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Extraction of Piperine from Piper nigrum (Black

Pepper) by Hydrotropic Solubilization
Article in Industrial & Engineering Chemistry Research May 2002
DOI: 10.1021/ie0107845

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2966

In d . E n g. Ch em . R es. 2002, 41, 2966-2976

Ex tra c tio n o f P ip e rin e fro m P i p er n i g r u m (B la c k P e p p e r) by

Hy d ro tro p ic S o lu biliza tio n
Girija Ra m a n a n d Vila s G. Ga ik a r*
M u m bai Un iversity In stitu te of Ch em ical T ech n ology, Un iversity of M u m bai, M atu n ga,
M u m bai 400019, In d ia

H ydr ot r opes, su ch a s sodiu m a lkyl ben zen e su lfon a t es a n d sodiu m bu t yl m on oglycol su lfa t e,
wer e u sed for t h e select ive ext r a ct ion of piper in e by cell per m ea biliza t ion of Piper n igru m fr u it s.
P en et r a t ion of t h e h ydr ot r ope m olecu les in t o t h e cellu la r st r u ct u r es a n d su bsequ en t cell
per m ea biliza t ion wer e h ypot h esized t o expla in t h e en h a n ced ext r a ct ion r a t es of a qu eou s
h ydr ot r ope solu t ion s. H ydr ot r ope m olecu les, a ft er a dsor pt ion on a cell wa ll, ca u se disor der in
it s st r u ct u r e a n d in t h e bila yer ed cell m em br a n e t o fa cilit a t e t h e r a pid ext r a ct ion of piper in e.
Th e h ydr ot r ope solu t ion sh owed select ive a n d r a pid ext r a ct ion of piper in e fr om bla ck pepper .
Th e r ecover ed piper in e wa s 90% pu r e a n d su bst a n t ia lly fr ee fr om oleor esin s. Th e t ype a n d
n a t u r e of t h e h ydr ot r ope, t h e h ydr ot r ope con cen t r a t ion , t h e t em per a t u r e, a n d t h e pa r t icle size
a ll h a d sign ifica n t effect s on t h e ext r a ct ion pr ocess.
In tro d u c tio n
Th e in cr ea sed in t er est in pla n t -der ived dr u gs in
r ecen t yea r s is beca u se of t h eir u n dispu t ed effica cy a s
ph yt om edicin es a n d beca u se a ct ive pr in ciples fr om
n a t u r a l pr odu ct s ser ve eit h er a s t em pla t es or a s in t er media tes for synthetic drugs.1 Despite the sophistication
of m oder n or ga n ic syn t h esis, it is n ot a lwa ys econ om ica lly fea sible t o syn t h esize dr u gs t h a t a r e sim ila r t o
t h ese a ct ive in gr edien t s. Accor din gly, m ost pla n t dr u gs
a r e cu lt iva t ed a n d a r e u sed clin ica lly a s sem ipu r ified
or pu r ified ext r a ct s. Th e ext r a ct ion a n d pu r ifica t ion
st eps ca n con st it u t e 50-90% of t h e fin a l pr odu ct cost
in su ch ca ses.
P iper in e (st r u ct u r e 1), wh ich is a m a jor a lka loid in
bla ck pepper ,2 exh ibit s a pot en t ch em o-pr ot ect ive effect
a ga in st pr oca r cin ogen s a n d a lso ba ct er iost a t ic, fu n gist a t ic, a n d in sect icida l a ct ivit ies.3 P iper in e pr ovides
pr ot ect ion a ga in st seizu r es in epilepsy a n d h a s been
ga in in g in cr ea sin g a t t en t ion a s a bioa va ila bilit y en h a n cer in t h e for m u la t ion s of sever a l dr u gs. 4,5 P iper in e,
beca u se of it s pr ot ect ive effect a ga in st r a dia t ion , ca n
a lso be a dm in ist er ed t o ca n cer pa t ien t s befor e r a diot h er a py.6 Th ese a pplica t ion s su ggest a n eed for pu r e
piper in e t h a t is fr ee fr om r esidu a l solven t s t o en a ble
it s dir ect u se in m edicin a l for m u la t ion s.

On a n in du st r ia l sca le, pepper is com m in u t ed in t o

fla kes or gr ou n d in t o coa r se powder a n d t h en ext r a ct ed
* To wh om cor r espon den ce sh ou ld be a ddr essed. F a x: 9122-4145414. E-ma il: vgg@udct.ernet.in, vgg_udct@ya hoo.co.in.

r epea t edly wit h a n or ga n ic solven t su ch a s a cet on e,

et h a n ol, or ch lor in a t ed h ydr oca r bon s. 7 Repea t ed solven t
ext r a ct ion of r a w pepper pa r t icles for lon g du r a t ion s
r esu lt s in t h e ext r a ct ion of ot h er com pon en t s, su ch a s
polysa cch a r ides, gu m s, a n d n on -fla vor su bst a n ces. As
a r esu lt , t h e solven t ext r a ct ion pr ocesses u su a lly give
com plex cr u de pr odu ct s. Th e cr u de ext r a ct h a s t o t h en
be pu r ified by m u lt ist ep t ech n iqu es su ch a s ch r om a t ogr a ph y or cr yst a lliza t ion . Apa r t fr om t h e poor ext r a ct
qu a lit y, difficu lt ies in h a n dlin g la r ge volu m es of in fla m m a ble vola t ile or ga n ic solven t s a n d r esidu a l solven t
t r a ces r em a in in g in t h e fin a l pr odu ct lim it t h e u se of
or ga n ic solven t s for pepper ext r a ct ion .
Su per cr it ica l flu id ext r a ct ion u sin g ca r bon dioxide is
a n ot h er opt ion for t h e ext r a ct ion of piper in e. 8,9 Th e cost
of t h e h igh -pr essu r e equ ipm en t n eeded t o obt a in su per cr it ica l ext r a ct ion con dit ion s, h owever , becom es pr oh ibit ively h igh a n d lim it s t h e a pplica t ion of su per cr it ica l
ext r a ct ion t o on ly h igh -va lu e a n d low-volu m e m a t er ia ls.
H igh -pr essu r e st ea m t r ea t m en t ca n a lso en h a n ce ext r a ct ion r a t es by a n osm ot ic sh ock; h owever , t h is
t ech n iqu e is r ela t ively slow a n d con su m es a la r ge
a m ou n t of st ea m .10 Ult r a sou n d t r ea t m en t h a s been
cla im ed t o in cr ea se t h e yield a n d m a ss-t r a n sfer r a t e in
sever a l solid-liqu id ext r a ct ion pr ocesses.11 Th e t r ea t m en t r u pt u r es t h e cell wa lls t h r ou gh st r on g dyn a m ic
st r essin g, wh ich r esu lt s fr om t h e spon t a n eou s for m a t ion
of bu bbles in a liqu id below it s boilin g poin t a n d t h e
colla pse of t h ese bu bbles wit h in a ver y sh or t t im e. Th e
effect of u lt r a sou n d is, h owever , loca lized, a n d it s
a pplica t ion t o a la r ge volu m e of r a w m a t er ia l m igh t be
en er get ica lly in efficien t .
In t h is r epor t , we pr esen t a h igh ly efficien t pr ocess
ba sed on t h e ph en om en on of h ydr ot r opy for t h e select ive
ext r a ct ion of wa t er -in solu ble ph yt och em ica ls fr om com plex n a t u r a l pr odu ct s. H ydr ot r opy r efer s t o t h e a bilit y
of h igh ly wa t er -solu ble bu t m ildly su r fa ce-a ct ive a m ph iph ilic or ga n ic sa lt s ca lled h ydr ot r opes 12 (st r u ct u r e
2) to increase the solubility of sparingly soluble or waterin solu ble or ga n ic com pou n ds in a qu eou s solu t ion s.

