Isolation and characterization of therapeutic molecules from locally available wild plants

1. 1
Ph.D. Research proposal presentation

2. Contents 2

3. 3
Review of literature
Ethnobotany and Ethnomedicine:
 The Sumerian Herbal of 2200 BC: First record of curative
plants
 In the 5th century BC, The Greek doctor Hippocrates listed
out some 400 herbs in common use.
 Rig Veda and Atharva Veda (4500-1600 BC): the ancient
texts of India
 Charaka Samhita and Susruta Samhita: Ayurvedic books
 The rural people from different socio-economical backward
communities in Goa have rich indigenous knowledge of
medicinal plants.

4. 4
What is the need to investigate ethanobotany &
ethanomedicine??
Diabetes mellitus: glucose metabolism disorder
 The worldwide prevalence and prominent adverse side-effects of
insulin and other conventional drugs
 Investigation of management strategies involving the use of plants
reported to be cost-effective antidiabetic agents with low side effects.
Viral diseases:
 Among the available antiviral regiments, the majority are nonspecific
for particular viruses.

5. 5
Cancer:
 One of the leading causes of morbidity and
mortality globally
 In 2018, an estimated 9.6 million deaths and
18.1 million new cancer cases, likely to move up
to 17 million deaths by 2030
 Plant biomolecules as therapeutics can be one of
the best sources for obtaining a range of newer
drugs
https://www.cancer.gov/about-cancer

6. 6
 NCI, USA, estimated that during 1981-2002, 61% of the 877
small-molecules as new chemical entities introduced as drug
worldwide were inspired by natural products.
 In 1997, out of 25 best-selling pharmaceutical drugs, 11 were
either biological/natural product or entities, worth 17.5 billion
USD.
 According to WHO, 80% of the population of developing
countries relies on traditional medicines, mostly plant-based.
 Lack of scientific evidence as acceptable in modern medicine.

7. 7
Aims and objectives
1. Study of pharmacological applications of selected locally
available wild plants.
2. Isolation and characterization of compounds with potential
bioactivities.
3. In vitro culturing of species with potential bioactivities.

8. 8
Methodology
 Collection of plant samples from different parts of Goa.
 Different techniques such as maceration, decoction, Soxhlet etc. along
with different solvents based on targeted bioactive compounds and
applications can be employed.
 This extract tested for different pharmacological applications such as
antidiabetic, anti-inflammatory, anti-oxidant, anti-cancer and anti-
viral.

9. 9
Botanical Name Antidiabetic Anti-Cancerous Anti-inflammatory
Bombax Ceiba Chetan et. al., 2013; Xu et. al.,
2017; Mohd et. al., 2017;
Khurshid et. al., 2018.
Tundis et. al., 2014. Wanjari et. al., 2016; Masood-ur-
rehman et. al., 2016.
Sesbania Seshan Ramdasv et. al., 2011; Vadivel et.
al., 2012.
------ Dande et. al., 2010; Subramanian et.
al., 2014; Fitriansayah et. al., 2017.
Ixora coccinea Maniyar et. al. 2011; Johnson et.
al., 2016; Shalini et. al., 2018;
Anila et. al., 2019.
Baliga et. al., 2012; Velliangeiri et.
al., 2018; Deeparani et. al., 2019.
Rikhabchand et. al., 2017.
Ziziphus oenoplia Maourya et. al., 2017. Kaur et. al., 2011; Siddiqui et. al.,
2015.
--------
Ziziphus mauritiana Cisse et. al., 2000; Jarald et. al.,
2009; Bhargava et. al., 2014.
Mishra et. al., 2011; Ashraf et. al.,
2015. --------
Zizyphus rugosa Mohamad et. al., 2013; Sichaem
et. al., 2017.
-------- Yadav et. al., 2010; Rambabu et. al.,
2010.
Tephrosia tinctoria Rajaram et. al., 2011 Adinaryana et. al., 2009; Rajaram
et. al., 2013. -------
calycopteris floribunda -------- Wall et. al., 1994; Huse-Ara et. al.,
2008.
--------
Buchanania lanzan Sushma et. al., 2013. -------- Shailasree et. al., 2012.
Helicteres isora Kumar et. al., 2008; Kumar et.
al., 2009
Varghese et al. 2011; Rattanamaneerusmee et. al., 2018

