10urnal of Scientific & Industrial Research
Vo1.58 , December 1999 , pp 990-994
Simple Method for Enrichment of Azadirachtin
from Neem Seeds
Sarojini Sinha*t, P S N Murthy, C V N Rao, G Ramaprasad, S Sitaramaiah ,
D G Kumar and Suresh Kumar Savant.
Division of Crop Chemistry and Soi l Sciences and Crop Protecti on,
Central Tobacco Research Institute, Rajahmundry 533 105
Received : 2 1 April 1999 ; accepted : 23 August 1999
Azadirachtin the principle ac tive constituent of Azadirachta indica ranges from 0.3 to 0.6 per cent. A simplifi ed procedure, when
co mpared to establi shed method s, has been adopted to enri ch azadirac htin •A' to 50 per cent using minimum poss ibl e steps without
resorti ng to chromatographic techniques. It in volves addit ional partiti on with CCI~ before final extracti on which removes most of
nimbi n, salanin. and allied triterpenoids. Enriched product find s immense utility in preparing formulat ions with high azadirachtin and
also in isolati ng pure azadirachtin by adopting preparati ve HPLC.
Introduction
°
Azadirachtin (C}) H44 16 ), a tetranortriterpenoid from
the neem tree (Azadirach ta indica A. Ju ss), was first isolated by Butterworth and Morgan 1 • It is hOl:monall y active in in sects2.3 and a powerful antifeedant and growth
disrupti ve agent. It is ovipositiona l deterre nt and a n excellent ne maticide. It has therefore arou sed worldwide
academ ic and industrial interests . More than hundred
compound s have been iso lated so far from va ri ous parts
of A. indica and related Meliaceae spec ies. These compounds and extracts of A. indica have potent and spec ifi c effects again st various in sect pests\ but none has
biolog ica l activi ty comparab le to azadirachti n. Formulati ons from neem ke rne l ex tract, stand ardi sed in te rm s
of azadirachtin conte nt, ho ld promise for use in plant
protection, with no adverse effects on th e e nvironmen t.
The purificat ion of azadirachtin is diffi cu lt to accompli sh due to the compl ex ity and similarity in structu re of
compounds found in th e seeds and folia ge of nee m tree .
Although azadirachtin is present in qu antiti es ran gin g
from 0 .3 to 0.6 per cent, its iso lat ion l .5 is tedious and
time consuming, involvin g partition s; co lumn c hromatography; preparative TLC; preparative HPLC; reversedphase column c hromatography, etc . Desp ite th ese meth-
* Present Address: Division of Agri cultural Chemica ls. Indian
Agricultu ral Research In stitute. Pusa, New Delhi 1100 12.
t Author fo r correspondence
ods, there is sti ll shortage of pure materi al ava ilabl e. The
best technique so fa r for purification of azadirac htin is
preparative HPLC(re f. 6). The present paper deal s with
enri c hment of azad irachti n us ing simp le la borato ry techniques lik e solvent ex traction and partiti o n.
Materials and Methods
Extraction and Enrichment ofAzadirachtin A ji'O/17 Neel1l
Seeds
A suspension o f 250 g of ground neem seed kerne l in
I I of hex ane was stirred at 40°C for 2 h and filte red .
The hex ane extract was concentrated in vacuo to g ive
106 g of neem o il. Th e de fatted ma rc was the n ex tracted
w ith I I methan o l in th e same manne r as n-hex ane extract ion. Th e ext ract was co nce ntrated u nd e r vacuum to
y ie ld 32 g (Tabl e I , I 25 g/k g seed) of da rk brown sem iso lid . The methanoli c extract was di sso lved in 100 ml
of 90 per cent aqueous methanol and part ition ed tw ice
with 50 ml hexa ne to remove re mainin g oil and o the r
non-po lar co mpoun ds. The aqueou s layer was th en dilu ted with water to make it I: I :: m ethan o l: water, saturated with sodium c hlori de and part iti o ned thri ce wi th
100 ml of the follow ing so lvent s:
(a)
E thy lace tate, dichloromethanc , ic hl oroethanc,
tolue ne, c hl orofo rm , carbontetrac hloriclc.
SINHA et al. : ENRICHMENT OF AZADIRACHTIN
99 1
Table I-Enrichment of crude methanoli c extract
Yield (g/kg)
Stage of extraction
Azadirachtin (per cent)
125-1 30
Melhanolic extract of defatt ed ma rc
1.5-2.2
15-20
10- 11
Extract aftcr passi ng ovcr full ers ean h
8-10
15-25
(lI a) Ethylacetate ex traction aft er rcmovi ng CCl.
