Various medicinal plants are rich in certain bioactive compounds that can help in restoration of immune system but these compounds may be unstable in gastro intestinal tract due to elevated pH and harsh conditions that render the biotherapeutic compound ineffective in GI tract. Various particulate systems such as nanoparticles had been used to improve the pharmacokinetic and pharmacodynamic properties of various drugs. In our present research we have used chitosan, a biodegradable polymer as carrier of plant extract (i.e Ethanolic extract of leaves of Ziziphus mauritiana) having immunomodulatory property and checked its immunorestorative efficacy in immunosuppressed hostIt was found that the oral administration of leaf extract of plant loaded in chitosan nanoparticles is an efficient immunorestorer in Swiss albino mice whose immune system had been suppressed by giving intraperitoneal injection of hydrocortisone(10mg/Kg body weight).The nanoparticles system enhances stability in harsh conditions in GI tract and may be a better vehicle in future for efficient drug delivery.Keywords: Chitosan, nanoparticles, immunorestoration, Ziziphus mauritiana, Hydrocortisone, Immunosuppressio

Bhatia, Aruna

Chopra, Dimple

Mishra, Tulika

Shard, Pankaj

English

International Journal of Drug Delivery

International Journal of Drug Delivery 3 (2011) 381-385 
http://www.arjournals.org/index.php/ijdd/index 
 
Original Research Article 
                                                            
ISSN: 0975-0215 
Chitosan nanoparticles as Carrier of Immunorestoratory plant extract: 
synthesis, characterization and Immunorestoratory efficacy 
Aruna Bhatia1*, Pankaj Shard1, Dimple Chopra2, Tulika Mishra1 
 
*Corresponding author: 
Aruna Bhatia 
1Department of Biotechnology, 
Punjabi University Patiala, 
Punjab, India 147002 
E-mail: 
pankajshard21@gmail.com 
2 Department of Pharmaceutical 
Sciences, Punjabi University 
Patiala, Punjab, India 147002 
 
 
 
 
 
 
 
 
 
 
 
Abstract 
Various medicinal plants are rich in certain bioactive compounds that 
can help in restoration of immune system but these compounds may be 
unstable in gastro intestinal tract due to elevated pH and harsh 
conditions that render the biotherapeutic compound ineffective in GI 
tract. Various particulate systems such as nanoparticles had been used to 
improve the pharmacokinetic and pharmacodynamic properties of 
various drugs. In our present research we have used chitosan, a 
biodegradable polymer as carrier of plant extract (i.e Ethanolic extract of 
leaves of Ziziphus mauritiana)  having immunomodulatory  property and 
checked its immunorestorative efficacy in immunosuppressed host 
It was found that the oral administration of leaf extract of plant loaded in 
chitosan nanoparticles is an  efficient immunorestorer in Swiss albino 
mice whose immune system had been suppressed by giving 
intraperitoneal injection of hydrocortisone(10mg/Kg body weight). 
The nanoparticles system enhances stability in harsh conditions in GI 
tract and may be a better vehicle in future for efficient drug delivery  
Keywords: Chitosan, nanoparticles, immunorestoration, Ziziphus 
mauritiana, Hydrocortisone, Immunosuppression 
 
Introduction 
For the past few decades there is a considerable 
research interest in the area of drug delivery 
systems as carriers for small and large molecules. 
Nanotechnology has been increasingly employed 
in drug delivery as it increases the drug 
dissolution rate, leading to enhanced drug 
absorption and bioavailability. Polymeric 
nanoparticles (NPs), prepared via coprecipitation 
of drugs and polymers are promising drug 
delivery vehicles for treating diseases with 
improved efficacy and reduced toxicity [1]. 
Particulate systems are found to be very efficient 
in drug delivery. DNAzyme and si RNA is used 
in gene therapy by encapsulating it in chitosan 
nanoparticles [2]. Water soluble chitosan 
nanoparticles are also used as carrier of proteins 
[3]. Particulate system like nanoparticles [4,5] 
has been used as a physical approach to alter and 
improve the pharmacokinetic and 
pharmacodynamic properties of various drug 
molecules. There are many materials known to be 
used for preparation of nanoparticles like 
cyclodextrins, chitosan, ceramics etc. but all the 
materials cannot be employed in in vivo systems 
because of their toxicity. Formulation of 
doxorubicin-polylactide nanoconjugates is 
employed for cancer drug delivery [6]. 
Bioadhesive nanoparticles of fungal chitosan are 
used for oral DNA delivery [7].On the other hand 
plants have been reported to have medicinal 
values, but still a number of plants remain 
unexplored for their immunomodulatory 
Bhatia et al. International Journal of Drug Delivery 3 (2011) 381-385 
 
