in vitro binding of lipid (olive oil and ghee) with chitosan under the

78
IN VITRO BINDING OF LIPID
(OLIVE OIL AND GHEE) WITH CHITOSAN UNDER
THE CONDITIONS MIMICKING THE GASTROINTESTINAL TRACT
N. J. Noor Izani*1, Md. Rafiquzzaman, M. D. Nur Aidah and M. Mohd Rafi
‫أﺟﺮﻳﺖ دراﺳ ﺔ ﻣﻌﻤﻠﻴ ﺔ ﻻرﺗﺒ ﺎط ﻣ ﺎدة آﻴﺘ ﻮزان ﺑﺎﻟﺸ ﺤﻮم ﺗﺤ ﺖ ﻇ ﺮوف ﺗﺤ ﺎآﻲ اﻟﻘﻨ ﺎة اﻟﻬﻀ ﻤﻴﺔ اﻟﻤﻌﻮﻳ ﺔ وﺧﺼﻮﺻ ًﺎ‬
‫ وﻗﺪ ﺗﻢ اﺳﺘﺨﺪام زﻳﺖ اﻟﺰﻳﺘﻮن واﻟﺴﻤﻦ ﻟﺘﻤﺜﻴﻞ ﻣﺤﺘﻮﻳﺎت‬.‫درﺟﺔ اﻟﺤﻤﻮﺿﺔ )اﻷس اﻟﻬﻴﺪروﺟﻴﻨﻲ( ﻟﻠﻤﻌﺪة واﻹﺛﻨﻰ ﻋﺸﺮ‬
‫ وأﻇﻬﺮت اﻟﻨﺘﺎﺋﺞ أن‬.‫ واﺳﺘﺨﺪام ﻣﺎدة آﻴﺘﻮزان )اﻟﻤﻌﺎﻟﺞ وﻏﻴﺮ اﻟﻤﻌﺎﻟﺞ( ﻟﻴﺮﺗﺒﻂ ﺑﺎﻟﺸﺤﻮم اﻟﻤﺬآﻮرة‬،‫اﻟﻐﺬاء ﻣﻦ اﻟﺸﺤﻮم‬
.‫آﻴﺘﻮزان اﻟﻤﻌ ﺎﻟﺞ أﻋﻄ ﻰ ارﺗﺒﺎﻃ ًﺎ أﻓﻀ ﻞ ﺑﺎﻟﺸ ﺤﻮم )زﻳ ﺖ اﻟﺰﻳﺘ ﻮن أو اﻟﺴ ﻤﻦ( ﺑﺎﻟﻤﻘﺎرﻧ ﺔ ﻣ ﻊ آﻴﺘ ﻮزان ﻏﻴ ﺮ اﻟﻤﻌ ﺎﻟﺞ‬
‫ وﻗﺪ أﺛﺮت ﻓﺘﺮة اﻟﺤﻀﺎﻧﺔ اﻟﻤﺼﺤﻮﺑﺔ‬.‫وأﻇﻬﺮ آﻼ اﻟﻨﻮﻋﻴﻦ ﻣﻦ آﻴﺘﻮزان ُأﻟﻔﺔ أﻋﻠﻰ ﺗﺠﺎﻩ زﻳﺖ اﻟﺰﻳﺘﻮن أآﺜﺮ ﻣﻦ اﻟﺴﻤﻦ‬
.‫ وﺣﺪﺛﺖ اﻟﺪرﺟﺔ اﻟﻘﺼﻮى ﻟﻼرﺗﺒ ﺎط ﺑﻌ ﺪ ﺳ ﺎﻋﺘﻴﻦ وﻧﺼ ﻒ ﻣ ﻦ اﻟﺤﻀ ﺎﻧﺔ‬،‫ﺑﺎﻟﺮج أﻳﻀًﺎ ارﺗﺒﺎط اﻟﺸﺤﻮم ﺑﻤﺎدة آﻴﺘﻮزان‬
‫وﺗﺒﻴﻦ أن ﻧﺴﺒﺔ ﻣﺎدة آﻴﺘﻮزان إﻟﻰ اﻟﺸﺤﻮم اﻟﻤﺴﺘﺨﺪﻣﺔ ﺗﺆﺛﺮ ﻋﻠﻰ آﻤﻴﺔ اﻟﺸﺤﻮم اﻟﻤﺮﺗﺒﻄﺔ ﻟﻜﻞ ﻏﺮام ﻣﻦ ﻣﺎدة آﻴﺘﻮزان‬
‫ ﻏ ﺮام ﺑﻴﻨﻤ ﺎ ﻓ ﻲ اﻟﺴ ﻤﻦ آﺎﻧ ﺖ‬0.14±7.1 ‫ إﻟ ﻰ‬0.28±4.6 ‫وﺗﺮاوح زﻳﺖ اﻟﺰﻳﺘﻮن اﻟﻤﺮﺗﺒﻂ ﺑﻜ ﻞ ﻏ ﺮام ﻣ ﻦ آﻴﺘ ﻮزان ﺑ ﻴﻦ‬
‫ واﻟ ﺮج ﻟﻤ ﺪة ﺳ ﺎﻋﺘﻴﻦ وﻧﺼ ﻒ‬،‫ ﻣﻦ آﻴﺘﻮزان إﻟﻰ اﻟﺸﺤﻮم‬100:1 ‫ ﻏﻢ ﻋﻨﺪ ﻧﺴﺒﺔ ﺗﺠﺮﻳﺒﻴﺔ ﻣﻘﺪارهﺎ‬0.28±6.2 - 0.28±3.6
‫ أن اﻟﺸﻜﻞ اﻟﻤﺴﻴﻄﺮ ﻟﻤﺎدة آﻴﺘﻮزان ﻋﻨﺪ درﺟﺔ‬pka ‫ وﺗﺸﻴﺮ ﻗﻴﻤﺔ اﻟـ‬.‫ درﺟﺔ ﻣﺌﻮﻳﺔ‬37 ‫ﻋﻨﺪ درﺟﺔ اﻟﺤﺮارة اﻟﻔﺴﻴﻮﻟﻮﺟﻴﺔ‬
.‫( هﻮ آﻴﺘﻮزان ﻏﻴﺮ ﻣﺸﺤﻮن ﺗﺮﺳﺐ ﻣﺘﺮاﻓﻘًﺎ ﻣﻊ اﻟﺸﺤﻢ وﻣﺪﻣﺼًﺎ ﻋﻠﻴﻪ‬7 ‫ﺣﻤﻮﺿﺔ اﻹﺛﻨﻰ ﻋﺸﺮ )ﺣﻮاﻟﻲ‬
