screening of potentially active drug candidate from...
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Screening of potentially active drug candidate from Solanum nigrum L. against inflammatory diseases and evaluation of
their cellular and molecular mechanism.
A project proposal submitted toA project proposal submitted toA project proposal submitted toA project proposal submitted to
Foods and InflammationFoods and InflammationFoods and InflammationFoods and Inflammation
Department of Biotechnology
Ministry of Science and Technology
Government of India
New Delhi
Dr. Dr. Dr. Dr. A. MaheshA. MaheshA. MaheshA. Mahesh,,,,
Lecturer
School of Biological Sciences,
Madurai Kamaraj University,
Madurai 625 021.
June 2011
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PROFORMA - I
PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH AND
DEVELOPMENT, PROGRAMME SUPPORT
(To be filled by the applicant)
PART I: GENERAL INFORMATION
1. Name of the Institute/University/Organisation submitting the Project Proposal:
Madurai Kamaraj University, Madurai.
2. State : Tamil Nadu
3. Status of the Institute : State University.
4. Name and designation of the Executive Authority of the Institute/University forwarding the application:
Registrar, Madurai Kamaraj University, Madurai 625021.
5. Project Title: Screening of potentially active drug candidate from Solanum nigrum
against inflammatory diseases and evaluation of their cellular and molecular mechanism. 6. Category of the Project (Please tick) : R&D 7. Specific Area : Foods and Inflammation
8. Duration : Three Years
9. Total Cost (Rs.) : `̀̀̀ 28,01,400/-
10. Is the project Single Institutional or Multiple-Institutional (S/M): S
11. If the project is multi-institutional, please furnish the following : Not Applicable.
Name of Project Coordinator :
Affiliation : Address :
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12. Scope of application indicating anticipated product and processes.
In recent years, the development of herbal therapeutic drug candidates from various medicinal
plants has received great attention from the medical research community. Herbal medicines have
been used for centuries against different ailments including inflammatory diseases and become a
focal point to identify, isolate and purify new entities to treat diseases. Herbal medicines are less
toxic, cheap and suitable for use over a prolonged period. However, the traditional use of
plants is not sufficient for ethical validation of their therapeutic effects, requiring scientific
studies to verify their real pharmacological potential.
Owing to the advantages and modern pharmaceutical demand in traditional medicines,
Solanum nigrum would be a potential drug for the inflammatory and cardiovascular
diseases. This study will help us to understand the cellular and molecular mechanism of
Solanum nigrum against inflammatory diseases. Solanum nigrum would be an effective,
cheapest, more accessible and safety drug for inflammatory and cardiovascular diseases.
Solanum nigrum is a leafy vegetable, used in India and African countries. Hence, it could be
served as a food supplement for the treatment of inflammatory diseases.
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13. Project Summary (Not to exceed one page. Please use separate sheet).
The present proposed work aims to screening a potentially active drug molecule from Solanum
nigrum L. against inflammatory and cardiovascular diseases. S. nigrum is a leafy vegetable has
immense medicinal properties. According to the estimation of WHO, 25% of the commonly used
medicines contain compounds isolated from plant origin. The basis of some modern medications
is indeed plants, such as aspirin from white Willow bark, digitalis from foxglove, warfarin from
sweet clover, antimalarial drug quinine from the bark of Cinchona officinalis, taxol isolated from
the Yew plant and digoxin from Digitalis purpurea (Kinghorn 1994). Hence, the search for
alternative anti-inflammatory drugs mainly from natural herbs is required. S. nigrum A leafy
vegetable will be used for the present proposed work and it has huge medicinal values.
Anti-inflammatory drug will be extracted and fractioned from S. nigrum. Isolated
bioactive fractions will be assessed their anti-oxidant potency using DPPH and Superoxide
radical scavenging method. In vitro Anti-inflammatory activity of S. nigrum will be evaluated on
activated macrophage cell line RAW 264.7. Cytokine inhibitory activity will be assessed in S.
nigrum treated macrophage cell line using the method of flow cytometric bead array.
Quantification of inflammatory marker such as NF-kB, COX-2 and TNFα Inhibitory activity will
be tested in S. nigrum treated macrophage cell line using Western blot and qPCR techniques.
Cardiovascular curative/protective activity will be performed in cardiovasculars animal model
treated with S. nigrum. The molecular level inhibition of inflammatory markers (NF-kB, COX-2
and TNFα) will be assessed in heart tissue of drug treated cardiovascular rat models by Western
blot and qPCR. Tissue oxidative status SOD, GPx, and GSH will be estimated in drug treated
cardiovascular rat model. Histopathological status of the heart architecture will be investigated in
drug treated cardiovascular rat models.
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PART II: PARTICULARS OF INVESTIGATORS
(One or more co-investigators are preferred in every project. Inclusion of co-investigator(s) is
mandatory for investigators retiring before completion of the project)
Principal Investigator:
14. Name : Dr. A. Mahesh
Date of Birth : 15/1/1980, Sex (M/F): M
Designation : Lecturer
Department : School of Biological Sciences
Institute/University : Madurai Kamaraj University
Address : Palkalai Nagar
Madurai- 625 021
Telephone: +91 98438 74873, Fax: +91 452-2459873, E-mail: [email protected]
Number of research projects being handled at present: NIL
Co-Investigator
15. Name : Dr. P. Gunasekaran
Date of Birth : 2.07.1954 Sex (M/F): M
Designation : Professor & Head Department of Genetics
Department : School of Biological Sciences
Institute/University : Madurai Kamaraj University
Address : Palkalai Nagar
Madurai- 625 021 Telephone: +91-452-2458478, Fax: +91 452-2459873, E-mail: [email protected]
Number of Research projects being handled at present: 4
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PART III: TECHNICAL DETAILS OF PROJECT
16. Introduction (not to exceed 2 pages or 1000 words)
Inflammation is the response of the tissue to an injury, infection, irritation and foreign body, such
as bacteria, parasites and viruses. In this context, the inflammatory response is a critical
protective reaction to irritation, injury, or infection, characterized by redness, heat, swelling, loss
of function and pain. It is a complex pathophysiological process mediated by a variety of
signaling molecules produced by activated immune-associated cells to efficiently remove the
injurious stimuli and initiate the healing process (Stables and Gilroy, 2011). However,
uncontrolled and persistent inflammation implicate in the pathogenesis of many diseases,
including cancer, diabetes, cardiovascular, neurodegenerative and other life-threatening and
debilitating diseases (Lawrence et al., 2002).
Cardiovascular disease remains a major contributor to the high cause of death occurring
among the population worldwide. In India, about 25 % of deaths in the age group of 25- 69 yrs
occur because of heart diseases. In urban areas, 32.8 % deaths occur because of heart ailments,
while 22.9 % people die of heart diseases in rural India (Raja et al., 2011). Inflammation plays a
role in all stages of cardiovascular disease, the underlying cause of approximately 80% of all
sudden cardiac death (Albert et al., 2002). Atherosclerosis causes ischaemia and infarction by the
chronic or acute occlusion of arteries, and inflammatory cells have been identified in
atherosclerotic lesions. Systemic markers of inflammation such as cytokines, C-reactive protein
(CRP), fibrinogen, and white cell count have previously been shown to be associated with
increased risk of cardiovascular events in apparently healthy populations and patients with
established vascular disease (Sabatine et al 2007). Although inflammatory markers do improve
prediction of cardiovascular events, their predictive value may be modest (Danesh et al 2004).
