 3000 2000 1500 1200 1000

CM- 1 900 800 700

Fig. 6. !R curve of fhe Ketone (ill)

407 (oe -fission to the =C=O group, 22)

394 (rf-3CH2, 8)

379 ( oC -fission to the =C=O group, 8'

364 (fragment !• 15)

72 (t.ragment m, (3 -fission to the =C•O groJ.p, -Mo-Le.fferty plus one ion 100)

58 <.a-ca2 , 42)

57 ( oc-fissi on to the =C•O group, 58)

54 (!-H20, 60) . 29 ( o<:-fissi on to the =0•0 group, 18)

The mass fragmentation pattern of the compound (III) is

quite in conf'orm:l. ty w1 th the structure asaigoed for it. Consequently

the original hydroxy ketone may reasonably be represented as (I).

Reduction of' the hydroxyketone (I) to {II) by Huans-Minlon reduction 1

The hydroxy ketone (I) was reduced by hydrazine hydrate in

alkaline sol uti on using diethylene glycol sol vent. The product

obtained after the reduction had mp. 98•, IR 3220 011-1 (Jig • 5)

Anal. Fou.nd t c, 82.14~ 1 H, 14·09~ J

Calc. for c3QB62o 1 C, 82.19~ J H, 14.12~ J

Ohromiu11 trioxide oxidation of compound (II) to oompoun4 (III) a

The compound (II) was oxidieed by chromium trioxide in acetic

acid and worked up in the usual way. The product formed had mp.92•,

IR 1700 cm-1 (Fig. 6)

WAVELENGTH (MICRONS) 3 4 5 6 7 8 9 10 11 12. 13 14 15

1--~----~----~------~----~----~'------~'----~~----~'------~-----L------~----~ 100 I '

......._ ~ ~

80

lJJ 60 '-.)

<: ~ I-

~ 40 (/)

<: oq: 0::: 1-

20

OL_~------~---~-----~~--~~~--~~---~~ 4000 3000 1500 2000 1000 1200

CM- 1 900 800 700

Fig. 7. I R curve of a/kano/s from c5 -c10 fractions

WAVELENGTH (MICRONS) 3 4 5 6 7 8 9 10 11 f2 1'3 14 15

,I

100 ,_~----~------~----~----~------~-----L----~~----J-----~------L------L----~

80

"'""' ~ a '-..J

l1j 60 LJ < ~ I--~ 40 U)

<: ~ ct: 1-

20

0 -

4000 3000 2000 1500 1000 1200 CM -f

·goo ' 800 700

Fig. 8. /R curve of ester from CHC/ 3 extract of M. m. Linn.

Anal. Found a o, 82.36~ a H, 13.72~ a Calc. for o3QH60o a c, 82.56~; H, 13.76~~

All the foregoing evidences have led in support of the structure (I)

for the hydroxy ketone.

An other major o omponent in the :tr acti on• c5-c1 0 was obt aintd from

chromatography over argentized silica gel and was found to be a

single component by TLC. lR spectrum {Fig, 7' gave absorption at

3220 cm-1 assignable to a hydroxyl group, It did not giYe any

colouration with tetra-nitro-methane. It also did not correspond to

the test for sterols and triterpenes. However GLC (rig. 1) revealed

that it was a mixture of 5 components with carbon content ranging froa

027 to 031 with 027,029' 0,1 predominating, only' minute traces Of

compounds with even number were present, These compounds were supposed

to be a mixture of alkanole as one of the compounds wa1 identical

with hentriaoontan-16-ol17 and was compared with the synthetic compound

by GLC study where both of them have been found to contain ea11e

carbon number with the same retention value. Hentria-oontan-16-ol

was prepared from palmi tone 18 by sodium borohydride reduction.

Treatment of the ohlorofo.rm extract 1

The residue from the ohl or oform extract which had a fishy odour

on being heated with dilute alkali gave profuse quantity of methyl­

amine. Chromatographic separation of the neutral moiety over alumina

gave @-sitosterol, mixture of hydrocarbons and a very small amount

of ester which Showed carbonyl absorption in the IR at 1740 om- 1 (~

disc) (Fig. 8). TLC of the ester showed two spote-oae very weak. The , ..

major spot ·.had R:t 0.60 (5~ ether to pet:r oleum ether) corresponding

100

80

.....-... ~ -...__,

l..t.J 60 u <: ~ 1---~ 40 U)

<: ~ a: ~ 20

3 4 WAVELENGTH (MICRONS)

5 6 7 B 9 10 11 1'2. 13 14 15

·~

ol_~~----L-----~----~~----~~~~--~~--~~~ 4000 3000 2000 1500 1000 1200 900 800 700

CM -l

Fig. 9. I R curve of Marsileaqenin A

lO

to esters of high molecular weight. The ket ODe mp.85-86• isolated

from this fraction by Chatterjee et a16 could not be traced..

Treatment of the ethanolio extract a

The ethanol! c extract gave copious froth with water ther ebJ

indicating the presence of a saponin in it~ On removing the solYeDt

a orude saponin was obtained aDd it was extracted repeatedly with

Butanol. :Butanol was distilled off under reduced pressure when the

mixture of saponin was obtained as a dark brown gummy mae a. !he

tongue loses -the taste !or sugar when the saponin is put over it.

This effect exists from half an hour to one hour. In this respect 16 it resembles the saponin of Gymnemagenin •

The crude saponin was hydrolysed by re!luxing with ~ ethanolic

hydrochloric acid for 10 hours. The alcohol was evaporated ott

keeping the acid strength more or lea·s constant on frequent addition

of water. The crude sapogenin was filtered and the residue was

washed repeatedly with water until free from acid, d:ried at 60•. The

crude genin on :repeated chromatography over Brockmann alumina aDd

subsequent erystallisati on from ethanol yielded a new tri terpene 2.S•

called ' marsileageninA', mp. 332-33•, [oc]D + 48• (ethanol), IR

spectrum (Fig. 9) of the compound showed bands at 3500 (hydroxyl

group) and 820, 830 om_, ( trisubsti tut ed double bond).

The hydrolysis of the saponin with ethanolic sulphuric acid was

avoided due to the fact that the crude sapogenin obtained after

hydrolysis becomes a tar-like material and it was dif!ioult to isolate

the pure genin from this product.

\

WAVELENG TI-l (MICRONS) 3 4 5 6 7 8 9 10

100 I

80 ...-....._ ~ 0

'--

lJj 60 C.> <: ~ 1--~ 40 U)

2: ~ 0:: t--

20

0 I

4000 3000 2.000 1500 1200 1000 CM -f

11

900

Fig. 10 IR curve of Marsi/eagemn A Hexaacetate

_,

12 13 1·:

I

800 700

~ .. ll

Molecular formula 1

Analytical sample of marsileageninA was analysed tol carbon and

hydrogen. Difficulties were realised in detecting the &JOleculal

weight from mass spectroscopy as it was not volatile upto 320•, but

the molecular weight was d ed uoed from the molecular weight of its

hexaacetate. The data given below clearly indicated a molecular

formula o3oa50o6 for marsileageninA.

Name of the compound

Mar sileageni nA

Found

colour reaction a

71.23

71·11

9 ·9

9·95

Kol. wt.

506

506

MarsileageniDA gave a dark red colouration with concentrated

sulphuric acid. It showed pink oolouration in the Id.ebermann-Burchard

test indicating it to be a triterpene. It also gave a yellow colour

with tetra-nitro-methane indicating the presence of unsaturation.

MarsileageninA Hexaaoetate 1

MarsileageninA was acetylated with acetic anhydride and pyridine 2.50

in the usual way and the product thus formed had mp. 285•,~] +35·5• D

(CHcl3), molecular formula c4~62o12 • It showed m bands at 1740 (br),

1242 (br) 011-1 (acetate carbonyl) and 830 c11.-1 (trisubstituted double

bond) (Fig. 10).

Thus the above results showed that all the six oxygen aio11e

in marsileageninA are present as hydroxyl :f'unct1o1us.

Monotrityl derivative of MarsileageninA 1

MarsileageninA was heated with trityl chloride 27 in pyridine

and the product formed had mp. 205-7°, molecular fcn=mula, o49H64o6•

The result showed the presence of one primary hydroxyl group

in marsileageninA.