10.1021/ie0107845 CCC: $22.00 2002 Am er ica n Ch em ica l Societ y

P u blish ed on Web 05/18/2002

In d. E n g. Ch em . Res., Vol. 41, No. 12, 2002

H ydr ot r opy is a collect ive m olecu la r ph en om en on

similar to micellar solubilization but with a much higher
ca pa cit y.13 It is a con sequ en ce of t h e t en den cy of
a m ph iph ilic h ydr ot r ope m olecu les t o a ggr ega t e a m on g
t h em selves a n d pr oba bly wit h ot h er h ydr oph obic m olecu les.14 Th ese a ggr ega t es a r e su pposedly m u ch sm a ller
t h a n su r fa ct a n t m icelles a n d fa r less cooper a t ive. An ot h er dist in gu ish in g fea t u r e of h ydr ot r opes, u n like
su r fa ct a n t s, is t h eir a bilit y t o differ en t ia t e a m on g
differ en t or ga n ic con st it u en t s of a m ixt u r e, even closely
r ela t ed su bst a n ces.12 It is t h is a bilit y of m olecu la r
r ecogn it ion t h a t sh ou ld be u sefu l for t h e pr efer en t ia l
ext r a ct ion of a com pou n d fr om n a t u r a lly occu r r in g r a w
materials. The high solubilization capacity of hydrotrope
solu t ion s sh ou ld lea d t o h igh ext r a ct ion ca pa cit ies for
ot h er wise in solu ble or ga n ic-a ct ive elem en t s.
H ydr ot r opes dem on st r a t e a r em a r ka ble pr oper t y of
disr u pt in g t h e la m ella r cr yst a llin e st r u ct u r e of su r fa ct a n t s in a qu eou s solu t ion s, pr odu cin g a con t in u ou s
isot r opic liqu id solu bilit y r egion .13 In su r fa ct a n t solu t ion s, t h e pr esen ce of su ch a la m ella r st r u ct u r e a t h igh
con cen t r a t ion s lim it s t h e solu bilit y of h ydr oph obic com pou n ds. Th ese st r u ct u r es a r e a n a logou s t o t h e ph osph olipid bila yer s of cell m em br a n es. We dem on st r a t e
here t he a bilit y of h ydr ot r opic solu t ion s, in a n a n a logou s
m a n n er , t o disr u pt pla n t cell st r u ct u r es a n d a id in t h e
ext r a ct ion of h ydr oph obic con st it u en t s fr om t h e com plex
biom a t r ix. Th e h ydr ot r opic effect is sign ifica n t a bove a
m in im u m h ydr ot r ope con cen t r a t ion (MH C) t h a t is a
ch a r a ct er ist ic of a given h ydr ot r ope, a n a logou s t o t h e
cr it ica l m icelle con cen t r a t ion (CMC) of a su r fa ct a n t .
H owever , beca u se h ydr ot r opes h a ve r ela t ively sh or t
h ydr oca r bon ch a in s or h ydr oph obic gr ou ps, t h eir MH Cs
a r e u su a lly in t h e m ola r r a n ge. Th e solu bilit y of a n
or ga n ic com pou n d in a h ydr ot r ope solu t ion r ises a lm ost
expon en t ia lly im m edia t ely a bove t h e MH C, bu t a t
h igh er con cen t r a t ion s of t h e h ydr ot r ope, it m igh t level
off t o a pla t ea u depen din g on t h e n a t u r e of t h e solu t e.
Dilu t ion of t h e sa t u r a t ed solu t ion wit h wa t er is u su a lly
su fficien t t o r ecover t h e dissolved solu t e, wh ich sepa r a t es ou t a s a n ot h er solid or liqu id ph a se wh en t h e
h ydr ot r ope con cen t r a t ion dr ops below it s MH C.
In t h is pa per , we explor e t h e ext r a ct ion of piper in e
fr om Piper n igru m u sin g h ydr ot r opic solu biliza t ion . Th e
object ive of t h e pr esen t wor k wa s t o develop a n efficien t
pr ocess for t h e ext r a ct ion of a ct ive in gr edien t s fr om
n a t u r a l pr odu ct s a n d t o u n der st a n d t h e r ole t h a t ea ch

2967

pa r a m et er pla ys in t h e ext r a ct ion . Disr u pt ion of t h e

cellu losic cell wa ll of t h e biom a t r ix a n d su bsequ en t
disor ga n iza t ion of t h e ph osph olipid bila yer s by t h e
h ydr ot r ope m olecu les, followed by dissolu t ion of t h e
cellu la r con t en t s, seem t o be t h e key st eps in t h is
ext r a ct ion pr ocess. A h igh ext r a ct ion efficien cy, la r ge
ca pa cit y of h ydr ot r ope solu t ion s, a n d h igh select ivit y
t owa r d piper in e a r e m a jor obser va t ion s.
We believe t h a t h ydr ot r opic ext r a ct ion ca n pr ovide a
com pet it ive a lt er n a t ive t o su per cr it ica l flu id ext r a ct ion .
Th e sca le of oper a t ion a n d t h e ea se of sca le-u p of
h ydr ot r opic ext r a ct ion a r e a t levels u n im a gin a ble for
su per cr it ica l flu id ext r a ct ion . Wh a t is a ch ieved wit h
pr essu r e in t h e su per cr it ica l flu id ext r a ct ion ca n be
a ch ieved wit h ea se u sin g t h e h ydr ot r ope con cen t r a t ion
in a qu eou s solu t ion s. Beca u se t h e solu bilit y en h a n cement is insignificant at lower hydrotrope concentrations,
sim ple dilu t ion by wa t er pr ovides a n ea sy r ecover y
m et h od, ju st a s does t h e r elea se of pr essu r e in su per cr it ica l flu id ext r a ct ion . Beca u se h ydr ot r opes a r e h igh ly
wa t er -solu ble sa lt s, con t a m in a t ion of t h e pr odu ct by
h ydr ot r ope m olecu les is m in im a l a n d ca n be r edu ced t o
below a n a ccept a ble level, if a n y, sim ply by wa sh in g
wit h wa t er . We a lso believe t h a t t h is m et h od sh ou ld
wor k wit h sim ila r efficien cies for ot h er n a t u r a l pr odu ct s
a lso. In a ddit ion , it wou ld be in t er est in g t o u n der st a n d
h ow h ydr ot r opes differ fr om su r fa ct a n t s in t h eir a ct ion
on cellu la r st r u ct u r es.
Ex p e rim e n ta l S e c tio n
Ma te ria ls a n d Ex p e rim e n ta l Me th o d s . Th e a r om a t ic su lfon a t e h ydr ot r opes sodiu m xylen e su lfon a t e
(Na XS; m ixed isom er s wit h a n et h yl ben zen e su lfon a t e
con t en t of a bou t 6%, a s qu ot ed by t h e m a n u fa ct u r er ),
sodiu m cu m en e su lfon a t e (Na CS), a n d sodiu m p-t olu en e
su lfon a t e (Na P TS), wer e pu r ch a sed fr om Na vdeep
Ch em ica ls Lt d., Mu m ba i, In dia , a n d wer e r ecr yst a llized
wit h m et h a n ol befor e t h e ext r a ct ion st u dies. n -Bu t yl
ben zen e wa s obt a in ed fr om H er dillia Ch em ica ls Lt d.,
Mu m ba i, In dia . It wa s su lfon a t ed wit h con cen t r a t ed
su lfu r ic a cid (98%) a n d t h en n eu t r a lized wit h sodiu m
h ydr oxide t o give sodiu m n -bu t yl ben zen e su lfon a t e.14
Sodiu m bu t yl m on oglycol su lfa t e (Na BMGS) wa s obt a in ed a s a 50% (w/v) solu t ion (fr om H u ls, Du sseldor f,
Ger m a n y). Wh ole pepper ber r ies wer e obt a in ed fr om
M/s. Ca n cor In dia Lt d., Coch in , In dia . Dich lor om et h a n e
(DCM) a n d m et h a n ol (H P LC-gr a de) wer e u sed a s solven t s for h igh -per for m a n ce liqu id ch r om a t ogr a ph y
(H P LC) a n a lysis. Cet yl t r im et h yla m m on iu m br om ide
(CTAB) a n d sodiu m la u r yl su lfa t e (SLS), obt a in ed fr om
Spect r och em , Mu m ba i, In dia , h a d a m a n u fa ct u r er s
st a t ed pu r it y of 99% a n d wer e u sed a s r eceived.
Con t in u ou s Soxh let ext r a ct ion wit h pet r oleu m et h er
wa s in it ia lly ca r r ied ou t for 48 h t o det er m in e t h e
piper in e con t en t of t h e r a w m a t er ia l, wh ich wa s 4.0%
(w/w).
Wh ole pepper fr u it s wer e fir st pu lver ized t o a coa r se
powder a n d t h en sepa r a t ed in t o ba t ch es of differ en t
sizes u sin g m ech a n ica l sieves. P a r t icles wit h a n a ver a ge
size 50 m wer e u sed for t h e ext r a ct ion st u dies u n less
st a t ed ot h er wise. Th e ext r a ct ion exper im en t s wer e
carried out in a fully baffled borosilicate cylindrical glass
vessel (9-cm h eigh t , 7.0-cm i.d.) equ ipped wit h a sixbla ded t u r bin e im peller (i.d. 2 cm ). Th is en t ir e a ssem bly
wa s kept in a con st a n t -t em per a t u r e ba t h du r in g exper im en t a t ion . A 10-g sa m ple of gr ou n d pepper wa s a dded
t o 0.1 dm 3 of h ydr ot r ope solu t ion of a kn own con cen t r a -

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In d. E n g. Ch em . Res., Vol. 41, No. 12, 2002

t ion in t h e r a n ge 0.05-3.4 m ol/dm 3 in t h e gla ss vessel.

