15.1.
1978 Specialia
f r o m w h e r e an overflow of m a t e r i a l begins to CA via
NCA I f r o m t h e 7th d a y o n w a r d s . C o n s e q u e n t l y t h e
n e u r o s e c r e t o r y m a t e r i a l b e c o m e s again m o r e a b u n d a n t
in t h e CA on t h e 7th day. This overflow of n e u r o s e c r e t o r y
m a t e r i a l r e n d e r s t h e g l a n d s i n a c t i v e (figure 3). The
m o u l t i n g of 2nd stage n y m p h s occurs f r o m 12th to 14th
day. T h e CA of 2nd i n s t a r n y m p h s r e a d y to moult, a n d
also t h o s e of f r e s h l y m o u l t e d 3rd stage n y m p h s , p r e s e n t
6r CA
6r
H6
' 3oolJm'
Fig. 5. Photomicrograph of the frontal section of the Corpora allata
of an adult female killed 4 h after the final nloult showing sparse
neurosecretory granules (Gr). HG, hypocerebral ganglion. Note the
scattered nuclei and relatively large size of the gland.
A f u n g i t o x i c principle f r o m the leaves of L a w s o n i a i n e r m i s L a m .
R. D. T r i p a t h i , H. S. S r i v a s t a v a * and S. N. D i x i t 1
Department o/ Botany, University o/ Gorahhpur, Gorahhpur, U. P. (I~r
Summary. D u r i n g a n t i f u n g a l screening of h i g h e r p l a n t s , t h e leaves of Lawsonia inermis were f o u n d to e x h i b i t s t r o n g
f u n g i t o x i c i t y . On c h e m i c a l i n v e s t i g a t i o n , t h e a n t i f u n g a l f a c t o r was f o u n d to be 2 - h y d r o x y - l , 4 - n a p h t h o q u i n o n e (Law-
sone). The m i n i m u m effective dose a g a i n s t t e s t o r g a n i s m was f o u n d to be 1000 p p m . L a w s o n e was f o u n d to e x h i b i t
fungicidal a c t i v i t y , w i d e i u n g i t o x i c s p e c t r u m a n d n o n p h y t o t o x i c i t y .
P l a n t s are k n o w n to c o n t a i n various a n t i m i c r o b i a l
s u b s t a n c e s 2,a. A l t h o u g h t h e a n t i f u n g a l a c t i v i t y of ex-
t r a c t s of h i g h e r p l a n t s is well d e m o n s t r a t e d 4-7, t h e
c h a r a c t e r i z a t i o n of t h e f u n g i t o x i c principle h a s received
little a t t e n t i o n . T h e leaves of Lawsonia inermis L a m .
were f o u n d t o e x h i b i t s t r o n g t o x i c i t y d u r i n g a n t i f u n g a l
screening of h i g h e r p l a n t s . The p r e s e n t c o m m u n i c a t i o n
deals w i t h t h e isolation, i d e n t i f i c a t i o n a n d f u n g i t o x i c
p r o p e r t i e s of t h e active principle.
Experimental procedures and results. 20 g of leaves as
well as s t e m , r o o t a n d b a r k were e x t r a c t e d s e p a r a t e l y
w i t h m e t h a n o l a n d s c r e e n e d for a n t i f u n g a l a c t i v i t y b y
a m o d i f i e d p a p e r disc m e t h o d 8 a g a i n s t t h e t e s t o r g a n i s m
Helminthosporium oryzae B r e d a de H a a n . Of v a r i o u s
p a r t s t e s t e d , only t h e leaves of Lawsonia inermis ex-
h i b i t e d f u n g i t o x i c i t y . F o r t h e isolation of t h e active
principle, 500 g of fresh leaves were e x t r a c t e d w i t h
m e t h a n o l several t i m e s until t h e last eluate b e c a m e
colourless. The s o l v e n t f r o m this e x t r a c t was e v a p o r a t e d
u n d e r r e d u c e d pressure. The residue was dissolved in
51
histological c h a r a c t e r i s t i c s of a n i n a c t i v e gland a n d con-
tain a b u n d a n t n e u r o s e c r e t o r y m a t e r i a l . This p a t t e r n is
r e p e a t e d in s u b s e q u e n t n y m p h a l i n s t a r s too.
