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Research Article

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IMMUNOADJUVANT POTENTIAL OF
AZARDICHTA INDICA AGAINST RABIES,
HEPATITIS AND DPT VACCINE ANTIGEN
Amit Gupta, Sushama R. Chaphalkar
Vidya Pratishthans School of Biotechnology (VSBT) Vidyanagari, Baramati, District Pune, M.S., India.

ABSTRACT
Aim: To study the immunoadjuvant potential of Azadirachta indica against different vaccine antigens (Rabies, hepatitis and
DPT).
Methodology: In order to study the IgG antibody titre of hepatitis B surface antigen (HBsAg), diphtheria-pertussis-tetanus (DPT)
and rabies vaccine antigen with variable doses of aqueous extract of Azadirachta indica and also estimate the human blood
counts (i.e. lymphocytes, monocytes and granulocytes) were evaluated.
Results: Azadirachta indica has anti-HBsAg, anti-DPT and anti-rabies antibody IgG titre as compared to standard vaccine. In
addition, vaccines containing Azadirachta indica increased the number of lymphocytes, monocytes and granulocytes count in
whole blood as compared to standard vaccine.
Conclusion: The study presents evidence that Azadirachta indica is a plant based adjuvant candidate of choice over commercial
vaccine which is available in the market and improving as well as maintaining the improved immune status against HBsAg, DPT
and rabies vaccine antigen.
Key Words: Azadirachta indica, Hepatitis B surface antigen, Diphtheria-pertussis-tetanus, Rabies

INTRODUCTION
The term medicinal plants which include various types of
plants used in herbalism and some of these plants including its fruit, seed etc have number of medicinal activities
or properties [1, 2, 3]. These plants may be considered as
rich resources of ingredients or raw materials which can
be used in synthesis as well as for drug development [2,
3, 4 5, 6]. Besides that these medicinal plants play a crucial role in the development of human cultures around
the whole world including India. Moreover, some medicinal plants consider as important source of animal or
human nutrition and as a result of that these medicinal
plants recommended for their therapeutic values [4, 5,
6, 7]. One of the medicinal plant i.e. Azadirachta indica
(commonly known as neem; class Dicotyledonous; order
Fagales; family Meliaceae) is a plant used widely in the
Indian system of medicine for its diverse medicinal properties [4, 5, 6, 7, 8]. Its extracts or fraction have vast
immunopharmacological activities and these are used
as raw materials for pesticide, medicinal purposes etc.

Each part of this plant has its own therapeutic importance and uses which include: anti-fertility antipyretic,
anti-inflammatory, anti-malaria, anti-rheumatic and others [2, 3, 4, 7, 8, 9, 10]. The neem has been reported to
contain several biologically active constituents such as
azadirachtin, meliantriol, salanin, as well as nimbin and
nimbidin. The aqueous extract of leaf from Azadirachta
indica showed potent immunostimulant or immunomodulatory activity as evidenced by both humoral as well as
cell-mediated immune responses [10]. Oral administration of leaf aqueous extract showed higher levels of IgM
and IgG along with increased titer of anti-ovalbumin
antibody. In addition, neem oil has been shown to possess immunostimulant activity by selectively activating
the cell-mediated immune mechanisms to elicit an enhanced response to subsequent mitogenic or antigenic
challenge [10]. The dry flowers are considered tonic and
stomachic [8, 9]. Stem, leaves and seed oil has shown
number of immunopharmacological activities that inspire to work on leaves to study its immunological potential for antigen specific immune response which has

Corresponding Author:
Author:
Corresponding
Amit
Gupta,
Vidya
Pratishthans
School of Biotechnology
(VSBT)
Vidyanagari,
District
Pune, M.S.,
India.
Anil Pawar, Assistant
Professor, Department
of Zoology, D.A.V
. College
for Girls,Baramati,
Yamunanagar
(Haryana);
Mobile:919467604205;
Email:
E-mail:sumanil27@yahoo.co.in
amitvsbt@gmail.com
Received: 16.6.2014
24.01.2015

Revised:
11.7.2014
Revised:
13.02.2015 Accepted:
Accepted:29.7.2014
02.03.2015

Int J Med Phar Sci | Vol 5 Issue 7 March 2015

Gupta et. al.: Immunoadjuvant potential of azardichta indica against rabies, hepatitis and dpt vaccine antigen

not yet exploited. Hence, the present study was designed


to study the protective action of leaves aqueous extract
of Azadirachta indica used as vaccine adjuvant candidate
on antigen (hepatitis, DPT and rabies vaccine) specific
immune response.