Macrophages are professional phagocytes that remove metabolic waste products,
pathogenic microorganisms and senescent cells. The cell surface is armed with various
recognition receptors specific for their targets. In the process of inflammatory response,
macrophages release nitric oxide (NO), a reactive molecule originated from the guanidine
nitrogen of l-arginine, catalyzed by nitric oxide synthases enzymes (NOS) and other cytokines
(Kock et al., 1990). It has also play an important role in inflammatory diseases relating to
overproduction of pro-inflammatory cytokines and tumor necrosis factor-α (TNF-α) (Holm et al.,
2009). Excessive NO production leads to the development of many inflammatory related
diseases (Skidgel et al., 2002). Prostaglandin E2 (PGE2), generated by specific COX-2 function,
was another anti-inflammatory parameter (Surh et al., 2001).
The non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most widely
used classes of drugs throughout the world for the treatment of inflammation, but their
undesirable side effects on gastric mucosa and cardiovascular system are well know
(Wallace and Vong, 2008). This has prompted us to search for novel compounds with
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therapeutic effect to reduce inflammatory diseases and relieve symptoms. Hence, the
search for alternative anti-inflammatory drugs mainly from natural herbs is required.
16.1 Origin of the proposal:
India is a richest country in the world with regard to genetic resources for medicinal plants. They
have been used as a source of safe and effective medicine since time immemorial. The revival of
interest in herbal medicine is due to increased awareness of the limited horizon of synthetic
pharmaceutical products, and the current widespread belief that ‘‘green medicine’’ is safe, less
toxic, cheap, more accessible and suitable for use over a prolonged period. It is noteworthy that
the use of medicinal plants or their crude extracts is increasingly becoming an attractive
approach as a complement and an alternative for treating various inflammatory disorders.
Solanum nigrum L. is a leafy vegetable, which belongs to the Solanaceae family,
commonly known as Makoi or black nightshade, usually grows as a weed in moist habitats in
different kinds of soils, including dry, stony, shallow, or deep soils, and can be cultivated in
tropical and subtropical agro climatic regions. Solanum nigrum possesses various compounds
that are responsible for diverse activities. The major active components are glycoalkaloids,
glycoproteins, and polysaccharides. It also contains polyphenolic compounds such as gallic acid,
catechin, protocatechuic acid (PCA), caffeic acid, epicatechin, rutin, and naringenin. The
glycoalkaloids include solamargine, solasonine, and solanine that belong to the tropane group of
compounds (Sikdar & Dutta, 2008).
In south India and African countries, Solanum nigrum used as a leafy vegetable and also
plant medicine (Sultana et al., 1995). It has also been used traditionally to treat various ailments
such as pain, inflammation and fever. The leaf, stem and roots are used as a poultice or wash to
treat cancerous sores, boiled, leucoderma and wounds while extracts of the plant are claimed to
possess anti-infammatory, antispasmodic and vasodilator effects. There is also report on the used
of the plant in the manufacture of locally analgesic ointments and the fruits, in particular, have
been used as an analgesic for toothaches (Ong, 2003). The fruit of S. nigrum has been reported in
the ancient Indian medicinal literature with beneficial effects in inflammation, tuberculosis,
diuretics etc (Chopra et al., 1956). Nevertheless, as similar as the most traditional medicines, the
poorness and difficulty in quality control and pharmacological interpretation was one of the
bottlenecks for further development of new drugs. Therefore, it is necessary to simplify it
successively through bioactivity-guided screening to achieve an optimized minimal
phytochemical composition and interpret its action mechanism clearer.
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16.2 (a) Rationale of the study supported by cited literature (b) Hypothesis (c) Key
questions.
Rationale: Nature has provided a complete store-house of remedies to cure all aliments of
mankind (Kokate et al., 2002). This is where, nature provides us drugs in the form of herbs,
plants and algae to cure the incurable diseases without any toxic effect. There is a growing
interest in herbal remedies because of their effectiveness, minimal side effects in clinical
experience and relatively low cost. Herbal drugs or their extracts are prescribed widely, even
when their biological active compounds are unknown (Gupta et al., 2005). Plants contain a
variety of chemically active compounds such as flavonoids, terpenoids, lignans, sulphides,
polyphenols, coumarins, saponins, alkaloids, terpenes, thiophenes, proteins and peptides, which
has proven inhibit the various inflammatory diseases.
Inflammation is associated with many pathophysiology of various clinical conditions like
arthritis, cancer and cardiovascular diseases. Because of increasing side effects of available
synthetic drugs, there is need to focus on the scientific exploration of herbal drugs having no side
effects. A number of natural products are used in various traditional medical systems to treat
relief of symptoms from pain and inflammation. Medicinal compounds derived from plant
extracts, are of lifelong interest to the pharmaceutical industry. For example, taxol is an
antineoplastic drug obtained from the bark of the Western yew tree, found to be useful in the
treatment of breast cancer (Slichenmyer and Von Hoff, 1991). Hence, the modern
pharmaceutics is interest in nature medicine, the present proposal aims to develop anti-
inflammatory and anti-cardiovascular drug from Solanum nigrum.
Key questions.
1) Can Solanum nigrum bioactive fractions used as a potential drug for the inflammatory
and cardiovascular diseases?
2) Can Solanum nigrum inhibit the inflammatory molecular biomarkers?
3) What is the cellular and molecular mechanism of Solanum nigrum on activated
macrophage cell?
4) How the Solanum nigrum enhance the protective effect of cardiovascular diseases in
vivo?
5) How the inflammatory markers useful for investigating anti-cardiovascular drug in vivo?
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16.5 Current status of research and development in the subject (both international and
national status)
International status
Recent studies from worldwide have demonstrated that S. nigrum has anti-inflammatory, anti-
cardiovascular diseases, anti-ulcer and hepatoprotective effects, as well as the ability to induce
apoptosis in several types of cancer cells. Hee et al. (2011) has been reported that, the
chloroform fraction of reduced NO through inhibition of iNOS synthesis and inhibited TNF-α
and IL-6 at the level of protein secretion and also suppressed p38, JNK and ERK1/2. S. nigrum
significantly inhibited the growth of SMMC-7721 and HepG2 cells and induced cell apoptosis.
Cell cycle analysis revealed that solamargine caused cell cycle arrest at the G2/M phase (Ding et
al., 2012). Ethanol and water extracts of S. nigrum showed hepatoprotective effects against
chemically-induced liver damage in rats (Hsieh et al., 2008). Alkaloids and polysaccharides
isolated from S. nigrum induced apoptosis in cervical cancer cells in vitro and inhibited tumor
growth in vivo (Li et al., 2009). Polyphenolic extract of Solanum nigrum inhibited
hepatocarcinoma cell growth by inducing G2/M phase of the cell cycle, meanwhile, the protein
levels of cell CDC25A, CDC25B, and CDC25C were clearly reduced (Wang et al., 2011).The
mechanisms of anti-inflammatory effect of S. nigrum remain unclear.
National status
In India, there are few reports from S. nigrum including anti-inflammatory activity (Arulmozhi et
al., 2010; Arunachalama et al., 2009; Dhirender et al., 2009). Supplementation of S. nigrum fruit
extract significantly improved the antioxidant status by decreasing the levels of TBARS and
altering the lipid profiles to near normal (Arulmozhi et al., 2010). Recently, principal
investigator has been reported in another leafy vegetable Taraxacum officinale enhanced the
activities of antioxidant enzymes (GSH, GPx, SOD and CAT) against the CCl4 induced oxidative
stress in mice (Mahesh et al., 2010), suggesting that the reduction of oxidative stress in this
scenario likely plays a role in the mechanism of its hepatoprotective effects. To the best of our
knowledge, no other group in India is exploring the bioactive fraction of S. nigrum for the
inflammatory studies.
