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High Pressure Liquid

Chromato-Graphic
Separation of Aryl-
Naphthalide Lignan Lactones
a a
K. S. Annapoorani , C. Damodaran & P.
a
Chandra Sekharan
a
Forensic Sciences Department Kamarajar Salai
Mylapore , Madras, 600, India
Published online: 05 Dec 2006.

To cite this article: K. S. Annapoorani , C. Damodaran & P. Chandra Sekharan

(1985) High Pressure Liquid Chromato-Graphic Separation of Aryl-Naphthalide
Lignan Lactones, Journal of Liquid Chromatography, 8:6, 1173-1194, DOI:
10.1080/01483918508067135

To link to this article: http://dx.doi.org/10.1080/01483918508067135

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 JOURNAL OF LIQUID CHROMATOGRAPHY,8(6), 1173-1194 (1985)

HIGH PRESSURE LIQUID CHROMATO-

GRAPHIC SEPARATION OF ARYL-
NAPHTHALIDE LIGNAN LACTONES

K. S. Annapoorani, C . Damodaran, and

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P. Chandra Sekharan
Forensic Sciences Department
Kamarajar Salai
Mylapore, Madras 600, India

AFY.5 T U C T
A high-pressure l i q u i d chromatographic system is
d e s c r i b e d for the s e p a r a t i o n o f few a r y l n a p h t h a l i d e
lignan l a c t o n e s . An o c t a d e c y l - s i l i c a ( S p h e r i s o r b
5 ODS) column w a s used w i t h methanol/water (73/27
volume $) i s o c r a t i c system as e l u e n t . Both HPLC-
f l u o m m e t r y and HPLC-UV d e t e c t i o n methods were
employed. The p r e s e n t technique is compared w i t h
o t h e r insirrumental methods developed e a r l i e r and its
u t i l i t y in chemotaxonomic a n d t o x i c o l o g i c studies
is d iscussed.

INTRODUC T ION
h r y l n a p h t h a l i d e lignan l a c t o n e s are n a t u r a l l y
o c c u r r i n g dimers in p l a n t s and are o f i n t e r e s t t o
chemists and medical s c i e n t i s t s because o f t h e i r
p o s s i b l e p o t e n t i a l use as a n t i - c a n c e r a g e n t s (1,2).

1173

Copyright 0 1985 by Marcel Dekker, Inc. 0148-3919/85/0806-1173$3.50/0

 1174 ANNAPOORANI,DAMODARAN, AND CHANDRA SEKHARAN

A few of them f i n d a p p l i c a t i o n i n i n d u s t r y as a n t i o x i d a n t s
and i n s e c t i c i d e s . A f e w o t h e m possess t h e r a p e u t i c ae
well as t o x i c p r o p e r t i e s ( 3 ) and f i n d use as a u i c i d a l and
homicidal agents-hence of i n t e r e s t t o c l i n i c a l and f o r e n s i c
toxicologists.
C l e i s t a n t h u s c o l l i n u s (Roxb. ) Benth. & Hook. a Euphor-
biacean shrub, found widely d i s t r i b u t e d i n t r o p i c a l
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c o u n t r i e s , is a h i g h l y poisonous p l a n t . !bough a l l p a r t s
of it are reported t o be t o x i c , a d e c o c t i o n o f t h e crushed
l e a v e s is mainly used a8 s u i c i d a l and c a t t l e poison and f o r
procuring criminal a b o r t i o n (4,5). Chemical c h a r a c t e r i z a -
t i o n of d i f f e r e n t p a r t s o f t h e p l a n t h a s l e d t o the
i d e n t i f i c a t i o n and i s o l a t i o n of c e r t a i n l i g n a n s (6,7)
including c l e i s t a n t h i n A , c l e i s t a n t h i n B, c o l l i n u s i n and
d i p h y l l i n (Fig.1). The presence of these compouIlds in t h e
l e a v e s h a a a l r e a d y been e s t a b l i s h e d . Reports on t h e
medicinal u t i l i t y of t h e above said compounds have been
w l l documented in o u r e a r l i e r r e p o r t s (8-11).
The f o u r lignans c i t e d e a r l i e r , d u e t o t h e i r high
a r o m a t i c i t y , e x h i b i t b r i l l i a n t luminescence when i r r a d i a t e d
under U.V. and serve as e x c e l l e n t model compounds t o b e
followed fluorome t r i c a l l y . Based on t h e i r photometric
p r o p e r t i e s (includ ing f l u o r e s c e n c e ) photometric (UY ald
v i s i b l e ) , fluorometric, solid s t a t e fluorodenaitometric
and photodensitometric methods were developed in o u r
l a b o r a t o r y (8-11 ). The s p e c t r a l c h a r a c t e r i s t i c s ale
presented i n Table 1. The p r e s e n t communication d e a l s
 ARYLNAPHTHfu.DE LIGNAN LACTONES 1175