 
 
382
property. Moreover, there is hardly any report 
where a bioactive plant extract has been delivered 
in nanoparticles. In our lab previous studies 
revealed that leaf extract of Ziziphus mauritiana 
was immunomodulatory. The present study was 
conducted with the aim to find out whether the 
extract efficacy can be enhanced by its delivery 
through non toxic biodegradable chitosan 
nanoparticles. 
 
Methods 
Preparation of Extract 
Aqueous: Ethanolic (1:1) extract of shade dried 
crushed leaves of Ziziphus mauritiana percolated 
in solvent and extract was filtered (whatmann 
paper), centrifuged (4000 rpm, 10 min) and 
concentrated under reduced pressure in a thin 
film evaporator at 50 ± 5 °C. 
 
Preparation of chitosan nanoparticles [8] 
Chitosan (0.17-0.18 % w/v) added to distilled 
water containing 2%  acetic acid and 1.2 % 
Tween 80 . The solution was placed on a 
magnetic stirrer for 68- 72 h with an intermittent 
sonication every 24 h in a bath sonicator. Sodium 
Sulphate 17% (w/v)  was added  and stirred for 1 
h on magnetic stirrer  and sonicated again for 15 
min . The nano sized particles were precipitated 
out. The size was found to be 395 nm.  
 
Loading of nanoparticles with leaf extract 
100 ml of nanoparticles solution was centrifuged 
at 5000 rpm for 15 min. The   wet pellet 
(6mg/ml) was taken and suspended in normal 
saline containing 0.5 mg/ml of (protein) 
powdered extract. The suspension was stirred on 
a magnetic stirrer at 300 rpm - 400 rpm for 4 h 
and later centrifuged to get loaded nanoparticles 
and protein content of supernatant was 
determined before and after loading of 
nanoparticles with plant extract by Lowry 
method. The protein content of supernatant was 
found to be 0.1 mg/ml which was initially 0.5 
mg/ml. Loading efficiency (LE) was determined 
using the formula  
          
  LE =Initial protein content in suspension –Final 
protein content in suspension × 100 
                                            Initial protein content 
in solution
The loading efficiency was found to be 80%.  
 
Animals 
Swiss albino mice (18-22 gm) of either sex were 
maintained on standard laboratory diet (Kisan 
Feeds Ltd., Mumbai, India) and having free 
access to tap water were employed in the present 
study. They were housed in the departmental 
animal house and were exposed to 12 hr cycle of 
light and dark. 
  
Induction of immunosuppression. 
Immunosuppressed state was produced by giving 
1 ml Hydrocortisone intraperitonially 
Experimental setup  
The animals of either sex were divided into six 
groups (n = 6) viz.:  
 Group I, untreated control;  
 Group II, Hydrocortisone (HC) treated; 
(HC only) 
 Group III, Plant extract (PE) treated (0.3 
mg/ml); (HC+P.E300) 
 Group IV, Plant extract treated (0.2 
mg/ml); (HC+P.E200) 
 Group V, Loaded nanoparticles (LNP’s) 
treated (3mg/day); (HC+LNP’s)  
 Group VI, Unloaded nanoparticles 
(ULNP’s) treated (3mg/day) ; 
(HC+ULNP’s) 
 