Detection o! number Of double bonds in marsileageninA 1

standard chloroform solution o:f' marsileageninA hexaacetate

was treated w1 th an excess of standa:rd per benzoic ac1d28 solution.

From the amount of consumption of perberizoic acid per mole o!

marsileageninA hexaacetate it was found that marsileageninA contains

one double bond,

Detection of number of oe-glyool systems in marsileyeninA 1

MarsileageninA was found to consume two moles o.t periodic acid

per mole of the compound. The products obtained after periodic acid

oxidation29 ''0 was found t~ give yellow oolouration with tet:ra­

nitro-methane and hence the double bond of the compound remained

intact during periodic acid oxidation.

The results from the above experiment showed the presence of

two oe-glycol systems in the compound.

Preparation of 11-keto marsileagenin! hexaacetate a

MarsileageninA hexaacetate was heated under reflux with chromium

trioxide fn glacial acetic acid and the 11-keto compound thus .formed

239 mJ-1 (logE 4·tz)

2.40 250 260

A cO

270

:A max

A cO

280 290 300

Fig.11. UV Curve of 11-l<eto Marsileagenin A Hexaacetate

oAc

-- oAc

CHz.oAc

oAc

310

I b ·-U)

t::: Q)

L:::l

-Q:l \..) -a_

a

100

80 ......... ~ 0 ~

Lu 60 \._)

<: ~ ~

~ 40 U)

<. ""l:: 0: ~

20

' WAVELENGTH (MICRONS) 3 4 5 6 7 8 9 10 11

0 v

4000 2000 1500 1200 t 1000 900 eM-

3000

Fig. 12 IR curve of /1-l<eto Marsileagenin A Hexaacetate

12 13 14 15 I

800 700

1a

was worked up in the usual way. The molecular :formula of the compound

was found to be c42H60o13 , mp. 287°, UVmax 239 nm (-log.E 4.12)

(lig. 11 ), IR ~"uJol 1735, 1240 (acetate carbonyl), 1670 ( oC,@ mo..x

flneaturated ketone), 830 om-1 (tr1substitute4 double bond) (Fig.12).

The above reaction indi~ated the presence o:r a 1211' double

bond in marsileageninA. The :following Tables (1 and 2) shows the

UV absorption maxima o:r some 11•keto .. compound belonging to 18~ and

18@ oleanane and ur sane series.

TABLE - 1

UV Maxima of 180C and 18@ oleanane epimere

!fame of the compound

3@ -Acetoxy-12-bromo-11-oxoolean-12-ene

3@ -Aoet oxy-11-oxoolean-12-ene

3@ -Acet oxy-11-oxoolean-12-ene-30 oic acid

2 -Bromo-3, 11-dioxoolean-12-ene-30 oio acid

3, 11-Dioxoolean-12-ene-30 oio acid

., @ -Hydro:xy-11-o:xoolean-12-ene

3@ -Hydroxy-11-oxoolean-12-ene oic acid

269

250

248

250

250

245

248

Methyl-3oC-aoet oxy-11-oxoolean-12-ene-24-oate 248

Methyl-3@ -acet oxy-11-oxolean-12-ene-28-oate 250

Methyl-3~ -aoetoxy-11-oxoolean-12-en-30-oate 248

Methyl-3~ -hydroxy-11-oxoolean-12-en-30-0ate 248

max ( nm) R e:f.

18@

267 31

245 32, .,,

244 34,35

245 36

245 36

243 .,7

242 34,35

244

248

243

243

38

35,39

34,40

,4,35

lt

TABLE - 2

UV Maxima of 180C and 18@ oleanane epimere and the corresponding

Vrsane derivatives

lame ot the oom.pound ~A. max (am)

'3@ -Aoet oxy-11-oxour s-12-ene 251

3@ -Aoetoxy-11-oxoolean-12-ene 250

3~ -Aoetoxy-11-oxo-18oC-olean-12-ene 245

Methyl-'3 ~ -acet o:xy-11-oxours-12-en-28-oate 250 '

Methyl- '3@ -aoet oxy-11-o:x:oolean-12-en-26-oate 250

Ref

41

32,33

42

4'3

35,39

Methyl-'3@ -aoet oxy-1 1-oxo-180C -olean-12-en-28-oate 248 42

5 ,8,14-Tri methyl-11-oxo-18~-novoleana-9( 10),

12-diene 256,287 42

5,8,14-Trimet~yl-11-oxo-novurea-9( 10), 12 diene 256,290 43

5 ,a, 14-Trimethyl-11-oxo-18oC-novoleana-9( 10 ),

12-diene-28-0io acid

5,8,14-Trimethyl-11-oxo-novursa-9(10)-12-diene

28-oio acid

5 ,a, 14-Tr1methyl-11-oxo-18~novoleana-9( 10)

12-diene-30-0ic acid

2-Benzylidiene-3,11-dioxoolean-12-ene

2-:Ben eylidiene-.,, 11-dio:x:ours-12-ene

3,11 ,ai oxoolean-12-ene

3,11,dioxours-12-ene

257,288 44

259,292 43

254,287 44

231,255,294 45

232,255,293 45

294

293

45

45

Relative Intensity in % --~

~

~ IJ1 0

\.() U1 0

- 0 ~

s:: cu (/) (J)

(JJ \:)

(1) C) ........ .., t:: 3' Cl \.11

- 1.)1

::X: (1)

X co I

Cl I ~ () I"(} .....

"<:: --1

~ tll ..,

0

(J) 0'1 :::.. 0 (1:) 0 1:\)

1..0 (t)

:::J -· ::::!

.h

I- 4 58 ( M+ - 5 A c 0 H)

~ 476 (M+-4AcOH-C 2HzO)

~

518 Uvt+ 4AcOH) t---

~---

,...._ 536 ( M+-3Ac01-i-Cz/-/zO)

1-

578 (M+-3AcOH) .

~---

~

F. ::::::. .::. . ::==._ -t-···--- 63B (M+-2AcOH)

.

1--- 683 ( f1+ -AcOH -C H3)

698 (M+-AcOH)

3 (13'

(1' 0

r--- /87 ( ?_ -·2 AcOH) !=-- ---

197 (Q 3AcOH-CH2 0Ac)

~ 1\J F" 1'\J 0

I=-r-- ;

t'V =--(Jl F 247 (~-AcOH) 0

t'V ~

(] r--- 270 ( Q- '3AcOH)

()j

302 (b-AcOH-CHzOAc-CH3) 0 0 ...=.-

r--

F- 3!5(Q-2Ac0H-CH3)

1::-

1-- 375 (g-AcOH-CH3) t::.=:

1--

1---- :::\qn f h -A~OH)

f:=:-

~

~

445 (M+ -4Ac0H-CH2.~Ac)

---~ -

241 m_}J(/ogE4·43) 250 m_)J (logE 4·46)

259 m_}J (logE 4·33)

\/ I t oAc

-- oAc I I

CH2oAc b ·-{I) oAc t:::::

AcO --/ ~ ~~~ Q)

C)

-A cO ___(__ ~ / I OJ \.) ·--/ '\. I Q_

0

230 240 250 260 270 280 290 300 310

A max

fig. 13 UV Curve of 611

:12

' 13

·'8 diene Marsi/eegenin A Hexaacetate

.• 1 s

The 11-keto compound of marsileageninA hexaacetate displayed

UV abeorpti on maximum at 239 nm against usual position at about

249 nm. This hypsochromic shift is reasonably attributed to the

presence of a number of acetoxyl groups in the molecule.

Selenium dioxide O:xidation of mare1leagen1n.A. hexaacet ate '

The hexaacetate in glacial acetic acid was refluxed with .

selenium dioxide and the prOduct was worked up in the us•al way.

The molecular formula of the compound was found to be c42H60o12,

mp • 285-86• X mo.)f. ~41, 250 and 259 nm (logE 4.43, 4.46 and 4. 38 ·

respectively) (Fig. 13).

The triple UV maxima of the selenium dioxide oxidation product

of marsileageninA hexaacetate is characteristic of trana heteroannular 46-48 12 49 diene system and is a characteristic of b.- oleanenes • It

should be mentioned here that tri-tetpenes of the ursane series

having 12t 13 double bond yields above short o! dienes only on heating

at a higher temperature (200°) with Se02 in benzylal.cohol.