CHEMICAL STRUCTURES AND R, VALUES OF

LIGNAN LACTONES FROM THE LEAVES OF C.COLLlNUS

COMPOUND STRUCTURE R,VA L uE

~

R = 3, 4 d i - 0 - methyl
xylore
OR
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CLElSTANTHlN A MOO
0.37

CLElSTANTHlN B
0.01

OlPHYLLlN
0.27

COLLlNUSl N
0.55

'TIC on kierrlgcl 60 G w i t h n- Hcptane-Chloroform

Ethanol ( 5 0 : 5 0 : 5 ) as mobile system.
FIGURE 1
 1176 ANTIAPOORANI, DAMODARAN, AND CHANDRA SEKHARAN

TABLE 1
SPECTRAL C H A I W ~ R R T I C SOP C.COLLINUS LIGNANS

uv Via i b l e * Flu0 m me t r y
mox Am a
AExci t
!
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Cleistanthin A 262 580 356 441

Cleistanthin B 262 580 322 440

Diphyllin 2 68 580 362 432

Collinusln 2 50 580 ** *it

with the h igh-preseure 1iquId chromatographic s e p a r a t i o n

o f t h e f o u r lignans, t h e " a c t i v e p r i n c i p l e s " of t h e
poisonous p l a n t C. c o l l i n u e .

In8trument :

A Pye Unicaa ( P h i l i p s , Cambridge, UK) Liquid Chromato-

graph (PU 4800) equipped with video chromatographic c o n t r o l
centre waa used.
 ARYLNAPHTHALIDE LIGNAN LACTONES 1177

Stand a d S o l u t ions :
Authentic samples of c l e i s t a n t h i n A , c o l l i n u s i n and
d i p h y l l i n were obtained from M/S C i b a (Bombay) a2ld
c l e i s t a n t h i n B f r o m O s m a n i a U n i v e r s i t y (Hyderabid). The
p u r i t y of the samples *re checked from t h e i r s p e c t r a l
and chromatographic data.
S t a n d d s o l u t i o n s of the f o u r l i g n a n s were prepared
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in e t h a n o l (500 pg/ml) and d i l u t e d s u i t a b l y as and &en

needed.
S o l v e n t s were o f a n a l a r / s p e c t r a l grade q u a l i t y and
used a f t e r degassing, Water used in t h e mobile phase i s
all-glass double d is t i l l e d water.
Chromatograph i c Cond i t ions :
Column: Spherisorb-5 ODs (25 mm x 4.6 mm i . d )
Mobile phase: i&thanol, Water (73-27) i s o c r a t i c system
Plow rate: 1 m l m i n - '
Oven temperature : 34OC
I n j e c t o r : 20 p1 c a p i l l a r y l o o p
Detection: a ) UV d e t e c t o r - Pye Unicam v a r i a b l e
wavelength (PU 4020) s e t a t 262 nm
b) Fluorescence d e t e c t o r : Pluorichrom (Varian,
U.S.A) equipped witb e x c i t a t i o n f i l t e r 340-380 nm and
emission f i l t e r above 400 nm. me lamp md gain were s e t
at low p o s i t i o n a d the a t t e n u a t o r at 1 (moat s e n s i t i v e ) ,
HPLC Separation:
Standard samples of the f o u r l i g n a n s (0.25 pg) were
injected M i v i d u a l l y u n d e r t h e described chromatographic
 1178 ANNAPOORANI, DAMODARAN,AND CHANDRA SEKHARAN