An in vitro binding study of chitosan with lipid was carried out under the conditions
mimicking the gastrointestinal tract, with special reference to the pHs of stomach and
duodenum. Olive oil and ghee were used to represent lipid components of diet, and
chitosan (treated and untreated) was used to bind with the mentioned lipids. The results
obtained from this study showed that the treated chitosan gave better binding to lipid
(olive oil or ghee) compared with the untreated chitosan. Chitosan (treated or untreated)
showed higher affinity towards olive oil than ghee. Incubation period accompanied with
shaking also influenced the binding of lipid to chitosan and the maximum binding was
attained after 2.5 hrs of incubation. The ratio of chitosan to lipid used was found to
affect the amount of lipid bound per gram of chitosan. Olive oil bound per gram of
chitosan was 4.6 ± 0.28 - 7.1 ± 0.14 g while ghee was 3.6 ± 0.28 - 6.2 ± 0.28 g under
1:100 experimental ratio of chitosan to lipid, and shaking for 2.5 hrs at physiological
temperature of 37 oC. Consideration of pKa value suggests that the predominated form
of chitosan at duodenal pH (~7.0) is uncharged chitosan, which is precipitated along
with the lipid adsorbed onto it.
Key words: Chitosan; olive oil; ghee; lipid binding; mimic gastrointestinal tract
School of Health Sciences, Universiti Sains Malaysia, 16150
Kubang Kerian, Kota Bharu, Kelantan, Malaysia.