10. 10

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Anti-diabetic assay
Alpha-glucosidase inhibition assay
To 50µl of different conc. of extract add
6.9pH, 100µl alpha-Glucosidase (1.0 U/ml)
Incubate for 10 min
Add 50 µl of 3.0 mM p-nitrophenyl
glucopyranoside (pNPG)
Incubate at 37 0C
for 20min.
Stop the reaction by adding 2ml of 0.1 M
Na2CO3
Record the absorbance at 405 nm
Alpha-amylase inhibition assay
To 250µl of different conc of extract add
6.9pH, 250µl of alpha-amylase (0.5 mg/ml)
Incubate at 25 0C
for 10 min.
Add 250µl of 1% starch solution prepared in
0.02 M sodium phosphate buffer (pH 6.9)
Incubate at 25 0C
for 10 min
Terminate the reaction by adding 500µl
dinitrosalicylic acid (DNS) reagent
Incubate for 5 min.
in boiling water bath
After cooling add 5 ml of D/W and measure
the absorbance at 540 nm
 %Inhibition =
𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙
− 𝐴𝑏𝑠𝑒𝑥𝑡𝑟𝑎𝑐𝑡
𝐴𝑏𝑠𝑐𝑜𝑛𝑡𝑟𝑜𝑙
x 100
 Concentrations of extracts resulting in 50% inhibition of enzyme activity (IC50) can be determined
graphically

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Anti-cancer assay
 RPMI medium along with supplements under controlled conditions, cancer cell lines are
grown.
Cell proliferation assay
Add aliquots containing 2.5 x 104 cells from medium in each well of 96 well microtiter
plate.

Incubate the cells with different conc. of extract for 48 h

After 48 h replace the medium with the medium containing 100µg/ml of MTT for 4 hrs
Aspire the supernatant and dissolve the MTT-formazan crystals in 100µl DMSO
Measure the absorbance at 570 nm
 Cell growth calculated by comparing the absorbance of treated and treated cells

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DNA Fragmentation
 Apoptosis and necrosis of the cancer cells can be confirmed using flow
cytometry and gel electrophoresis (to identify the fragmentation pattern of
the DNA)

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Characterization of bioactive compounds
 The bioactive compounds present in the plant extracts will be separated
and characterized through chromatographic techniques such as GC,
HPLC and TLC
 The nonchromatographic techniques such as immunoassay and spectral
analyses such as UV-vis/fluorescence, IR, NMR, FTIR spectroscopy can
be employed.

15. 15
Sr.
no. Activity
1st
year
2nd
year
3rd
year
4th
year
5th
year
1.
Literature review
2.
Screening of plants
3.
Extract preparation
4.
Use of extract for different pharmacological
applications
5.
In-vitro culturing of species with potential
bioactivities
6.
Characterization of bioactive compounds
7.
Statistical analysis
8.
Thesis writing
Timeline

16. 16
References
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University.
 http://www.greekmedicine.net/whos_who/Dioscorides.html
 Jain S. K., (1968). Medicinal plants. Natural book Trust, India, 1968; 16: 14-15.

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Contd…….
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 Kazeem M. I., Adamson J. O., and Ogunwande I. A., (2013). Modes of Inhibition of 𝛼-
Amylase and 𝛼-Glucosidase by Aqueous Extract of Morinda lucida Benth Leaf, BioMed
Research International Volume 2013, Article ID 527570
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Ziziphus mauritiana against Cancer Cell Lines and Ehrlich Ascites Carcinoma, Evidence-Based
Complementary and Alternative Medicine Volume 2011, Article ID 765029,
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over the period 1981-2002, J Nat Prod. 2003;66:1022–1037

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 Newman, D., & Cragg, G. (2007). Natural products as sources of new drugs over the last 25
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Contd…….

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