Th e su spen sion wa s a git a t ed vigor ou sly a t 1100 r pm for
a per iod of 2 h a t 30 C. Th e solu t ion wa s t h en a llowed
t o set t le for a n ot h er h ou r a n d wa s su bsequ en t ly filt er ed
u n der va cu u m wit h in 10 m in . A clea r br own -color ed
liqu id wa s obt a in ed a s t h e filt r a t e. Th e solid r esidu e,
wh ich con sist ed m ost ly of t h e st a r ch con t en t of pepper ,
wa s soft a n d pu lpy bu t did n ot h in der t h e filt r a t ion
pr ocess. Th e ca ke wa s fu r t h er wa sh ed wit h h ydr ot r ope
solu t ion (0.01 dm 3 ) of t h e sa m e con cen t r a t ion a s in t h e
ext r a ct ion st a ge t o r em ove ext r a ct r esidu e a dh er in g t o
it , if a n y. Th e wa sh solu t ion wa s a dded t o t h e fin a l
ext r a ct .
Th e filt r a t e wa s t h en dilu t ed wit h wa t er a t 30 C t o
br in g t h e h ydr ot r ope con cen t r a t ion below it s MH C.
P iper in e pr ecipit a t ed fr om t h e solu t ion a s fin e cr yst a ls
over a per iod of 1 h . Th e su spen sion wa s t h en cen t r ifu ged a t 2822g for 15 m in t o sepa r a t e t h e solid pr odu ct
fr om t h e r em a in in g solu t ion . Th e pr ecipit a t e wa s dr ied
a n d a n a lyzed for pu r it y u sin g H P LC wit h a 5-m
Novopa k C-18 colu m n . Th e colu m n wa s in it ia lly r in sed
wit h m et h a n ol a n d DCM a n d t h en equ ilibr a t ed wit h t h e
elu t in g solven t (DCM/MeOH 100:4).15 Th e colu m n wa s
m ou n t ed on a Tosh o H P LC ch r om a t ogr a ph equ ipped
wit h a 20-L loop in ject or . Th e m obile ph a se flow r a t e
wa s 0.6 m L/m in , a n d t h e det ect ion wa velen gt h wa s 343
n m . Th e a n a lysis wa s isocr a t ic a n d wa s ca r r ied ou t for
15 m in .
Extraction of piperine from pepper was also conducted
separately with aqueous solutions of CTAB and SLS (0.5
m ol/dm 3 con cen t r a t ion ) in a n iden t ica l m a n n er for
ver ifica t ion of t h e ext r a ct ion a bilit y m ea su r em en t s a n d
for com pa r ison .
Solubiliza t ion experimen ts were sepa ra tely con du ct ed
by su spen din g pu r e piper in e in a qu eou s h ydr ot r ope
solu t ion s a t differ en t con cen t r a t ion s r a n gin g fr om 0.2
t o 2.0 m ol/dm 3 . Th e solu t ion wa s equ ilibr a t ed wit h
excess solid piper in e for sever a l h ou r s a t a con st a n t
t em per a t u r e of 303 K u sin g a m a gn et ic st ir r er . Th e
su spen sion wa s t h en cen t r ifu ged, a n d t h e cen t r ifu ga t e
wa s ext r a ct ed in t o DCM for a n a lysis. Th e con cen t r a t ion
of dissolved piper in e in t h e solu t ion wa s est im a t ed
spect r oph ot om et r ica lly a t 343 n m u sin g a H it a ch i UVvisible spect r oph ot om et er .
In a sepa r a t e exper im en t , a n a qu eou s h ydr ot r ope
ext r a ct wa s ca r efu lly weigh ed a n d dr ied. Th e t ot a l
in or ga n ic ph osph or u s con t en t wa s t h en est im a t ed by a
m odified F iske-Su bba r a o m et h od 16 u sin g sodiu m m olybda t e a n d h ydr a zin e su lfa t e a t 650 n m . A sa m ple of
feed h ydr ot r ope solu t ion of t h e sa m e con cen t r a t ion wa s
ta ken a nd subjected to the sa me procedure for reference.
To det er m in e wh et h er r edu cin g su ga r s wer e pr esen t ,
t h e h ydr ot r ope ext r a ct wa s t r ea t ed wit h F eh lin gs solu t ion .17 Th e a m in o a cid con t en t of t h e h ydr ot r ope ext r a ct
solu t ion wa s est im a t ed qu a lit a t ively u sin g Nin h ydr in
r ea gen t .18 Th e h ydr ot r ope ext r a ct (2 cm 3 ) wa s ext r a ct ed
with buta nol, a nd this buta nol extra ct wa s used for thinla yer ch r om a t ogr a ph y (TLC). Th e TLC pla t e wa s developed in a bu t a n ol/a cet ic a cid/wa t er (4:1:1) m ixt u r e.
Th e TLC pla t e wa s spr a yed wit h Nin h ydr in r ea gen t t o
det er m in e t h e pr esen ce of a m in o a cids.
Re s u lts a n d D is c u s s io n
F igu r e 1 sh ows r epr esen t a t ive ch r om a t ogr a m s of
st a n da r d pu r e piper in e, piper in e ext r a ct ed h ydr ot r opica lly, a n d piper in e ext r a ct ed wit h pet r oleu m et h er .
H ydr ot r opica lly ext r a ct ed piper in e is r ela t ively m or e

F ig u re 1. Com pa r it ive H P LC ch r om a t ogr a m s of ext r a ct ed piper in e (m obile ph a se ) DCM/MeOH (100:4), flow r a t e ) 0.6 m L/
m in , det ect ion wa velen gt h ) 343 n m ): (a ) pu r e piper in e, (b)
h ydr ot r opica lly ext r a ct ed piper in e, (c) Soxh let -ext r a ct ed piper in e

pu r e t h a n pet r oleu m et h er ext r a ct ed piper in e wh er e

ot h er oleor esin s a r e pr esen t in a la r ge per cen t a ge. Th e
select ive ext r a ct ion of piper in e in t o a qu eou s h ydr ot r ope
solu t ion s pr om pt ed fu r t h er st u dies of ext r a ct ion u sin g
differ en t h ydr ot r opes a n d differ en t oper a t in g pa r a m et er s. Th e ext r a ct ion of piper in e sh ou ld be t h e r esu lt of
ch a n ges in t h e st r u ct u r e of t h e pepper cells t h a t occu r
a s a r esu lt of t h e pr esen ce of h ydr ot r ope in t h e solu t ion .
Bot h su r fa ct a n t s a n d or ga n ic solven t s a r e kn own t o
ca u se cell per m ea biliza t ion , a lt h ou gh t h e exa ct sit e a n d
m ech a n ism of t h eir a ct ion a r e a poin t of deba t e in m a n y
ca ses.19 Or ga n ic solven t s, su ch a s t olu en e, ca u se con sider a ble da m a ge t o t h e cyt opla sm ic m em br a n e, wh ile
t h e ou t er m em br a n e r em a in s r ela t ively in t a ct . 19 Solven t s su ch a s DMSO pr om ot e t h e loss of ph osph olipids
in the cell membrane, whereas cationic surfactants, such
a s CTAB, a ct on t h e ph osph olipid la yer t h r ou gh t h eir
h ydr oph obic ch a in s a n d a r e kn own t o disr u pt it com plet ely.19-20
To dissolve piper in e, a h ydr ot r ope solu t ion h a s t o
pen et r a t e a n d dest a bilize t h e cell st r u ct u r e. Sect ion s of
whole Piper nigrum fruits were microscopically observed
befor e a n d a ft er t r ea t m en t wit h h ydr ot r ope solu t ion s
for 30 m in . Th e im a ges wer e a n a lyzed wit h a P r oP lu s
Im a ge An a lyzer .
In t h e ca se of sa m ples soa ked wit h Na BMGS solu t ion ,
t h e cells wer e ver y t u r gid a n d swollen , bu t t h eir sh a pe
wa s con sider a bly in t a ct (pa n els a a n d b of F igu r e 2).
Th is su ggest s a n in cr ea sed per m ea bilit y of t h e cell wa ll,
a s piper in e wa s ext r a ct ed fr om t h ese cells wit h ou t t h eir

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2969

F ig u re 2. (a ) In t a ct sect ion of P. n igru m . (b) Sect ion of P. n igru m a ft er soa kin g in BMGS solu t ion . (c) In t a ct sect ion of P. n igru m . (d)
Sect ion of P. n igru m a ft er soa kin g in Na NBBS solu t ion . (e) In t a ct sect ion of P. n igru m . (f) Sect ion of P. n igru m a ft er soa kin g in Na CS
solu t ion . (g) In t a ct sect ion of P. n igru m . (h ) Sect ion of P. n igru m a ft er soa kin g in Na P TSA solu t ion . (i) In t a ct sect ion of P. n igru m . (j)
Sect ion of P. n igru m a ft er soa kin g in Na XS solu t ion .