Mass spectrum of MarsileageninA Hexaacetate J

The mass spectrum of the hexaaoetate (Jig. 14' of marsileageninA

showed the characteristic retro Diels-Alder fragmente50 involving

12t13 double bond of the oleanenes or the lll!senes.

+ . 01\.~

01\e.

--~

Ac.O

ReO

A eO

AeO

Cl

mje. 307

b

mfe 450

+ .

The other significant peaks appeared at m/e a

6$3 (lt -AcOH-cH3)

6:58 (It--2Ao0H'

578 (rf-3Ao0H)

536 (wr-3AcOB-c2H5o' 516 (I+ -4Ao0H)

476 (x+-4AcOH-o2H5o)

458 (M+ .. 5Ac0H)

445 (M+-4AoOH-cH20Ao)

385 ( Jt -5!cOH-oH20Ac}

247( a-Ao'OH) -187(,!-2Ao0R)

- 16

390(b-Ac0H) -375(~Ao0B-CH3 )

315 (,k-2AcOB-cH3)

270( b-:5Ao0H) -

The mass spectrum of the hexaaoetate supports the presence of i2.

/J -oleanane skeleton. The mass spectrum did not show the moleoula't

ion peak, but it showed the rt -AoOH peak at m/e 698 due to the

facile loss of a molecule of acetic aoid in the 1onisin1 chamber

whiCh is a phenomenon of common occurrence. The mass spectrum showed

the retro Diels-Alder fragments a at m/e 307 and b -AcOH at m/e·390 . - -indicating that two hydroxyl groups are present in the part containing

rings A/B and four hydroxyl groups are present in the part containing

rings D/E in marsileageninA. The mass fragmentation pattern suggests

the primary acetoxyl group to be at C-28 as there is pronounced 7'

unit loss from fragment l• As has been mentioned previously,

marsileageninA contains two oe -glycol systems. Jaw only four hydroxyl

groups are present· in the part containing rings D/E of which one

17

primary hydroxyl group is present at c28 • It follows, therefore,

that one oc -glycol system and another secondary hydroxyl are

present in this part. The presence of a ·«:: -glycol eystea at

a15 ,c16 was ruled o~t, because it has been mention,ed by "Yarioue 5 ~

workers 1 that hydroxyl group at c15

, whether a.xial or equatorial

is resistant to acetylation under ordinary condition. But

marsileageninA furnished a hexaacetate under normal condition.

consequently the p1esence of~ oe-glycol system at o21

,a22

seems

to be certain52 • The presence of a 16~- hydroxyl is re"Yealed :troa

NE spectrum of the hexaacetate and corroborated by the :tact that

the hexaacetate is· easily formed, whereas the 16oC-hydroxy is

reported to be highly hinde~ed53 • Furthermore, the secoad oC -glycol

system seems to be present at c2, c3

• The presence of a hydroxyl

group at c3 is more probable from b1ogenatic point of view and

as there are two hydroxyl groups in the part containiag rings ~B,

constituting a oe•glycol system, the .other hydroxyl group must be

present at c2. The stereochemistry of the hydroxyl group is e'Yideat from. the

NMR spectrum of the hexaacetate •

signals ara shown in Table-3.

TA:BLE - 3

Tertiary methyls

. The eigni!i oant NMR

0 ·91 (311, e)

1 .02 ( 6H,e)

1 .11 (9B, e)

1. 35 (3H, e)

18

TABLE- 3 (Contd.)

ocooa, 2.0 (6H,s)

2.10 (6H,a)

2.14 (6H,a>

c17-cH20Ae 3 .g6 ( 2H, br s)

C-2H 4 .ea < 1H .. m)

c-3H ' 4.84 (1H,d,Ja10Hs)

C-21H 5.0 (1H,d,J=11 Hz)

o-22H 5.39 (1H,4,J=11 Hs)

C-12H 5 ·41 ( 1H,a)

C-16~! 5.75 ( 1H,t-like)

The lOR data discloses the existence of seven tertiary methyls . 12 (eliminating the possibility o! ~ ursene skeleton). One olefinic

hydrogen and six acetoxyl functions, uong which one is attached to

primary and the othe~ five are connected to secondary }Qdroxyls. The

broad singlet at b 3.96 is assigned to methylene prot on a ot C-28

acetoxymethyl group. The AB quartet at 5.0 and .at.5.39 is better

ascribed to the trane-diaxial prot one c:£ to acetylated 112 glycol

system at c-21 and c-22 rather than C-15 and c-16. The presence ot

triplet like multiplet at 5·75 is attributed to C-16 axial proton54. \

The downf'ield shift is due to the deshielding effect by 1t3 inter­

action with the 0-27 methyl groups. Diequatorial relationship of

the glyo ol eyst em at c2 and ~3 i e revealed by the appeaJ: ance ot

signals at 4.98 (1H,m) and 4.82 (1H,d,J=10 Hz) ascribable to diaxial

OH HO

I Barrigenol R1

III Theasapogenol A

HO

\ \

\

' '

\ \

V Gymnemagenin

'

OH

-- OH

CH 20H

'OH

OH

-- OH

i

HO~

Ho--

HO

I I

H CH20H

II Protoascigenin

W Tang(nol

;<1 oH

-- OH

C H'2. OH

TI Marsi/eagenin A

Chart ill Naturally occuring Hexahydroxy Triterpenes

Name of the

compound

Barrigenol R1

P.r ot oascigenin

Theasapogen olA

Tanginol

Gymnemagenin

llareileageninA

[oe]D & mp.

+ '37° (dioxane)

'308-10•

+ 31·5•(dioxane)

'31 0°

+14° (py.)

301-'3°

_,.go (EtOH)

28'3-4•

+ 53.1• (MeOH)

329-'31 °

+ 48• ( BtOH)

'332-3•

I

TABLE - 4

Acetate

mp. & [oe]D

Hexaacetate -28°(CHC~)

186-87.5°

Hexaacet ate -3 .5o ( CHol3 '

140-41•

Pentaacetate + 29°( CHC13 )

174-178°

Hexaacetate -63• ( OHC13 '

150-3•

source

Tea. ~yngium

Aesculus

turbinata seed

Thea sinensis

seed

'Barr ingt oni a

acutangula

Hexaacetate + 35 .5• (CHC~) Gymnemaeylveetre

290-1•

Hexaaeetate + 37.5• (CH013 ) Marsilea minuta

285• !d.nn

Re:C.

55

56

57.58

59,60

16.61

13

19

protons at c2 and c, respectively.

All the foregoing evidences have led to the conelueion that

marsileageninA may b.e formulated as olean-12-en-2 «: , 3<?, 16 ~ ,

21 @ , 22oC , 28-hexol.

Hexabydroxy triterpenes· isolated from ti:t!erent plute are

presented in the Table-4 and Chart-III.

Identification of sugars s

:For identit:ioation of sugars in the saponin obtaiaed :trom

ethanolio extract of marailea minuia, a portion of the ethanolio

extract of the plant was b¥drolyeed with ~ ethanolic sulphuric

acid for ten hours under refiux. The alcohol was evaporated off

keeping the acid strength more or less oonat ant on tr equent

addition of water. !hie was filtered and the filtrate wae neutrali­

sed with barium carbonate. The barium sulphate thus precipitated

and then excess of barium carbonate was added and :til tered off.

The filtrate was deioniaed, dried aad examined :tor its sugar

constituents by descending paper partition chromatography using

n-ButanolsP.yridineaWater (6a4a3) as solvent at the upper layer and

aniline oxalate as the developing agent. The sugars thus identified

were D-glucoae, D-galaotoee, D•xylose, D-arbinose, ~rbamnos~ by

comparison with their authentic specimens.