16.6 The relevance and expected outcome of the proposed study
The advantages in modern green pharmaceutical concerning, anti-inflammatory drug will
be screened from Solanum nigrum, and their inflammatory activity will be assessed in activated
macrophage cell line. Further, the drug will be investigated against cardiovascular in vivo model
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too. This study will help us to understand the molecular mechanism of Solanum nigrum on
inflammatory diseases. This would be effective, cheapest, more accessible anti-
inflammatory and anti-cardiovascular safety drug from Solanum nigrum. It could also be
served as a food supplement for the treatment of inflammatory diseases.
16.7 Preliminary work done so far
Presently, principal investigator has involved in isolation and fraction of anti-oxidant rich
bioactive compounds from various medicinal plants and food crop including Solanum nigrum.
Preliminary phytochemical analysis revealed that, Saponin glycosides and phenolic acid rich
in leaf crude extract Solanum nigrum (Table 1). The leaf crude extract Solanum nigrum shows
excellent results in anti-oxidant activity (Data not shown). Recently, principal investigator has
also reported in another leafy vegetable, the enhanced activity of the antioxidant enzymes (GSH,
GPx, SOD and CAT) against the CCl4 induced oxidative stress mice treated with sesquiterpene
lactones and ethanolic root extract of Taraxacum officinale (Mahesh et al. 2010). Also the
Scientific evidence suggests that, Saponin glycosides and phenolic acids are the anti-oxidant rich
compounds from the medicinal plants. Polyphenols and Saponin glycosides, currently sold as
dietary supplements and/or herbal remedies have antioxidant, antimutagenic, antiestrogenic,
anticarcinogenic and anti-inflammatory effects that might potentially be beneficial in preventing
disease. Epidemiologic findings revealed that polyphenols have cardioprotective effects, which
include inhibition of platelet aggregation and vascular relaxation through the production of nitric
oxide which decrease LDL oxidation and prevent atherosclerosis (Aron and Kennedy 2008). S.
nigrum chloroform fraction of reduced NO through inhibition of iNOS synthesis and inhibited
TNF-α and IL-6 at the level of protein secretion and also suppressed p38, JNK and ERK1/2 (Hee
et al., 2011).
Table 1: Qualitative phytochemicals analysis of leaf extracts of Solanum nigrum
Phytochemicals analyzed
Solanum nigrum
Aqueous Methanol Chloroform
Alkaloids ++ - -
Cardio active aglycons + + -
Saponin glycosides ++ ++ -
Flavonoids + + -
Phenols ++ ++ -
Sterol + + ++
Proteins ++ - -
Carbohydrates + - -
++ Strongly presence, + presence, - not presence
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Subsequently, the author investigated whether the NO inhibition was due to the reduced number
of cells or due to the change in iNOS levels. The effect of each fraction (50 µg/ml) on LPS/IFN-
γ-stimulated iNOS synthesis was measured using Western blotting. The chloroform fraction
completely inhibited iNOS; the methanol extract showed partial inhibition and the water fraction
showed little influence (Fig. 1a). And also, it was observed the changes in concentration of iNOS
protein expression in response to the chloroform fraction (Fig. 1b).
Figure 1: Effect of the S. nigrum fractions on iNOS expression assessed by Western blotting.
17. Specific objectives
The primary objective of the present proposal is to extraction and fraction bioactive molecule
from Solanum nigrum. Determination of antioxidant activity in fractioned compound and
evaluate their anti-inflammatory and anti-cardiovascular activity
� Quantification of inflammatory cytokines such as, IL-2, IL-4, IL-5, IL-6, IL-10, IL-
17A, TNF, and IFN-γ using flow cytometric bead array in activated macrophage cell
treated with Solanum nigrum.
Methods: Macrophage cells (RAW 264.7) will be seeded to 48 well culture plates with
treatment of S. nigrum fraction for 24 and 48 h. Flow Cytometric Bead Array assay will
be performed for the quantification of cytokine expression in activated macrophage
treated with S. nigrum. In this study, inflammatory cytokine inhibitory activity of S.
nigrum will be known.
� Investigate the inflammatory biomarkers such as NF-kB, COX-2 and TNFα
inhibitory activity of Solanum nigrum in activated macrophage cell line.
Methods: Total protein and RNA will be isolated from activated macrophage treated with S.
nigrum fraction. cDNA will be synthesized using total RNA. The transcriptional level
expression of NF-kB, COX-2 and TNFα will be quantified using qPCR. The protein level
expression of NF-kB, COX-2 and TNFα will be quantified using western blotting
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techniques. This study will help us to understand the anti-inflammatory molecular
mechanism of S. nigrum in activated macrophage.
� Evaluation of cardiovascular protective effect of Solanum nigrum in vivo.
Determination of tissue anti-oxidative enzymes such as SOD, GPx, and GSH in
cardiovascular animals treated with Solanum nigrum fraction.
Methods: Isoproterenol will be injected in Male albino rats to induce Cardiovascular
experimental model. The intoxicated rat will be treated with S. nigrum for 15 days. The
end of the day, heart tissue will be separated by surgical and the tissue anti-oxidant such
as SOD, GPx, and GSH levels will be quantified. Cardiovascular oxidative status will be
clearly known in S. nigrum treated animal model.
� Investigation of inflammatory biomarker NF-kB, COX-2 and TNFα inhibitory
activity at molecular level in cardiovascular animals treated with Solanum nigrum
fraction.
Methods: Isoproterenol will be injected in Male albino rats to induce Cardiovascular
experimental model. The intoxicated rat will be treated with S. nigrum for 15 days. The
end of the day, heart tissue will be separated by surgical and the total protein and RNA
will be isolated. cDNA will be synthesized using total RNA. The transcriptional level
expression of NF-kB, COX-2 and TNFα will be quantified using qPCR. The protein level
expression of NF-kB, COX-2 and TNFα will be quantified using western blotting
technique. This study will help us to understand the molecular mechanism of S. nigrum in
Cardiovascular experimental model
18.1. Work Plan:
18.1a. Plant extraction and fraction
Solanum nigrum will be collected and verified with the voucher specimen. The powdered
plant material (100 g) will be extracted with 600 ml of methanol (1:6 w/v) by maceration at room
temperature. The extract will be filtered using Whatman No. 1 filter paper and vacuum
concentrated at 40°C using a rotary evaporator. Further, the extract will be fractioned with polar
and non- polar solvents.
18.1b. In Vitro determination of Anti-Oxidant activity
18.1b.1. DPPH (2,2-Diphenyl-1-picrylhydrazyl) method
DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay will be carried out
as described by Brand et al. (1995). Various concentrations (10, 50, 100, 500 and 1000 lg/ ml) of
methanolic extract and its fraction will be used. The assay mixture contained in total volume of 1
ml, 500 µl of the extract, 125 µl prepared DPPH and 375 µl solvent. Ascorbic acid will be used
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as positive control. After 30 min incubation at 25 °C, the decrease in absorbance will be
measured at 517 nm. The radical scavenging activity will be calculated from the equation:
% of radical scavenging activity = Abscontrol __
Abssample = Abscontrol X 100
18.1b.2. Superoxide anion radical scavenging
Superoxide anion radical scavenging assay will be carried out as described by Ilhami et
al. (2005). The reaction mixture consisting of 1ml of nitro blue tetrazolium (NBT) solution (156
mM NBT in phosphate buffer, pH 7.4), 1 ml NADH solution (468 mM NADH in phosphate
buffer, pH 7.4), and 1ml of sample solution of HTF extract will be mixed. The reaction will be
started by adding 100 ml of phenazine methosulfate (PMS) solution (60 mM PMS in phosphate
buffer, pH 7.4) to the mixture. The reaction mixture will be incubated at 25oC for 5 min and the
absorbance will be measured at 560 nm against blank sample and compared with standards.