5-6
25-3 5
(la) Ethylacetatc cx tractio n
(b)
solubles and after passing over full ers earth
Tabl e 2- Extracti on of methanoli c ex tract with different solvent s
Solvent
Yi eld , g/kg
Azadirac htin ,
per ce nt
Pure azt. , g/kg
Recovery,
per cent
I(a) Ethy lacetate
13.50
17. 12
2.33
77.9 8
(b) Chloroform
12.65
15.78
2.00
66.54
(c) Dichloroethane
10.3 8
18.06
1. 87
62.49
(d) CCl.
4.50
4.00
0. 18
6.00
(e) Toluene
3.50
4.00
0. 14
4.67
8.00
11 .00
0.88
29.33
II
Dichl oromethane
(b)
Carbontetrachloride partiti on in g follow ed by
e thy lacetate, dichlorome thane, dichloroeth a ne
toluen e and chloroform.
Each extraction was monitored by TLC. T he o rganic
layers were azeotroped with isopropanol and rotary
evaporated in vacuo and dissolved in minimum amount
of ethyl acetate and dropped in hexane unde r constant
stirring to yield a light yell ow to whitish coloured enriched dry amorphous powder. Yield and purity of the
fractions obtained by adopting the procedures (a) and
(b) are presented in Table 2 and 3, respective ly, and th e
scheme of isolation is depicted.
Thin Layer Chromatography
Normalphase a nalytical TLC was performed on 3 x
Scm s ilica gel (O.S mm thickness) coated g lass p lates
activated at 60°C for 12 h. The plates were d eve loped in
solvent system, as presented in Tabl e 4. The developed
plates were air dried , spra yed with a vanillin: sulphuri c
acid: ethanol (3 g: I.Sml : 100ml) reagent and heated to
100°C . The compo und s we re detected by th e characteristic co lou rs of the spots : azadirachtin (green), nimbin
fy iol et to black ) a nd sa lanin (blue). The R r va lues are
repcrted in Tabl e 4.
HPL C Analytical Conditions
Azadirachtin A co nte nt in enric hed powde r was dete rmined on HPLC, cons isting of Shimadzu Model LCSA chromatograph li nked to C-R7 Ae data processor.
The peak s were detected with Shimadzu SPD -IOAY
(UY) detector at 217 nm. The samples were reso lved o n
a 2Scm X 4.6mm ID , RP- IS, ODS column (S mp pa rticles) usi ng isocratic run of 60 per cent aqueous methanol at a f low rate of I mllmin . Using thi s syste m
azadirachtin A is found to have a retention time of 5 .S 6. 1 min , within approximately 300-600ng detection limit.
The pe rcentage of azadirachtin A in tes t sampl es was
estimated, us in g th e standard of 9S per cent purity proc ured from SPIC Science Foundation , Centre fo r
Agrochemicals Researc h, Chennai , by adopting exte rnal standa rd method .
Estimation ofA zadirachtin in Neem Seeds and Enrich ed
Powders
Neem seeds from different agro-climatic zones we re
collected a nd decorticated . The kemel were g round a nd
ex tracted directl y with methan ol and filte red to get a
transparent so luti o n free from suspe nd ed particles. Thi s
solution was anal ysed on HPLC fo r azadi rachtin a nd the
J SCI IND RES VOL.58 DECEMBER 1999
992
Table 3- Extraction in different solvents after CCI. parti tion and full ers earth
treatment
Yield, glkg
Solvent
"
III
Azadirac htin , pe r cent
II
Mean
Pure azt,
g/kg
III
Mean
Recovery,
percent
Ethylacetate
6.8
5.8
5.2
5.9
30.3
35.5
40.7
35.5
2. 1
70.2
Chl oro form
4.0
5.0
5.8
4.9
24.7
33.0
41.2
33.0
1.6
54. 2
Dichl oroeth ane
2.9
3.2
3.0
3.0
45 .0
52.3
48.7
1.5
49.2
Toluene
3.2
2.0
3.0
2.7
52.0
62.2
52. 0
1.4
47.3
Dic hl oro meth ane
3.5
5.4
4 .0
4 .3
3 1.2
23.8
27. 5
1. 2
39.4
Table 4-T LC -
41.7
Crude and enri ched ex tracts of neem
Spots
Crude ex tracts
R, valu e
CHCI) : Acetone (8 :2)
2
0.2 1, 0.36 All gree n
0.36
Ether
3
0.22, 0.34, 0.48 All gree n
0.48 gree n
Eth ylacetate
3
0.42, 0.58 , 0.7 1 All gree n
0.7 1 green
C HCI ) : C H)CN (4: I)
3/2
(0.22, 0.25)* ,0.35 All gree n
0.35 green
Solvent syste m
results are presented in Tab le 5 .