c o n d i t i o n s and the r e t e n t i o n times were recorded. !l%iswae

repeated t o observe t h e r e p r o d u c i b i l i t y In r e t e n t i o n time
and f o l l o t e d by a mixture of a l l t h e f o u r (0.25 kg each).
The above procedures were repeated for both HPLC-fluoro-
metry alla HPLC-UV methods.
P r e p a r a t i o n of Leaf E x t r a c t :
C.collinue l e a f e x t r a c t wa8 prepared from d r i e d
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l e a v e s ( 1 g ) as describe6 e a r l i e r ( 9 ) and t h e f i n a l
residues taken in an a l i q u o t of e t h a n o l and subjected t o
HPLO.
Recovery f r o m SRiked Biospeclmens:
Blood samples ( R a b b i t ) were spiked w i t h a known
q u a n t i t y of the mixture of f o u r lignnans and processed as
reported (9). An a l i q u o t o f the f i n a l extract i n ethanol
w a s subjected t o HPLC s e p a r a t i o n .

F@SUL!l!S
The ' W l C cr1romatograms o f the mixture of f o u r l i g n a m
as shown by the HPLC-fluorometry and WLC-UV d e t e c t o r s are
shown in Fig. 2 and 3. The r e t e n t i o n time and t h e
c a p a c i t y r a t i o ( k ' ) value o f the four compounds are
given i n Table 2.
C a l i b r a t i o n Curve
In o r d e r t o e s t a b l i s h the l i n e a s i t y of t h e methods
developed, c a l i b r a t i o n graphs were c o n s t r u c t e d for a l l
t h e four compounds i n d i v i d u a l l y as the s e n s i t i v i t y
d i f f e r e f o r the four l i @ a n s . The l i n e a r i t y extends in
the range of 0.05 t o 0.4 Pear d i p h y l l i n , 0.1 t o 0.7 pg
 ARYLNAF'HTHALIDE LIGNAN LACTONES 1179
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HPLC - FLUORESCENT CHROMATOGRAM OF MIXTURE OF AUTHENTIC

SAMPLES ( 0 . 2 5 p g eoch) ON REVERSEDPHASE COLUMN (SPHERISORB-
5 ODs ) . MOBILE PHASE : - METHANOL : WATER ( 7 3 : 2 7 )
PEAK IDENTIFICATION :- 1 CLEISTANTHIN 8
2 DlPHYLLlN
3 CLEISTANTHIN A

FIGURE 2

f o r c l e i s t a n t h i n B and 0.4 t o 1.2 pg f o r c l e i s t a n t h i n A

in RPLC-fluoromtry. In t h e c a s e o f HPLC-UV t h e range is
.05 t o 0.5 pg f o r c l e i s t a n t h i n B, 0.1 t o 0.7 pg f o r
c l e i s t a n t h i n A and 0.2 t o 1.2 pg f o r c o l l b u s h and
diphyllh.
 1180 A"OoRAN1, DAMODARAN, AND CHANDRA SEKHARAN
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-'
TIME (rccl

HPLC - ULTRAVIOLET CHROMATOGRAM OF MIXTURE OF

AUTHENTIC SAMPLES ( 0 . 2 5 pg each 1 ON REVERSELlPHASE
-
COLbMN ( SPHERISORB 5 ODs I . MOBILE PHASE : -
METHANOL : WATER (73 : 2 7 )
PEAK IDENTIFICATION : - I CLEISTANTHIN B
2 DlPHYLLlN
3 COLLINUSIN
4 CLElSTANTHlN A