*
To whom correspondence should be addressed
E-mail: [email protected]
Saudi Pharmaceutical Journal, Vol. 17, No. 1 January 2009
IN VITRO BINDING OF LIPID WITH CHITOSAN
Introduction
Chitosan is a natural product prepared from the
exoskeletons of crustaceans (1-2) such as crabs,
shellfish, shrimps, lobsters etc.
Chitosan was
isolated from mushroom and described for the first
time by Braconnot (2) in 1811. Chemically, chitosan
is represented as a β-(1,4)-D-glucosamine polymer
(Fig. 1). However, 5-30% free amino (-NH2) groups
in chitosan remain as acetylated form (2). Thus,
chitosan is not a single chemical entity rather a copolymer of D-glucosamine and N-acetyl-Dglucosamine (3). It has been shown through
toxicological studies that chitosan is a less or
79
with ineffective ingredient because of lower cost of
the raw materials (15). In either case, the lipid
binding will be sharply reduced and thus it may not
bring desirable benefit to its user. On the other hand,
Nauss et al (6) suggested for studying the interaction
of individual lipid with chitosan. We, therefore, have
sought to demonstrate how the purity of chitosan
(treated or untreated chitosan), incubation period
(accompanied with shaking), and the ratio of
chitosan to lipid influence the adsorption of lipid
(olive oil or ghee) on chitosan. The results have been
also discussed in this paper in the light of the
findings of the other workers.
Materials and Methods
Fig. 1. Chitosan
non-toxic (2, 4, 5) substance and because of its fatbinding properties (6-8) it has been approved as a
food additive recently in several developed countries
(9).
The ability of chitosan to bind with
triglycerides, fatty acids and other sterol compounds
gives it a commercial value as a dietary supplement.
Chitosan is not metabolized (10) and thus it has no
nutritive and caloric value, which further attracts it
to the obese people. The use of chitosan as dietary
supplement (2, 11) was in the highest level
amounting to 1,000 tons in 2000 among its use in
numerous other fields.
Though chitosan is able to exhibit numerous
biological properties such as healing of ulcer or
lesions (12), inhibition of bacterial growth (13, 14),
etc. however, the degree of binding of lipid by
chitosan is the important consideration to its users
especially to the obese people. Usually, chitosan is
5-30% N-acetylated (2). N-acetylated part of
chitosan structurally resembles the repeating unit of
chitin, which (i.e. chitin) is much less effective in
binding with lipid as it is evident from its less
hypolipidemic activity (7, 8) compared to that of
chitosan. Manufactures may produce their product
using poor quality chitosan (having less degree of
deacetylation or virtually chitin) or chitosan diluted
Saudi Pharmaceutical Journal, Vol. 17, No. 1 January 2009
1. Chitosan:
A commercial preparation of chitosan was
bought from a local pharmacy in Malaysia. Obtained
chitosan was processed further as described later in
this section and accordingly the prepared samples
were termed as untreated chitosan and treated
chitosan. Cornstarch (Maizena, Malaysia) was used
as a control in this study.
2. Lipids:
Ghee or clarified butter (Barkath, Malaysia) and
olive oil (Bertolli, Italy), were the lipids used in this
study. Both ghee and olive oil were purchased from
the local market of Malaysia.
3 Chemicals:
Chemicals used were hydrochloric acid (Merck,
Germany), sodium hydroxide (Merck, Germany),
potassium chloride (Merck, Germany), sodium
chloride (HmbG chemicals), sodium hydrogen
phosphate (BDH, UK) and potassium dihdrogen
phosphate (APS chemicals). All the purchased
chemicals were of GR and AR grades.
4. Preparation of untreated and treated chitosan
samples:
The chitosan pellets of the commercial product
mentioned above was ground into powder before it
was used in the experiment of the present study. The
particle size was in the range of 25-250 μm. This
powdered chitosan has been referred as an untreated
chitosan hereafter in this paper.