18.1c. In Vitro Anti-inflammatory assays
18.1c.1. Cell culture
Murine macrophage cell line RAW 264.7 will be used in this study. Cells will be
maintained in high glucose DMEM with 10% FBS and no antibiotics, with passage every 2–3
days using standard aseptic techniques. Cells from 70–90% confluent flasks with >90% viability
will be seeded into 96-well culture plates at 5X105cells ML
-1.
18.1c.2. Cell proliferation assay
Cell proliferation will be determined by MTT assay (Campling et al., 1988). Murine
macrophage cells (RAW 264.7) will be seeded to 48 well culture plates in the presence of
Solanum nigrum fraction for 24 and 48 h. After treatment, the medium will be changed and 20 µl
of MTT (5 mg/ml of PBS) will be added to the fresh medium. After 2 h incubation at 37 °C, 100
µl of DMSO will be added to each well and plates will be agitated for 1 min. Absorbance will be
read at 570 nm on a multi well plate reader. Percent inhibition of proliferation will be calculated
as a fraction of control.
18.1c.3. Quantification of Cytokines Using Flow cytometry
Murine macrophage cells (RAW 264.7) will be seeded to 48 well culture plates with
treatment of Solanum nigrum fraction for 24 and 48 h. The cell suspension was centrifuged
(225g, 10 min, RT), and the supernatants were harvested and dispensed into 0.5-mL aliquots in
microcentrifuge tubes. An aliquot was immediately tested for cytokines, IL-2, IL-4, IL-5, IL-6,
IL-10, IL-17A, TNF, and IFN-γ using a flow Cytometric Bead Array assay. Flex Set following
the protocols described in the BD CBA Human Th1/Th2/Th17 Kit Instruction Manual.
18.1c.4. Immunoblot analysis
Murine macrophage cells (RAW 264.7) will be seeded to 48 well culture plates with
treatment of Solanum nigrum fraction for 24 and 48 h. Cells harvest will be used for preparation
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of whole cell extract. The harvested, control and treated, macrophage cells will be washed with
PBS and suspended in lysis buffer (20 mM Tris, 1 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5%
sodium deoxycholate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, 10
µg/ml leupeptin, 20 µg/ml aprotinin and phosphatase inhibitor cocktail 1 and 2 with 100 fold
dilution). After 30 min of shaking at 4 °C, the mixtures will be centrifuged (10,000 g) for 10
min, and the supernatants will be used as the whole cell extracts. The protein content will be
determined according to the Bradford method (Bradford, 1976). 100 µg of protein from each
treatment will be resolved on 12% SDS-PAGE gels (COX-2, NF-kB, and TNFα) along with
protein molecular weight standards, and then transferred onto nitrocellulose membranes. The
membranes will be blocked with 5% w/v nonfat dry milk and then incubated with the primary
antibodies in 10 ml of antibody-diluted buffer (1X Tris-buffered saline and 0.05% Tween-20
with 1% milk) with gentle shaking at 4 °C for 8–12 h and then incubated with peroxidase
conjugated secondary antibodies. Signals will be detected by using western blot detection
reagents.
18. 1C.5. qRT-PCR analysis
Total RNA will be extracted from Solanum nigrum treated RAW 264.7 cells using TRI
reagent. The cDNA will be synthesized from 1 µg of purified RNA using M-MuLV reverse
transcriptase according to manufacturer’s recommendations (Product # EPO451, Fermentas).
Produced cDNA will be diluted and will be used in qRT-PCR. The COX-2, NF-kB, and TNFα
gene specific primers will be used in qRT-PCR. qRT-PCR reactions will be done in a 96- well
plate containing both RAW 264.7 cell (as reference) and RAW 264.7 treated with Solanum
nigrum. The qRT-PCR will be performed using ABI prism 7500 Taqman real time PCR
machine. The results will be calculated using the quantification cycle (Cq) method (delta delta
Cq) according to AB prism default settings. Standard error of mean (s.e) will be also calculated
of three biological replicates.
18.1d. IN VIVO ANTI-CARDIOVASCULAR ACTIVITY
18.1d.1. Animals
Male albino rats (150-170g) will be used throughout the experimental study. The animals
will be housed in polypropylene cages with sterile inert husk materials as bedding. The
experimental animals will be maintained under controlled environment condition at 23±2˚C and
relative humidity of 55±10% with a 12 h dark/light cycle. They will be allowed to acclimatize
for one week and will be provided a free access to standard pellet diet and water ad libitum.
18.1d.2. Experimental design
The animals will be divided into five groups consisting of six animals each for different
experiments. Group I rat will be served as placebo control, received an injection of vehicle only.
Group II (intoxicated group): Isoproterenol (ISO) will be dissolved in physiological saline and
injected into rats subcutaneously (85 mg/kg daily for 2 consecutive days) to induce experimental
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cardiovascular model. Group III and IV cardiovascular rats will be treated with methanolic
extract and active fraction respectively for fifteen days. Group V will be received anti-
inflammatory drug. The end of the day, animals will be anaesthetized with pentobarbital sodium
(35 mg/kg, i.p.) and sacrificed by cervical dislocation and used for the further experiments.
18.1d.3. Determination of tissue anti-oxidant biochemical biomarkers
18.1d.3.1. Determination of reduced glutathione GSH assay
GSH level was measured by the method of Ellman (1959). The homogenate (720 µl) was
double diluted and 5% TCA was added for the precipitation of protein content of the
homogenate. After centrifugation (10,000g for 5 min) the supernatant was taken, DTNB solution
(Ellman's reagent) was added to it and the absorbance was measured at 412 nm. A standard
graph was drawn using different concentrations of a standard GSH solution (1 mg/ml). With the
help of the calibration curve, GSH contents of the kidney homogenates of experimental animals
were calculated.
18.1d.3.2. Measurement of Glutathione Peroxidase (GPx) Activity
The glutathione peroxidase activity was measured by the method of Rotruck et al. (1973).
The homogenate was added to the mixture containing 0.4 ml of phospohate buffer (0.4 M, pH
7.0), 0.1 ml of sodium azide (10 mM), 0.2 ml of reduced glutathione (4 mM) and 0.1 ml of
hydrogen peroxide solution (30 mM) and made up to 2 ml with water and incubate for 10
minutes at room temperature and add 3 ml of Na2HPO4 to terminate the reaction and finally add
1 ml of DTNB. The color developed was read at 412 nm in spectrophotometer.
18.1d.3.3. Measurement of Superoxide Dismutase (SOD) Activity
The activity of SOD was measured by the modified method of Kakkar et al. (1984).
About 5 µg of total protein from kidney homogenate was mixed with sodium pyrophosphate
buffer, PMT and NBT. The reaction was started by the addition of NADH. The reaction mixture
was incubated at 30°C for 90 seconds and stopped by the addition of 1 ml of glacial acetic acid.
The absorbance of the chromogen formed was measured at 560 nm.
18.1d.4. Immunoblotting analysis
The heart will be removed surgically and washed with physiological saline. About 200
mg of heart tissue will be homogenized in 10 ml of 100 mM KH2PO4 containing 1mM EDTA,
pH 7.4 and centrifuged at 12,000g for 30 min at 4°C. The protein content will be determined
according to the Bradford method (Bradford, 1976). 100 µg of protein from each treatment will
be resolved on 12% SDS-PAGE gels (COX-2, NF-kB, and TNFα) along with protein molecular
weight standards, and then transferred onto nitrocellulose membranes. The membranes will be
blocked with 5% w/v nonfat dry milk and then incubated with the primary antibodies in 10 ml of
antibody-diluted buffer (1X Tris-buffered saline and 0.05% Tween-20 with 1% milk) with gentle
shaking at 4 °C for 8–12 h and then incubated with peroxidase conjugated secondary antibodies.
Signals will be detected by using western blot detection reagents.
