“Anti-oxidant and Anti-microbial activity of alcoholic extract fractions of Delonix elata barks”

SYNOPSIS FOR

M.PHARM DISSERTATION

SUBMITTED TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA

BY

SATHYA CHETHAN PABBITHI

I M. PHARM

DEPARTMENT OF PHARMACOGNOSY

PES COLLEGE OF PHARMACY

BANGALORE – 560050, KARNATAKA.

(2011 – 2012)

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BANGALORE.

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR P.G. DISSERTATION

1. / Name of the candidate and address(in block letters) /

SATHYA CHETHAN PABBITHI

S/O NAGENDRA PRASAD
NAGENDRA MEDICALS & GENERAL STORES ,R.S ROAD,
TANAKALLU.PIN-515571
ANANTAPUR(DIST),AP
2. /

Name of the institution

/

PES COLLEGE OF PHARMACY

50 FEET ROAD, HANUMANTHANAGAR, BANGALORE - 560050
3. /

Course of study and subject

/

MASTER OF PHARMACY IN

PHARMACOGNOSY

4. /

Date of the admission

/ 05.06.2010

Title of the topic:

“ANTI-OXIDANT AND ANTI-MICROBIAL ACTIVITY OF ALCOHOLIC EXTRACT FRACTIONS OF DELONIX ELETA BARKS”
BRIEF RESUME OF THE INTENDED WORK
6.1 NEED FOR THE STUDY :
About 80,000 species of plants are utilized for treating various diseases in different systems of Indian medicine. Since 1990s there has been a growing shift in interest towards plant as significant sources for new pharmaceuticals, companies show interest in plant derived drugs mainly due to the current widespread belief that ‘Green medicine’is safe and more dependable than the costly synthetic drugs, which have adverse side effects.
As per the world Health Organization (WHO) report, 80% of the world population presently use herbal medicine for some aspect of primary health care.1 Since last decade, the rise in the failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic microbial infectious agents has led to the screening of several medicinal plants for their potential anti-microbial activity.2,3 With the advancement of modern medicinal technology, it is now easier to identify specific botanical constituents and assess their potential antimicrobial activity. Many herbs contain dozens of active constituents that combine to give the plants its therapeutic value.
Antioxidant-based drugs/formulations for theprevention and treatment of complex disorders likeatherosclerosis, stroke, diabetes, Alzheimer’s disease andcancer have appeared during the last few years.4 Thishas attracted a great deal of research interest in naturalantioxidants. Subsequently, a worldwide trend towardsthe use of natural phyto-chemicals present in berry crops,tea, herbs, oilseeds, beans, fruits, and vegetables hasincreased.5
As there were some preliminary work carried out on extractsof Delonix Species forantimicrobial and anti oxidant activity, in this study we have taken up fractionation of the hydro-alcoholic extract in order to look for the active fraction/isolated compound responsible for the activity.
REVIEW OF LITERATURE :
Habitat :
Deionix eleta growsthroughout india.
Morphology description :6
Delonix elata is erect tree 6-9 m. high;bark tolerably smooth, ash coloured. Leaves abruptly 2-pinnate,10-20cm.long; main rhacis slender; pinna,e 4-8 pairs, opposite. Leaf lets 10-20 pairs,subsessile, 3 by 8mm., closely set along the rhachis, linear ablong, rounded and usually apiculate at the apex, glabrous, caduceus. Flowers in terminal few-flowered corymbiform racemes ; pedicels stout, finely pubscent. Calyx 2-2.5 cm. long , coriaceous, silky-pubscent outside; segments linear-oblong, acute. Petals suborbicular, yellow, scarcely exserted, the upper a little smaller and of a deeper colour than the others, the margins of all much curled. Filaments often 6.3 cm. long, villous and thickened at the base. Pods 12.5-18 by 2-3.2 cm., attenuated at both ends, reticulately veined ,glabrous.
Synonyms ofDelonix elata :
English :creamy peacock flower, flamboyant tree, tiger bean, white gul mohur
Gujarati : sandesra
Tamil : padenarayan, vadanarayana
Telugu :Chinna seribiseri, Chitti keshwaramu.
Kannada :kempukenjiga, nirangi
DELONEX ELATA:
1.Kingdom : Plantae
2.Division : Magnoliophyta
3.Class : Magnoliopsida
4.Order : Fabaceae
5.Subfamily :Caesalpiniodae
6.Tribe : Caesalpinieae
7.Genus : Delonix
Chemical constituens:6
The phyto constituents such as Alkaloids, Tannnins, Triterpenoids, steroids,Glycosides.