After ten long years, however, Ike. A· Chatterjee et al

corrected 11 that marsilin isolated :from Marailea minuta Itnn was

not a ma.orooyolio ketone as reported ea:rlier 6• It has bee1 claimed

by them that marsilirl has e.lso been isol.a.ted trom another plan-t

• Ipomoea :!istulosa tt but the molecular formula wae changed trom

~29Hs4o to c56H112o2 • They corrected that mareilin is a high

molecular ester. The m spectrum of the compound here showed peak

tor carbonyl absorption at 1735 c11-1 and l:r.fi 1peotrum of the compound

gave signals at 'r 9·1 (6H}, 8.75 [ H of (OB2)11

] , 7·75 (2H,OH2-c.o), 5 ·9 (2H,OR2-o·'· Behaviour tower~s chemical reagents wae e111ilar as

described in earlier literature. In this later communication it has

been reported to be an ester of triaoontanol and hexacoeanoio acid

which were obtained by the hydrolysis of the compound with (21)

potassium hydroxide solution. This compound wae synthesised froa ita

hydrolysed products and also from erotic acid and 1-triacontanol.

100

80 ....-.._ ~ 0

'-

Lt.! 60 l.l <: ~ 1--~ 40 ~-(/)

<::: "'(

0::: 1- 20

3 WAVELENGTH (MICRONS)

4 5 6 7 8 9 10 11 12 13 14 15 I

OL---~-----------L----------~--------~----------~------~~------~----------~--~ 4000 3000 2000 1500 1QOO 1000

CM - 1 900 800 700

Fig. /6 . I R curve of the hydrocarbons

21

EzyERIMEITAL

The stems and the leaves of Marsilea minuta Linn was

out irrt o pieces, air dried and powdered. !he material ( 640 g)

was euocessi vely extracted in a one litre sexhlet extract~ w1 th

petroleum ether (60-80°) tor 50 hours, with chloroform te.r 30

hours and with ethanol (95~) toz 50 hours.

Treatment of the petrolium-ether Extract a

The petr olium ether extract Ol'l removal of the solvent and

proper d:y·ing yielded a dark green solid (5 g). The solid was

die solved in 50 ml of petroleum ether ( 40-60•) containing about

6 m.l of benzene and chromatographed over a column of neutral

Brockmann alumina (200 g), the elution being carried out eucoessivel1

w1 t h petroleum ether ( 40-60° ) , Petl: oleum ether c Benzene ( '11 ) . .

(1s1), Benzene, Benzene·Ether C5a1) (111), Ether, Ether-Kethanol

( 111) and Methanol. Each fraction contains 10 ml of eluent. !he ,

reeults of the chromatography are shown in table-5.

Each fraction from A1-A20 ( 105 ~) •as tXBllined separately by

TLC when a single strip was obtained. These !racti one showed no

absorption at 1720-1740 011.-1 or 3400....3600 011.-1 in the m apectrua

(Fig. 16) coofirming the absence of carbonyl and cydroXJl containiag

compounds. These tractions did not give any colouratioa with

tetranitr ometbane indicating the absenoe o:t any unsaturation in the

compound. Moreover, they did not respond to Liebermann-Burohard

reaction eliminating the possibility of any eteroids or tri-terpene.

22

TABLE - 5

Fractions Eluent a llatW!e o! the products Remarks

· 11-A2o Petroleum Ether White solid, a.p.S0-81• Mixture of

( 40-60°) 105 mg hydrocarbons

bJ m & GLC

report

A21-A45 -do- White solid, m.p. 88°, Mt.xture 15 mg

A46-Aao Petroleum Ether- White solid, m..p.91• Jlixture 'by

Benzene (3a1) 150 rag TLO & GLC

A~u-.&.110 Petroleum Ether- White solid,m.p.91• -do-

Benzene ( 111) 105 mg

A111-A1,0 Benzene :tlil '

A131•A140 Benzenes Ether Little .yellow solid ( ,, 1) 8 mg m.p. 73•

A141-A147 Ben Eenes Ether Very little yellow aolid

( h 1' 5 mg, m.p. 75•

A148-A150 Ether Nil

A151•A153 Ether : Methanol Little oily liquid, 12 mg

A154-A156 Methanol Little Oily liquid, 8 llg

These fractions were then mixed together and examined with the help

o! GLC when it was :found. that they contain mixture o:f seYen bydrOca!boru

', i I, 'I

I I

lr

I'

~so.' vent

10

I IJ

I I I I I

20

7/ME IN 'M(NUTES --..::;,.

27 2 9 31 33

CARBON NUMBER --~ ~:;. Standard compounds J o Hydrocarbons

FIG. /7. GLC OF THE HYDROCARBONS

3C

23

:from o2rc33 with c27,o29 ,o,1 and o33 predominating (Fig. 17). These

were examined with three standard hydroca:bons contatninl c27,o29 and o31 ca:r bon at oms.

Fraoti one A21-A45 ( 15 mg) gave only a Vely small quantity at

material and was f'oond to be a mixture by TLC.

Fractions A4g-A110 eluted with petroleum et~er (40-60•) and

benzene gave one elongated distinct strip in TLC. GLC reYealed that

it was a mixture Of several components. !heee f:raotione were mixed

together and ohromatographed over .A.rgentized silica ae described

in Table.-6.

TABLE - 6

Fractions Eluent a Nature Of the products Remarks

B,-:s,o Petroleum Ether Nil

B11-B20 Petroleum ethera White eo114, m.p. g,o, Jrtl. :xt ur e o! Benzene ( 3a1) 40 Jig hydrocarbon by

IR & GLC repor-t

B21Bso -do- 'fbi te solid, m •. p.95° lixture of

120 1lg bJdr OXJ C Ollp OUDd

by IR & GLC•

Bef .. %5 Petroleum. ether 1 111 Benzene ( 1 a1 )

Bso-B105 Benzene Whi t'e solid,a.p.102-10'• GLC indicated 40 mg oae c omponen1

o out a1 ni ng :50 car 'bon at oae, Ill showed t~ preeence o! H

B -B Ben zenea Ether 1'11 &•O=Ogr. 106 115 (1a1)

24

Preparation of Silver nitrate Impregnated Silica sel 1

A 2C),C solution ·of silver nitrate (80 ml) wae heated on a steam

bath with silica gel (40 gm) !or hal! an hOUI• cooled and filtered.

fhe adsorbant was dried at 120• tor 16 hrs. and cooled ira a deei-

coat or.

Reobromatograpby of the fractions A40A110 over .Argent1ze4 silica t

Fracti one A40A110 (255 mg) was d1ssolyed in 1 ral ot pe"trolium

ether containing a few drops of Benzene and adsorbed in 5 gu o!

argentized silica gel. The adsorbed material was cbroma:tcrcraphed

over a column of argenti 2:ed silica gel ( 15 ga) and elute4 w1 th

different solvents (Table-6) and each traction collected in 10 ml

portions.

Fractions B11 -B20 (40 mg) eluted with petrolium ether (40-60•)

and benzene C5:1) gave a solid m.p. 93°. No peak for carbonyl or

hydroxyl group was found in the IR spectrum and 1 t was found to be

a mixture of hydrocarbon by GLC. These hydrocarbons are the same as

found in A1-'2o• Fr acti one ~1-Bao eluted w1 t h petr olium ether ( 40-60•) and

benzene (h1) gave a solid m.p. 95°0. IR (Fig. 7) absorption at -1 :5600-3400 c• shows the presence of hydro;ql c out ainin1 c om.pound e

and GLC revealed that it is a mixture of ee-.eral components. It was

then rechromatographed over .Argentized eilica gel as deseri 'bed in

Table-7.

25

Jraotione Ja6-B105 eluted with benzene aDd pet~olium ether

(40-60•' was found to be a single component b7 TLC and GLO (F1g.1).

The product obtained !rom these :t'racti ons after oryetallieati on

from petrolium ether and benzene (1s3) yielded a white crystalline

solid m.p. 102• [oc.]:s. 9o(CBC1') Jt 452. !hie compound 4td n'"

give any colouration with tetranitromethane aor it ga'Ye any teet

far eter ols and tri-terpene.

Infrared spectrum (Nujol)(Fig. 5) of the compound showed

absorption at 3210 cm-1 (..OH stretching due to polymeric association), . .

-1 -1 -1 1700 om (ac-0 oaxbonyl ), 1455 ca ( -c~ bending), 1375 CJl ( -c-cH3 bending), 1066 cpt1 (·C-0 stretching of secondary alcOhol), doublet

727 and 718 cm-1 •( OH2 )0 bending ( n > 4) vi bl ati on due to 1tr sight

chain methylene groupe.