15
18.1d.5. qRT-PCR analysis
The heart will be removed surgically and washed with physiological saline. Total RNA
will be extracted from heart tissue of rat treated with Solanum nigrum using TRI reagent. The
cDNA will be synthesized from 1 µg of purified RNA using M-MuLV reverse transcriptase
according to manufacturer’s recommendations (Product # EPO451, Fermentas). Produced cDNA
will be diluted and will be used in qRT-PCR. The COX-2, NF-kB, and TNFα gene specific
primers will be used in qRT-PCR. qRT-PCR reactions will be done in a 96- well plate containing
both RAW 264.7 cell (as reference) and RAW 264.7 treated with Solanum nigrum. The qRT-
PCR will be performed using ABI prism 7500 Taqman real time PCR machine. The results will
be calculated using the quantification cycle (Cq) method (delta delta Cq) according to AB prism
default settings. Standard error of mean (s.e) will be also calculated of three biological replicates.
18.1d.6. Histopathological examination
The cardiac apex will be excised and fixed in 4% buffered paraformaldehyde solution.
Tissues will be embedded in paraffin, sectioned at 4 µm and stained with hematoxylin and eosin
(H&E). The sections will be examined by light microscopy for histoarchitectural changes, and
photomicrographs taken.
18.2 Connectivity of the participating institutions and investigators (in case of multi-
institutional projects only)- Nil
18.3 Alternate strategies (if the proposed experimental design or method does not work
what is the alternate strategy)
Alternative strategy for In vitro anti-inflammatory study
� The anti-inflammatory experiment can be done with the methods of Carrageenan-induced
paw edema, Histamine-induced paw edema, Serotonin-induced paw edema, Acetic acid
induced paw edema using animal model.
� Cytokine level will be quantified in the method using ELISA. Inflammatory marker NF-
kB, COX-2 and TNFα levels will be assessed by reverse transcriptase PCR.
Alternative strategy for In vivo cardio-protective study
� Cardio-protective study can be done by atherosclerosis animal model and the serum
enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST),
alkaline phosphatase (ALP) and C-reactive protein will be determined.
� Inflammatory marker NF-kB, COX-2 and TNFα levels in atherosclerosis animal treated
with S. nigrum will be assessed by reverse transcriptase PCR.
16
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Nutr Food Res 52: 79‑104.
Arunachalama G, Subramanian N, Pazhani PG, Karunanithi M, Ravichandran V. (2009). Evaluation of anti-inflammatory Activity of Methanolic Extract of Solanum nigrum (Solanaceae). Iranian Journal of Pharmaceutical Sciences 5(3): 151-156.
Arulmozhi V, Krishnaveni M, Karthishwaran K, Dhamodharan G, Mirunalini S. (2010). Antioxidant and antihyperlipidemic effect of Solanum nigrum fruit extract on the experimental model against chronic ethanol toxicity. Pharmacogn Mag 6: 42-50.
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Gupta RK, Kesari AN, Murthy PS, Chandra R, Tandon V, Watal G (2005). Hypoglycemic and antidiabetic effect of ethanolic extract of leaves of Annona squamosa L. in experimental animals. J. Ethnopharmacol 99(1): 75-81.
Hee K, Ha-Deok J, Ho-Young C. (2011). The Chloroform Fraction of Solanum nigrum
Suppresses Nitric Oxide and Tumor Necrosis Factor-α in LPS-Stimulated Mouse Peritoneal Macrophages through Inhibition of p38, JNK and ERK1/2. The American Journal of
Chinese Medicine 39: 1261-1273.
Holm S, Mackiewicz Z, Holm AK, Kontinen YT, Kouri VP, Indahl A, Salo J, (2009). Pro-inflammatory, pleiotropic, and anti-inflammatory TNF- α, IL-6, and IL-10 in experimental porcin intervertebral disk degeneration. Veterinary Pathology 46: 1292–1300.
Hsieh CC, Fang HL, Lina WC. (2008). Inhibitory effect of Solanum nigrum on thioacetamide induced liver fibrosis in mice. J. Ethnopharmacol 119: 117–121.
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Lawrence T, Willoughby DA, Gilroy DW. (2002). Anti-inflammatory lipid mediators and insights into the resolution of inflammation. Nature Reviews Immunology 2: 787–795.
Li J, Li QW, Gao DW, Han ZS, Lu WZ. (2009). Antitumor and immunomodulating effects of polysaccharides isolated from Solanum nigrum L. Phytother Res 23: 1524–1530.
Mahesh A, Jeyachandran R, Cindrella L, Thangadurai D, Muralidhara Rao D. (2010). Hepatocurative potential of sesquiterpene lactones of Taraxacum officinale on carbon tetrachloride induced liver toxicity in mice. Acta Biologica Hungarica, 61: 175-190.
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Atherothrombotic risk factors & premature coronary heart disease in India: A case-control study. Indian J Med Res 134: 26-32.
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Med 2: 39-45.
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Sultana S, Perwaiz S, Iqbal M, Athar M. (1995) Crude extract of hepatoprotective plants,
Solanum nigrum and Cichorum intybus inhibit free radical‑mediated DNA damage. J
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Wang HC, Chung PJ, Wu CH, Lan KP, Yang MY, Wang CJ. (2011). Solanum nigrum L. polyphenolic extract inhibits hepatocarcinoma cell growth by inducing G2/M phase arrest and apoptosis. J Sci Food Agric 91: 178-85.
18
19. Timelines: (Please provide quantifiable outputs)
Period of study Achievable targets
6 Months
Appointment JRF, Purchase chemicals and instruments. Collection, extraction and fraction of Solanum nigrum and
evaluation of their antioxidant activity.
12 Months Quantification of cytokines using flow cytometric bead array
and MTT proliferation assay in macrophage cells treated with Solanum nigrum.
18 Months qRT-PCR quantification of COX-2, NF-kB, and TNFα expression in macrophage cells treated with Solanum nigrum.
24 Months Immunoblot quantification COX-2, NF-kB, and TNFα expression in macrophage cells treated with Solanum nigrum.
30 Months Assessment of Histopathological and oxidative enzyme activity in Solanum nigrum treated cardiovascular animal model
36 Months qRT-PCR and Immunobloting investigation of inflammatory biomarkers COX-2, NF-kB, and TNFα in Solanum nigrum
treated cardiovascular animal model
20. Name and address of 5 experts in the field
S.No. Name Designation Address
1
Dr. D. Muralidhara Rao
Associate professor
Department of Biotechnology Sri Krishnadevaraya University, Anantapur 515003, (A.P).
2
Prof. Veeresh P. Veerapur
Professor and Head
Department in Quality Assurance, Sree Siddaganga College of Pharmacy B.H Road Tumkur-572 102. Karnataka
3
Dr. J. Arunakaran Associate Professor
Department of Endocrinology, Dr ALM PGIBMS, University of Madras,
Taramani, Chennai 600 113. Tamilnadu.
4
Prof. K. Anand Kumar,
Professor, Department of Biotechnology, University of Hyderabad,
Prof. C. R. Rao Road, Gachibowli, Hyderabad-500046 (A.P), India
5
Dr. S. Narkunaraja Associate Professor &
Head
Department of Bio-Medical Science, Bharathidasan University,
Tiruchirappalli – 620 024. Tamilnadu.
19
PART IV: BUDGET PARTICULARS
Budget (In Rupees)
A. Non-Recurring (e.g. equipments, accessories, etc.)
S.
No.
Item Year 1 Year 2 Year 3 Total
1.
2 3 4 6
Centrifuge 5430R
Price in € 7370
Auto clave (Table Top) BioClave-16Lit Equitron Dry Bath Model 8550.1.C-10 WITH COOLING MODEL Spinot Magnetic Stirrer With hot plate Rotospin Rotary mixer
5,25,000
2,64,000 58,500 15,600 35,800
Sub-Total (A) `̀̀̀ 8,98,900
Justification:
Equipment: Centrifuge 5430R is important for collecting macrophage cells by centrifugation
and also needed for isolation of DNA, RNA and proteins from macrophage cells after
completion of drug treatment.