flavonoids, so-flaflavones, flavones,anthocyanine, coumarines, lignins,vitamin-A,vitamin-E,vitamin-C.
ß-Amyrin, hesperitin and neohesperidin have been newly isolated from the dried roots.
The seed oil of Delonexelata contains small amounts of sterculic acid and Malvalic acid.
Uses :7
1.Root decoction is used for abdominal pains and in treatment of scorpion bite.
2.Leaf extract areused asin anti-inflammatory.
4. It is used as Bronchitis and pneumonia in infants.
5. used as a carminative..
6. The twigs are used for the toothache.
8. The plant is used in the treatment of rheumatism and flatulence.
6.2 Literature :
Preliminary studies on phytochemicals and Anti-microbial activity of Delonex elata and Prosopsis cineraria was carried out by Sivanarayan, V and Suriyavathana. In their study they have concluded the Delonix elata and Prosopis cineraria were deserved to have antimicrobial activity and can be used for medicinal purposes.8
Delonex elata (L.) was subjected for qualitative test which indicated the presence of phenolic compounds and TLC system confirmed the same.Authors also studied the microscopical character of the leaf and determined some physic chemical parameter for the leaf.9
In vitro Antioxidant Properties of Asteracantha longifolia, Delonix elata, Passiflora edulis was carried out.10
The antibacterial activity of organic solvent extracts of few plants were determined by disc diffusion and broth dilution techniques against gram-positive bacterial strains and gram-negative bacterial strains.Results revealed that the chloroform and methanol extract of D. elata exhibited significant antibacterial activity against gram-positive and gram-negative strains with minimum bactericidal concentration (MBC) ranging from 1.5 to 100 mg/ml.11
Jahan I and co authors had reported lupeol , epilupeol , β-sitosterol , stigmasterol and p-methoxybenzaldehydefrom the petroleum ether and dichloromethane fractions of methanolic extract of the stem bark of Delonix regia. Antimicrobial screening of the extracts was conducted by the disc diffusion method.12
A galactomannan was isolated from seeds of Delonix regia by Tamaki Y et. al., The ratio of D-mannose to D-galactose was estimated approximately to be 4:1 by HPLC and H1-NMR spectra, and methylation analysis of the galactomannan indicated that it was composed of 1,4-linked beta-D-mannose , 1,4,6-linked beta D-mannose, and terminal alpha D-galactose.13
Delonix regia seed gum used as a binder in paracetamol tablets. Acacia and tragacanth were used official gum standards. The mechanical properties ,i.e. tensile strength (TS) and brittle fracture index (BFI) ,showed that with an increase in concentration of the gum binder ,the tensil strength increased while the BFI was reduced. The result suggest that Delonix regia seed gum may be useful as a binder ,its use at a low concentration improved the balance between the binding and disintegration properties of tablets.14
Water extracts from Delonix regiaflowers was studied which contain polyphenols bioproducts. Three major anthocyanins from this extract have been characterized. The molecular structures were confirmed from LC-SM analysis15.
A plant decoction (AM-1) formulated from Jatropha curcas, Gossypium hirsutum, physalis angulata and Delonix regiawas subjected for the evaluation of efficacy and safety for the treatment of malaria. The results indicated that AM-1 could be used to treat malaria. The obtained data Warrant further studies in a large number of malaria subjects with monitoring for possible drug interaction.16
A lectin from Delonix regia seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethyl aminoethyl - sepharose and reverse phase HPLC on C18 column.17
6.3 OBJECTIVES OF THE STUDY
1. Collection of Delonix elata bark.
2. Extraction of plant material by hydro-alcohol.
3.Development of TLC and HPTLC profiles.
4.Fractionation ofextract by column chromatography.
5. Anti-oxidant and Anti-microbial Activity of the Fractions / isolated compound.
7.MATERIAL AND METHODS
7.1 SOURCE OF DATA:
Whole work is planned to generate data from laboratory studies i.e.,experiments are performed as described in reference , and text books available within college, IISC, Bengalouru, RGUHS-Digital library (helinet).
WEBSITES :