Anal. Found. o, 79-70,CJ

B.echromatography of the fraction J21 -B80 over a:rgentized 1ilica gel 1

Fractions B21 -B80 (140 mg) was dissolved in 1 ml o! lenzene and

adeatbed i~ .3 gms of argentized silica gel and dried in a 'Yacuum

desiccator. The adscr'bed material was cbromatographed O"fe! a.J:geatized

silica gel (12 g) and eluted with different solvents (!able-7) aid

each fraction collected in 10 m1 portions.

'f!BLE - I

26

TLC Report

One c ompoaen"t

Two components in

equal amoun"t

Two components. one in trace amoun-t

One c ornponent

Two components, one

1 n trace amount

One component

Fractions c5-c10 were found to be a single component by TLC.

However GLC (Fig. 1) revealed that it was a mixture of 5 components

w1. th carbon content ranging from c27-c 31 1 odd number of oar bon at 0118

prevailiag, only minute traces of compound w1 th e'YeD number were

present. The mixture of these compounds did not give, any coloutati on

with tetra-nitromethane and did not give a1JY test for sterols or

27

-1 triterpenes. IR spect~a showed absorption at 3510 oa thereby

indicating the presence or eydroxyl group in the compounds. Jlo.reover

one of the compounds was seen to be identical with hentria-oontan-. .

16-ol17 in the GLC (Fig. 1) report. Hentriaoontan-16-ol was prepared

synthetically from Palmitone18 by sodium borobydride reduetion, Other

.tour components were supposed to be alkanols ot this k1D4 as it has

been :t'ound that certain natural plant generally contains a symmetri­

cal class ot compounds.

Synthesis of di-n-tet~adecll ketone (Palmitoae) 1

Palmi tone was prepared by the method o! Kippings ( 1890). 2 gas

of pentadeoylic acid (Dlp 52°' was taken in a 100 ml. beaker. fhe

beaker was kept in a metal bath. The temperature of the bath was

kept at 205-210°. Phosphorous pentaoxide (;g) was added to it

slowly in small portions with constant stirring !oz about 5 minutes.

The molten mass was then poured into water to decompose the excess of

phosphorous pentaoxide. An excess of sodium hydroxide solution was

then added to 1 t to convert the excess pentadeoyl acid to soap and the . temperature of the mixture was then raised to its boiliDI pOint. On

cooling the product sol1d~t.ie4 at the surface as a waxy cake. It was

then taken out and washed well w1 th water. The product ( 1.5 g) waa

dried and then dissolved in 2 ml of petroleua ether {40-60•)-Benzene

( 111) and ads~rbed in neutral Br OokllaDD alullina (5 g) and dried iD

the usual wq. The adsorbed material wae ohrom.atographed oYer neutral

Brockmann alumina (20 g' and eluted with dit.terent sol'YeDts

(Table-8) end each :t:raoti on collected in 10 ml portions.

TABLE •' 8

I

Z8

Eluent a Fractions Wt. of the leaa:rka materials

Petrolium Ethe:r a Benzene , .. , 0.18 g Brown seal solid

( 1011'

Petrolium Ether a Benzene 4-8

( 4a1)

Petroleum ether t Benzene 9-16

(1a1)

Benzene

Benzene a Chloroform

(1t1)

Chloroform

17•22

23-30

,,_,, Chloro:tormamethanol { 111) 3•-'a Methanol 39-42

0.21 g

0.95 g

0.15 g

0.06 g

111

trace

o.oa

'lhit e solid, mp 82•

Brown semi solid

Gummy

Br own g\tll'JIY

The product obtained :trom the .traoti on 9-16 were llixed together

and crystallised from Benzene. TUl gaYe a single apo~.

IR 1705 om-1, mp. 82-8,•.

Anal. Found I C, 82 .65~ J H, 1:5 ·7~ J

Calc. tor c31B62o, c, 82.66- 1 H, 13.77~ 1

Z9

Preparation of Hentriaoontan-16-ol 1

Palmitone (80 mg) was suspended in 10 ml of methanol and to

this sodium borohydride ( 100 mg) was added. Tbe mixture was kept

!or 15 hours at room temperature. Hydrochloric acid (0.7 Ill) and

water (10 ml) was then added to it and the reaction llixtYre was

warmed on the steam bath !or 10 minut ee and then dllut ed w1 th

water. The solid was collected by filtration and washed with water,

dried, dissolved ~n ~nimum volume of benzene and ads~ bed in 5 gms.

o:t Brookma:nn alumina. The adsorbed material was chromatogre.phed over

a column o:t Brockmann alumina (15 g) and eluted With different

solvents (Table-9) and each :traction collected in 10 ml portions

TABLE ... 2

Eluent a Jtaoti one Wt .o:t the Reaarke residue

Petroleum ethe% (40-60°) 1-4 5mg gua

Petroleum ethersBenzene 5-12 50- ·White solid ( :511)

Petroleum ethersBenzene 1,·16 10 Ill Brown solid (1a1)

Benzene 17-20 111

:Ben zene• Ether (1t1) 21-23 l'il

Ether 24-25 Nil

Chloroform 26-28 trace

Methanol 29-30 trace

The fraction a 5-12 was recrystallised from petroleum ether 1

Benzene ( 1t1' as white plates and showed a single spot on T LC on

silica gel G. IR 3400-3200 cm-1 mp. 78•

Aaal. Found. 1 O, 82.'5" 1 B, 14.18" I

Calc. f~ o31H64o, c, 82·3~ 1 H, 14.15~ 1

P:eparati on of OXime of the Rydrog ketone a

HYdroxy ketone (50 mg), hydroxylamine hydrochloride (100 D!),

ethanol (3 ml) and pyridine (0.3 ml) were heated on a water bath

under renux condition for one hour. The alcohol was e'Yaparated,

diluted with water, eol>led in ice and filtered. !he residue wae

washed with water and d:ried and then c:rystellieed troa 80" ethanol

yield 30 mg,mp. 106°.

Anal. Found 1 1, ~·J

Calc. for o,oft61N02 I I, 3·1" C

Preparation of Acetate of the PYdr oxy ketone t

The hydroxy ketone (50 mg' was taken With aceti o anhydride ( 0.8

ml) and two dxope of py:idine ·and the mixture was heated 01 water bath

foz one hour. Working up in the usual way the product was cxystal:1eed

:!% om a mixture of chloro:torm and ethanol. Yield. 40 mg

mp. ga• [ae J ~o + .,. ( oHol3'

Anal. Pound 1 c, 79·70faH, 12.51" J

Calc. far e32! 62o, t 0, 79.76~ 1 H, 12.55" 1

31

Reduction of the &drozy ketone (I) to (II) bx H•ans-M:l.nln Reduction 1

100 mg of the hydroxyke~one (I), 200 mg of KOH, 1 Dll of diethylene

glycol and 1 ml of 85~ hydraziDe hydrate were refluxed for one hour.

Water was then drained of! from the condenser and the te~~perature

was allowed to rise to 200•. Refiuxing was continued for .2 hours more.

The reaction mixture was cooled, diluted with water and poured slowly

into 6lf HCl. Working up in the usual way the product obtained was

crystallised from petroleum ether-Benzene (1a1). GLC gave a single . . .

peek. Yield 80 mg, mp. 98°, IR 3220 cm-1 •

Anal. Found 1 c, 82 ·14~J H, 14 ·09" J

Calc. for c,o~~620 a C, 82·19" J H, 14.12" 1

Chromium trioxide OXidation of compound (II} to compoual (III.) 1

compound II (50 mg) was taken in glacial acetic acid ( 10 Til) am

wae heated to refiu:x and then ch:romium trioxide (50 11g) 4issolved in

acetic acid (85", 5 ml' was added gradually over a period of one hour •.

:Re!luxing was oont .inued for another one houx and the prOduct was then

diluted With water. The precipitate was filtered sad washed well with

water and dried. It was crystallised from Benzene as plates. GLC ga'fe

a single peak. Yield 35 mg, mp. 92°, IR 1700 ca - 1•

Anal. Found a c, 82.3~ J H, 13·7~ J

Calc. for c3oH60o a c, 82.56", H, 13·7'",

32

1reatmen't ot the Ohlorotorm Ext:raot 1

letpyl amine from Chloroform extract 1

The chloroform extract on removal of the solvent and proper

drying yielded a deep green· solid ( 30 g) which posses !isl!ly odour •

The solid ( 10 g) obtained from chloroform -extract wae heated

with 5" sodium hydroxide (100 ml) in a round bottom flask. !he

issuing gas was absorbed into 21 hydrochloric acid. When the issuing

of the gas is complete, the acid was completely removed ua4er reduced

pressure. The residue was then crystallised fl011 water ae needles.