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com plet e r u pt u r e. Th is sh ou ld a ccou n t for t h e ver y

select ive ext r a ct ion bu t low yields of piper in e obt a in ed
wit h Na BMGS solu t ion s, a s descr ibed below. In t h e
sect ion s soa ked in Na NBBS solu t ion s (pa n els c a n d d
of F igu r e 2), t h r ee la yer s of t h e per ica r p a ppea r ed
cla r ified. Th e ou t er per ica r p h a d a fibr ou s a ppea r a n ce,
in dica t in g slow bu t defin it e degr a da t ion of t h e la yer . At
the edges, the testa, containing a reddish brown pigment
a n d t h e cellu lose la yer , seem ed t o h a ve for m ed a n
isot r opic ph a se, wit h t h e la yer s in t er m ixin g wit h ea ch
ot h er . E ven t h e h ya lin e la yer of t h in wa lled cells wa s
en la r ged. Beca u se t h e per ica r p cells a r e pa cked wit h
piper in e cr yst a ls, a h igh ext r a ct ion efficien cy a n d select ive ext r a ct ion wer e obt a in ed wh en Na NBBS wa s u sed
a s t h e h ydr ot r ope, a s descr ibed below. In t h e ca se of
NaCS (panels e and f of Figure 2), substantial disruption
of t h e cellu la r st r u ct u r e a n d disor der in g of t h e per ica r p
occu r r ed. Cellu la r debr is wa s visible, a lon g wit h h igh ly
swollen cells. Beca u se t h e in n er pa r t of t h e per ica r p a lso
seem ed t o h a ve been r u pt u r ed, ot h er oleor esin s wer e
a lso ext r a ct ed fr om t h e disr u pt ed oil cells. Na P TSsoa ked sect ion s sh owed a n in cr ea sed r u pt u r e of t h e
pa r en ch ym a t ou s cells of t h e m esoca r p a n d t h e br own
pigm en t ed cells of t h e t est a bu t n ot of t h e cells of t h e
per ica r p (pa n els g a n d h of F igu r e 2). Na P TS, h owever ,
exh ibit ed a low ext r a ct ion efficien cy a s br own pigm en t
a n d ot h er cellu la r con st it u en t s wer e ext r a ct ed m or e
t h a n piper in e. Na XS-soa ked sect ion s (pa n els i a n d j of
F igu r e 2) sh owed on ly m in im a l ch a n ges in t h e epider mal la yer of the perica rp; even the thick-wa lled sclereids
seem ed in t a ct .
F r om t h e m icr oscopic obser va t ion s a n d pr elim in a r y
ext r a ct ion exper im en t s, wh er e a good a m ou n t of piper in e wa s ext r a ct ed, t h e ext r a ct ion efficien cy of t h e
h ydr ot r ope solu t ion s wa s clea r ly eviden t . F or t h e ext r a ct ion of piper in e, h ydr ot r ope sh ou ld fir st a dsor b on t o
t h e su r fa ce of t h e cells a n d t h en pen et r a t e t h e cell
st r u ct u r e t o a ccess piper in e. Th e pen et r a bilit y of t h e
h ydr ot r ope ca n be r ela t ed t o t h e pa r a m et er 21 pC 20 /A m in ,
wh er e pC 20 is t h e n ega t ive loga r it h m of C 20 , i.e., t h e
m ola r con cen t r a t ion of a n a m ph iph ile in t h e a qu eou s
ph a se r equ ir ed t o r edu ce t h e su r fa ce t en sion of a qu eou s
solu t ion s by 20 dyn /cm . It is a m ea su r e of t h e su r fa ce
activity and adsorption efficiency of the amphiphile. A min
is t h e h ydr a t ed cr oss-sect ion a l a r ea u n der given con dit ion s, wh ich ca n be est im a t ed fr om t h e su r fa ce t en sion
da t a . Alt h ou gh a ll h ydr ot r opes u sed in t h is wor k h a ve
t h e sa m e pola r gr ou p, t h e h ydr a t ed cr oss-sect ion a l a r ea
ca n depen d on t h e m a n n er in wh ich t h e m olecu les a r e
pa cked a t t h e in t er fa ce or in t h e a ggr ega t e, so t h e fa ct or
pC 20/A min ca n be u sed t o expla in pen et r a bilit y. Th e pC 20/
A m in va lu es for Na NBBS a n d Na CS a r e h igh a t 0.018
a n d 0.02, r espect ively, in dica t in g bet t er pa ckin g of t h ese
m olecu les in t h e a dsor bed st a t e. F or Na XS, Na P TSA,
a n d Na BMGS on t h e ot h er h a n d, t h e pC 20 va lu es a r e
n ea r ly 10 t im es lower a t 0.0039, 0.0053, a n d 0.0065,
r espect ively. Th ese va lu es a pt ly r epr esen t t h e m icr oscopica lly obser ved effect s of t h e r espect ive h ydr ot r opes
on cellu la r st r u ct u r es.
Th e sect ion s t a ken fr om t h e wh ole fr u it s soa ked in
h ydrot rope solut ion s sh owed br oken per ica r ps a n d ot h er
epidermal layers and the presence of cellular debris. The
effect s of differ en t h ydr ot r opes on t h e st r u ct u r e of t h e
pla n t cells wer e a lso differ en t . Beca u se t h e a ct u a l
ext r a ct ion of piper in e wa s ca r r ied ou t u sin g pu lver ized
pepper fr u it s, t h er e wa s a n in it ia l br ea kdown of t h e

ou t er la yer s of t h e fr u it s, lea vin g t h e in n er epider m a l

la yer s dir ect ly exposed t o t h e h ydr ot r ope solu t ion s.
P iper in e, a lt h ou gh pr esen t t h r ou gh ou t t h e Piper
n igru m fr u it , is la r gely con cen t r a t ed wit h in t h e per ica r p
cells. Th e cell wa lls, m a de u p of cellu lose, pr esen t a n
or der ed st r u ct u r e. Th e pr im a r y cell wa ll con sist s of
glu cose polym er s, ea ch of wh ich con t a in s r ou gh ly 6000
glu cose u n it s, wh er ea s in t h e secon da r y wa ll, t h e
n u m ber in cr ea ses t o 13 000-16 000 u n it s.22 Cellu lose
ch a in s for m cr yst a llin e st r u ct u r es ca lled m icr ofibr ils,
ea ch wit h a dia m et er of 20-30 n m con t a in in g a bou t
2000 m olecu les in a lt er n a t e cr yst a llin e a n d n on cr yst a llin e sect ion s.22 Th e cr yst a llin e sect ion for m s t h e t h r eedim en sion a l cellu lose m a t r ix t h r ou gh t h e for m a t ion of
t h e h igh est possible n u m ber of h ydr ogen bon ds. Th e cell
wa ll a s a wh ole is h et er ogen eou s beca u se it sh ows t wo
ph a ses on swellin g wit h wa t er a n d det er m in es t h e r a t e
of en t r y a n d exit of com pou n ds wit h r espect t o t h e cell.22
We expect t h a t , a s a fir st st ep, h ydr ot r ope m olecu les
a dsor b on t h e cellu losic cell wa ll. Th e r edu ct ion of
su r fa ce for ces a t t h e in t er fa ce im pr oves t h e cell wa lls
wet t a bilit y, a n d su bsequ en t ly, wa t er a n d h ydr ot r ope
m olecu les ca n pen et r a t e ea sily in t o t h e cellu losic st r u ct u r e a n d a ccess t h e cell m em br a n e.
Th e cell m em br a n e, r espon sible, a lon g wit h t h e cell
wa ll, for m a in t a in in g a ba la n ce bet ween t h e ou t side
bu lk solu t ion a n d t h e in side of t h e cell, is m a de u p of
ph osph olipid bila yer s in t er sper sed wit h pr ot ein s su ch
a s ext en sin s a n d a qu a por in s. E xt en sin s a r e m a de u p of
sh or t lin ea r ch a in s of on e t o fou r su ga r u n it s a t t a ch ed
t o a polypept ide ba ckbon e. Th e pr esen ce of a qu a por in s
in cellu la r m em br a n es fa cilit a t es a t r a n s-cellu la r pa t h wa y for wa t er flow.23 Two h igh ly h ydr oph obic h ydr oca r bon s t a ils of ph osph olipids cr ea t e a h ydr oph obic
en vir on m en t wit h in t h e m em br a n e. Th e h ydr ot r ope
ext r a ct solu t ion sh owed t h e pr esen ce of in or ga n ic ph osph or u s (0.0156 m ol/dm 3 ), wh ich in dica t es pr oba ble
dest a biliza t ion of t h e ph osph olipid la yer .16 Th e t r ea t m en t of t h e ext r a ct wit h F eh lin g solu t ion ga ve a r u st br own -color ed pr ecipit a t e, qu a lit a t ively in dica t in g t h e
pr esen ce of ca r boh ydr a t es wit h r edu cin g gr ou ps t h a t
r edu ced cu pr ic ion s t o cu pr ou s ion s. 17 Th e pr esen ce of
reducing sugars and amino acids in the extract indicated
a pa r t ia l, if n ot com plet e, br ea kdown of t h e cell wa ll
polym er s a n d t h e pr ot ein s su ch a s ext en sin s in t o t h eir
r espect ive a m in o a cids.18 Dissolu t ion of cellu lose in t o
t h e h ydr ot r ope solu t ion s a lso ca n n ot be r u led ou t ,
a lt h ou gh it ca n n ot be t h e sole m ech a n ism of t h e
obser ved en h a n ced ext r a ct ion r a t es, a s it wou ld h a ve
ca u sed ext r a ct ion of ot h er species t oo in t o t h e h ydr ot r ope solu t ion s.
Th e pen et r a t ion of h ydr ot r ope in t o t h e cell wa ll a n d
m em br a n e st r u ct u r e pr oba bly in du ces m olecu la r disor ga n iza t ion a n d a lt er s t h e per m ea bilit y of t h e m em br a n e
by dissolvin g a t lea st som e of t h e cell wa ll com pon en t s.
We believe t h a t t h e h ydr ot r ope is ca pa ble of t h is
disor ga n iza t ion , a s is eviden t fr om t h e disor der in g of
liqu id la m ella r st r u ct u r es of su r fa ct a n t s in a qu eou s
solu t ion s in t h e pr esen ce of h ydr ot r opes. 24 Th e liqu id
la m ella r st r u ct u r es a r e r em in iscen t of t h e cellu la r
m em br a n e st r u ct u r es. By in du cin g a ch a n ge in t h e
m olecu la r or ga n iza t ion of t h e cell m em br a n e, a h ydr ot r ope a lt er s t h e per m ea bilit y of t h e m em br a n e in su ch
a m a n n er t h a t piper in e is m a de ea sily a ccessible t o t h e
hydrotrope solution. The schematic representation of the
ph en om en on of h ydr ot r opic ext r a ct ion is sh own in
F igu r e 3.