The CA of f r e s h l y m o u l t e d a d u l t f e m a l e s (1 h a f t e r final
moult) are loaded w i t h n e u r o s e c r e t o r y m a t e r i a l a n d are
m u c h smaller t h a n t h o s e of 4 h old females. The CA of
1 h old f e m a l e s h a v e a low v o l u m e of c y t o p l a s m a n d h i g h l y
c r o w d e d nuclei a n d c a n t h e r e f o r e be r e g a r d e d as i n a c t i v e
(figure 4). B u t o n l y 4 h a f t e r t h e final moult, t h e CA of
a d u l t females s h o w a n o t a b l e increase in size, h a v e m o r e
c y t o p l a s m a n d less c r o w d e d nuclei, a n d c o n t a i n o n l y a few
n e u r o s e c r e t o r y g r a n u l e s (figure 5). I t s e e m s t h a t , as soon
as m o s t of t h e n e u r o s e c r e t o r y m a t e r i a l h a s gone o u t f r o m
t h e CA, the r e s t r a i n t or i n h i b i t i o n is over a n d the CA
resume their activity.
H o w e v e r , t h e p r e s e n t histological findings c a n be inter-
p r e t e d in a n o t h e r w a y also. I t m a y well be t h a t t h e s m a l l
a m o u n t of b r a i n h o r m o n e r e a c h i n g t h e CA p e r h a p s s t i m u -
lates t h e glands for a c e r t a i n t i m e ; t h e n , as t h e t i m e for
m o u l t i n g a p p r o a c h e s , t h e a c t i v i t y of t h e CA w h i c h h a s to
be r e s t r a i n e d is c h e c k e d b y t h e o v e r f l o w a n d c o n s e q u e n t
p r e s e n c e of a b u n d a n t n e u r o s e c r e t o r y m a t e r i a l in t h e s e
glands. The o t h e r p o s s i b i l i t y is t h a t t h e p r e s e n c e of small
a m o u n t of b r a i n h o r m o n e does n o t s t i m u l a t e t h e g l a n d s
b u t j u s t allows t h e m to do a c e r t a i n degree of t h e i r func-
tion. The s e c r e t o r y cells of t h e CA are allowed to p r o d u c e
a c e r t a i n a m o u n t of juvenile h o r m o n e u n d e r t h e super-
vision of b r a i n NSC (which m a y n o t be w o r k i n g com-
p l e t e l y i n d e p e n d e n t l y of t h e CA) for a c e r t a i n period
a f t e r w h i c h t h e o v e r f l o w a n d h i g h c o n c e n t r a t i o n of b r a i n
n e u r o s e c r e t o r y m a t e r i a l r e s t r a i n s t h e i r activity. I n a n y
case, t h e i n v o l v e m e n t of b r a i n n e u r o s e c r e t o r y m a t e r i a l
in t h e c o n t r o l of t h e a c t i v i t y of CA seems to be fairly
certain.
2 May 1977
distilled w a t e r a n d e x t r a c t e d w i t h n - b u t a n o l a n d chloro-
form separately. The solvents from the n-butanol and
t h e c h l o r o f o r m fractions a n d w a t e r from t h e a q u e o u s
f r a c t i o n were r e m o v e d b y e v a p o r a t i o n u n d e r r e d u c e d
* Department of Life Science, University of Indore, Indore M.P.
(India).
1 Acknowledgment. Authors are thankful to Dr Nitya Nand,
Central Drug Research Institute, Lucknow, for providing faci-
lities to carry out chemical investigations. Our thanks are
particularly due to Dr R. S. Kapil, Dr Geeta Devi Tripathi
and Dr S. C. Tripathi for their help rendered in various ways.
2 L.G. Nickell, Econ. Bot. 13, 281 (1959).
3 P . N . Thapliyal and Y. L. Nene, J. Scient. ind. Res. 26, 289
(1967).
4 K. Gilliver, Ann. appl. Biol. 34, 136 (1947).
5 M.L. Dhar, M. M. Dhar, B. N. Dhawan, B. N. Mehrotra and
C. Ray, Indian J. exp. Biol. 6, 232 (1968).
6 J.M. Nieolls, Ann. Bot. 3d, 229 (1970).
7 S.N. Dixit and S. C. Tripathi, Curt. Sci. 4d, 279 (1975).
8 S.C. Tripathi and S. N. Dixit, Experientia 33, 207 (1977).
52 Speeialia EXPERIENTIA 34/1

