World Journal of Pharmaceutical Research

Amit et al.

World Journal of Pharmaceutical Research

SJIF Impact Factor 5.045

Volume 4, Issue 2, 1063-1071.

Research Article

ISSN 2277 7105

FLOW CYTOMETRIC ANALYSIS OF IMMUNOADJUVANT

ACTIVITY OF EMBLICA OFFICINALIS ON HUMAN WHOLE BLOOD
Amit Gupta*1 and Sushama R Chaphalkar1, 2*
1

Department of Immunology, Vidya Pratishthans School of Biotechnology (VSBT)

Vidyanagari, Baramati, District Pune, Maharashtra, India.

Director, Vidya Pratishthans School of Biotechnology (VSBT) Vidyanagari, Baramati,

District Pune, Maharashtra India..

Article Received on
26 Nov 2014,

ABSTRACT

Revised on 21 Dec 2014,

Accepted on 15 Jan 2015

for its potential ability as an adjuvant effect on the immune responses

In this study, alkaloid fraction from Emblica officinalis was evaluated

to hepatitis and diphtheria-pertussis-tetanus (DPT) antigen on human

*Correspondence for

whole blood using flow cytometry. Cells were treated with variable

Author

doses of alkaloid fraction (0.625 2.5 mg) in presence or absence of

Dr. Amit Gupta

hepatitis and DPT vaccine antigen. Hepatitis and DTP vaccine

Department of

containing alum used as standard for these studies. The results showed

Immunology, VSBT,
Baramati, District Pune,
Maharashtra, India.

that the DPT mediated blood counts were significantly enhanced by

alkaloid fraction at lower doses (0.625 mg) compared with DPT
control group. Moreover, alkaloid containing hepatitis antigen showed

Dr. Sushama R
Chaphalkar

no adjuvant effect on human whole blood.

Director, VSBT,
Baramati, District Pune,

KEYWORDS: Emblica officinalis, hepatitis, DPT, alum.

Maharashtra, India.

INTRODUCTION
A body of evidence showed that numerous bioactive molecules isolated as well as
purified from various medicinal plants have shown immunomodulation and anti-cancer
effects. Previous studies have shown that bioactive molecules evoke stronger immune
responses (antibody titre and cytokines) than alum or others.[1,

2, 3, 4, 5]

Thus, the bioactive

molecules from various medicinal herbs are becoming an attractive material as

pharmaceutical products and may provide an opportunity to develop a new adjuvant for
vaccine antigen. Indeed, a wide range of bioactive molecules have been isolated from various
medicinal plants and these molecules have been shown to possess immunomodulatory

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activity through their ability to modulate macrophage function.[3, 4] Appropriate enhancement

of innate immune functions by bioactive compounds can then augment host defense
responsiveness. Thus, because of their low toxicity and high potency of these molecules,
plant bioactive molecules represent one of the ideal or best candidates for therapeutics with
immunomodulatory action.
Vaccine adjuvant research totally belongs to immunopharmacological field that is advancing
very fast or rapidly, reflecting at a very high rate in which new or proposed adjuvant
candidates are being discovered. The requirement as well as challenge for safe and effective
adjuvant for current vaccine antigen cannot be overstated. Newly developed or proposed
vaccines are usually based on target antigen, they are usually weak immunogenic, costly and
induce poor immunological responses. However, adjuvants can override such immunological
inadequancy and help in mounting protective immune responses.[1, 2, 6, 7] There is a significant
interest in developing as well as synthesizing the new vaccine adjuvant that combine the
safety advantages of subunit or recombinant protein based vaccines with enhanced efficacy.
However, many of them are not highly immunogenic whether administered parenterally or
mucosally and approved adjuvants are ineffective in triggering the innate as well as adaptive
immunity, thus the identification of plant based adjuvant candidates capable of facilitating
antigen delivery to immune responsive cells and functioning as adjuvants.[3, 4, 5]
Emblica officinalis (commonly known as Indian gooseberry or amla) belongs to the family
Euphorbiaceae.[8] Various part of this medicinal plant are used in the treatment of number of
diseases such as respiratory disorders, diabetes, heart and eye disorder, scurvy and ageing.[9,
10, 11]