Yield 0.3 g mp. 224°

Anal. :round a 1, 20.6~ 1

CH6J01 requires N, 20.74" 1

Pzeparation o! 2a4-dinitromet&l aniline derivative a

The hydrochloride (0.2 g) obtained from the above experiment is

taken in 5 co of ethaDOl and 5 oo ethanolic eoluti on of 2a4

dinitrochlorobenzene (10}C) was added and warmed a little on a water

bath. Solid obtained on cooling was filtered, washed witlt. a little

water and reoryetallieed :f'rom ethanol as needles, yield 0.1 g,mp.

174-75°.

Anal. :round 1 1, 21 .45" J

Calc. for C-r!:5H704 t ll, 21 .32~ 1

The solid {20 g) obtained fro the chloroform extract was

dissolved in minimum volume of ohl roform and adsorbed in 25 gaa

33

of neutral Brockmann alumina and ied. The adsorbed material was

chromatographed over a column of neutral alumina (80 g) 8M eluted

with different solvents (Table 10) and each fraction oolleeted ia

50 m1 portions.

TABLE - 10

Eluente Fractions Wt, Of the Remarks :residues

Petroleum ether 1 111

(40-60°) 2 111

'3 0.15 g White gua

4 0.0'3 g " 5 111

Petroleum ether 1 6 trace Yellow gum

Benzene ( 1t1)

7 0.24 tt

8 o.og • 9 0.06 White gua

.10 trace • 11 111

TABLE-10 (Contd.)

Eluent s Fractions Wt. of the Remark• residues

Benzene 12 o.og colourless gum

13 0,15 col our lees gum + White oryst ale

14 0.45 White crystals

15 0.24 .. 16 0.15 .. 17 0.12 " 18 o.Og " 19 t:raoe

Benzene s ether ( 1s1) 20 111

21 111

Ether 22 111

Ether 1 Methanol(90a1 0) 23-24 111

25 111

Methanol 26-27 trace Brownish gum

Ieolati on of @ -si tost er ol 1

Fraction nos. 14-18 were mixed together. It responded to

Li ebe:rmann Bur chard test indicating the presence Of et er ol aDd on

crystallisation from methanol afforded colourless needles (0.8 g)

34

· 2.5 nuJol o! @-eitoseterol mp. 135-136• [oe]l>-'.55° (CHcl3),IR ~"'o.x3400 ca·1•

No depression in melting point was observed on admixture w1 th

35

eD authentic specimen of @-sitosterol

Anal. Found 1 c, 83.85~ ' B, 12.26~ '

Oalc • tor o24H; 00 1 C, 8,.99~ J H, 12.15" J

Acetzlation of <!-si toaterol 1

~-sitosterol (50 mg) in pyridine (5 Ill) was heat ... oD a

steam 'bath atte't addition of acetic anhydride (0.5 ml) fer 2 brs.

Working up in the usual way the product was o:ryetellieed f:roa

ohl or of orm-11et han ol ( 111 ) yielded ~ •a1 t ost er ol ac et at • 1 n fiat . . . 2S

needles (35 mg), mp. 125-26•, [o<] I> -36° (oHol3)

Anal. Found 1 o, 81 .4~ J H, 11 .. 4~ ;

Calc. far c31 ~~; 2o2 t c, 81.5~ 1 H, 11.48~ ;

Ben zQ1lati on Of @ -ai t oat er ol t

@-sitosterol (50 mg) was dissolved in pyridine (1.2 11.1) and

benzoyl chloride (0.25 ml) was added to it. The result inc ldxture

was stoppered and left over night. Working up in the usual wq the

prOduct on crystallisation trom ethanol afforded a benzoate. 2.5

mp. 142-143°, {~]I>_,,, (ohl~ro:tora)

Anal• :Pound a O, 8., .36~ f B, 10;.59" I

calc .• tO% o,6Bs 4o2 a o, a., ·39~ , H, 10 ·4~ '

Treatment o:t Ethanolio Extract ·•

The ethanolic extract on removal of the solvent and p:rope:r

drying yielded a dark browa gummy substance (80 g). It ga'Ye auoh

troth when a small portion of it was shaken with water indicating

36

the presence of a saponin in the extract. '.rhe saponin ia auch

hygroscopic. f.he solid waa repea:tedly extracted with Butaaol. Butanol

was distilled off under. reduced pressure when the oru4e eaponin was

obtained as a brown solid mass (60 g).

Bldrollsis of the Ethanolic EXtract t

'.rhe ethanolio extract (55 g) was dissol~ed in 6~ ethanolio

bydtoohlorio acid ('380 ml) in a 500 m1 round aottom flux aad it

was refluxed for 16 hours. After the hydrolysis is oyer alcohol

was distilled off on a water bath keeping the volume of the distilling . '

product same on .frequent add.ition of water. It was then .filtered,

washed well W1 th water till free· of acid and then the resitue was

dried. The dried resid'ae (30 g) was then dissolved in 20 ml o.f

ethanol and mixed up w1 th Brockmann alumina ( 30 g) and dried. !rhe

adsorbed aaterial was ch:tomatographed over a column ot neu-tral alumina

(600 g) and eluted with different eolYents (Ta~le-11) aDi eaoh

fraction collected in 100 ml portions.

The .fraction numbers 16-25 obtained from the above ohromatograpey

were mixed together. It was brown in cOlour. It was charcoalieed by

dissolving in methanol and crystallised from this eolveat. A. faint

brown colour still remained in the product and T~ ga"fe the eXistence

of th:zee components in this product With the pr'edominanoe of one and

the other two are in minor quantities. The material (3.2 g) was then

dissolved in 5 ml of ethanol and mixed up w1 th Brockmann alullina

(10 g). It was then rechromatographed on a column of neu:tral Brockmann

alumina ( 100 g). Elution being carried out with ohl oro form aad methanol

TABLE -11

Eluents Fraction it. of the Re11ark1 numbers materials

Petroleum Ether (40-60°' 1-4 1·5 g gum

Petroleum ~her• 5-6 Nil Beozene ( 3a 1)

Petroleum Ether a Benzene 7-8 111

(1•1)

Benzene 9•10 111

Benzene a Ob.l or oform ( 3a1' 11•12 Nil

Ben zenea Chloroform ( 1a 1) 13•14 111

Chloroform 15-16 Ni:l.

Chloroforma Methanol(9a1) 17-25 3·5 g Brown solid mp. 30-4-,05.

Ohl.or oforma Methanol( 3J 1' 26-30 0.12 g

Cbl or of orm 1 Methanor (1a1)31-'32 trace

Methanol '33-34 trace

used successively in ~ gradient elution procedure (Table•12) and

each fraction collected in 50 ml portions.

3?

Fractions 16-30 were mixed together and crystallised from ethyl

alcohol as sand like cryatalls. 'It gave a sin&].e spot in the TLO

plate. It gave deep red colouration with oonceatr at ed eulpb.uric acid

38

TABLE - 12

Eluente Fraction Wt. o! the Reaarks numbers residues

Cbl. or otor m 1-2 10 mg White gua

Chl or o:f'orma Methanol 3-5 30 mg White eol14 mp.180•

(98t2)

-do- 6-9 20 mg White solid mp.165•

Ohl or ot or ma Methanol 10-15 15 JJg White solid mp ·320-25•

(95 15'

-do- 16-30 1·5 g White solid mp.330-31•

Ohl or ofornu let han ol 31-34 20 Jig Brown soli4 11p .225-28•

(911'

Chloro!ormaMethanol 35-40 25 Jig Jr on ••mi.-solid

< ''1' Ohl oro tor taaMet han ol 41-42 trace

( h 1'

Methanol 43-44 111

and gave pink to violet oolouration in Id.eberaann Burchard test, .2. !5.

mp. 332-33° (DL)D + 48~(Et0R}

Anal. Found a 0~ 71.23~ J H, 9.91~ J

Calc. for a301fs0o6 a c, 71.11~1 H, 9·95",

Preparation o! marsileageninA hexaacetate a

MarsileageninA (300 mg) was suspended in acetic anhydride (6 ml)

and pyridine (3 ml) was added to it. The mixture was heate4 on a wate:r:

39

bath for 4 hrs. under reflux condition. After the reaotiOI was over

the mixture was dropped into ioe-cold water. The solid product was

washed well w1 th water. The solid was then extracted with ether and

the etherial layer was washed successively with dilute sodium bicar­

bonate solution and then with water. The etherial solution was then

dried by adding anhydrous sodium sulphate. Etherial solution was

separated from sodium sulphate and. ether was removed. The residue

(2.45 g) was dissolved again in dry ether (5 ml) and· then mixed llp

with Brockmann alumina (10 g). It was then chromatographed Ol'er a

column of Brockmann alumina (50 g). Elution was carried out wiih

petroleum ether (40-60° ), Benzene, chloro!ora and methanol uaed

successively in a gradient elution procedure (Table-13). Each

fraction of the eluents contains 75 m1 of solvent.