pressure. The residues o b t a i n e d f r o m t h e 3 f r a c t i o n s love, S. m e l o n g e n a cv. P u s a p u r p l e long. The c o m p o u n d

were s u b j e c t e d to a n t i f u n g a l t e s t i n g b y p o i s o n e d food
t e c h n i q u e S. The n - b u t a n o l f r a c t i o n e x h i b i t e d fungi-
t o x i c i t y a t 1~ c o n c e n t r a t i o n . No a c t i v i t y could be
d e t e c t e d in t h e c h l o r o f o r m a n d a q u e o u s fractions. T h e
n - b u t a n o l f r a c t i o n w a s a c e t y l a t e d w i t h acetic a n h y d r i d e
in t h e p r e s e n c e of d r y p y r i d i n e , a n d t h e a c e t a t e d e r i v a t i v e
was h y d r o l y s e d w i t h Ba(OH)z to o b t a i n t h e b a r i u m salt.
This salt was t h e n s u b j e c t e d t o c a t i o n e x c h a n g e c o l u m n
c h r o m a t o g r a p h y , using a Dowex-50 resin a n d 75%
m e t h a n o l . T h e eluate was t r e a t e d u n d e r r e d u c e d pres-
sure to r e m o v e t h e s o l v e n t , a n d t h e b a r i u m - f r e e residue
(FI) was collected. T h e F 1 f l a c t i o n was f o u n d to be 100%
effective a t 0.75% c o n c e n t r a t i o n in a n t i f u n g a l tests.
I t gave a d e e p blue colour w i t h ferric chloride a n d a
red b r o w n colour w i t h 5~ s o d i u m b i c a r b o n a t e solution
i n d i c a t i n g t h e p r e s e n c e of some c o m p o u n d h a v i n g
p h e n o l i c h y d r o x y l g r o u p a n d a n a p h t h o q u i n o n e ring.
Since t h e p r e s e n c e of 2 - h y d r o x y - l , 4 - n a p h t h o q u i n o n e
was r e p o r t e d earlier TM,n a n d t h e F~ f r a c t i o n e x h i b i t e d
t h e p r e s e n c e of s o m e h y d r o x y - n a p h t h o q u i n o n e , it was
c o n s i d e r e d desirable t o isolate t h e c o m p o u n d f r o m t h e
leaves of L a w s o n i a a n d to t e s t its f u n g i t o x i c i t y . The
c o m p o u n d was isolated according a p r o c e d u r e d e s c r i b e d
earlier TM. I t s i d e n t i t y w i t h 2 - h y d r o x y - l , 4 - n a p h t h o q u i n o n e
was c o n f i r m e d b y m . p . d e t e r m i n a t i o n (m.p. 195-196~
d e c o m p o s i t i o n ) , m i x e d m.p. d e t e r m i n a t i o n (no d e p r e s s i o n
in m.p.) as well as UV, I R a n d N M R s p e c t r a [UV in
e t h a n o l Amax 236, 272 n m , I R K B r , 3185, 1381, 874,
810, 782, 739, 1600, 1451, 1684, 1645 c m -~, N M R d 8.0,
7.6, 6.271 . T h e c o m p o u n d c o m p l e t e l y i n h i b i t e d t h e
mycelial g r o w t h of t h e t e s t o r g a n i s m a t a m i n i m u m
i n h i b i t o r y c o n c e n t r a t i o n (MIC) of 1000 p p m . To a s c e r t a i n
w h e t h e r t h e f u n g i t o x i c a c t i v i t y of t h e a c t i v e principle
is f u n g i s t a t i c or fungicidal, t e s t s were m a d e a c c o r d i n g
to G a r b e r a n d H o u s t o n ' S . T h e c o m p o u n d was f o u n d t o
be fungicidal. I t was t e s t e d for its f u n g i t o x i c i t y a g a i n s t
17 o t h e r fungi (table) a n d f o u n d to be a c t i v e a t v a r i o u s
c o n c e n t r a t i o n s a g a i n s t all fungi t e s t e d . F o r t e s t i n g p h y t o -
t o x i c i t y d i f f e r e n t c o n c e n t r a t i o n s of L a w s o n e viz., 1000,
2000 a n d 4000 p p m were s p r a y e d on a l t e r n a t e d a y s for
a w e e k on a b o u t 15-day-old p l a n t s of C h e n o p o d i u m a m a -
ranticolor a n d Z e a m a y s cv. S a n k a r M a k k a - G a n g a - 2-
a n d 20-day-old p l a n t s of S o l a n u m l y c o p e r s i c u m cv. Marg-
did n o t p r o d u c e a n y visible s y m p t o m s of i n j u r y or o t h e r
t y p e of a b n o r m a l i t y . L a w s o n e also h a d no significant
effect on t h e p e r c e n t g e r m i n a t i o n a n d on radicle a n d
p l u m u l e l e n g t h of m a i z e seedlings.
D i s c u s s i o n . D i f f e r e n t p a r t s of a p l a n t h a v e b e e n r e p o r t e d
to e x h i b i t f u n g i t o x i c a c t i v i t y due t o t h e d i s t r i b u t i o n
of t h e active p r i n c i p l e in d i f f e r e n t p a r t s la. H o w e v e r ,
in L a w s o n i a i n e r m i s , t h e a c t i v i t y was f o u n d t o be con-
fined to t h e leaves only, while o t h e r p a r t s viz., stem,
r o o t a n d b a r k did n o t e x h i b i t a n y a c t i v i t y . Chemi-
cally our f u n g i t o x i c a c t i v i t y was s h o w n to be identical
to 2 - h y d r o x y - l , 4 - n a p h t h o q u i n o n e (Lawsone), w h i c h in
earlier o b s e r v a t i o n s of K a r a w a y a et al. 14 has been s h o w n
to be a b s e n t f r o m s t e m , r o o t a n d b a r k of t h e p l a n t . The
role of s u b s t i t u t e d c~-naphthoquinones as a n t i f u n g a l
a g e n t s is well e s t a b l i s h e d . 2 - m e t h o x y - l , 4 - n a p h t h o q u i -
n o n e 15, 5 - h y d r o x y - l , 4 - n a p h t h o q u i n o n e 1~ (juglone) and
2 - m e t h a l - 5 - h y d r o x y - l , 4 - n a p h t h o q u i n o n e1~ h a v e b e e n re-
c o r d e d as a n t i f u n g a l f a c t o r s f r o m t h e flowers of I m p a t i e n s
b a l s a m i n a , leaves, p e r i c a r p s a n d b a r k of t h e J u g l a n s
regia a n d leaves of C e r a t o s t i g m a w i l l m o t t i a n u m respec-
tively. H o w e v e r , t h e isolation of 2 - h y d r o x y - l , 4 - n a p h t h o -
q u i n o n e as f u n g i t o x i c principle f r o m t h e leaves of L a w s o n i a
i n e r m i s has b e e n d o n e for t h e first time. The lawsone
h a s been f o u n d to be m o r e p o t e n t t h a n its p o s i t i o n isomer
juglone 18 w h i c h was earlier c o n s i d e r e d to be one of t h e
s t r o n g e s t f u n g i t o x i c n a t u r a l p r o d u c t s f r o m h i g h e r p l a n t s 19.
Furthermore, the present compound has been found
to be n o n p h y t o t o x i c , while p h y t o t o x i c a c t i v i t y has
b e e n r e p o r t e d for j u g l o n e ~~ I t m a y be n o t e d t h a t t h e
s u b s t i t u t i o n of a h y d r o x y l - g r o u p in t h e second p o s i t i o n
i n s t e a d of t h e f i f t h in a e - n a p h t h o q u i n o n e s y s t e m leads
to m o r e a n t i f u n g a l p o t e n c y a n d n o n p h y t o t o x i c i t y .
O O
I[ II
V\/
II I II
O OH O
2- h y d r o x y - 1 , 4 - n a p h t h o - 5 - h y d r o x y l - 1,4 -naph t h o -
q u i n o n e (lawsone) q u i n o n e (juglone)