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2971

F ig u re 3. Sch em a t ic r epr esen t a t ion h ydr ot r opic ext r a ct ion of piper in e. On t r ea t m en t wit h h ydr ot r ope solu t ion , t h e wa t er m olecu les
pen et r a t e in t o t h e cell wa ll a n d t h r ou gh t h e t r a n spor t a qu a -por in s, ca u sin g swellin g of t h e m em br a n e pr ot ein s, a n d t h e h ydr ot r ope
m on om er s a lso pen et r a t e in t o t h e cellu la r st r u ct u r e. Cell wa ll a n d cell m em br a n e disor ga n iza t ion occu r s, lea din g t o t h e r elea se of piper in e
fr om wit h in t h e cell in t o t h e h ydr ot r ope solu t ion .

Beca u se h ydr ot r opes sh ow in cr ea sed solu bilit ies for

h ydr oph obic su bst a n ces a bove t h eir r espect ive MH Cs,
piper in e ca n be ea sily solu bilized a n d ca r r ied in t o t h e
ext er n a l h ydr ot r ope solu t ion s. Beca u se of t h e st r u ct u r a l
ch a n ges in t h e biom a t r ix, t h e r a t e of ext r a ct ion of
piper in e sh ou ld u n der go a su bst a n t ia l en h a n cem en t ,
wh ich in deed is obser ved. Th e effect s of h ydr ot r opes a n d
t h eir con cen t r a t ion s wer e in vest iga t ed fu r t h er for t wo
differ en t t ypes of h ydr ot r opes, a r om a t ic su lfon a t es a n d
glycol su lfa t es.
Substituted aromatic sulfonates such NaNBBS, NaCS,
Na XS, Na P TS, a n d a lin ea r a liph a t ic su lfa t e su ch a s
Na BMGS h a ve a t ypica l a m ph iph ilic st r u ct u r e, in clu ding a st r on gly ionic h ydr oph ilic gr ou p a n d a h ydr ophobic
gr ou p con sist in g of a n a lkyl gr ou p wit h or wit h ou t a n
a r om a t ic r in g. Alt h ou gh t h e pr esen ce of a n a r om a t ic
r in g wa s on ce con sider ed essen t ia l for t h e h ydr ot r opic
effect ,24 a lkyl glycol su lfa t es defy t h ese expect a t ion s a n d
a r e ext r em ely good h ydr ot r opes. Th e su bt le differ en ces
in h ydr ot r opy displa yed by t h e m em ber s wit h in ea ch
cla ss a n d bet ween t h e cla sses ca n be a t t r ibu t ed t o t h e
differ en t sizes of t h eir h ydr oph obic pa r t s, n u m ber s of
-CH 2 - gr ou ps in t h e h ydr oca r bon side ch a in s, a n d
efficien cies of in t er m olecu la r pa ckin g in t h eir selfa ggr ega t es.
F igu r e 4 sh ows a dist in ct r ela t ion sh ip bet ween t h e
h ydr oph obic ch a in len gt h of a h ydr ot r ope a n d t h e efficien cy of ext r a ct ion in t h e or der Na NBBS > Na CS >
Na XS > Na P TS. Th e per cen t a ge ext r a ct ion is defin ed
h er e a s t h e per cen t a ge of piper in e in it ia lly pr esen t in
t h e r a w m a t er ia l t h a t wa s ext r a ct ed in t o t h e h ydr ot r ope
solu t ion . Th e effect ive h ydr oph obic ch a in len gt h va r ies
fr om C 5 for Na P TS t o C 8 for Na NBBS. H ydr ot r opic
solu biliza t ion is a collect ive m olecu la r ph en om en on ,
possibly occu r r in g by t h e in t er ca la t ion or co-a ggr ega t ion
of solu t e wit h h ydr ot r ope m olecu les a n d t h e selfa ggr ega t ion of h ydr ot r ope m olecu les in a qu eou s solu tions, that is a prerequisite for increased solubilization. 13
It is, t h er efor e, n ot su r pr isin g t h a t t h e solu biliza t ion
ca pa cit y is gover n ed by t h e h ydr oph obic fu n ct ion a lit y,
i.e., t h e a lkyl gr ou p on t h e a r om a t ic su lfon a t es. Th e
hydrophobic volume (v, 3) provided by a hydrotrope ca n

F ig u re 4. Com pa r ison of ext r a ct ion efficien cy of h ydr ot r opes for

piper in e (t em per a t u r e ) 300 K, con cen t r a t ion ) 2 m ol/dm 3 , per iod
of ext r a ct ion ) 2.0 h ): 2, expect ed % piper in e ext r a ct ed; st r iped
ba r , a ct u a l % piper in e ext r a ct ed; dot t ed ba r , % pu r it y.

be est im a t ed fr om it s effect ive ca r bon ch a in len gt h s

(n c)25

v ) (27.4 + 26.9n c)

(1)

wh er e n c r epr esen t s t h e n u m ber of ca r bon a t om s t h a t

ca n en t er in t o t h e for m a t ion of t h e h ydr oph obic spa ce
analogous to the hydrophobic core of surfactant micelles,
u su a lly ign or in g on e ca r bon close t o t h e pola r gr ou p.
Th e sh a pe of t h e h ydr oph obic r egion of t h e h ydr ot r ope
a ggr ega t es is n ot con clu sively kn own , bu t a dissolved
solu t e does exper ien ce a r edu ced m icr opola r it y a n d
in cr ea sed m icr oviscosit y in h ydr ot r ope solu t ion s.13 Th e
h ydr oph obic r egion of t h e h ydr ot r ope a ggr ega t es seem s
t o be a ccom m oda t in g t h e dissolved solu t es. Th ese
h ydr oph obic volu m es wer e est im a t ed t o be 215.7 3 for
Na NBBS, 188.8 3 for Na CS, 161.9 3 for Na XS, a n d
135 3 for Na P TS.
Th e in cr ea sed solu biliza t ion wit h in cr ea sin g h ydr oph obic volu m e in dica t es t h a t h ydr ot r opic solu biliza t ion

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n ea r ly 230 t im es t o 3.221 10 -3 m ol/dm 3 . On ly a bove

t h e MH C of Na NBBS (0.1 m ol/dm 3 ) is t h e in cr ea se in
t h e solu bilit y sign ifica n t . Sim ila r solu bilit y beh a vior is
eviden t in ot h er h ydr ot r ope solu t ion s.
Th e solu bilit y of a solu t e in a h ydr ot r ope solu t ion (S )
is u su a lly cor r ela t ed by a n expon en t ia l r ela t ion , in a
m a n n er a n a logou s t o sa lt in g-ou t , t o t h e h ydr ot r ope
con cen t r a t ion (C t ) a n d t h e solu t e solu bilit y in wa t er (S w )

log(S /S w ) ) K s C t

F ig u re 5. Solu bilt y of piper in e in differ en t h ydr ot r opes (t em per a t u r e ) 300 K): ), Na NBBS; 2, Na CS; 0, Na P TSA; O, Na XS; *,
Na BMGS.

cou ld be a con sequ en ce of t h e h ydr oph obic dom a in s

pr esen t wit h in t h e h ydr ot r ope a ggr ega t es, wh ich ca n
pr ovide a m icr oen vir on m en t com pa t ible wit h t h e h ydr oph obic n a t u r e of piper in e. P iper in e is n ea r ly wa t er in solu ble ot h er wise. It is a lso possible t h a t t h e a ggr ega t ion beh a vior of t h e h ydr ot r ope it self is fu r t h er
pr om ot ed by t h e pr esen ce of a ver y h ydr oph obic solu t e.
Th e solu biliza t ion is t h en a n a logou s t o co-a ggr ega t ion
of t h e h ydr ot r ope m olecu les a n d t h e solu t e, givin g r ise
t o select ivit y in h ydr ot r opic solu biliza t ion . If t h e solu t e
in t er ca la t es ea sily bet ween t h e h ydr ot r ope m olecu les in
t h e a ggr ega t es, t h en it s solu biliza t ion sh ou ld be bet t er .
Beca u se t h e h ydr oph obicit y of t h e a r om a t ic su lfon a t es
in cr ea ses wit h in cr ea sin g a lkyl gr ou p len gt h , t h ey
displa y a n in cr ea sin g t en den cy for t h e solu biliza t ion of
n on pola r m olecu les.26
Na BMGS is a sh or t -ch a in a liph a t ic su lfa t e t h a t is
st r u ct u r a lly ver y differ en t fr om t h e con ven t ion a l a r om a t ic su lfon a t es bu t t h a t st ill sh ows a ver y h igh
select ivit y t owa r d piper in e (F igu r e 4). H ydr ot r opic
ext r a ct ion is a ph en om en on , a s discu ssed ea r lier , t h a t
pr oba bly in volves a dsor pt ion of h ydr ot r ope on pla n t
cells, pen et r a t ion in t o t h e cellu la r m a t r ix, a n d t h en
solu biliza t ion of t h e a ct ive com pon en t . Th e su r fa ce
t en sion va lu es of h ydr ot r ope solu t ion s a r e 37 dyn /cm
for Na BMGS, 50-53 dyn /cm for Na P TS a n d Na XS, 43
dyn /cm for Na CS, a n d 40.3 dyn /cm for Na NBBS a t t h eir
m in im u m h ydr ot r ope con cen t r a t ion s.13 Na BMGS sh ows
t h e lea st su r fa ce t en sion , bu t t h e decr ea se is gr a du a l
over a la r ge con cen t r a t ion r a n ge, a n d t h e m olecu les
occu py la r ger a r ea s a t t h e in t er fa ce t h a n a r om a t ic
su lfona t es, pr oba bly beca u se of th e ga u ch e con for m a t ion
a t t h e bu t yl gr ou p join in g t h e glycol pa r t or t h e fla t
or ien t a t ion of t h e m olecu le a t t h e in t er fa ce. 13 F or t h e
pen et r a t ion of a h ydr ot r ope in t o t h e biom a t r ix, a lower
su r fa ce t en sion is u sefu l in over com in g t h e su r fa ce
ca pilla r y for ces wit h in t h e cellu la r su r fa ce.27 In t h is
r ega r d, Na NBBS a n d Na BMGS h a ve excellen t wet t a bilit y ch a r a ct er ist ics a s com pa r ed t o Na XS a n d Na P TS.
F igu r e 5 sh ows t h e solu bilit y of piper in e in differ en t
h ydr ot r ope solu t ion s. In wa t er , piper in e is solu ble a t a
1.4 10 -5 m ol/dm 3 con cen t r a t ion . In 0.5 m ol/dm 3
Na NBBS solu t ion s, t h e piper in e solu bilit y in cr ea ses by