In addition, this medicinal plant also has anti-bacterial and astringent properties. The

number of preclinical studies has shown that Emblica officinalis exhibits strong antioxidant
activity;[8]

immunomodulatory;[9]

anti-inflammatory;

antiulcer;

hepatoprotective

and

anticancer actions.[10, 11] However, there is very little information about the clinical evidence
to support the use of Emblica officinalis as an adjuvant for vaccine antigen.
To achieve the objective for adjuvant development against bacterial infections is normally
described in three groups of vaccines. Firstly, it involves well established and commonly
used vaccines such as BCG and DPT vaccines. Secondly, it includes newer vaccines or
vaccines that are under development, for instance vaccines against number of pneumococcal
diseases, Haemophilus influenzae and enteropathogenic E. coli. Thirdly, it covers vaccines
whose realization at present appears to be difficult or hardly feasible, e.g. vaccines against
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enterotoxins of enteropathogenic organisms; gonorrhea or organisms of hospital infections. In

this short communication, our group focused on the alkaloid isolated from the Emblica
officinalis on human whole blood was treated with first group of well established vaccines
such as DPT (for eliminating bacterial infections) and also analyzed the effect on hepatitis
vaccine (for eliminating viral infections) as well.
The extract powder obtained from the fruits of Emblica officinalis was subjected to
qualitative and quantitative investigation of metabolites through HPTLC. The solvents and
other purified reagents, HPTLC plates (10 x 10 cm) were purchased from Qualigens and
Merck. The result is depicted in Fig. 1. The following HPTLC figure for the extract powder
of Emblica officinalis sample whose 10 l was injected shows six peaks at Rf values 0.04,
0.20, 0.56, 0.62, 0.65, 0.80. The maximum concentration is obtained at Rf value 0.20
indicating presence of phytoconstituents eluting out at 200 nm with the mobile phase ethyl
acetate and n-butanol.
In order to find out the adjuvant activity of alkaloid fraction with hepatitis vaccine and DPT
vaccine antigen (Serum Institute of India, India) containing alum. Human blood samples were
collected from Mangal Pathology laboratory, Baramati, District Pune, Maharashtra, India at
different time intervals for flow cytometric studies. Human whole blood was treated with
20 g hepatitis and DPT formulated with one of the following delivery vehicles: Phosphate
buffered saline and alkaloid fraction (0.625, 1.25 and 2.5 mg). Phosphate buffered salinetreated sample were included as control. Control adjuvant (standard) was alum (0.5 and 1.45
mg). In every experiment, one tube was kept as control, whilst another tube received standard
vaccine for comparison of the study and authenticity of the experiment.
Flow cytometry analysis of human whole blood for examine and counting the number of cells
i.e. lymphocytes, monocytes and granulocytes count which are suspended in a stream of
fluid. To examine the forward and side scatter gating of human whole blood with variable
doses of alkaloid ranging from 0.625 2.5 mg for data acquisition of 10000 events and
fraction or separation of cell populations representing different phenotypes analyzed using
cell quest software. Briefly, 50 l of human whole blood was pipetted directly into a falcon
tube containing phosphate buffered saline or with containing concentrations of alkaloid and
then incubated at carbon dioxide incubator (37 C, 5 % CO2) for 2 h. After incubation, RBCs
were lysed using 2 ml of red cell lysis buffer and incubated the sample for 30 minutes. After