The s t r o n g f u n g i t o x i c i t y a n d t h e a b s e n c e of p h y t o t o x i c i t y
of lawsone i n d i c a t e s t h e p o s s i b i l i t y of its e x p l o i t a t i o n
Effect of 2-hydroxy-I,4-naphthoquinone on the percent inhibition as a n effective fungicide.
of growth of different fungal organisnls

Fungi tested Concentration of Lawsone (ppm)

1000 2000 4000

A lternaria solani 60 100 100

A. tenuis 100 100 100 9 R.K. Grover and J. D. Moore, Phytopathology 52, 876 {1962).
A spergillus niger 65 1O0 100 10 J . B . Lal and S. B. Dutt, J. Indian ~hem. Soc. 10, 1577 (1933).
A. [lavus 35 75 100 11 H . E . Cox, Analyst 63, 397 (1938).
A. wentii 100 100 100 12 R.H. Garber and B. R. Houston, Phytopathology 49,449 (1959).
A. taedius 40 75 100 13 R.P. Scheffer and J. C. Walker, Phytopathology 4d, 94 (1954).
A bsidia ramosa 1O0 1O0 100 14 M.S. Karawaya, S. M. Abdel Wahhab and A.Y. Zaki, Lloydia
A. corymbi/era 100 100 100 32, 76 (1969).
Cladosporium herbarum 44 80 100 15 J . E . Little, T. J. Sporston and M. W. Foote, J. biol. Cheul
C. sphaerosperraum 41 76 100 174, 335 (1948).
Penicillium/uniculosum 5 15 100 16 R. Fischer and F. Stauder, Pharm. Zentralhalle Dtl 72, 97 (1932).
P. oxalicura 0 19 100 17 M. Masuko, H. Egawa, A. Ueyama, K. Koshimizu, K. Kobata
P. rubrura 13 16 100 and J. Fumoto, Ann. phytopath. Soc. Japan 36, 286 (1970).
P. variabile 12 20 100 18 K. Steffen and H. Pesehel, Planta reed. 27, 201 (1975).
Acrophialophora [usispora 100 100 100 19 C.H. Fawcett, and D. M. Spencer, A. Rev. Phytopath. 8, 403
Circinella umbellata 84 100 100 (1970).
Syncephalastrum racemosum 41 74 100 20 J. Dekker, in Antibiotics Fungicides, vol. 2, p. 580. Ed. D. C.
Torgeson, Academic Press New York and London 1969.