The e nriched powders obta ined in differe nt e xtracti ons were ana lysed o n HPLC fo ll o wed by a simp le clean
up procedure . E nriched powde r with azadirac ht in content of approximate ly I mg was accurate ly weig hed and
di ssolved in 1Oml of HPLC grade metha no l and th e data
o bta ined are presented in Tab les ! -3.
Results and Discussion
T he a mount of azadirac htin in neem seed kerne ls is
influe nced by many factors like humidity, c limate, expos ure to sunli g ht , sto rage, seed dryin g, p H and the
method of ex traction7 . M eth ano l ex tracti on of neem seeds
·is the most e ffi c ie nt way to recover azad irac htin from
seed x. Methan o l ex traction of neem seeds fro m differe nt
loca ti o n s in So uth Ind ia s h owe d a va ri a ti o n in
azadirachtin content between 0. 2 to 0.65 per cent (Table 5) , max imum busy in Vedasandur sample.
Iso latio n of azad irac htin , described in ea rli e r l 5 , involves adsorpti on c hromatograph y and tedi ous preparat i ve HFLC. Th e me th od ad o pted by S c hroede r a nd
Nakani shi) for e nri ch ment of azadirac htin in vo lves partitio n be tween 90 per cent aqueous meth ano l : hexane
Enriched azad iracht in
R, value
green
Tab le 5- Azad irac htin va ri ati on in neem seed
Source of seed
Azadirac htin (per cent)
II
II I
Vedasa nclur
0.45
0. 38
0.65
Kandukur
0.20
0. 14
0. 18
Samalkota
0.25
0.28
0.23
Hyderabad
0. 30
0.26
and e th y lacetate : wate r, fo ll owed by sil ica ge l filtrati on, vac uum liqu id c hro m atog rap hy w ith eth y lace tate :
hexane (3: I), fl ash c hro matog ra ph y and subsequ e ntly
c rysta ll isat ion, to o bta in pure azadirac hti n. The present
meth od in vo lves so lvent e xtrac tio ns fo ll owed by puri ficati o n ste ps to enric h azadirac ht in by so lvent -so lvent
partiti o ns. It is an easy me thod to iso late azadirachtin o f
hi g h purity fro m neem seeds. M ethano l/a lco ho l extracti o n of the defatted marc affo rded a conce ntrate of 125 g/
kg (approx) cont aining 2.2 pe r ce nt of azad irac htin (Table I) acco mpani ed by polar co mpound s Iike sugars and
prote ins a lo ng w ith th e useful te rpe no id s. D e fa tted
e th y lacetate e nri ched ex trac t is semi -~o lid stic ky p1a~s
SINHA et at. : ENRICHMENT OF AZADIRACHTIN
993
Nee] '~ kem,1 powd" (250g)
hexane lL
hexane extract concentrated to get neem oil (106g)
Residue (140g)
methanol lL
1------+
Residue (discard)
Methanolic extract (concentrated & dissolved in 100 ml of 90 per cent aq.
MeOH & partitioned)
hexane (SOml X 2)
1--_ _ _ _ _ _ _ _
hexane layer (Sg oil)
Aqueous methanolic layer (diluted to 1:1 ::MeOH:water)
Carbontetrachloride (100ml X2)
Carbontetrachloride layer (concentrated &
dropped in hexane, 1.1Sg)
Aqueous layer (saturated with NaCl, 20g)
ethylacetate I chloroform I dichloroethane I
dichloromethane I toluene (100m I X 3)
1 - : - - - - - - -....
Aqueous layer (discard)
Organic layer (dried over Na,SO" concentrated & dropped in
hexane)
Scheme of Isolation
whic h contain s approx imate ly I 0 pe r cent azadirachtin
(Table I ,1a) . It can be used in preparin g azadirac htin based
formul ation s up to 10,000 ppm . The po lar co mpoun ds
like sugars and prote ins were e liminated in aqueous phase
when partiti oned with organi c so lvent. T he organi c phase
was d ri ed over a nh yd ro us sodium sul p hate, filt e red
throug h full ers ea rth and d ropped in hexane to get a dry
powder enric hed to 15-20 pe r cent azadirachtin co nte nt
(Tabl e I ,Ib). O n an ave rage a threefo ld enri c hme nt of
azad irachtin (35 per cent) in crude meth ano lic ex tracts
was achi eved by the inte rcepti on of carbo ntetrachl o ri de
ex trac tion pri o r to enrichment with eth ylacetate (Table
I ). T he enric hment of azadirachtin in c rude methano li c
ex tracts with di fferent so lvents afforded max imum recovery of azadirachtin in ethylacetate (7 7 .98 per cent)
al0 lowest in toluene (4 .67 per cent, Table 2). Thu s, to lu ene Cmnot be used directly for e xtraction of azadirachtin
fro m crude meth ano lic ex tract but o nl y afte r th e re ma in in g o il , sugars and prote in s are re moved fro m thi s extract, whi c h can be acco mpli shed thro ug h procedure (b) ,
as described in Materials and Methods . It in vo lves
carbonte trachl oride ex tracti o n of the 50 per cent aqu.