The commercial chitosan as obtained is usually
not 100% pure despite it is labeled as pure and also
its degree of deacetylation may be poor. Therefore, a
80
NOOR IZANI ET AL
simple procedure, as follow, was adopted to remove
any additive materials from the chitosan and to
improve its deacetylation. Three grams of chitosan
(ground to powder) was added to 20 mL of 0.1 M
HCl solution (pH 2.0) and was mixed for one hour at
room temperature. Then, the tubes were centrifuged
at 2500 rpm for 15 minutes. The supernatants
containing dissolved materials were aspirated to new
tubes and then neutralized with NaOH solution to
pH 7.0. The tubes were then centrifuged at 2500 rpm
again for 15 minutes. The sediments formed were
collected and dried in an oven at 55 ± 5 oC. The
dried whitish powder was assumed as the partially
pure chitosan and it has been referred as treated
chitosan hereafter in this paper.
adjusted to 7.0 ± 0.2 using NaOH and HCl as
needed. This step was done to mimic the pH
condition of duodenum of gastrointestinal tract. The
tubes were placed in a shaker bath for 30 minutes at
37 oC and then centrifuged at 2200 rpm for 20
minutes. The unbound lipid (olive oil or ghee) at the
uppermost layer of the tubes were aspirated onto
tarred weighing boat and then was dried placing it in
a hot air oven at 55 ± 5 oC, which was subsequently
cooled to room temperature and weighed. Finally,
the amount of bound lipid (olive oil or ghee) was
calculated and recorded. Samples were run in
duplicate or triplicate.
5. In-vitro lipid adsorption on chitosan:
It was carried out following the method used by
Rockway (15) with slight modification. In this
experiment, 0.05 g chitosan (sample) and cornstarch
(control) were weighed separately and placed into
two separate labeled 50 mL Falcon tubes,
respectively. Then 2.5 mL of 0.1 M HCl solution
was added into each of the tubes and vortexed well
for about 20 minutes at room temperature. The pH
was adjusted to about 2.0 in order to mimic the
condition of the gastric juice in stomach. Lipid
(olive oil or ghee) 0.05 to 15 g, depending on the
experiment, was added into each of the tubes and
was vortexed again for about 2 minutes. The tubes
were capped tightly and placed in a shaking water
bath for 2 hours (except the experiments where
incubation period was varied) at the physiological
temperature 37 oC. Shaking was important to make
a homogeneous mixture of the contents of the tubes.
After 2 hours, the tubes were removed from the
shaker bath and 16 ml of 10 mM PBS (Phosphate
Buffer Solution) of pH 7.4 was added to each tube
and mixed well with a vortex mixer. The pH was
Chitosan through adsorption of lipid on it may
influence lipid absorption and metabolism in the
gastrointestinal tract. So, we devised an experiment
mimicking the gastrointestinal tract (2, 15) with
special reference to pHs of the stomach and
duodenum to see the influence of chitosan on lipid
absorption and metabolism there. Olive oil and ghee
were used to represent lipid components of diet, and
chitosan was used to exert its in vitro action on lipid
absorption and metabolism in the mimicked
gastrointestinal tract system in the present study
through adsorbing lipid on it. Cornstarch was used
as a control. It was observed that the amount of
bound olive oil or ghee with cornstarch was very
negligible (data not shown) but binding of lipid
(olive oil or ghee) with chitosan (treated or
untreated) was increased and finally reached to
maximum followed by a bit decreasing trend with
the increase of lipid proportion to chitosan (Table 1
and Table 2). Nauss et. al. (6) observed similar
saturation of lipid binding on chitosan where as
Rodriguez and Albertengo (1) found that when
the dietary oil (sunflower oil) content was
Results and Discussion
Table 1. Binding of Lipid (Olive Oil) with Chitosan Under Varying Proportion of Lipid (Olive oil).
Weight of chitosan
Weight of lipid
used (g)
(olive oil) used (g)
0.05
0.05
0.5
1.0
5.0
10.0
15.0
Saudi Pharmaceutical Journal, Vol. 17, No. 1 January 2009
Chitosan : lipid
(olive oil) (w/w)
1:1
1:10
1:20
1:100
1:200
1:300
Bound lipid (Olive Oil) g/g of
Chitosan, Mean ± SD
Untreated chitosan Treated chitosan
0.6 ± 0.00
0.6 ± 0.00
3.2 ± 0.28
4.0 ± 0.28
3.8 ± 0.57
5.2 ± 0.28
4.6 ± 0.28
7.1 ± 0.14
4.4 ± 0.28
10.0 ± 0.28
4.0 ± 0.57
9.6 ± 0.28
IN VITRO BINDING OF LIPID WITH CHITOSAN
81
Table 2. Binding of Lipid (Ghee) with Chitosan Under Varying Proportion of Lipid (Ghee).