(2)

wh er e t h e Set ch n ow con st a n t (K s ) r epr esen t s t h e efficien cy of a h ydr ot r ope. Th is expr ession , h owever ,
ca n n ot r epr esen t t h e sa t u r a t ion lim it s obser ved in
h ydr ot r ope solu t ion s. An a ssocia t ion m odel wa s r ecen t ly
pr oposed for h ydr ot r ope solu biliza t ion t h a t con sider s
a ggr ega t ion of t h e h ydr ot r ope m olecu les in a st epwise
m a n n er a n d t h en solu biliza t ion a s t h e co-a ggr ega t ion
of a solu t e wit h t h ese a ggr ega t es.28 Th e t ot a l con cen t r a t ion of h ydr ot r ope (C t ) is r ela t ed t o t h e h ydr ot r ope
m on om er con cen t r a t ion (H 1 ) t h r ou gh eq 3 u n der t h e
a ssu m pt ion t h a t t h e a ggr ega t ion con st a n t decr ea ses
wit h in cr ea sin g a ggr ega t ion n u m ber (n ) a s K n ) K 2 /n ,
wh er e K 2 is t h e dim er iza t ion con st a n t for h ydr ot r ope
m olecu les.

C t ) H 1 [2 exp(K 2 H 1 ) - 1]

(3)

Th e in cr ea sed solu bilit y of a solu t e (S ) du e t o t h e

a ssocia t ion of solu t e m olecu les wit h t h e h ydr ot r ope
a ggr ega t es ca n t h en be cor r ela t ed wit h t h e h ydr ot r ope
con cen t r a t ion by

S ) 2(K s /K 2 )[S w ]{exp(K 2 H 1 ) - (1 + K 2 H 1 )} (4)

wh er e t h e con st a n t K s ch a r a ct er izes t h e in t er a ct ion
bet ween t h e h ydr ot r ope a ggr ega t es a n d t h e solu t e
m olecu les. A h igh er va lu e of K s sign ifies a st r on ger
int er a ct ion of t he solut e wit h t he hydr ot r ope a ggr ega t es.
Alt h ou gh t h e a ssocia t ion m odel is a n a ppr oxim a t ion
of t h e a ct u a l a ggr ega t ion pr ocess of a h ydr ot r ope a n d
su bsequ en t solu t e solu biliza t ion , it explicit ly con sider s
h ydr ot r opy a s a n a ggr ega t ive ph en om en on . In t h e
pr esen t ca se, t h e a ssocia t ion of piper in e wit h h ydr ot r ope
a ggr ega t es is r eflect ed in t h e va lu es of K s , wh ich
r epr esen t t h e st r en gt h s of t h e in t er a ct ion s of piper in e
wit h differ en t h ydr ot r opes. Th e solu bilit y da t a wer e
fit t ed t o t h e m odel eqs 3 a n d 4 t o est im a t e K s va lu es for
t h e differ en t h ydr ot r opes. Th e K s va lu es fou n d a r e 368
dm 3 /m ol, t h e h igh est , for Na NBBS; 320.9 dm 3 /m ol for
Na CS; 301.7 dm 3 /m ol for Na XS 281.3 dm 3 /m ol for
Na P TS; a n d 232.4 dm 3 /m ol for Na BMGS. Th e h ydr ot r ope-h ydr ot r ope a ssocia t ion con st a n t s (K 2 ), h owever ,
h a d ver y low va lu es of 0.101, 0.11, 0.11, 0.06, a n d 0.09
dm 3 /m ol for Na NBBS, Na CS, Na XS, Na P TS, a n d Na BMGS, r espect ively. Th e h igh ly ion ic n a t u r e of t h e
h ydr ot r ope wou ld n ot per m it a close pa ckin g of t h ese
m olecu les, a s is eviden t fr om t h e dim er iza t ion con st a n t
va lu es. In st ea d, t h e pr esen ce of piper in e m igh t a u gm en t
t h e h ydr ot r ope a ssocia t ion , a s t h e pr ocess is a kin t o coa ggr ega t ion .
Th e expect ed ext r a ct ion of piper in e wa s est im a t ed on
t h e ba sis of t h e sa t u r a t ion solu bilit ies of piper in e in
differ en t a r om a t ic su lfon a t e solu t ion s a t 2.0 m ol/dm 3
con cen t r a t ion . Th er e wa s ver y lit t le devia t ion in t h e
expect ed va lu es of ext r a ct ion fr om t h e a ct u a l exper im en t a l va lu es for t h e given set of con dit ion s (F igu r e 4).

In d. E n g. Ch em . Res., Vol. 41, No. 12, 2002

2973

F ig u re 7. E xt r a ct ion of piper in e wit h differ en t con cen t r a t ion s of

a qu eou s Na BMGS solu t ion (t em per a t u r e ) 300 K, t im e ) 2.0 h ,
solid loa din g ) 10% w/v, speed of a git a t ion ) 1100 r pm ): ), %
piper in e ext r a ct ed; 4, % pu r it y.
F ig u re 6. (s) E xpect ed a n d (poin t s) a ct u a l ext r a ct ion of piper in e
wit h a qu eou s Na BMGS a n d Na NBBS solu t ion s (t em per a t u r e )
300 K, t im e ) 2.0 h ): 0, 2.0 m ol/dm 3 Na NBBS; 2, 1.7m ol/dm 3
Na BMGS.

Ex tra c tio n o f P ip e rin e w ith Hy d ro tro p e S o lu tio n s (N a B MGS a n d N a N B B S ). F igu r e 6 sh ows t h e

effect of t h e Na BMGS con cen t r a t ion on t h e ext r a ct ion
of piper in e fr om P. n igru m fr u it s. Above t h e m in im u m
h ydr ot r ope con cen t r a t ion of Na BMGS,13 i.e., 0.8 m ol/
dm 3 , a s t h e con cen t r a t ion of Na BMGS wa s in cr ea sed
fr om 1.0 t o 2.5 m ol/dm 3 , t h e per cen t a ge ext r a ct ion of
piper in e in cr ea sed fr om 20 t o 60%. Beca u se h ydr ot r opy
is m a in ly oper a t ive a bove t h e m in im u m h ydr ot r ope
con cen t r a t ion , t h e ext r a ct ion of piper in e r ose m a r kedly
a t con cen t r a t ion s a bove t h e MH C of t h e h ydr ot r ope a n d
t h en leveled off a t h igh er h ydr ot r ope con cen t r a t ion s.
Below t h e MH C, h owever , t h e in cr ea se wa s m a r gin a l.
At a h ydr ot r ope con cen t r a t ion of 1.7 m ol/dm 3 , t h e
solu bilit y of piper in e in t h e a qu eou s h ydr ot r ope ph a se
wa s 5.6 10 -3 m ol/dm 3 , a n in cr ea se by a fa ct or of 400
over it s wa t er solu bilit y. Th e pu r it y of piper in e, h owever, decrea sed slightly when the hydrotrope concentrat ion wa s in cr ea sed t o 2.0 m ol/dm 3 a n d a bove.
Na NBBS h a s a n MH C of 0.1 m ol/dm 3 . Th e ext r a ct ion
exper im en t s wer e, t h er efor e, con du ct ed a t con cen t r a t ion s ju st below a n d well a bove t h is va lu e. As t h e NBBS
con cen t r a t ion wa s in cr ea sed fr om 0.05 t o 2.4 m ol/dm 3 ,
t h e a m ou n t of piper in e r ecover ed by pr ecipit a t ion fr om
t h e ext r a ct solu t ion by dilu t ion wit h wa t er a lso in cr ea sed fr om 57 t o 95% a n d r em a in ed con st a n t t h er ea ft er (F igu r e 6). Th e dilu t ed h ydr ot r ope solu t ion r et a in ed in ea ch ca se t h e r em a in in g a m ou n t of piper in e,
a s t h e ext r a ct ion fr om t h e r a w m a t er ia l wa s com plet e.
Th e r ecover ed piper in e a m ou n t s a r e clea r ly in dica t ive
of a h igh ly efficien t ext r a ct ion by Na NBBS solu t ion s.
No sign ifica n t decr ea se in t h e pu r it y wa s obser ved wit h
t h e in cr ea se in h ydr ot r ope con cen t r a t ion , wh ich r em a in ed con st a n t a t 92-93%. F igu r e 6 a lso com pa r es t h e
a ct u a l ext r a ct ion of piper in e wit h t h e expect ed ext r a ct ion a s det er m in ed fr om t h e solu bilit y of piper in e in
Na NBBS solu t ion s. Th e a ct u a l r ecover y is sligh t ly lower
t h a n t h e expect ed ext r a ct ion of piper in e. Th is sligh t
reduction in the recovery could be due to a sma ll amount
of piper in e r em a in in g in t h e solid ca ke. Th e opt im u m
con cen t r a t ion of Na NBBS r equ ir ed for ext r a ct ion wit h
opt im u m r ecover y a n d pu r it y wa s fou n d t o be 2.0 m ol/