FIGURE 3
 ARYLNAPHTHALIDE LIGNAN LACTONES 1181

PABLE 2
RE!FENTION TIME AD CAPACITY -
RATIO ( k')
VALUES OF C.COIILINUS LIGNANS ON SPHERLSORB -
5 ODs COLUMN
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Q e n a . i t i v it x
The limit o f d e t e c t i o n of these compounds in both the
methods, along w i t h a cornpa-ison of s e n s i t i v i t y obtained
for d i f f e r e n t techniques, is provided in Table 3.
ReDroducibility
The p r e c i s i o n of t h e method waa v e r l f i e d by c a r r y i n g
o u t t h e experiments r e p e a t e d l y over a period o f time an3
the r e p r o d u c i b i l i t y is expressed 88 $ C.V. i n Table 4.
HPLC of Leaf E x t r a c t :
The HPLC-fluorometry artl chromatogram of the l e a f
e x t a o c t o f C.collFnua are shown i n Fig. 4 and 5. The peaks
corresponding t o the four lignans are marked. As e v i d e n t ,
the leaf e x t r a c t c o n t a i n s B few more f l u o r e s c l n g and UV
absorbing compounds, y e t t o be i d e n t i f i e d . A thin-layer
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COMPARISON OF S E N S I T I B I T Y IN DIFFERENT !PECHNIQUES

Cleistanthh A 1 0.25 0.4

Cleistanthin B 1 0.1

D iph y l l in 9.1 0.25

it No response upto 600 pg/3ml

++ No loaponse upto 10 pg
 ARYLNAPHTHALIDE UCNAN LACTONES 1183

2
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HPLC-FLUORESCENT CHROMATOGRAM OF C . COLLINUS

LEAF EXTRACT ON REVERSED PHASE COLUMN ( SPHERISORB-
5 ODS 1, MOBILE PHASE : - METHANOL : WATER ( 73 : 2 7 ) .
PEAK IDENTIFICATION : 1 CLEISTANTHIN B
2 DlPHYLLlN +UNKNOWN COMPOUND
3 CLEISTANTHIN A
FIGURE 4
 1184 ANNMOORANI, DAMODARAN, AND CHANDRA SEKHARAN

m
-
a,
N

2
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T I M E (tee)

HPLC- ULTRAVIOLET CHROMATOGRAM OF C. COLLINUS LEAF

EXTRACT ON REVERSEDPHASE COLUMN ( SPHERISORB 5 0 0 s ) . -
MOBILE PHASE :- METHANOL : WATER (73 : 27 1.
PEAK IDENTIFICATION :- 1 CLEISTANTHIN B
2 DlPHYLLlN + UNKNOWN COMPOUND
3 COLLINUSIN
4 CLEISTANTHIN A
FIGURE 5
 ARYLNAPHTHALIDE LIGNAN LACTONES 1185

I
t I
I
e 0 I
I ua ? I
I c m
I
I
I
I
I
I
I
I
I
I
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I
I ua 0 a3
I
I
l-

F
'9
0
(u

N I
I
I
I
I
I
I
I
I I
I
I
21 I
e
I
b?
W
0
I
I
*
a3 Lc
?
M
e ua
I
I
I I d 0 l- d I
I 4 l I
I
I
I
I
I
I
3
I
1
I
*
M
ua
N
*
(u
l
M
- I
I
I d 0' d d I
I
I
I
I
I
I
I
I
I
I
m I
I