Weight of
Weight of lipid
chitosan used (g) (ghee) used (g)
0.05
Chitosan : lipid (ghee)
w/w
0.05
0.5
1.0
5.0
10.0
15.0
1:1
1:10
1:20
1:100
1:200
1:300
Bound lipid (ghee) g/g of Chitosan,
Mean ± SD
Untreated chitosan
Treated chitosan
0.6 ± 0.00
0.6 ± 0.00
1.9 ± 0.14
3.7 ± 0.14
3.2 ± 0.28
3.9 ± 0.42
3.6 ± 0.28
6.2 ± 0.28
3.5 ± 0.42
5.8 ± 0.28
3.2 ± 0.28
5.5 ± 0.14
Table 3. Percent of Fatty Acid Content in the lipids Olive oil and Ghee.
Lipid
Olive oil
Ghee
Lauric
acid
0.0
0.2
Myrisitic
acid
0.0
4.7
Fatty acid content (in %)
Stearic
Plamitoleic
acid
acid
3.0
0.7
20.4
2.7
Palmitic
acid
11.1
27.1
Oleic
acid
74.8
39.1
Linoleic
acid
9.0
2.1
Linolenic
acid
0.6
0.8
Table 4. Effect of Incubation (Accompanied with Shaking) Period on Binding of Olive Oil (1 g) with Treated
Chitosan (0.1 g).
Chitosan : Olive oil used
(w/w)
1:10
Incubation period (hrs)
Olive oil bound (g) / (g)
chitosan, Mean ± SD
2.4 ± 0.14
3.4 ± 0.07
5.2 ± 0.21
5.3 ± 0.00
0.5
1.5
2.5
3.5
Table 5. Amount of Lipid Bound per Gram of Chitosan in Different Studies.
Amount of chitosan used, Chitosan : lipid
(Type of chitosan)
used (w/w)
0.05 g
1:100
(Commercial, untreated)
Incubation period Temperature
(hrs)
in oC
2.5
37
Lipid (g) bound/ g of
chitosan
4.6 ± 0.28
(for olive oil)
3.6 ± 0.28
(for ghee)
7.1 ± 0.14
(for olive oil)
6.2 ± 0.28
(for ghee)
264.1 ± 104.9
(for olive oil)
Source of data
Present study
0.05 g
(Commercial, treated)
1:100
2.5
37
0.05 g
(Raw or finished product)
1:400
2.5
37
0.001 mg
(Commercial but treated
wit 4% Acetic acid and
then freeze dried,
deacetylation 90%)
1g
(Prepared in lab, degree of
deacetylation 78 to 95%)
1:1 to 40
0.25
20
4-5
(for cholate, glycocholate,
taurocholate, dodecylsulfate,
& ox bile)
Reference 6
1:~10
1.5
37
3.5-5.1
(for sunflower oil)
Reference 1
Saudi Pharmaceutical Journal, Vol. 17, No. 1 January 2009
Present study
Reference 15
82
increased, the entrapped oil percentage was
decreased. To explain the results of this study it is
assumed that fixed amount of chitosan (0.05 g)
contained obviously fixed number of binding sites.
When low amount of olive oil or ghee was used then
binding sites in chitosan remained unbound, which
were filled with the increased amount of olive oil or
ghee and ultimately resulted in a saturation point.