dm 3 a s no significant increa se in piperine extra ction wa s

observed at a 2.4 mol/dm 3 hydrotrope concentration. The
solu bilit y da t a a lso in dica t ed t h a t NBBS wa s t h e m ost
effect ive h ydr ot r ope in dissolvin g piper in e. E ven a t
con cen t r a t ion s a s low a s 0.5 m ol/dm 3 , a lm ost 70%
ext r a ct ion of piper in e cou ld be a ch ieved in 2 h .
At lower con cen t r a t ion s, close t o t h e MH C, som e
h ydr ot r ope m on om er s ca n st ill becom e in cor por a t ed in t o
t h e cell wa ll/m em br a n e st r u ct u r e, wh ich ca n ca u se t h e
m em br a n es t o lose t h eir in t egr it y.29 At con cen t r a t ion s
m u ch a bove t h e MH C, t h e solu biliza t ion ca pa cit y of t h e
h ydr ot r ope a ggr ega t es is m u ch h igh er , i.e., a lm ost a s
a n expon en t ia l fu n ct ion of t h e h ydr ot r ope con centra tion.
As t h e solu biliza t ion wit h in t h e cell m a t r ix is ea sier a n d
m or e r a pid, t h e t r a n spor t of piper in e ba ck in t o t h e
ext er n a l liqu id solu t ion sh ou ld be fa st er , a n d it s pu r it y
sh ou ld a lso possibly be bet t er , a s ot h er oleor esin s
dissolve less r ea dily in t o h ydr ot r ope solu t ion s in t h e
sa m e a m ou n t of t im e.
F igu r e 7 sh ows t h a t , for 1.7 m ol/dm 3 Na BMGS solu t ion , t h e per cen t a ge ext r a ct ion of piper in e in cr ea sed
fr om 10% in t h e fir st 15 m in t o 59% a ft er 2.0 h a n d
r em a in ed con st a n t t h er ea ft er . Th e sigm oida l n a t u r e of
t h e ext r a ct ion cu r ve in dica t es t h a t t h e ext r a ct ion is a
secon d-or der pr ocess, i.e., in it ia lly, t h e r a t e of ext r a ct ion
is slow, followed by a fa st er ext r a ct ion st a ge befor e a
pla t ea u of lim it in g va lu e is r ea ch ed. Th e fin a l ext r a ct ion
lim it is det er m in ed by t h e solu bilit y lim it of piper in e in
t h e h ydr ot r ope solu t ion u n der given con dit ion s. It
a ppea r s t h a t t h e ext r a ct ion n eeds t o over com e t wo
r esist a n ces in t h e pr ocess. On e m igh t h a ve t o con sider
t h en t h e cellu la r st r u ct u r e of t h e com plex biom a t r ix t o
u n der st a n d t h ese r esist a n ces. Th e or der ed st r u ct u r e of
t h e cells wou ld r esist t h e pen et r a t ion of h ydr ot r ope
m olecu les fir st in t o t h e cellu losic la yer a n d t h en fu r t h er
in t o t h e ph osph olipid bila yer befor e it ca n a ccess t h e
piper in e disper sed t h r ou gh cell.
Th e r a t e of ext r a ct ion sh ou ld depen d on t h e ea se wit h
wh ich t h e h ydr ot r ope ca n pen et r a t e in t o t h e biom a t r ix
a n d a lso on t h e solu bilit y of piper in e in t h e h ydr ot r ope
solu t ion . In t h e ca se of Na NBBS a t a con cen t r a t ion of
0.05 m ol/dm 3 , below it s MH C, 45% piper in e wa s ext r a ct ed wit h in 10 m in , a n d t h e ext r a ct ion r em a in ed
fa ir ly con st a n t even a ft er 2 h , wit h on ly a sligh t in cr ea se
t o 57%. At h igh er Na NBBS con cen t r a t ion s, h owever , a

2974

In d. E n g. Ch em . Res., Vol. 41, No. 12, 2002

F ig u re 8. Kin et ics of ext r a ct ion of piper in e wit h differ en t

con cen t r a t ion s of Na NBBS (t em per a t u r e ) 300 K, solid loa din g
) 10% w/v, speed of a git a t ion ) 1100 r pm ): 9, 0.05; ), 0.5; O, 1.0;
2, 2.0; 0, 3.0; 4, 3.4 m ol/dm 3 .

la g t im e of 30 m in wa s obser ved, wit h ju st 25% ext r a ct ion of piper in e. Ra pid ext r a ct ion of piper in e wa s
a ch ieved in t h e n ext 1 h . F in a lly, a ft er 2 h , t h e
extraction reached 95% (Figure 8). The rate of extraction
is det er m in ed by pen et r a bilit y, wh ich depen ds on t h e
h ydr ot r ope m on om er con cen t r a t ion a n d t h e solu bilit y
of piper in e in t h e h ydr ot r ope a ggr ega t es. At h ydr ot r ope
con cen t r a t ion s of 0.05 a n d 0.5 m ol/dm 3 , t h e per cen t a ge
ext r a ct ion followed a r ect a n gu la r sh a pe wit h a fin it e
in it ia l r a t e, wh ich is t ypica l of a fir st -or der pr ocess.
Beca u se h ydr ot r ope is pr esen t pr edom in a n t ly in m on om er ic for m a t t h ese con cen t r a t ion s, it s a dsor pt ion on
t h e solid su r fa ce is pr efer en t ia l, wh ich a lso lea ds t o a
h igh er pen et r a t ion r a t e. At t h e h igh er h ydr ot r ope
con cen t r a t ion s of 1.0 a n d 2.0 m ol/dm 3 , h owever , t h e
extraction showed a sigmoidal behavior with almost zero
initial slope, typical of a second-order process. The initial
r esist a n ce t o solu biliza t ion pr oba bly a r ises beca u se few
h ydr ot r ope m olecu les a r e pr esen t a s m on om er s r a t h er
t h a n a ggr ega t es, wh ich a voids a dsor pt ion on t h e solid
su r fa ce. On ce t h e pen et r a t ion t a kes pla ce, h owever ,
solu biliza t ion is fa st er .
With the increased concentration of NaNBBS, a larger
osm ot ic pr essu r e a lso develops a cr oss t h e cell wa ll.
Th u s, a t h igh er con cen t r a t ion s, h ydr ot r ope solu t ion
en t er s t h e cell m a t r ix r ela t ively slowly. Aft er t h e in it ia l
la g per iod, du r in g wh ich t h e h ydr ot r ope m on om er s
pen et r a t e in t o t h e cell wa ll a n d dest a bilize t h e liqu idcr yst a llin e n a t u r e of t h e bila yer , t h e t u r gidit y of t h e cell
wa ll is lost . Furth er pen etr a tion of a qu eou s solu t ion in t o
t h e cellu la r m a t r ix a n d t r a n spor t of piper in e ou t t o t h e
ext er n a l h ydr ot r ope m ediu m a r e t h en r a pid st eps.
F igu r e 9 sh ows a com pa r ison of t h e ext r a ct ion pr ofiles
of Na NBBS wit h t h e ca t ion ic su r fa ct a n t cet yl t r im et h yla m m on iu m br om ide (CTAB) a n d t h e a n ion ic su r fa ct a n t
sodiu m la u r yl su lfa t e (SLS) a t t h e sa m e con cen t r a t ion .
At a con cen t r a t ion of 0.5 m ol/dm 3 , SLS sh owed a
sigm oida l ext r a ct ion pa t t er n , bu t a ft er 2 h , on ly 15%
ext r a ct ion of piper in e wa s obser ved. CTAB, on t h e ot h er
h a n d, ga ve a som ewh a t bet t er ext r a ct ion of 35% wit h in
2 h . In com pa r ison t o t h ese su r fa ct a n t s, Na NBBS
dem on st r a t ed fa st er a n d bet t er h ydr ot r opic ext r a ct ion .
Th e su r fa ct a n t s CTAB a n d SLS in du ced per m ea bilit y

F ig u re 9. E xt r a ct ion of piper in e wit h su r fa ct a n t s SLS a n d CTAB

(con cen t r a t ion ) 0.5 m ol/dm 3 , t em per a t u r e ) 300 K, solid loa din g
) 10% w/v, speed of a git a t ion ) 1100 r pm ): 0, SLS; 4, CTAB; O,
Na NBBS.