.a .a
I
I
3
I 0
d
d
A
c
I
I
3
I
0
d
0
V
a
d
FI
I
I
 1186 ANNAPOORANI, DAMODARAN, AND CHANDRA SEKHARAN

chromatogram of the l e a f e x t r a c t along with the f o u r

lignans on ltleselgel 60 0 is shown in Fig.6. The e l u a t e s
from HPLC-column corresponding t o t h e f o u r ligpana were
monitored by Tw: and photometry t o check t h e i r purity.
Recovery f r o m Spiked BioloRical Samples :
The percentage recovery of the lignans fmm 8piked
blood samples is shown i n Table 5.
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DISCUSS LON
As shown in Fig.2 and 3, the f o u r compounds were well
separated under the cond i t i o n s described. D i f f e r e n t binary
and ternary solvent aystema such as a c e t o n i t r i l e - w a t e r ,
ethanol - water, chloroform - methanol, acetonitrile -
methanol - water were t r i e d as mobile phase. But methanol -
water offered the best resolution. D i f f e r e n t r a t i o of water
in the mobile phase, f r o m 5 to 40$ were t r i e d and 27s was
found t o give the optimum s e p a r a t i o n , both f r o m the view of
s e n s i t i v i t y and resolution, Increaeing the water content
in the mobile ph-e decreased the s e n s i t i v i t y , espec i a l l y
i n HPLC-fluorometry.
Hollrever, no d e f i n i t e explanation could be offered f o r
the order of r e s o l u t i o n as there is no c o r r e l a t i o n between
molecular s t r u c t u r e and r e t e n t i o n time, A s explained by
K i m and AyreS ( 1 ) in the eeparation of aryletrahydro-
naphthalene, the r e l a t i v e o l d e r o f a f f i n i t y towards
hydrophobic c e n t r e s could be one of the c o n t r i b u t i n g
f a c t o r e i n deciding t h e order of e l u t i o n f r o m t h e column.
As seen in Fig.2 only c l e i s t a n t h i n s A, B and
a i p h y l l i n alone were detected b u t not c o l l i n u e i n in FPLC-
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FIGURE 6

U V PHOTOGRAPH OF THIN LAYER CHROLltl!TOGR.APHIC N3SOLUTION

OF C.COUIXUS LIGNNKN LACTONES

From L to R

1. Cleistanthin A

2 . Cleistanthin B

3&4. Leaf extract of C.collinus+

5. Collinusin

6. Diphyllin

*C.collinua leaves wele collected from the

d i f f e r e n t places separated by a distance

or 200 km.
 Downloaded by [Tufts University] at 14:02 31 October 2014

Ta
m 5
RJ3COVERY OF C.COLLINUS LICNtLNS PROM SPII[ED BLOOD SAMPLE BY F@VERSED PHASE HIGH
FERFORMA.NCE LIQUID CHROI’LlTOGRiiPHY FLUOROMETRY*
-

Specimen
 ARYLNAPHTHALJDE LIGNAN LACTONES 1189

fluorometry. T h i s wae the case up t o a c o n c e n t r a t i o n o f

10 pg. T h i s observation is c o r r o b o r a t i v e w i t h o u r e a r l i e r
one in spectrofluorolaetry w h e r e then? was no response up
t o a c o n c e n t r a t i o n of 600 pg/3 m l ae given i n T a b l e 3.
As i n s p e c t r o f l u o r o m e t r y , d i p h y l l i n o f f e r d t h e m a x i m u m
s e n s i t i v i t y in IIPLC-fluommetry followed by c l e i e t a n t h i n B
and c l e i s t a n t h i n A. It is known t h a t t h e i n t e n s i t y of
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fluorescence and s e n s i t i v i t i o f the f o u r l i g n a n s d i f f e r in

s o l u t i o n and s o l i d s t a t e (on s i l i c a gel) (10). Collinusin,
however, could be d e t e c t e d at aa low a8 0.25 pg l e v e l w i t h
the i n t e n s i t y of f l u o r e s c e n c e remaining unaltered for
s e v e r a l days fluorodensitornetrically. In the caae of
HPLC-UP, a l l t h e four l i g n a n s could be monitored crt 262 nm
simultaneously, c l e i s t a n t h i n B o f f e r i n g t h e maximum
s e n s i t i v i t y followed by c l e i s t a n t h i n A , c o l l i n u s i n and
diphyllin. O f the two methods HPLC-fluorometry is t h e
preferred one d w t o t h e absence o f i n t e r f e r e n c e from
endogenous compounds (Fig. 7 ) .
tlnalysis o f the f r a c t i o n s c o l l e c t e d from the column
e f f l u e n t by t h i n - l a y e r chromatography ( 1 0 ) revealed t h a t
d i p h y l l i n could n o t be estimated i n the l e a f e x t r a c t
(Fig. 4 a d 5) d u e t o the i n t e r f e r e n c e of another
f l u o r e s c i n g compound whose s t r u c t u r e h a s n o t been
established. Attempts are being under way t o overcome
t h i s d i f f i c u l t y by using a g r a d i e n t system and a d s o r p t i o n
columns. But t h i s i s s t i l l n o t a s e r i o u s problem in
c l i n i c a l and f o r e n s i c t o x i c o l o a becuase c l e i s t a n t h i n A
 1190 ANNMOORANI, DAMODARAN,AND CHANDRA SEKHARAN
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- TIME (sac)