Minor equipment: These basic minor equipments include a Dry Bath, Spinot Magnetic Stirrer,
Rotary mixer are needed often for performing experiments.
B. Recurring
B.1 Manpower (See guidelines at Annexure-III)
S.
No.
Position
No.
Consolidated
Emolument
Year 1 Year 2 Year 3 Total
1 Research fellow
` 16,000/- p.m years 1 & 2 and `
18,000/- p.m for 3 year + 15% HRA
2,20,800 2,20,800 2,48,400 6,90,000
Sub-Total (B.1) = ` 6,90,000
Justification:
Manpower: One research fellows is essential for carry out the work outlined in the proposal.
20
B.2 Consumables
S.
No.
Item
Year 1 Year 2 Year 3 Total
1. Chemicals, glassware,
plastic ware, molecular biology
kits, primers and fine chemicals for
research purpose
3,00,000 3,00,000 2,00,000 8,00,000
Sub-Total (B.2) = ` 8,00,000
Justification:
Consumables: The proposed work requires a large number of chemicals, molecular biology
grade reagents and enzymes. These are needed for the maintenance of murine macrophage cell
line (RAW 264.7), animal maintenance, flow cytometry kit, PCR, protein purification, DNA
extraction, RNA isolation, real-time PCR analysis, western blot analysis. Disposable 24, 48 and
96 well plate, petri dish, Pipette tips, microcentrifuge and high-speed centrifuge tubes are also
needed for the experiments.
Other items Year 1 Year 2 Year 3 Total
B.3 Travel
30,000 25,000 30,000 90,000
B.4 Contingency
25,000 25,000 25,000 75,000
B.5 Overhead
(If applicable)
86,370 85,620 75,510 2,47,500
Sub-total of B
(B.1+B.2+B.3+B.4+B.5)
6,62,170 6,56,420 5,78,910 19,02,500
Grand Total (A + B) 28,01,400
Grand Total = `̀̀̀ 28,01,400
Justification:
Travel: Grant is requested under this head to meet the travel expenses for presenting the project
proposal and progress in each year. This grant will be utilized for attending national symposiums
and workshops too.
Contingency: Purchase of local items like liquid nitrogen (for plant DNA and RNA extraction),
gloves, aluminum foil, cotton rolls, tissue paper, Whatmann No.1 filter papers, sterile blades,
stationary items, compact discs for the data storage and custom clearance charges for imported
chemicals and equipment. Also the charges for cleaning glassware and the lab have to be met
from this head.
21
PART V: EXISTING FACILITIES
Resources and additional information
1. Laboratory: a. Manpower: NIL
b. Equipments:
1) Flow cytometry, 2) Live Cell Imaging System,
3) LC/MS-MS, 4) Animal cell culture chamber,
5) light microscope, 6) florescent microscope,
7) Ultra centrifuge, 8) Ultracentrifuge, 9) speed vacuum, 10) ice machine, 11) –20oC and -70oC freezers, 12) PCR machines, 13) Chemilumiscence Gel Doc system, 14) 2 D Gel electrophoresis unit, 15) Sonicator, 16) Bioanalyzer, 17) Microarray analyzer 18) Spectrophotometer.
2. Other resources such as clinical material, animal house facility, glass house.
Experimental garden, pilot plant facility etc.
Animal Tissue Culture room, Animal house, etc.,
22
23
PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS
Provide the following information for the key personnel in the order listed on PART II. Follow this format for each person. DO NOT EXCEED THREE PAGES
Name : Dr. A. Mahesh Designation : Lecturer
Department/Institute/University : School of Biological Sciences, Madurai Kamaraj
University.
Date of Birth : 15/01/1980 Sex (M/F): M SC/ST : No
Education (Post-Graduation onwards & Professional Career)
Sl No.
Institution Place
Degree Awarded
Year Field of Study
1 Bharathidasan University/ St. Joseph’s College, Tiruchirappalli
M. Sc 2003 Plant Biotechnology
2 Bharathidasan University/ St. Joseph’s College, Tiruchirappalli
Ph. D 2008 Plant Biotechnology and Pharmacology
3 Agricultural Research Organization, The Volcani Centre, Israel
Post-doctoral fellow
Jan 2009- June 2011
SiRNA silencing vector developed against Ricin for Castor bean
4 Bar- Ilan University, Israel
Post-doctoral fellow
July 2011-Sep 2011
Functional genomics analysis in Cucumber plant- a reverse genetics approach
A. Position and Honors
Position and Employment (Starting with the most recent employment)
Sl No. Institution Place
Position From (Date) To (date)
1 Madurai Kamaraj University Lecturer Sep 2011 Till date
2 Bar- Ilan University,
Israel
Post-doctoral fellow July 2011 Sep 2011
3 Agricultural Research
Organization, The Volcani Centre, Israel
Post-doctoral fellow Jan 2009 June 2011
Honors/Awards
� Konan Zoref Post- doc fellowship, Bar-Ilan University, Ramat- Gan, Israel (2011-2012)
� University Research Fellow, Bharathidasan University, Tiruchirapalli, India (2005-2008)
� Reviewer, Journal of Pharmacy and Pharmacology, Wiley-Blackwell Publications (Since
2010)
24
� Reviewer, Pharmaceutical Biology, Taylor & Francis Publications (Since 2008)
� Reviewer, Natural Product research, Wiley-Blackwell Publications (Since 2010)
� Associate Editor, Biotechnology, Asian Network for Scientific Information (2007-2011)
� Associate Editor, International Journal of Pharmacology, Asian Network for Scientific
Information (2007-2011)
Membership in Scientific societies
• Life Member – Biotechnology Research Society of India (BRSI)
• Life Member – The Indian Science Congress Association
Professional Experience and Training relevant to the Project
Phytochemical investigation
The principal investigator has been working on the extraction and characterization of plant based
pharmaceutics, such as secondary metabolites and protein from various medicinal plants. He has
well experienced in TLC, column chromatography, and spray detection of secondary metabolites
such as, polyphenols, terpenoids, Alkaloids, and flavonoids, etc. He has also working on column
based purification of bioactive principles for NMR, GC/MS, LC/MS, UV and FTIR analysis. He
also published many research article related to the proposed in various peer reviewed journal.
Pharmacology work
The principal investigator has been working on drug development and delivery in vivo during his
Ph.D., programme. He is well expertise in experimental animal (mice, rat and rabbit) handling
and breeding. He has well experiences in developing animal models such as, Cardiovascular
diseases, Hepatoxic, Nephrotoxic, Diabetic and Gastric ulcer for drug developmental research.
And also, he developed serum enzyme and tissue oxidative biochemical biomarkers assays for
herbal drug research.
Animal cell culture
Presently, principal investigator maintaining the Cancer cell line such as, Hepatocellular
carcinoma (HepG2), Breast cancer (MCF7) and Prostate cancer (PC-3). He is also started
working drug development research against various cancer and inflammatory model in vitro. He
has also experience for rat phagocytes isolation and culturing in vitro and he also is maintaining
phagocyte culture.
Molecular biology
He is involved in developing flow cytometry assays and live cell imaging for cancer drug
development. In addition, he had experience in molecular biology techniques and genomics
research in post-doctoral studies. He developed siRNA construct against ricin for producing ricin
free castor bean in ARO, Israel. He has constructed the Ricin sense and antisense repeated
orientation with spacer for expressing the siRNA targeted to down-regulation of Ricin toxin in
25
Ricinus communis. He confirmed the siRNA production by qPCR, RT-PCR, confocal
microscopy visualization, Northern blotting and Western blotting. He was working in reverse
genetic for assessing the functional genome in Cucumber in Bar-Illan University, Israel. There,
he learned computational such as, BLAST, SNP analysis, sequence alignment, pair wise
sequence alignment, M13 florescent tagged PCR amplification, licor gel electrophoresis.