TABLE - 1' Eluents Fraction Wt. o! the Remarks

numbers residues

Petroleum ether (40-60o) 1-2 trace gum

Petroleum ether sBen zene 3-4 lil

( 311'

Petroleum ether a Benzene 5-6 llil

(1a1)

Benzene 7-8 111

Benzene 1 Chloroform 9-10 Nil (9515'

Benzene' Chlor o.form ( 9 a1) 11-12 :111

fABLE - 13 ( contd.)

Eluents Fraction 'It. of the :Rem.a:rks numbers residues

Benr;eneaOhloro:torm ( 3&1) 13-16 50 mg White aol14 :ap .2,5•

-do- 17-18 111

Benzene a Chloroform 19-28 210 mg White eolid mp.284•

( 111)

Chloroform 29-30 Nil

Chl or of orm 1 Methanol 31-32 Nil

(9a1')

Chloroform a Methanol 33-34 lfil

( 311'

Ohl or of orm 1 Methanol 35-36 trace Brown o111 residue

{1J1)

Methanol 37-'38 111

Thi fractions obtained !rom 19-28 was crystallised .trom aethanol

as needle shaped crystals. It gave single spot on Ttc plate. mp.285•,

lR 1740 (br,, 1242 (br) cm·1 (acetate carbonyl) and 830 c•-1 (tri-7.50

substitute~ double bond~ [c,L]J> +_35~5o (CHcl3).·

Anal. Found 1 c, 66.;9~ J H, 8.26~ 1

Oalo. for o42a62o12 a Ot 66.4~; H, 8.24~ 1

Kydrolzeis of mareileageninA hexaacetate 1

MareileageninA hexaaoetate (50 mg} was taken in 5~ methanolio

XOH (5 m1' and the mixture was kept under refiux on a water bath for

1rsileagenin A He xaacetate

oAc

--oAc

oAc

oAc

--OAC

OAC

AcO __

AcO --

AcO

1 oAc

. -- oAc

, CH20AC

l oAc

I R i) max 1735, 1670 em-'

UV :\max 239 nm

'oAc

UV A max 241 ,250,259 nm

+ AcO __

A cO

RDA >

a

b

mje 450

+ '-':::: c Hz

mje :307

+ ~oAc

.

~--OAc

OAC

41

one hour. Then the product was dropped in water and eolid obtained

wae filtered and washed well with water till it wae oomple'tely free

of alkali • It was then orystalli sed !:rom ethanol when sand like

crystals were obtained. It gave single spot on TUJ. axed melting

point with previous sample of marsileageninA remained undepressed ,_s·

mp • ,,,. • [ oC J » .,. 48 ° (EtOH)

, Anal. Pound 1 Ot 71 ·2~ ' I, 9·93- I

oalc. for c3oa50o6 • o, 71.11- 1 H, 9·95~,

Preparation of c£·,@ unsaturated ketone from aareileyenia! hexaaoetate t

The hexaacetate (50 mg} was taken in glacial aoetic acid ( 10 al)

and was heated to reflux and then chrOilium trioxide (50 mg) dissolved

in acetic acid (85", 5 ml) was added gradually over a period of one

hour. Re:fluxing was continued tor another one hour and the pr educt

was then diluted wi'th water. The precipitate was filtered and waehecl

well with water and dried. The solid was clissolvecl in 0.5 ml of methanol

and mixed up with alumina ( 2 g) and ohr ome.t ogr aphed over Br octunn

alumina (10 g' eluating euooess1Yely with different solvents and eaoh

fraction oolleoted in 10 ml por'ti.ons (!able-14) •

Fractions 10-18 were orystallieed t.rom methanol as colourless 2-So

tleedles and was found to be a single eompound by TLC mp. 287• [c£]D + 20•

UV (EtOH) 239 nm (log f 4·12) \. nt.tJol 1 IR ')) 1735, 1240 011- (acetate carboayl>

!'\'lo-x

1670 oa-1 ( ae, ~ uosaturated lcetoae)

830 cm-1 (tri-subetituted dou~le boad)

A.nal. Found 1 o, 65 .28" 1 H, 7. 79"'

Calc. tor o42860o13 1 o, 65.2~ J H, 7·8~ 1

100

so --

" ~ 60 '--l!j '-> <: ;:s 40 1--~ {/)

<: ~ 20 1'-

3 '

WAV£LENGT!-i (MICRONS) 4 5 6 7 8 9 10 11

I

0 v v

\J

4000 3000 2000 1500

Fig.t5.~:::.'t:i2 ,13: re

1200 CM- 1

1000 900

diene Marsileagentn A hexaacetate

12 13 14 15

.-~-

800 700

42

TABLE - 14

I

Eluents Fraction Wt. of the Reaarks

numbers Residues

Petr oleu~ ether (40-60•) 1-2 l{il

Petroleum ether a Benzene 3-4 lil

( 3a1)

Petroleum ether:Benzene 5-6 Nil

( 1 11)

Benzene 7-9 tract

Ben zenea Ether ( 4a1' 10-18 35 mg fbi te etlid

Ben zen ea Ether ( 1 a1) 19-20. 5. -do-

Ether 21-22 111

Ethers Chloroform. ( 111 ) 23-24 5mg Brown semi solid

Chloroform 25-27 trace Brown oily residue

Chl or otorma Methanol ( 111' 28-30 trace -do-

Methanol 31-32 111

Selenium dioxide oxidation of MarsileageninA hexaacetate 1

Marsileagen1nA hexaacetate (50 mg) was taken in glacial acetic

acid (3 ml) and mixed up with freshly subliaed selenium dioxide (50 mg)

The solution was then refluxed for 4 hours. It was then filtered hot

and the filtrate was diluted with water when a solid product wa1

eepar at ed • It was filtered and washed well w1 th water and dried • The

43

sOlid was dissolved in 0.5 m1 ot methanol and mixed up w11)1. Brookmana

alumina (2 g). It was then ohromatographed OYer a colum.a •t Brockmann

alumina ( 10 g) and eluted with different eolyente (Table-15). Each

fraction contains 10 ml of solvents.

tABLE - 15

Eluent a

Petroleum ether ( 40-60°)

Petroleum ether 1 Benzene ( 1a 3)

Petroleum ether 1 Benzene ( 111)

Benzene

Benzene a Ether ( 4s1'

Benzene 1 Ether (1•1'

Ether

Ether s Ohl or oform ( 311 )

Ether 1 Chloroform ( 111)

Chloroform

Chloroform 1 Methanol (3a1'

Chloroform s Methanol ( 1a1)

Methanol

Fraction Wt. ot the Remarke 1 numbers residue •

1-2

3-4

5-8

9-16

17-20

21-22

23-24

25-26

27-28

29-30

31-32

33-34

35-36

111

Nil

5mg

trace

111

lil

lfil

Nil

111

.11

Nil

Wh1 te gum+

crystal

White crystal

-do-

gua

Fractions 9-16 were mixed together and orystallieed from •ethanol

as colourless needle shaped crystals. It was :round to be a single

'O'V (EtOH) 241 nm (log E

250 nm (log f

259 nm (logE

Anal. Found t c, 66.57~ 1 R, 7·~ J

Oalc. for c4iB60o12 1 o, 66.44~ 1 H, 7·99- 1

Acetylation of Ma.rsileageninA ,at room temperature a

44

MareileageninA (50 ~) was treated With acetic anhydride and

two drops of pyridine. The mixture w8 s kept for 24 hours at rooa

temperature. The mixture was poured into water and t~e solid which

separated, was worked up in the usual wq and was or7etallieed two

times from methanol when a single spot was obtained 111 TUl. mp. 2.5

285• ~ [a<:) D + 35 ·5°

No dep~eeeion in melting pOint was observed on admixture w1 th

marsileageninA hexaacetate prepared earlier. . .