MATERIALS AND METHODS


Plant material

The fresh green leaves of Azadirachta indica were collected from the garden of our institute in the month
of December 2014. The collected leaves were properly
washed with tap or distilled water and those leaves diseased/dried leaves were discarded. The fresh leaves were
crushed and macerated in liquid nitrogen to form fine
powder. The powder is dissolved in phosphate buffered
saline and filtered through a Whatman No. 1 filter paper.
The aqueous extract was subjected to high-performance
thin-layer chromatography (HPTLC) analysis for quantification of primary as well as secondary metabolites.

HPTLC study

This study has been used for quantitative analysis of


azadirachtin, as marker compound in the leaves aqueous
extract of Azadirachta indica. The presence of saponin,
terpenoids, flavonoids and phenolics in the phytochemical profile of aqueous extract of leaf of Azadirachta indica. The percentage of Azadirachtin in the aqueous extract sample was found to be 2.2% and retardation factor
(Rf) value of secondary metabolites i.e. terpenoid (0.96)
and saponin (0.38). The saponin was found in the peak
value between 490-665. These HPTLC results showed
indicative of the excellent reliability, reproducibility, accuracy and precision of the method.

ELISA

Indirect Elisa was performed using standard rabipur,


hepatitis and DPT vaccine (1:1000 dilution) as coating
antigen. Aqueous extract of Azadirachta indica and antiHBsAg, DPT as well as rabies serum were used for the
estimation of IgG antibody titre. Horse anti-serum used
as secondary antibody and optical density measured at
450 nm.

Human blood samples and estimation of blood


counts

The human blood samples included in this study were


analyzed at the VSBT, Baramati, Maharashtra, India, between May to June 2014. Control blood samples collected from Mangal Pathology Laboratory, Maharashtra,
India.
For the estimation of variable doses of formulation of
Azadirachta indica (1.25 10 mg) in human whole blood
Int J Med Phar Sci | Vol 5 Issue 7 March 2015

using flow cytometer. Data acquisition of 10000 events


(cell population) using forward and side scatter gating
applied to represent different phenotypes analyzed using
cell quest software. In this experiment, 100 l of human
whole blood was taken in each tube. Add serial dilutions
of formulation on human whole blood and then incubated the samples in dark for 2 h at 37C carbon dioxide incubator. After incubation, add 2 ml of red cell lysis
buffer and then incubate at room temperature for 10
min. The samples were spinned or centrifuged at 2500
rpm for 10 minutes and the supernatant was aspirated/
discarded and washed two times with 1x phosphate buffered saline. After centrifugation, pellet dissolved in PBS
and observed the cells through flow cytometer [11, 12].

Statistical analysis

The difference between the control and treated groups


is determined by one way ANOVA test. All the values are
expressed as Mean S.D. S.E.

RESULTS
Effect of aqueous extract on antibody titre

To determine the dose range for the aqueous extract of


Azadirachta indica, a dose response experiment was performed by indirect Elisa using rabies, HBsAg and DPT
vaccine as a coating antigen. The anti-rabies, anti-HBsAg
and anti-DPT IgG titre was assayed by ELISA and titres
obtained as shown in Fig. 1. Anti-rabies, anti-HBsAg and
anti-DPT serum were used as standard for the study.
Moreover, antibody titres induced by Azadirachta indica
against rabies, HBsAg and DPT at a dose range of 2.5 mg
and these were still higher than those elicited by antirabies, anti-HBsAg and anti-DPT serum.

Effect of formulation of Azadirachta indica on


human whole blood samples

The effect of formulation on the number of lymphocytes,


monocytes and granulocytes count as shown in Fig. 2.
The results showed that the aqueous extract showed
increased the number of lymphocytes, monocytes and
granulocytes as compared to standard vaccine which is
available in the market.

DISCUSSION
In India, hepatitis, rabies and DPT persists as a common
human disease despite effective vaccines having been
employed for almost 30 - 40 years. Since, plants were
considered as alternative sources of vaccine adjuvants, to
be mainly orally or intrapertioneally or subcutaneously
administered. Despite 20 30 year attempts, no real anti-rabies, anti-HBsAg and anti-DPT plant-based vaccine
containing adjuvant except alum has been developed.
Plant-based vaccine adjuvants seemed an excellent tool
2