Follow up: All the groups were treated 
intraperitoneally with hydrocortisone (1ml /day) 
on day 0 and day 5th, and the respective drug 
orally. All the groups were immunized with 1% 
BSA (200 μl) intraperitoneally on day 7th, 12th  
and 17th of hydrocortisone treatment. All the 
animals were treated with 30 μl of carregeenan  
(20 mg/kg body weight/animal) dissolved in 
normal saline into the planter side of left foot and 
same volume of sterile normal saline was injected 
into the planter side of right foot Thickness of the 
footpad with vernier caliper was measured on day 
19th, 20th and 21st . The animals were sacrificed 
Bhatia et al. International Journal of Drug Delivery 3 (2011) 381-385 
 
on day 21st of the Hydrocortisone treatment and 
their spleen was excised in MEM for 
immunological assays. The numbers of cells were 
adjusted to 2Χ 106 viable cells/ml. The cells were 
employed to assess the immune status of the 
animals employing the various techniques:   Nitro 
blue Tetrazolium Chloride (NBT) reduction test 
[9], Inducible Nitric Oxide Synthase (iNOS) test 
[10], Bactericidal activity [11] and Delayed type 
of hypersensivity (DTH) response. 
 
Results and Discussion 
Characterization of nanoparticles 
Stability of nanoparticles in suspension is directly 
proportional to zeta potential. The zeta potential 
of nanoparticles was found to be 30mV. The 
observed positive zeta potential might be due to 
residual amine group which act as carrier of plant 
extract. 
Immunorestoration efficacy of plant extract 
delivered as such and through nano delivery 
Delayed type hypersensivity 
The delayed type hypersensivity response was 
more pronounced in all the groups after 48 hours 
(Table 1). The maximum response was observed 
in the animals treated with LNP’s.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The unloaded particles showed the lower 
response as compared to only extract treated 
groups showing that the activity observed was 
due to plant extract as LNP’s showed the 
maximum activity.  
 
Nitro Blue Tetrazolium test 
The maximum NBT index observed in LNP’s 
treated group as compared to all other groups 
(Table 2). The plant extract treated groups also 
showed the rise in activity in a dose dependent 
manner. LNP’s treatment increased the 
bioactivity by 2.3 folds as compared to PE 0.3 
mg/ml group. 
 
Inducible Nitric Oxide Synthase (INOs) 
The results of INOS activity were parallel to 
those of NBT. The inducible nitric oxide synthase 
activity was highest in Ziziphus mauritiana leaf 
extract loaded chitosan nanoparticles group as 
compared to other groups (Table 2). This shows 
that leaf extract loaded on nanoparticles has 
better bioavailability as compared to when  only 
plant extract is taken directly through oral route. 
 
Bactericidal activity:  
The bactericidal activity also followed the same 
trend. The LNP’s showed the maximum activity 
as compared to all other groups (Table 2). 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Figure 1: Comparison of increase in activity (number of folds) as compared to only 
HC treated control. DTH- Delayed type hypersensivity, NBT –Nitro Blue Tetrazolium,INOs- Inducible 
Nitric Oxide Synthase, PE – Plant Extract 
 
 
 
 
 
383
Bhatia et al. International Journal of Drug Delivery 3 (2011) 381-385 
 
 
Table 1: DTH response of different group of mice after 24h and 48h 
 
Table 2: Effect of test material on Immune response 
Legend: HC-only hydrocortisone treated; HC+ULNP’s- hydrocortisone and unloaded nanoparticles treated; HC+P.E1- 
hydrocortisone and 0.2 mg/ml plant extract treated; HC+P.E2- hydrocortisone and 0.3 mg/ml plant extract treated; HC+LNP’s- 
hydrocortisone and plant extract(300ppm) loaded nanoparticles treated; (↑) Number of times increase in activity as compared to 
only hydrocortisone treated group 
 