F ig u re 10. E xt r a ct ion of piper in e u sin g Na BMGS a n d Na NBBS

a t differ en t t em per a t u r es (con cen t r a t ion ) 2.0 m ol/dm 3 , t im e )
2.0 h ): 4, % piper in e ext r a ct ed (Na BMGS); 2, % pu r it y piper in e
(Na BMGS); O, % piper in e ext r a ct ed (Na NBBS); b, % pu r it y
piper in e (Na NBBS).

in t h e cell wa ll bu t did n ot pr om ot e t h e com plet e

ext r a ct ion of piper in e.
H en ce, it is pr oposed t h a t h ydr ot r opic ext r a ct ion
pr oceeds in t wo st eps: fir st , pen et r a t ion of h ydr ot r ope
m olecu les in t o ext er n a l cell wa ll, wh ich offer s a m a jor
r esist a n ce t o m a ss t r a n sfer of solu t ion in t o t h e cell, a n d
secon d, solu biliza t ion of piper in e a n d it s ba ck t r a n sfer
t o t h e ext er n a l solu t ion . Th e in it ia lly obser ved ext r a ct ion cou ld r esu lt fr om piper in e bein g dir ect ly exposed
t o t h e h ydr ot r ope solu t ion u pon com m in u t ion of t h e r a w
m a t er ia ls.
F igu r e 10 sh ows t h e a ct u a l r ecover y of piper in e fr om
Na BMGS solu t ion s, wh ich in cr ea sed fr om 43 t o 56%,
wit h a sign ifica n t decr ea se in t h e pu r it y, wh en t h e
ext r a ct ion wa s con du ct ed a t eleva t ed t em per a t u r es for

In d. E n g. Ch em . Res., Vol. 41, No. 12, 2002

Ta ble 1. Effe c t o f P a rtic le S ize o n th e Ex tra c tio n o f
P ip e rin e a
pa r t icle size (m )

% piper in e

% pu r it y

53
180
600
710

90.14
92.14
95.12
96.15

98.3
97.3
90.2
89.12

a Con cen t r a t ion ) 2.0 m ol/dm 3 , t em per a t u r e ) 300 K, t im e )

2.0 h , solid loa din g ) 10% w/v.

t h e sa m e per iod. F igu r e 10 a lso sh ows t h e effect of

t em per a t u r e, in t h e r a n ge 30-65 C, on t h e degr ee of
ext r a ct ion of piper in e u sin g Na NBBS solu t ion s. Th er e
wa s a ver y sligh t in cr ea se in t h e per cen t a ge ext r a ct ion
in 2 h , bu t t h e pu r it y of t h e ext r a ct decr ea sed su bst a n t ia lly fr om 92 t o 70%. It is pr efer a ble, t h er efor e, t o
con du ct t h e ext r a ct ion a t a m bien t con dit ion s. At h igh er
tempera tures, more lysis of the cell structure might take
pla ce, a n d t h e cell wa ll m igh t becom e m or e per m ea ble
t o t h e h ydr ot r ope solu t ion . Beca u se of bot h t h e br ea kdown a n d t h e solu biliza t ion of t h e cellu lose polym er s
wit h in t h e cell wa ll, t h e con t r ibu t ion of t h e polym er s t o
t h e fir m n ess of t h e cell wa ll cou ld be r edu ced. Th e
en h a n ced cell r u pt u r e a t eleva t ed t em per a t u r es a lso
fa cilit a t es diffu sion of u n desir a ble oleor esin s in t o t h e
ext er n a l bu lk h ydr ot r ope solu t ion , r esu lt in g in decr ea sed select ivit y. Th e decr ea se in pu r it y a t h igh er
t em per a t u r es cou ld a lso be du e t o in cr ea sed solu biliza t ion of ot h er oleor esin s fr om t h e cells by t h e h ydr ot r ope
solu t ion . At a t em per a t u r e of 30 C, pr oba bly on ly
dest a biliza t ion of t h e liqu id la m ella r st r u ct u r e of cell
m em br a n e t a kes pla ce, wh ich en a bles m or e select ive
t r a n spor t of piper in e in t o h ydr ot r ope solu t ion s.
Beyon d t h e cr it ica l speed of a git a t ion , t h e m a jor
r esist a n ce t o m a ss t r a n sfer lies wit h in t h e pa r t icle. To
su bst a n t ia t e ou r r esu lt s on in t r a pa r t icle r esist a n ce, t h e
pa r t icle size of t h e r a w m a t er ia l wa s va r ied fr om 50 m
to a mesh size of 710 m (Table 1). A significant decrease
in t h e pu r it y of t h e ext r a ct ed piper in e fr om 98 t o 89%
wa s obser ved on r edu ct ion of t h e pa r t icle size. Th e
in cr ea sed r u pt u r in g of t h e cell wa lls u pon size r edu ct ion
in cr ea sed t h e a ccessibilit y of t h e h ydr ot r ope solu t ion t o
t h e cellu la r m a t r ix. Th is m igh t lea d t o solu biliza t ion of
ot h er solu t es a lon g wit h piper in e. F in e gr in din g is
expen sive, bu t it pr ovides m or e r a pid a n d possibly
t h or ou gh lea ch in g. H owever , it su ffer s t h e disa dva n t a ge
t h a t t h e weigh t of t h e liqu id a ssocia t ed wit h t h e set t led
solid m igh t be h igh , so a con sider a ble a m ou n t of solven t
is u sed t o wa sh t h e solid r et en t a t e/ca ke fr ee of solu t e,
m a kin g t h e r esu lt in g solu t ion qu it e dilu t e. Coa r sely
gr ou n d pa r t icles, on t h e ot h er h a n d, lea ch m or e slowly
a n d possibly less t h or ou gh ly, bu t on dr a in in g, t h ey
r et a in r ela t ively lit t le solu t ion , r equ ir e less wa sh in g,
a n d t h u s pr ovide a m or e con cen t r a t ed fin a l solu t ion .
Co n c lu s io n
H ydr ot r ope solu t ion s per m ea bilized t h e per ica r p of
Piper n igru m fr u it s a n d, t h er efor e, fa cilit a t ed t h e select ive ext r a ct ion of piper in e. Th e h ydr ot r ope m olecu les
pr oba bly a dsor b on t h e cellu losic cell wa ll, disor ga n ize
it s st r u ct u r e, a n d t h en pen et r a t e in t o t h e cell m em br a n e, a ssist in g in disor der in g t h e a m ph iph ilic lipid
bila yer a n d per m ea bilizin g it t o en a ble t h e ea sy r elea se
of piper in e. Th e pr esen ce of ph osph or u s r edu cin g su ga r s
a n d a m in o a cids in t h e ext r a ct ph a se su ggest s t h e
br ea kdown of cellu lose, a s well a s m em br a n e pr ot ein s,
to some extent. A substantial enhancement in extraction

2975

r a t e wa s obser ved a s a r esu lt of t h e st r u ct u r a l ch a n ges

in t h e biom a t r ix t r igger ed by t h e h ydr ot r ope a ct ion . Th e
pr im a r y st ep of t h e ext r a ct ion pr ocess in volved pen et r a t ion of t h e h ydr ot r ope in t o t h e ext er n a l cellu losic cell
wa lls, wh ich offer m a jor r esist a n ce t o m a ss t r a n sfer of
t h e h ydr ot r ope solu t ion . Th e secon d st ep con st it u t es t h e
solu biliza t ion of piper in e a n d it s ba ck t r a n sfer in t o t h e
ext er n a l solu t ion . Th e pr ocess wa s opt im ized wit h
r espect t o con cen t r a t ion , t em per a t u r e, a n d pa r t icle size
r equ ir ed for ext r a ct ion of piper in e. In a ddit ion , piper in e
cou ld be r ecover ed by sim ple dilu t ion wit h wa t er in
h igh ly pu r e for m a n d cou ld be dir ect ly a pplied for
for m u la t ion s a s it wa s fr ee fr om a n y con t a m in a t ion .
H ydr ot r opic ext r a ct ion sh ows t r em en dou s pot en t ia l for
cell per m ea biliza t ion a n d select ive ext r a ct ion of bioa ct ive com pou n ds on a com m er cia l sca le. Alt h ou gh we
h a ve h ypot h esised t h e m ech a n ism of h ydr ot r opic ext r a ct ion in t er m s of dest a biliza t ion of t h e cellu la r wa ll
st r u ct u r e, fu r t h er in vest iga t ion s a r e n eeded t o con fir m
t h is t h eor y.
Ac k n o w le d g m e n t
We a ckn owledge fin a n cia l su ppor t for t h is wor k t o t h e
Cou n cil for Scien t ific a n d In du st r ia l Resea r ch (CSIR),
Gover n m en t of In dia .
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R eceived for review Sept em ber 21, 2001

R evised m an u script received Ma r ch 19, 2002
Accepted Ma r ch 24, 2002
IE 0107845