1. Nimisha Tiwari*, Dinesh Kumar Patel and Anirban Pal
Molecular Bioprospection Division
CSIR-Central Institute of Medicinal and Aromatic
An immunological agent that increases the antigenic
A substance admixed with an immunogen in order to
elicit a more marked immune response
3. Adjuvants are important ...........
1. To bolster the immune response of any antigens by
delivering in native form.
2. To reduce the multiple immunization protocol for
3. To enhance the immune response of immune
compromised adults and weakened immune system of
5. Plant based Immuno-adjuvants
Plant-based vaccines, which are easy to produce and
administer, and require no cold chain for their heat stability.
More recently, it has been shown that even highly
immunogenic, enveloped plant-based vaccines can be
produced at a competitive and more efficient rate than
10. Saponins comprise a large family of structurally related
compounds containing a steroid or triterpenoid aglycone
(sapogenin) linked to one or more oligosaccharide
moieties by glycosidic linkage .The carbohydrate moiety
consists of pentoses, hexoses, or uronic acids.
11. Surface active triterpenoids and steroidal saponins are
the favourable options as adjuvants for vaccines with
low immunogenicity (against the microbial pathogens).
Saponins impart :
1. High antigenicity at low concentration
2. Simultaneously stimulate the innate and humoral
12. Recent contemporary vaccines made from purified antigens,
recombinant proteins and DNA are found to be of low
immunogenicity due to the lack of innate immune response
imparting the depletion of immune response as compared to the
DNA vaccines for example, are not cost effective and are often
termed to be low immunogenic.
Existing adjuvants, namely FCA and alum have been accounted
for severe lesions and toxicity imparted to some sensitive hosts.
Medicinal plants derived bioactives play an important role in
the development of potent therapeutic agents and are potent
immuno-modulators also being cost effective .
15. Spectrophotometric Analysis
Two reagents, i.e., (A) p-anisaldehyde :Ethyl acetate = 0.5:99.5 and
(B) H2SO4: Ethyl acetate = 1:1
Saponin rich fractions ethyl acetate and mixed with (A) and (B) in the ratio of
The mixture was stirred and incubated at 60⁰C for 10 minutes in a water bath.
The solutions were cooled at room temperature for 10 minutes and the
absorbance was recorded at 430 nm.
The reference curve was obtained using diosgenin.
Thin layer chromatography
Solvent system 4.5 ml n-butanol+0.75ml Acetic acid+1ml water
16. Haemolytic assay
Collected blood was washed three times with sterile saline solution
(0.89% w/v NaCl) by centrifugation (180×g/5 min.)
The cell suspension was prepared by diluting the pellet (0.5ml) in saline
A volume of 0.5ml of the cell suspension was mixed with varying
concentrations (5-1000 µg/ml).
The mixtures were incubated for 30 min at 37ºC and centrifuged at
The free hemogloblin of the supernatants was measured spectro-
photometrically at 412 nm.
Saline and distilled water were included for minimal and maximal
haemolytic controls respectively.
18. Immunological studies.........
In-vitro study In-vivo study
•Cellular immunity assessed by the LPS
stimulated murine macrophages treated
with the saponin rich fractions.
•Humoral immunity demonstrated by
assessing the production of IgG against
ovalbumin in combination with the
19. In-vitro studies
Peritoneal macrophage culture and treatment
Cytokine expression analysis
Expression of TNF-α, IL 6 from lipo-polysaccharide (LPS)
stimulated peritoneal macrophages
Cell viability through MTT assay
20. •In-vivo immunisation (OVA, OVA+FCA,OVA+QuilA, OVA+Plant
•IgG determination of OVA- specific antibody in plant
saponins rich fractions in comparison to the existing
adjuvant FCA and Quil A.
All the three plants’ saponin rich fractions showed significant in-vitro activity against LPS
stimulated macrophages inferencing the strong cellular response.
Macrophages when treated with the above saponins could significantly inhibit the
production of Tumour necrosis factor (TNF), while IL-6 could be lowered only by Asparagus
Tinospora cordifolia and Asparagus racemosus were found to increase the antibody levels,
elaborating, Asparagus racemosus significantly increased the IgG titres more promptly
The fractions didnot show any local lesions or granulomatous reactions and saponins were
found to be non-cytotoxic to primary macrophages.
These plants are potent stimulators of innate immunity.
27. Future aspects
Since all the three plants showed significant cytokine
depleting titres, they can be further assessed to employ
potent immuno-modulatory studies against pathogenic
microbes and parasites.
Asparagus racemosus, potent lead in the IgG stimulation will
be assessed against the immunogenic microbial pathogens.
Evaluation of immune-modulatory activity of AR and further
stepwise purification and identification of the active
compounds from the potent fractions displaying preliminary
Assessing the bioactives for cellular and humoral immune
responses considering examples of microbial pathogens and
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