1. Nimisha Tiwari*, Dinesh Kumar Patel and Anirban Pal
Molecular Bioprospection Division
CSIR-Central Institute of Medicinal and Aromatic
Plants, Lucknow.

2. Adjuvants
 An immunological agent that increases the antigenic
response.
 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
protective immunity.
3. To enhance the immune response of immune
compromised adults and weakened immune system of
children.

4. Can plants be of any utility in
vaccines??

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
conventional strategies.

7. Asparagus racemosus Bacopa monnieri
Tinospora cordifolia

8. Asparagus racemosus
•Galactogogue
•Antitussive
•Antiplasmodial
•Antileshmanial
•Antibacterial
•Hepato-protective
•Diuretic
•Anti ulcer
•Anti diarrheal
•Antenatal tonic
•Cardioprotective
•Anticancerous
•Antiepileptic
•Antidepressant.
Bacopa monnieri
•Used against cognitive
dysfunctions
•Efficient role against
memory impairment
•Widely used as
antidepressant
•Anticonvulsive
•Antiepileptic
•Anti-ulcerogenic
•Antioxidant
•Anxiolytic
•Also protective against
gastric ulcers.
Tinospora cordifolia
possess positive effects
against debility, fever,
dyspepsia, urinary diseases
It also exibit certain
properties like
•hypoglycaemic,
•Antioxidant,
•Anti-arthritic,
•Anti osteoporotic,
•Antimicrobial,
•Hepatoprotective.

9. Why “Saponins” for the
Adjuvant??????????????????

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
immune response.

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
traditional vaccines.
 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 .

13. Methodology

14. Extraction of the saponin rich fractions

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
2:1:1.
 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
(100ml).
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
90×g/10 min.
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.

17.  DPPH Assay for antioxidant studies
 Antibacterial studies -Minimum Inhibitory
Concentration (MIC)

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
saponins.

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
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)

20. •In-vivo immunisation (OVA, OVA+FCA,OVA+QuilA, OVA+Plant
saponins)
•IgG determination of OVA- specific antibody in plant
saponins rich fractions in comparison to the existing
adjuvant FCA and Quil A.

21. Results

22. Identification and Qualitative analysis of saponin rich extracts
0
20
40
60
80
100
120
Saponin Quil A Asparagus Tinospora Bacopa 1st
part
Bacopa 2nd
Part
50%Hemolysis
Spectrophotometric
quantification(µg/ml)
Saponin 30.43
QuilA 24.78
Asparagus 20.455
BacopaI 4.75
Tinospora 77.63

23. DPPH activity
0
20
40
60
80
100
120
140%Survival
MTT
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Saponin QuilA Asparagus Bacopa2 Tinospora Ascorbic Acid
Antioxidant activity

24. In-vitro immunomodulatory studies
-150
0
150
300
450
600
750
900
Concentrationinpg/ml
TNF-α
0
500
1000
1500
2000
2500
3000
3500
4000
Concentrationinpg/ml
IL6

25. In-vivo Quantification of humoral immune
response
1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
IgG analysis
DAY 24
DAY 42
DAY 56

26. Conclusion
 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
and Tinospora.
 Tinospora cordifolia and Asparagus racemosus were found to increase the antibody levels,
elaborating, Asparagus racemosus significantly increased the IgG titres more promptly
(p<0.001) .
 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
activity.
 Assessing the bioactives for cellular and humoral immune
responses considering examples of microbial pathogens and
formation ISCOMs.

28. References:
 [1]Singh M, O’Hagan DT. Recent advances in veterinary vaccine adjuvants. Int J Parasitol
2003;33(May (5–6)):469–78.
 [2] O’Hagan DT, MacKichan ML, Singh M. Recent developments in adjuvants forvaccines
against infectious diseases. Biomol Eng 2001;18(October (3)):69–85.
 [3] Aucouturier, J., Dupuis, L. and Ganne, V. (2001). Adjuvants designed for veterinary and
human vaccines. Vaccine, 19(17),:2666--2672.
 [4] Francis, G., Kerem, Z., Makkar, H. and Becker, K. (2002). The biological action of saponins in
animal systems: a review. British Journal of Nutrition, 88(06).587--605.
 [5] Sparg, S., Light, M. and Van Staden, J. (2004). Biological activities and distribution of plant
saponins. Journal of ethnopharmacology, 94(2).219--243.
 [6] Singla, R. and Jaitak, V. (2014). SHATAVARI (Asparagus Racemosus Wild): A review on its
cultivation, morphology, phytochemistry and pharmacological importance. International
Journal of Pharmaceutical Sciences & Research, 5(3).
 [7] Brahmachari, G. (2009). Natural products. 1st ed. Oxford, U.K.: Alpha Science International.
 [8] Gohil, K., Patel, J. and others, (2010). A review on Bacopa monniera: Current research and
future prospects. International Journal of Green Pharmacy, 4(1).1.
 [9] Saha, S. and Ghosh, S. (2012). Tinospora cordifolia: One plant, many roles. Ancient science
of life, 31(4).151.

29. Acknowledgements............
 Dr. Anirban Pal
 Department of Science and Technology
 CSIR-CIMAP, Lucknow

30. Thank you....