 
The possible reason for this potentiation of 
bioactivity of extract by delivery through 
nanaoparticles might be due to the fact that when 
given as such (unloaded) the extract might get 
degraded or digested by the gastro intestinal 
juices or enzymes etc. Nanoparticles are stable 
under elevated pH range [12] of gut and might 
not get degraded by the digestive enzymes or it 
may be due to the reason that there residence time 
in the gut is very less that is why they did not 
give time to intestinal juices to act upon. The 
Immunorestoratory activity of loaded 
nanoparticles was found to be higher as compared 
to plant extract, showing that nanoparticles can 
be the better vehicles for localized drug delivery. 
The higher immunomodulatory efficacy   may be 
due to control release of the plant extract locally. 
In short, the high diffusibility and better stability 
accounts for their overall immunomodulatory and 
Immunorestoratory efficacy. 
Effective oral drug administration is desirable but 
challenging owing to the nature of gastro 
intestinal tract. The highly acidic pH in the 
stomach, the enzymes and mucus in the gastro 
intestinal tract are complicating factors for the 
effective particle delivery [11]. The recent 
development in the field of pharmacy and 
medicine reveals that drugs / bacteria delivered in 
nanoparticles show better activity in the body but 
none of the reports in literature shows the 
delivery of plant extract in nanoparticles system. 
It is for the first time that we have employed 
Average footpad thickness after 
24h ( in mm) 
Average footpad thickness after 48h  
( in mm) 
Groups 
Test Control Test Control 
(HC+P.E200) 
 
1.69↑ 1.35 2.01↑ 1.39 
UNLP’s +HC 2.04↑ 1.68 2.36↑ 1.61 
(HC+P.E300) 
 
1.89↑ 1.39 2.38↑ 1.49 
LNP’s +HC 2.41↑↑↑ 1.22 2.90↑↑↑ 1.16 
HC only 
 
1.48↑ 1.30 2.67↑ 1.22 
Activity HC HC+ULNP’s HC+P.E1 HC+P.E2 HC+LNP’s 
NBT index 17.28 25.53 (↑1.47) 29.09 (↑1.67) 58.29 (↑3.95) 68.08 (↑9.14) 
iNOS  - 
 
30.76 (↑1.44) 41.93 (↑1.72) 50.86 (↑2.03) 71.87 (↑3.55) 
Bactericidal 16.38 29.91 (↑1.62) 32.19 (↑1.96) 55.98 (↑3.41) 53.27 (↑3.25) 
 
 
384
Bhatia et al. International Journal of Drug Delivery 3 (2011) 381-385 
 
plant extract loaded nanoparticles as therapeutic 
agents in animals. 
In the present study, attempts were made to 
entrap leaf extract from a common medicinal 
plant Ziziphus mauritiana in nanoparticles made 
of chitosan, a biodegradable and biocompatible 
polymer and in vivo studies were conducted to 
see the Immunorestoratory efficacy of Ziziphus 
mauritiana leaf extract loaded nanoparticles in 
immunosuppressed mice. 
Delayed type of hypersensivity, Nitro blue 
tetrazolium reduction and Inducible nitric acid 
synthase tests were used as parameters to study 
the immune status of host. Results revealed that 
immunological response was highly pronounced 
when plant extract was given loaded on 
nanoparticles as compared to plant extract alone. 
 
Conclusion 
Delivery of plant extract through nanoparticles as 
a vehicle is a better way  to improve the efficacy 
of result in expression of bioactivity of plant 
extract as an immunomodulator as well as an 
immunorestorer. 
 
Competing interests: No competing Interests 
as declared by authors  
Authors Contribution: AB designed the 
experiment. Experimentation was done by PS. PS 
did manuscript drafting and carried out extraction  
of plant extract of  Ziziphus mauritiana  by the 
method given by TM and  formulated 
nanoparticles by the method given by DC and 
characterization of nanoparticles such as particle 
Size ,zeta potential is also done by PS. AB 
directed the Study and drafting of manuscript by 
many revisions. 
 
Acknowledgements: This work was 
supported by the Department of Biotechnology 
Punjabi University Patiala, Punjab, India 147002. 
 
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Chitosan nanoparticles as Carrier of Immunorestoratory plant extract: synthesis, characterization and Immunorestoratory efficacy

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Chitosan nanoparticles as Carrier of Immunorestoratory plant extract: synthesis, characterization and Immunorestoratory efficacy

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