Anal. Found a C, 66 .45~ J H, 8 .22~ J

Oalo. for o42ft62o12 a c, 66.46~ J H, 8.24~1

ll:eparation of mono-trityl derivative o! M'a:reileyeninA 1

KareileageninA (50 mg' was dissolved in pyridine (0.5 ml) and

tri tyl chloride ( 150 mg) was added to 1 t. !he mixture was retluxed

on a water bath tor :5 hours. It was dropped in c•ld wa'ter, filtered

and washed. The residue was crystallised !roa etayl alcohol. It gaYe

single spot on TIC plate arp. 205 -7•

45

Anal. Found 1 o, 78·5~ J H, 8.51~ 1

Calc. for c49H6406 I c, 78.6~' B, 8.55~ •

Estimation of number of double bonds in marAt.leaseninA pexaaoetate 1

Marei~eageninA hexaaoetate (50 mg) was dissolved in 10 ml

chloroform. To this 5 ml of perbenzoic acid solution dissolved in

chloroform was added to it. A blank s.olution was .similarly prepared

by taking 10 ml of chloroform and 5 ml of perbenzOic acid in a flask •

• Both the !laske were shaken well and kept at 0°C in the refrigerator

for 15 days. Then the flask containing the mareileageninA hexaacetate

was taken out and to this solution sodium iodide ( 1 gm in 25 ml of water

and acetic acid (2 m1 ' were added. The solution was ehaken and kept

s:toppered in the dark for 5 minutes. The liberated iodine was titrated

with standard (1/100) sodium thiosulphate solution. The blank solution

was similarly titrated fOllOWing the same procedure. !he result of the

titrations is given below.

Amount of MarsileageninA hexaacetate = 50 mg

strength of sOdium tbiosulphate solution= (N/100)

Volume of sodium thi oeulphate solution required to titrate the

liberated iodine by unreacted per benzoic acid in the flask containing

mareileageninA hexaacetate = 87·3 ml.

Volume of sodium thiosulphate solution required to 'titrate the

liberated iodine by per benzOic acid in the blank flask:100 111.

Amount of perbenzoic acid oonsume6 by 50 mg of mazaileageninA

hexaaoetate ::: 100-87 ·3 ::: 12.7 ml of (1/100) sodium thiosulphate

solution;; 12.7 ml. or (N/100) perbenzoio acid solution.

46

Calculated amount of (N/100) perbenzo:l.o acid solution requi:t:ed

to r eaot w1 th one double bond in 50 mg ot mar eileageni nJ. hexaaoet at e•

13.2 ml

Found t 12.7 ml ot (1/100) per benzOic acid solutio•.

The value indicates the presence of one double bond in

me.t"SileageninA.

Eetimation of number of o<: -glycol system in maJa±leageninA s

Me.rsileageninA (50 mg' was dissolved in methanol ( 10 ml) and

periodic ac~d solution (3 ml, 0.5 K aqueous m.ethanolic eolution) as

added to it~ The mixture was shaken and kept for 24 hour• at r ooa

temperature. A blank solution was similarly kept by takiag aethanol

(10 ml' and periodic acid solution (3 ml, 0.5 11 aqueous aethanolio

solution). After 24 hours the solution containing marsileageninA

was mixed up with an excess of sodium arsenite solution (5 ml of 0.21). I.

Sodium bicarbonate ( 1 g' and water ( 25 ml) in presence of few crystals

of :potassium iodide were added and the fiask was .haken and kept for

fifteen minutes. !he excess of arsenite was back titrate4 with 0.01

(N) iodine solution using starch as indioato:. Blank solution was

similarly titrated. From the difference in titre values ),etween the

blank and compound. reaction mixture the periodate uptake was

calculated. The result o! the ti trations is given below.

Amount of marsileageninA • 50 mg

strength of iodine sol uti on = (ll/100)

Volume of iodine solution required to titrate e:xoeae of sodiu11

arsenite in the solution containing maxsileageninA = 15,.4 ml.

41

Volume of iodine solution required to titrate exeeaa sodiua

arsenite in .the blal'lk flask:: 110.4 ml

Difference in the volume of iodine solution • 15J.4•110.4c

43.0 ml

Calculated difference in the volume of iodine solution :f'o: oae

oe -glycol system = 19.7 ml.

Calculated difference in the volume of iodint solution !o:r two ce..­

glycol syet em = 39 .4 ml.

!he above result shOWed the presence Of two d:. -glycol system

in mareileageninA.

Hydrolysis of,the saponin mixture from ethanolic extract aDd

identification of suga:re t

The crude saponin (5 g' obtained from ethanolic extract was

refluxed with 5~ methanolic sulphuric acid (50 ml) on a eteam bath

for 10 hours. Methanol was evaporated off in the usual wq by repeated

addition of water 1 keeping the acid strength more or leal constant,

It was then filtered and the filtrate was nutrallised by llhaldng

successively with barium carbonate to pH 5.5-6.0 using uniTersal pH

indicator paper and filtered. The f~ltrate was deionieed w1 th Amber lite

m-120 (H) and Amberli te IR-45 ( OH'. The deiontsed filtrate was

reduced to a smaller volume (4 ml} under reduced pressure and

lypholieed. The syrup thus obtained was exald.ned for sugars by

descending method of paper chromatography with the apparatus uae4

by Hough & Jones on Whatman Jo. 1 chromatography papez. !he follOWing

solvent systems (v/v) were used for the ohromat ography

A62 = Ethyl aoetateaPyridineaWater; (8a2s1 h

B = 1!,-ButanolaPyridinetWatera (6a4a:5);

o63 = s.-Butan ol' acetic aoidtWateJq (4t h5 h n64 = .!!-Butanolabenzeneapyridineawater;(5a1a3z3h

(R:r values wexe measured using solvent B)

48

!he solvent was allowed to run :tor 18 b%s. The paper was then

dried in air and sprayed with saturated solution of anil1•• oxalate. 65

The paper was then kept at 110-20• in a hot air oven !or 10 minutes.

The sugars present were identified to_be D-gluooee (R! 0.19),

D-galaotose (R:r 0.16), D-Iyloee (R:r 0.23}, D-arabinose (!.t 0.29),

L-rhamn~se (R:r 0.39) by direct ~omparison with respective authentic

samples.

49

REFERENCES

1. IC. 1:. Gupta, Mareilea, Botanical Monograph lo. 2, pw'bliehed

by CSIR, New Delhi, India. ( 1962).

2. IC. P. Biewae and A. K41 Ghosh, Bha.ratya Bonouahadi (Jengali),

Calcutta University, ,l, 622 (1952).

3. SUpplement to Glossary of Indian Medicinal Plants b7 Chopra

and Verma, publication & Information Dixeotorate (CSIR),

New Delhi, P• 65 (1969).

4• The Wealth of India, Council of Scientific & Industrial

Research, lew Delhi, Vol. VI ( L-M), p 306. . .

5 • M. r.. Chatterjee 4: s. Pal, Bulletin of the Calcutta School

of Tropical Medicine, Vol. IX, lfo. 3, p. 123, Jul.J (1961 ).

6. A. Chatterjee, c. P. Dutta, B. Choudhury, P. K. Dlf, C. D. Dey,

C. Chatterjee and s. R. Mukherjee, Science & Cul tu:e, ~.

619 (1963). . . .

1. P. K. Dey, Mira Chatterjee and Chitralekha Chatterjee.

Nattttewisaenschaften, 22, 693 (1963). -8. Chitralekha Chatterjee, P. x. Dey aad o. D. Dey, Xaturwissens­

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