rn e th a no li c laye r w hi c h le ad s to th e e lim inati o n of
nimbin , sa lani n, fa tty ac ids and oth e r co nco mi tant s ubstances bes ides a littl e azad irac htin (4-6 per cent), thu s
e nri c hing azadirac htin in th e aq ueous phase . T he aqueous phase has to be saturated w ith sod ium ch lo ride to
avo id formation of e mul sio n and th e n extracted wi th
e th y lace t a te / c hl o r ofo r m / d ic hl o roe than e /
d ic hl orometh ane / to lue ne. To lue ne el imin ated maj o rity of the mo re po lar and co loured co m poun ds and a 40
- 50 pe r cent pure azadirachtin -A ri c h com pound was
obta ined w ith 47 .3 per cent recovery (Tab le 3). In the
crude extract, most of the trite rpeno id s we re ide ntified
994
J SCI INO RES VOL.58 DECEMB ER 1999
an d w hil e e nric hing to 10 per cent azadirac htin-A the
peaks e lutin g be tween 0-10 min were m a rg in ally eliminated. H o w e ve r, qu a lit a ti ve d iffe re nces in th e fin a l
fractionat io n of crude e xt rac ts ( in terce pt ed b y
carbontetrachloride pmtiti oni ng) with eth ylacetate I c hl orofo rm I dichl oroeth ane I dic hl oro metha ne I to lue ne were
not obse rved mu c h but q uan tit at ive d iffe re nces we re
perceptib le. Thu s, depe nd in g o n th e purity required, the
solvents for f in a l extractions coul d be se lected . Recovery of 60 per ce nt of the so lvents used for ex tracti o ns
and part it io ns was recorded .
Th e crude me th ano lic ext rac t w hen masce rated w ith
d ic h lorome th a ne, f iltered a nd concen t rat e d und e r
vacuum , y ie lded a da rk brown semi -so li d wit h II per
cen t azad irac htin (Table 2, II). Thi s is a direct me th od to
iso late e nric hed azadirachtin fro m crude extracts fo r preparing neem fo rm u la ti o ns but beca use of the sti cky nature of the ex trac t, f ilterat io n was c um bersome a nd a lso
the recovery was quite low (29 .33 per cent ).
F ulle r 's earth f iltrati o n e li m inates major ity o f th e co lo ured com pound s a lo ng w ith a little azadirac htin , he nce
the recoveri es a re a ffec ted . Thu s, thi s step fin ds utility
w he n there is need to iso late pure azadirac hti n 'A' using enric hed powde r o n pre pa rati ve HPLC o r to pre pa re
fo rm u lat io n s with hi g h azadirachtin (5, I 0 per cent) concentra ti o n . Gov indc ha ri et al.(' ac hi eved 99 per cent purifi catio n of azadirachtin- A thro ug h pre pa rati ve HPLC
using c rude ex tracts, w hil e the present procedure affo rds
e nri c hment up to 50 per cent by s impl e so lvent pa rt iti o ns. T he iso lati o n of pure azadirachtin is m uc h m o re
eas ie r a nd eco no mi ca l us in g e nri c hed 50 pe r ce nt materi a l in w hi ch eac h run ta kes o nl y 30 min as aga in st 90100 min w ith defatted c rude ex tracts. Th is avo id s con-
ta min a ti o n of the co lumn , th e re by e nh a nc in g th e li fe
w hil e m ainta inin g th e effi c ie ncy.
H owe ve r, va ri ations i n th e y ie ld a n d purit y of
azadirachtin in th e e nric hed powder are probab ly due to
the geographi ca l location of the p lant, time o f ha rves t,
and durati on of seed storage. It is ev ident that th e y ie ld
a nd purity depend s upon the amo u nt o f azad irachtin
present in the seed and th e re fo re on the sou rce of the
seed .
Acknowledgements
The autho rs a re g ratefu l to ]ndian Counci l Agricultura l R esea rc h, New De lhi for financial ass is tance .
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