B. Publications (Numbers only) .................
Books : 7, Research Papers, Reports : 17, General articles : NIL
Patents : NIL, Others (Conferences at International / National level) : 48
Selected peer-reviewed publications (Ten best publications in chronological order)
*corresponding author
1. *Mahesh, A., Jeyachandran, R., Muralidhara Rao, D. and Thangadurai, D. (2012).
Gastroprotective effect of Desmodium gangeticum roots on gastric ulcer mouse models.
Brazilian Journal of pharmacognosy, (On line Published). http://dx.doi.org/10.1590/S0102-
695X2012005000081.
2. *Sathish Climer, C., Mahesh, A., Sinilal, B., Muralidhara Rao, D. and Thangadurai, D.
(2012). Protective effect of Indigofera aspalathoides roots on N-nitrosodiethylamine
induced hepatocarcinogenesis in mice. Indian Journal of Pharmaceutical Sciences.
(Accepted).
3. Gobinath, S., Mahesh, A., Thangadurai, D. and Muralidhara Rao, D. (2012). Protective
effect of Caralluma umbellate Haw. against paracetamol induced oxidative stress and
hepatotoxicity in rat. Acta Biologica Hungarica. (Accepted).
4. *Vimaladevi, S., Mahesh, A., Dhayanithi, N.B. and Karthikeyan, N. (2012). Mosquito
larvicidal efficacy of phenolic acids of seaweed Chaetomorpha antennina (Bory) Kuetz.
Biologia. 67(1): 212-216.
5. *Mahesh, A. and Jeyachandran, R. (2011). Agrobacterium rhizogenes-mediated hairy root
induction in Taraxacum officinale and analysis of sesquiterpene lactones. Plant biosystems,
145(3): 620-626.
6. *Mahesh, A., Jeyachandran, R., Cindrella, L., Thangadurai, D. and Muralidhara Rao, D.
(2010). Hepatocurative potential of sesquiterpene lactones of Taraxacum officinale on
carbon tetrachloride induced liver toxicity in mice. Acta Biologica Hungarica, 61(2): 175–
190.
7. *Mahesh, A., Shaheetha, J., Thangadurai, D. and Muralidhara Rao, D. (2009). Protective
effect of Indian honey on acetaminophen induced oxidative stress and liver toxicity in rat.
Biologia, 64(6): 1225-1231.
8. Jeyachandran, R., Mahesh, A., Cindrella, L., Sudhakar, S. and Pazhanichamy, K. (2009).
Evaluation of antibacterial activity of plumbagin and the root extracts of Plumbago
zeylanica L. Acta Biologica Cracoviensia Series Botanica, 51(1): 17–22.
26
9. Jeyachandran, R., Cindrella, L. and Mahesh, A. (2009). Pharmacological evaluation of
Sphaeranthus indicus Linn. using albino rats. Afr. J. Trad. CAM, 6 (Supplement):456-457.
10. Jeyachandran, R., Mahesh, A. and Cindrella, L. (2007). DEN - induced cancer and its
alleviation by Anisomeles malabarica (L.) R. Br. ethanolic leaf extract in male albino mice.
International Journal of Cancer Research, 3(4): 174-179.
List maximum of five recent publications relevant to the proposed area of work.
1. *Mahesh, A., Jeyachandran, R., Muralidhara Rao, D. and Thangadurai, D. (2012).
Gastroprotective effect of Desmodium gangeticum roots on gastric ulcer mouse models.
Brazilian Journal of pharmacognosy, (On line Published). http://dx.doi.org/10.1590/S0102-
695X2012005000081.
2. *Mahesh, A., Jeyachandran, R., Cindrella, L., Thangadurai, D. and Muralidhara Rao, D.
(2010). Hepatocurative potential of sesquiterpene lactones of Taraxacum officinale on carbon
tetrachloride induced liver toxicity in mice. Acta Biologica Hungarica, 61(2): 175–190.
3. *Mahesh, A., Shaheetha, J., Thangadurai, D. and Muralidhara Rao, D. (2009). Protective
effect of Indian honey on acetaminophen induced oxidative stress and liver toxicity in rat.
Biologia, 64(6): 1225-1231.
4. Jeyachandran, R., Mahesh, A., Cindrella, L., Sudhakar, S. and Pazhanichamy, K. (2009).
Evaluation of antibacterial activity of plumbagin and the root extracts of Plumbago zeylanica
L. Acta Biologica Cracoviensia Series Botanica, 51(1): 17–22.
5. Jeyachandran, R., Mahesh, A. and Cindrella, L. (2007). DEN - induced cancer and its
alleviation by Anisomeles malabarica (L.) R. Br. ethanolic leaf extract in male albino mice.
International Journal of Cancer Research, 3(4): 174-179.
BOOK CHAPTERS
1. Jeyachandran, R., Mahesh, A. and Cindrella, L. (2010). Glucosamine- a novel medicine
for Arthritis. In: Role of Biotechnology in Medicinal and Aromatic plants, Vol- XXI, Ali
Khan, I. and khanum, A (eds). Ukaaz publications, Hyderabad. 246-272
2. Jeyachandran, R., Mahesh, A. and Cindrella, L. (2010). Epidemiology of Hot water
epilepsy. In: Role of Biotechnology in Medicinal and Aromatic plants, Vol- XXI, Ali
Khan, I. and khanum, A (eds). Ukaaz publications, Hyderabad. Pp 273-296
3. Mahesh, A., Jeyachandran, R., Cindrella, L. Thangadurai, D. and Pinheiro de Carvalho,
M.A.A. (2008). Phytochemical, Pharmacological and Tissue Culture Studies on
Taraxacum officinale Weber (Asteraceae). In: Biotechnology for food, Environment and
Agriculture, Vol. I. Thangadurai, D. (eds.), Agrobios Press, India. pp. 235-251.
4. Jeyachandran, R., Mahesh, A. and Cindrella, L. (2008). Hepatoprotective Medicines, In:
Role of Biotechnology in Medicinal and Aromatic plants, Vol- XIX. Ali Khan, I.,
Khanum, A. and Ahad khan, A. (eds). Ukaaz publications, Hyderabad. pp 114-142.
27
5. Jeyachandran, R., Mahesh, A. and Cindrella, L. (2008). Nephroprotective Plant
28
BIOGRAPHICAL SKETCH OF CO- INVESTIGATOR
Name : Prof. P. Gunasekaran
Designation : Head of the Department of Genetics
Department/Institute/University : School of Biological Sciences, Madurai Kamaraj
University.
Date of Birth: 2.07.1954 Sex (M/F): M SC/ST : No
Education (Post-Graduation onwards & Professional Career)
Institution
Place
Degree
Awarded
Year Field of Study
Haryana Agricultural
University, Hisar
Ph. D., 1980 Microbiology
Madurai Kamaraj
University, Madurai
M. Sc., 1976 Biology
Madurai Kamaraj
University, Madurai
B. Sc., 1974 Chemistry
A. Position and Employment
� Co-ordinator, School of Biological Sciences, Madurai Kamaraj University – Since July 2008
� Special Officer, Madurai Kamaraj University, Madurai - Since April 2008. � Senior Professor and Head, Department of Genetics, Madurai Kamaraj University,
Madurai - Since 2007. � Head, Department of Genetics, Madurai Kamaraj University, Madurai, India, since 2001.