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centrifugation at 1800 rpm for 10 minutes, the supernatant was removed or aspirated and
washed two- three times with phosphate buffered saline. After centrifugation, pellet dissolved
in PBS and observed the cells through flow cytometer.[12, 13, 14]
Hepatitis and DPT vaccine is commonly used as a model for specific T and B cell mediated
immune function. When this human whole blood were firstly exposed to hepatitis and DPT
vaccine containing alum, it is possible that protein antigens such as hepatitis and DPT
activate both B and T cells population, which results in the induction a highly efficient B cell
differentiation pathway through specific structures (germinal centers, GCs) in which antigenspecific B cells proliferate and differentiate into antibody-secreting plasma cells or memory B
cells.[7] The short life span of these plasma cells results in a small increased in the blood
count, which eventually returns to baseline levels or control. In secondary immune responses,
challenging exposure to proposed adjuvant candidate alkaloid from Emblica officinalis
reactivates immune memory and results in a rapid increase of blood count especially
lymphocytes, monocytes and granulocytes count. However, the addition of alum or alkaloid
in human whole blood, the results showed that the alkaloid fraction in case of DPT vaccine
showed remarkable increased in the number of blood counts as compared to alum containing
DPT and control (Fig .2) where as in hepatitis vaccine, alkaloid showed negligible effect on
human whole blood (Fig. 2). Furthermore, presence of alkaloid on human whole blood in
presence or absence of DPT or hepatitis vaccine, it showed (Fig. 3) at higher doses showed
rapidly decline in blood counts. In contrast, alum is one of the approved adjuvant for human
use and it increased only humoral response and poorly elicited the cell mediated immunity.
The main challenge for the science of adjuvants is how to selectively induce the appropriate
type of immune response to protective antigens and to provide optimal effect against each
type of infection.[3, 4]
In this study, DPT vaccine is the most effective method in the prevention and control of this
disease i.e. Corneybacterium diphtheria, Bacillus pertussis and clostridium tetani and it has
been used successfully for decades.[7] The primary vaccination of infants against these
disease does not always afford long term protection suggest the need for a better immune
strategy to maintain adequate levels of specific immunity to these diseases.[7] There are
multiple antigens which is already present in the DPT vaccine encoded as combined vaccines.
To increase the immunogenicity of DPT vaccine antigen, alum is used as an adjuvant for
human use. The relatively poor efficacy of DPT vaccines in immune responses, especially in

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animals has limited their practical use. However, we provide the strategy to increase the
efficacy of these antigens or to improve the immune response induced or supplementing with
the alkaloid isolated from Emblica officinalis. In this study, protein antigen i.e. DPT in
adjuvant alum or alkaloid was used as a boosting agent and significantly enhanced immune
response. Blood count especially lymphocytes, monocytes and granulocytes at lower
concentration elicited in the group protein DPT containing alkaloid boosted showed
significant increases than those in group protein boosted containing alum and control. This
prime and boost strategy of enhancement of lymphocytes, granulocytes and monocytes count
has proven to be very useful in improving the immunogenicity of DPT vaccines against
number of diseases.

Fig 1. HPTLC of aqueous extract of Emblica officinalis.

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Fig. 2. Effect of proposed adjuvant candidate alkaloid isolated from Emblica officinalis
on hepatitis and DPT vaccine.
Human whole blood was incubated with variable doses of alkaloid (0.625, 1.25 and 2.5
mg/ml) in presence or absence of alum. After incubation, cells were lysed using lysis buffer
and washed with phosphate buffered saline. After centrifugation, pellet dissolved in PBS and
observed the cells i.e. lymphocyte, monocyte and granulocyte count through flow cytometer

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Fig.3. Effect of alklaoid on human whole blood.

Human whole blood was incubated with variable doses of alkaloid (0.625, 1.25 and 2.5
mg/ml). After incubation, cells were lysed using lysis buffer and washed with phosphate
buffered saline. After centrifugation, pellet dissolved in PBS and observed the cells i.e.
lymphocyte, monocyte and granulocyte count through flow cytometer.
CONCLUSION
Alkaloid fraction is a strong Th2 adjuvant for DPT vaccine antigen in human whole blood.
Alkaloid-adjuvanted with DPT vaccine antigen is better than that of DPT adjuvanted with
alum. The DPT-specific blood counts induced by alkaloid fraction associated with increased
in the number of lymphocytes, monocytes as well as granulocytes count. Furthermore,
alkaloid fraction at higher doses showed rapidly decline in the number of blood counts. Thus,
alkaloid fraction is a potent enhancer of antigen-specific humoral immune responses, thus
showing promise as immune adjuvant for vaccines against extracellular infectious agents
such as bacteria, protozoa etc.
ACKNOWLEDGEMENT
Authors are thankful to Dr Gandhi and Mr. Shekhar, Mangal Pathology Laboratory,
Baramati region, District Pune, Maharashtra, India and Devdan Chopade, research associate
VSBT Baramati for giving and collecting human samples for research work.
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