Results as shown in the Table 1 and Table 2 also
indicated that in case of treated chitosan the amount
of lipid (olive oil or ghee) bound per gram of
chitosan was increased in a more pronounced way,
and moreover saturation was reached relatively with
more amount of bound lipid (olive oil or ghee)
compared to that of the bound lipid found in
experiments with untreated chitosan. Though the
difference of amount of bound lipid per gram of
chitosan between treated and untreated chitosan was
not much different (6) (Table 1 and Table 2) but
there was significantly a difference between the
effects of the two chitosans used. This observation
suggested that during treatment of chitosan, as
described in the experimental section, deacetylation
took place and thus treated chitosan contained more
binding sites, which resulted more binding of lipid in
it compared to that in equal amount of untreated
chitosan. From the Table 1 Table 2, it is also clear
that binding of ghee with chitosan (treated or
untreated) was not as much as in case of olive oil.
Fatty acids content of olive oil and ghee have been
cited from literature (16) in Table 3, which shows
that olive oil differs from ghee especially in stearic
acid (~ 7 times less) and linoleic acid (~ 4 times
high) content. Therefore, we assumed that the
difference of content of the mentioned fatty acids
might have relevance to the difference in binding of
olive oil and ghee with chitosan. Kristbergsson (3)
recently reported that interactions between chitosan
and lipid largely depended on, among other aspects,
the type of lipid used.
In another experiment tubes containing 1:10
chitosan to lipid were shaken at 37 oC under stomach
pH followed by duodenum pH but the total
incubation period was varied from 0.5 hour to 3.5
hours. Results of lipid bound per gram of chitosan
found from this experiment are tabulated in Table 4.
This table thus shows the effect of incubation period
with shaking on the binding of olive oil with
chitosan. It is clear from the results that binding of
olive oil with chitosan was increased with the
increase of incubation period, which was in the
Saudi Pharmaceutical Journal, Vol. 17, No. 1 January 2009
NOOR IZANI ET AL
line of our expectation. Similar result was obtained
for binding of ghee with chitosan (data not shown).
Nomanbhay and Palanisamy (17) observed that
efficacy of modified chitosan in removing chromium
from water was increased (from 60% to 90%) when
the contact time of chitosan with chromium was
increased (from 30 minutes to 180 minutes). Though
their experimental model was different from us but
their finding of influence of contact time on the
binding of adsorbent with chitosan is parallel and
supporting our observation.
In order to compare our findings with other
workers, we searched the literature and compiled the
data in the Table 5. The amounts of olive oil bound
per gram of untreated and treated chitosan found
from our experiment were 4.6 ± 0.28 g and 7.1 ±
0.14 g, respectively and the amount of ghee bound
per gram of untreated and treated chitosan were 3.6
± 0.28 g and 6.2 ± 0.28 g, respectively (Table 5),
which is very similar to the findings of other
workers (1, 6) except to the findings of Rockway
(15) Nauss et. al. (6) concluded that chitosan binds
4-5 times of its weight that means 4-5 g of lipid was
bound per gram of chitosan (Table 5). Recently,
Rodriguez and Albertengo (1) showed that 3.5-5.1 g
of lipid (sunflower oil) was bound per gram of
chitosan (Table 5). Rockway (15), in the contrary,
found the amount of lipid (olive oil) on average
bound per gram of chitosan product as 264.1 ± 104.9
g except for Absorbitol (a commercial chitosan
product), which showed even higher binding
capacity of lipid (~ 776 g of lipid (olive oil)/g of
Absorbitol, data not shown). Rockway’s (15) finding
is thus does not show similarity with our and others
(1, 4) findings. Rockway (15), however, used very
high 1:400 chitosan to olive oil ratio; even then their
results seems unexpectedly high to us because use of
1:300 chitosan (treated one) to olive oil ratio in our
experiment resulted only ~10 g of olive oil binding
per gram of treated chitosan (Table 1). The reason
behind the unexpectedly high binding of olive oil
with chitosan found by Rockway was not discussed
in his report and it is not clear to us too. However, it
needs to be addressed in future to understand in
depth the phenomenon of lipid binding with
chitosan.