METHOD OF COLLECTION OF DATA :
COLLECTION OF PLANT :
The bark of plantDelonex elata will be collected from wild source.
PRELIMINARY EXTRACTION :
Extraction will be carried out by using hydro-alcohol.
DEVELOPMENT OF TLC AND HPTLC :
After preliminary extraction the hydro Alcoholic extract will be subjected to TLC and HPTLC studies.
ISOLATION OF MARKERS :
The alcoholic extract will be subjected by column chromatography for the fractionation.
ACTIVITY OF FRACTIONS/ISOLATED COMPOUNDS :
After fractionation isolating the compound from column chromatography the compound will be subjected for the anti-oxidant and anti-microbial activities.
ANTI-OXIDANT MODELS :
1. Nitric oxide scavenging activity :
This method is based on the inhibition of nitric oxide radical generated from sodium nitroprusside in buffer saline and measured by Griess reagent. In presence of Scavengers, the absorbance of the chromophore is evaluated at 546nm.21
2. Super oxide dismutase scavenging activity :
This activity will be measured using NBT (Nitro blue tetrazolium reagent). The method is based on generation of super oxide radical (O2-) by auto oxidation of hydroxylamine hydrochloride in presence of EDTA will be detected colorimetrically at 560 nm.19
3.Reduction of ferric ions by o - phenanthroline colour method :
Fe++ ions reacts rapidly with 1,10-O-phenanthroline and forms red colured complex, which is exceptionally stable. This complex has a strong absorption in the visible spectrum at a wavelength of 510 nm. Extracts reacts with Fe+++ to reduce and convert it to Fe++.The degree of coloration indicates the reduction potential of the extracts. The change in the absorbance will produced at 510 nm has been used as a measure of ferric ions reducing activity.20
4.Reduction of 1,1-Diphenyl -2-picryl Hydrazyl (DPHP) :
This activity will be measured using DPPH (1-1-Diphenyl 2-picryl hydrazyl). The method is based on the reduction of colored solution of DPPH in presence of test drug measured at 516 nm.18
ANTI-MICROBIAL MODELS :
1.Antibacterial –Cup plate method :
The antibacterial activity of hydro alcoholic extract fractions will be performed using Agar cup-platemethod. 20ml of sterile nutrient agar medium will bepoured into sterile petri-dishes and allowedto solidify. The petri dishes will be incubated at 37°C for24 hours to check for sterility. The medium will beseeded with the organisms by pour plate method using sterile top agar (4 ml) contained 1ml culture.22
2.Antifungal Activity :
Hydroalcoholic extract of the plant will be prepared. About 15 ml of the medium will be poured into each petriplate and allowed to solidify. Five mm disc of 7-day-old culture of the test fungi will be placed at the center of the petriplates and incubated at 25±2 ºC for seven days. After incubation the colony diameter will be measured in millimeter. For each treatment four replicates will be maintained.23
7.3 DOSE THE STUDY REQUIRE ANY INVESTIGATION TO BE CONDUCTED ON PATIENTS OR ANIMALS?
-NO-
7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?
-NOT APPLICABLE-
Bibilography :
  1. Sujatha S. complementary and alternative therapies in palliative care. J cancer painsymptom pallition. 2005;1:25-9.
  2. Colombo M, Bosisio E. Pharmacological activites of chelidomium majus L.Pharmacol Res. 1996;33:127-34.
  3. Iwu M, Duncan A, Okunji C. New Antimicrobials of plant Origin. Alexandria Ashspress; 1999.
  4. Devasagayam T, Tilak J, Boloor K. Freeradicals and antioxidants in human health.JAPI. 2004.;52:794-804.