Gupta et. al.: Immunoadjuvant potential of azardichta indica against rabies, hepatitis and dpt vaccine antigen

for mass prevention. Orally administered plant-associated primary as well as secondary metabolites used as vaccine adjuvant candidates were reckoned as alternatives,
or at least supplements for, injection vaccines derived
from yeast or mammalian cells [13, 14, 15]. Other premises also indicated that plants could be a potent source of
adjuvants for vaccine antigen. The preparations of vaccine adjuvants from various medicinal plants and these
were considered to be advantageous in terms of cost,
safety as well as naturally free from microbial toxins and
pyrogens or human and animal pathogens. However,
oral/ intrapertioneally/ subcutaneously administration
of primary or secondary metabolites extracted from medicinal plants used as adjuvant for vaccine antigens were
thought to be the largest benefit for the people. In this
case, plant-based metabolites were to be used as edible
vaccines [16, 17]. They were to be produced locally or
distributed in India or abroad without the cold chain
storageas tubers, grains, or dried fruits, which can be
stored and transported at ambient or normal temperature. Generally, plant-based vaccine adjuvants were assumed to be cheap and easy to available, both in terms of
production, distribution and application, particularly for
developing countries [16, 17]. A leading project of this
idea soon became a plant based vaccine adjuvant candidate against rabies, hepatitis and DPT disease.
The rabies, hepatitis and DPT vaccine antigen were tested with variable doses of aqueous extract of Azadirachta
indica using human volunteers donating anti-coagulant
EDTA blood for the estimation of lymphocytes, monocytes and granulocytes count. When these vaccines were
exposed to human whole blood in presence or absence
of aqueous extract, there is a dose response change in
the number of lymphocytes, monocytes and granulocyte count which is determined through flow cytometry.
Flow cytometry is a contemporary analytical method
for the assessment of qualitative as well as quantitative
information of biological and physical characteristics
of prokaryotic and eukaryotic cells [18]. Today in immunopharmacology, flow cytometry is routinely used in
clinical laboratories for the assessment of the immune
status of healthy animals and human [19]. However, the
main application of this method is important in immunopharmacology such as hematology, toxicology etc [19,
20]. Several studies related to hematology have shown
that the reduction of monocytes and granulocytes count
contributes to the incidence of infections in our immune
system. The number of research papers or articles which
are published and reported in the decline of number of
monocytes as well as granulocytes count, neutrophil migration, phagocytosis capacity and hydrogen peroxide
production [11, 12, 20]. In addition, aqueous extract
tested against these vaccine antigens i.e. rabies, hepatitis
and DPT for the estimation of IgG antibody titre. These
antigens were recognized strongly by aqueous extract

containing primary as well as secondary metabolites


and antibody-secreting cells from vaccines. Additionally,
there was good correlation between the aqueous extract
of medicinal plant-made vaccine adjuvant candidate and
the conventional vaccine antigen both by Elisa assay. The
conclusion was that the aqueous extract candidate had
good vaccine adjuvant potential, but needed good preservatives for its long term use in a clinical trial.

CONCLUSION
The development of an effective plant based adjuvant
against rabies, hepatitis and DPT has been a great challenge for medical science. Medicinal plant production
platforms offer wonderful prospects for improving the
access to these vaccines. Thus, it seems a reasonably
safe prediction that viral or bacterial vaccines containing
plant based adjuvant will be approved more widely for
at least animal use in the near future and possibly also
for use as therapy for existing bacterial as well as viral
infections. Hopefully, approval for prophylactic as well
as therapeutic vaccines for humans such as for seasonal
influenza will follow in the slightly longer term.

ACKNOWLEDGEMENT
Authors acknowledge the immense help received from
the scholars whose articles are cited and included in
the references of this manuscript. The authors are also
grateful to authors / editors / publishers of all those articles, journals and books from where the literature for
this article has been reviewed and discussed. In addition,
authors are thankful to Dr Gandhi and Mr. Shekhar Mandhre, Mangal Pathology Laboratory, Baramati region,
District Pune, Maharashtra, India and Devdan Chopade,
research associate VSBT for giving and collecting human
samples for research work.

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Figure 1: ELISA assay. Indirect Elisa was performed using standard rabipur vaccine (1:1000 dilution) as coating antigen. Aqueous extract of Azardichta indica, anti-rabies serum, anti-HBsAg and anti-DPT serum from different companies were used for
the estimation of anti-rabies, anti-HBsAg and anti-DPT antibody titre. Horse anti-serum used as secondary antibody and optical
density measured at 450 nm. The difference between the control and standard is determined by one way ANOVA test.

Int J Med Phar Sci | Vol 5 Issue 7 March 2015

Gupta et. al.: Immunoadjuvant potential of azardichta indica against rabies, hepatitis and dpt vaccine antigen

Figure 2: Flow cytometric analysis of aqueous extract of Azardichta indica on lymphocytes, monocytes and granulocytes count.
EDTA human whole blood were treated with aqueous extract in presence of rabies, HBsAg and DPT vaccine antigen and then
lysed the cells with red cell lysis buffer and wash the cells with phosphate buffered saline and analyzed the samples through flow
cytometer. Values are expressed in Mean S.E. of fifty four human whole blood samples.

Int J Med Phar Sci | Vol 5 Issue 7 March 2015

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