We noticed that chitosan emulsified (1) olive oil
and ghee at low i.e. stomach pH (~2), however, it
was precipitated (1, 6) out through white flocculus
formation at higher i.e. duodenal pH (7.0 ± 0.2). In
the experimental system fatty acids may be
IN VITRO BINDING OF LIPID WITH CHITOSAN
83
generated from the partial hydrolysis of lipid (olive
oil or ghee). pKa value of long chain fatty acid is 9.0
(18) while chitosan is 6.2 (1, 19-20). The extent of
protonated chitosan and the anion of fatty acid
existing at different pHs can be calculated and
presented as a scheme (Scheme 1: (a) and (b)). We,
therefore, speculated that chitosan emulsified olive
oil and ghee at low i.e. stomach pH (~2) through
ion-dipole electrostatic interaction between –NH3+
part of Chi-NH3+ (dominated form of chitosan at pH
~2, Scheme 1a) and the electron rich -COOH part of
R-COOH (dominated form of fatty acid at pH ~2,
Scheme 1b) or electron rich ester part of triglyceride
(not shown in the scheme) of olive oil or ghee.
Precipitation of chitosan that took place through
white flocculus formation at duodenal pH (~7.00)
was most possibly due to the transformation of
major proportion of cationic chitosan (Chi–NH3+) at
that pH to uncharged chitosan (Chi-NH2) and
precipitation was accompanied along with the lipid
bound on it. Since chitosan (pKa=6.2) and lipid
(olive oil or ghee) and/ fatty acids (pKa=9; formed
from lipid) were expected to be virtually in
unionized form at duodenal pH(~7) then adsorption
of lipid on chitosan at that pH was mainly likely to
be the result of dipole-dipole, hydrogen bonding and
hydrophobic interactions, rather than ionic
interaction solely. Alkhamis et al. (21) predicted that
adsorption process between unionized chitosan and
unionized adsorbent such as allopurinol (pKa=10.25)
at neutral pH (=7) could be due to the force like
hydrogen bonding while Parfitt and Rochester (22)
also suggested hydrogen bonding and even van der
Waals forces for binding process between chitosan
and adsorbent. However, further studies are needed
to ascertain about the interactions and mechanism of
adsorption of lipid on chitosan in in vitro and hence
in in vivo gastrointestinal system to understand more
about the effect of chitosan in lipid absorption and
metabolism.
pHs
:
2
Protonated
Chitosan : Chi–NH3+
%
:
100
Unprotonated
Chitosan : Chi-NH2
% :
0
4.2
5.2
Chi–NH3+
99
Chi–NH3+
91
Chi–NH3+
50
Chi–NH3+
9
Chi-NH2
1
Chi-NH2
9
Chi-NH2
50
Chi-NH2
91
6.2
7.2
(a)
Fatty acid
%
Fatty acid
Anion
%
: R-COOH
:
100
-
: R-COO
:
0
R-COOH
100
R-COOH
100
-
-
R-COO
0
R-COO
0
R-COOH
99.8
-
R-COO
0.2
R-COOH
~98
-
R-COO
~2
(b)
Scheme 1. Relative Forms and Percentages of (a) Chitosan and (b) Fatty Acid, at Different pHs
(Abbreviations: Chi-, backbone of chitosan polymer; R- , aliphatic chain of fatty acid)
Conclusion
Cornstarch failed while chitosan is reaffirmed to
bind with lipid such as olive oil and ghee
quantitatively to an extent, which depends on degree
of deacetylation in chitosan, contact period between
Saudi Pharmaceutical Journal, Vol. 17, No. 1 January 2009
chitosan and lipid, and proportion of lipid to
chitosan in in vitro gastrointestinal system. This
binding property of chitosan can be extended to in
vivo to explain its hypolipidemic and weight loss
effect. Consideration of pKa values of chitosan and
fatty acids leads to suggest that chitosan emulsifies
84
NOOR IZANI ET AL
lipid at low such as at stomach pH through iondipole electrostatic chemical interaction. Flocculus
formation and sequestering of lipid by chitosan is
accomplished at higher such as duodenal pH due to
precipitation of uncharged chitosan along with the
lipid (olive oil or ghee) adsorbed on it. Adsorption
could be the result of mainly dipole-dipole,
hydrogen bonding and hydrophobic interactions
between chitosan and lipid rather than, as it is
usually thought for, ionic interaction solely.
10.
11.
12.
13.
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