  5. Lee K, Shibamoto T. Antioxidant properties of the Aroma compounds isolated from soyabean and mung beans. J Agri Food Chem. 2000:4290-3.
  6. Kiritikar KR, Basu BD. Indian Medicinal Plants: International Book Distibutors Booksellers & Publishers; 1999.vol 2, Page 852
  7. Rokhade K, Nalawadi U. Improving Germination Of Poinciana Elata Seeds. SouthIndian Horticulture. 1989;37(6):359-60.
  8. Sivanarayan V, Suriyavathana. Preliminary studies phytochemical and anti-microbial activity of Delonix elata AND Prosopis cinerariaInternational Journal of Current Research. 2010;8:066-9
  1. Wijayasiriwardena c, Sharma pp. pharmacognostical investigation of delonix elata (l.). Ayu. 2009 jan-mar;30:68-72.
  2. Doss A, Pichai A, Dhanabalan R. In vitro Antioxidant Properties of Certain IndigenousMedicinal Plants From Western Ghats of India. The Internet Journal of Nutrition andWellness 2009;7.
  3. Pavithra P, Janani V, Charumathi V, Indumathy R, Sirisha P, Rama S, et al. Antibacterialactivity of plants used in Indian herbal medicine. 2010(1):22-8.
  4. Jahan I, Rahman M, Kaisar M, Islam M, Wahab A, Rashid M. Chemical and biologicalinvestigations of Delonix regia (Bojer ex Hook.) Raf. 2010 Jun 1;60(2):207-15.
  5. Tamaki Y, Teruya T, Tako M. The chemical structure of galactomannan isolated fromseeds of Delonix regia. Biosci Biotechnol Biochem. 2010 May 7;74(5):1110-2.
  6. Adetogun G, Alebiowu G. Properties of Delonix regia seed gum as a novel tablet binder.Acta Pol Pharm. 2009 Jul-Aug;66(4):433-8.
  7. Adje F, Lozano Y, Meudec E, Lozano P, Adima A, Nzi G, et al. Anthocyanincharacterization of pilot plant water extracts of Delonix regia flowers. 2008 Jun1;13(6):1238-45.
  8. Ankrah N, Nyarko A, Addo P, Ofosuhene M, Dzokoto C, Marley E, et al. Evaluation ofefficacy and safety of a herbal medicine used for the treatment of malaria. Phytother Res.2003;17(6):697-701.
  9. Pando S, Macedo M, Freire M, Toyama M, Novello J, S M. Biochemical characterizationof a lectin from Delonix regia seeds. J Protein Chem. 2002 May;21(4):279-85.
  10. Vani T, Rajani M, Sarkar S, Shishoo C. Antioxidant properties of the ayurvedicformulation triphala and its constituents. Int J Pharmacogn. 1997;35:313-7.
  11. Gabriel A, Agbor J, Vinson J, Oben J. Nagogang comarative analysis of the in vitroantioxdant activity activity of white and black pepper. Pharmacol Res. 2004;49:1-5.
  12. Sreejayan N, Rao MNA. Nitric oxide scavenging by curcuminoids. Journal PharmPharmacology 1997;49:105.
  13. Seejayan N, Rao MNA. Free radical scavenging activity of curcumminoids. DrugResearch. 1996;46:169
  14. Gerard J, Tortora., Berdell R, Funke., Cristine L. Microbiology An introduction. International Journal of Pharma Research and Development8th edition:591-2.
  15. Satish S, Mohana DC, Ranhavendra MP, Raveesha KA. Antifungal activityof some plant extracts against important seed borne pathogens of Aspergillus sp. Journal of Agricultural Technology. 2007;3(1):109-19.

08. / NAME OF THE CANDIDATE / SATHYA CHETHAN PABBITHI
09. / SIGNATURE OF THE CANDIDATE / (SATHYA CHETHAN PABBITHI)
10. / REMARKS OF THE GUIDE
10.1 / NAME AND DESIGNATION OF THE GUIDE / G.MURUGANANTHAN,
ASST.PROFESSOR
DEPARTMENT OF PHARMACOGNOSY
PES COLLEGE OF PHARMACY, BANGALORE-50.
10.2 / SIGNATURE
10.3 / CO-GUIDE (IF ANY) / Not Applicable
10.4 / SIGNATURE / .
Not Applicable
11. / HEAD OF THEDEPARTMENT / Prof. Dr. K. Lakshman
11.1 / SIGNATURE
12. / REMARKS OF THE PRINCIPAL
SIGNATURE / Prof. Dr.S.MOHAN

1