� Professor of Microbiology, Department of Microbiology, Madurai Kamaraj University, Madurai, India, since 1991
� Invited Professor and Divisional Chair, Department of Biotechnology, Division of Biological and Health Sciences Universidad Autonoma Metropolitana, México, 1995-97
� Invited Professor, Faculty of Sciences at Luminy, University of Provence, Marseille, France. 1994
� Reader in Microbiology, Department of Microbiology, Madurai Kamaraj University, Madurai, India, 1987
� Invited Associate Professor, Faculty of Sciences at Luminy, University of Provence, Marseille, France, 1987
� Lecturer in Microbiology, Department of Microbiology Madurai Kamaraj University, Madurai, India, 1981
� Lecturer cum Curator in Microbiology, Department of Microbiology, Punjab University, Chandigarh, India, 1980
Honors/Awards (During 2000’s)
� Member, Twelth Five year Plan Working Group of Higher Education - Government of Tamilnadu - 2012-2017.
� Member, Monitoring Commitee of Distance Education Progrmme - Goverment of Tamilnadu - 2012
� Visitors Nominee - Central University of Patiala - Nominated by President of India - 2012
29
� Member, Monitoring Commitee of Goverment of Tamilnadu - Unapproved Institutions offering / awarding degree / diplomoa certificates without the prior approval of UGC, AICTE and DEC - 2011.
� Government of Tamilnadu Nominee - UGC Expert Commitee to evaluate the academic performance of SFR College for women, Sivakasi - 2011
� Outstanding Service Award - International Board of the American Soceity for
Microbiology (ASM) - 2011 President, Biotechnology Research Society of India � 76th Indian Science Congress-Madurai Kamaraj University Eminent Scientist Award
(Natural Sciences) – 2009 � International Editor, Journal of Microbiology and Biotechnology – 2009
� President, Association of Microbiologists of India – 2009 � Life Time Achievement Award, Indian Association of Applied Microbiologists, India -
2008. � Chairman, Advisory Monitoring Committee, Centre for Biofuels, Technology
Information, Forecasting and Assessment Council (TIFAC), DST, India – 2008 � Fellow of the National Academy of Biological Sciences (FNABS) – 2008. � American Society of Microbiology (ASM) International Professorship – 2007. � AMI-Prof.S.R.Vyas Award for the contribution in Microbiology – 2006. � Fellow of Association of Microbiologists of India (FAMI) – 2006. � Fellow of Biotechnology Research Society of India (FBRSI) – 2004. � Taskforce Member, Department of Biotechnology, New Delhi (2004-2007).
B. Publications (Numbers only)
Books : 28, Research Papers, Reports : 132, General articles : NIL Patents : NIL, Others (Please specify) : NIL
Selected peer-reviewed publications (Ten best publications in chronological order)
1. Babu, A., Jeyasubramanian, K., Gunasekaran, P. and Murugesan R. (2012) Gelatin nanocarrier enables efficient delivery and phototoxicity of hypocrellin B against a mice tumour model. Journal of Biomedical Nanotechnology (In Press).
2. Sujatha, K., Mahalakshmi, A., Sridhar J. and Gunasekaran P. (2012) Pseudomonas sp. as
a Source of Medium Chain Length Polyhydroxyalkanoates for Controlled Drug Delivery: Perspective. International Journal of Microbiology doi:10.1155/2012/317828.
3. Sridhar, J., Sabarinathan, R., Siva Balan, S., Rafi, ZA., Gunasekaran, P. and Sekar, K. (2011) Junker: An Intergenic Explorer for Bacterial Genomes. Genomics Proteomics
Bioinformatics 9: 179-182.
4. Ganesh Babu, MM., Sridhar J. and Gunasekaran P. (2011) Global transcriptome analysis of Bacillus cereus ATCC 14579 in response to silver nitrate stress. Journal of
Nanobiotechnology, 2011, 9: 49.
5. Lavanya Pushpam, P., Rajesh, T. and Gunasekaran, P. (2011) Identification and characterization of alkaline serine protease from goat skin surface metagenome. Applied
Microbiology and Biotechnology Express, 2011, 1: 3.
6. Jayashree, S., Rajendhran, J., Jayaraman, K., Kalaichelvan, G. and Gunasekaran, P. (2011) Improvement of riboflavin production by Lactobacillus fermentum isolated from yoghurt. Food Biotechnology 25: 240-251.
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7. Rajnish, KN., Asraf, SS., Manju, N. and Gunasekaran, P. (2011). Functional characterization of a putative β-lactamase gene in the genome of Zymomonas mobilis. Biotechnology Letters, 2011, 33: 2425-2430.
8. Asraf, SS., Rajnish, KN. and Gunasekaran, P. (2011). Bioinformatics and biosynthesis
analysis of cellulose synthase operon in Zymomonas mobilis ZM4. IIOAB Journal 2: 1-7.
9. Rajesh, T., Anthony, T., Saranya, S., Pushpam, PL. and Gunasekaran, P. (2011).
Functional characterization of a new holin-like antibacterial protein coding gene tmp1 from goat skin surface metagenome. Applied Microbiology and Biotechnology 89: 1061-
1073.
10. Rajendhran, J. and Gunasekaran, P. (2011). Microbial phylogeny and diversity: Small subunit ribosomal RNA sequence analysis and beyond. Microbiological Research 166: 99-110.
Selected Book Chapter’s (Ten best Book Chapter in chronological order)
1. T Rajesh, J Rajendhran and P Gunasekaran (2012). Isolation of Metagenomic DNA and RNA from Soil: Microbial Diversity Analysis and Construction of Metagenomic Library. Basic Protocols in Molecular Microbiology. (Accepted)
2. T Rajesh, J Rajendhran and P. Gunasekaran (2011). Genomics Strategies for Environmental Bioremediation. In: Microbes in Environmental Management and Biotechnology. (Ed: SatyanarayanaT). Springer Press. 701-718.
3. T Rajesh and P Gunasekaran (2011). Structural Model for a Holin-like Protein Tmp3
Exhibiting Broad Spectrum Antibacterial Activity. Science and Technology against Microbial Pathogens. Research, Development and Evaluation. World Scientific Publishing
Group.
4. T Rajesh, T Anthony, J Rajendhran and P Gunasekaran (2011). Alternate Expression Systems for High-level expression of Membrane Active Antibacterial Proteins. Advances in Bioprocesses in Food Industries, Volume IV. Asiatech Publishers.
5. T Rajesh, J Rajendhran, P Lavanya pushpam and P Gunasekaran. (2011). Methods in Metagenomic DNA and RNA Isolation from Soil. Handbook of Molecular Microbial Ecology II: Metagenomics in Different Habitats. Ed: Frans J. de Bruijn, Ed., John Wiley & Sons, New Jersey.
6. SS Asraf, KN Rajnish and P Gunasekaran (2011). Structure and functional analysis of the putative beta-galactosidase in Zymomonas mobilis. Recent Trends in Computational
Biology and Computational Statistics Applied in Biotechnology and Bioinformatics. Ed: Ajit Kumar Roy.
7. SS Asraf and P Gunasekaran (2011). Current trends of ß-galactosidase research and application. Current Research, Technology and Education Topics in Applied Microbiology
and Microbial Biotechnology. Microbiology book series - Number 2. Pg: 880-890. Antonio Mendez-vilas (Ed.) Formatex Research Center, Badajoz, Spain. ISBN (13) 978-
84-614-6195-0
8. T. Rajesh, J. Rajendhran, PL Pushpam and P. Gunasekaran (2010). Methods in Metagenomic DNA, RNA andProtein Isolation from Soil. In: Handbook of Molecular Microbial Ecology: Metagenomics in Different Habitats, John Wiley & Sons, NY, USA.
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9. T Rajesh and P