HPLC - FLUORESCENT CHROMATOGRAM OF

01 RABBIT BLOOD ( BLANK, 2 0 pt EXTRACT 1
b) RAEEIT BLOOD ( 20rI SPIKED WITH MIXTURE OF
C . COLLINUS LIGNANS 0.50 pq EACH 1
COLUMN 1- SPHERISORE -5 ODs
MOBILE PHASE :- METHANOL : WATER (73 : 27 1
PEAK IDENTIFICATION : - 1 CLEISTANTHIN B
2 DlPHYLLlN
3 CLEISTANTHIN A
FIGURE 7
 ARYLNAF'HTHALIDE LIGNAN LACTONES 1191

3
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HPLC - FLUORESCENT CHROMATOGRAM OF " ODUVIN" ISOLATED

FROM THE LEAVES OF C . COLLINUS.

COLUMN : - SPHERISORB 5 ODS-

MOBILE PHASE :- METHANOL : WATER ( 73 : 2 7 )
PEAK IDENTIFICATION :- 1 CLEISTANTHIN B
2 DlPHYLLlN
3 CLEISTANTHIN A

FIGURE 8
 1192 ANNAPOORANI, DAMODARAN, AND CHANDRA SEKHARAN

is the major c o n s t i t u e n t of C , c o l l i n u s leaf and a l s o

reported t o be hi&bly t o x i c ( 6 ) . Exteneioe i n v e s t i g a t i o n
w i t h the crude poisonous p r i n c i p l e s i s o l a t e d e a r l i e r ,
such aa "oduvinll ( 1 2 ) , " p r i n c i p l e A " , * p r i n c i p l e B1' ( 1 3 )
e t c . , showed the presence of d l e i a t a n t h i n A t o be the
major c o n s t i t u e n t . The HPLC- fluorometry chromatogram of
loduvin' is shown in Fig,8. The importance of q u a n t i f y i n g
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c l e i s t a n t h i n A is again emphasized from t h e f a c t t h a t t h e

bio-d i s t r i b u t i o n s t u d i e s a f C.collinus l i g n a n s in animals
R v e a l e d the presence o f c l e i s t a n t h i n A even after 2 4 h.
T h i s f r a c t i o n c o l l e c t e d f r o m the column e f f l u e n t was
t e s t e d f d r its p u r i t y by !I!IIC w i t h d i f f e r e n t systems a 4
by s p e c t r a l d a t a .
The p r e s e n t HPLC method can thus be r e a d i l y adopted
in c l i n i c a l and f b r e n s i c toxicology. F o r chemotaxonomical
and pharmacological stud ies f l u o r o d e n s i t o m e t r i c m e t h o d is
suggested .
Our thanks are due t o Mr.G.Kurnaravelu, I.F.S. f o r the
i d e n t i f i c a t i o n sf C.collinug l e a v e s ;
Drs. T.R.Govindachari (Amrutan jan), N.Viswanathan (Ciba)
and G.6rimannarayana (Oemania U n i v e r s i t y ) f o r g i f t s of
a u t h e n t i c samples; t o o u r staff f o r t e c h n i c a l and
s e c r e t a r i a l assistance.
Acknowledgmehts are a l s o due t o the Bureau of
P o l i c e Research and Development, New D e l h i for providing
f i n a n c i a l a s s i s t a n c e IDone o f us (K.S.A.).
 ARYLNAPHTHALIDE LIGNAN LACTONES 1193

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