The document summarizes research on the watery fluid found at the floral base of Spathodea campanulata. Key findings include: 1) The fluid was found to be stable for over 48 hours at room temperature and over 6 months refrigerated. It had a viscosity of 0.8645cp, density of 0.9426g/ml, and pH of 6.4. 2) Analysis found minerals including calcium, magnesium, iron, chromium, zinc, and manganese. Phytochemical screening indicated the presence of steroids, minerals, proteins and organic acids. 3) Antimicrobial testing of the dried fluid residue showed activity against all tested microorganisms, including

1. Asian J. Pharm. Ana. 2011; Vol. 1: Issue 1, Pg 19-21 [AJPAna.]
19
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RESEARCH ARTICLE
Standardization and Antimicrobial Activity of Watery Fluid at Floral Base
of Spathodea campanulata (Pal)
S.G. Killedar*, K.I. Kope, S.B. Sangle and M.S. Tamboli
Bharati Vidyapeeth College of Pharmacy, Kolhapur (M.S.), India
*Corresponding Author E-mail: sureshgk_64@rediffmail.com
ABSTRACT:
Traditionally seeds, bark, leaves of Spathodea campanulata Pal are used as laxative and antiseptic. Bark is also used
for swollen cheeks and cleaning newly born babies. Molluscicidal, antioxidant and anti malarial activities of bark are
reported. Central wood is toxic and used for hunting wild animals by Uganda tribes. Flower base contains watery fluid
and no scientific data available on this fluid hence the present work was undertaken to evaluate the watery fluid and its
medicinal properties.
Fresh flowers were collected locally early in the morning then punctured at base with pointed needle and fluid was
collected in cleaned screw cap bottles. The collected fluid was then subjected for stability studies. Fluid was analyzed
for viscosity, density, pH, florescent analysis and total solid content using standard official methods. Mineral content
was determined by acid digestion method using atomic absorption. Watery fluid was studied for secondary metabolites
by TLC studies. Antimicrobial activity was performed by cup plate method against selected strains of bacteria and fungi.
The watery fluid was found stable up to 48h and six months in atmospheric condition and at refrigeration respectively.
The viscosity, density and pH were found to be 0.8645cp, 0.9426g/ml and 6.4 respectively. Undiluted fluid has shown
milky, greenish and yellowish green fluorescence at short, long and day wavelengths respectively. Total dissolved
solids were found to be 3.15% w/v and sedimentation time was 2h. Mineral contents found were Ca, Mg, Fe, Cr, Zn,
Mn, Ni. TLC studies showed presence of steroids with blue fluorescence at Rf 0.9, and 0.7. Dried residue has showed
good antibacterial activity against all the tested organisms but had shown weak antifungal activity.
KEYWORDS: Standardization, Antimicrobial activity, Spathodea campanulata, Watery fluid, floral base.
INTRODUCTION:
Spathodea campanulata also known as African tulip is
evergreen, stout, deciduas tree grows up to 15-35m in
height with elliptical leaves and red flowers found in most
of the tropical regions. The plant is locally known as
pichkari. Plants produce flowers in the month of Feb –
March which are reddish brown in color and hence called as
flame of forest1
. Traditionally seeds, bark, leaves are used
for laxative, antiseptic and other disorders. Bark is also used
for swollen cheeks, cleaning newly born babies and
antimalarial2
. Molluscicidal, antioxidant and anti malarial
activities of bark are reported3
. Central wood is toxic and
used for hunting wild animals by Uganda tribes. Pollen
grains are poisonous to bees, while seeds are edible. Flower
base contains watery fluid and used to make fun by children
to play water holy. No scientific data available on this fluid
hence the present work was undertaken to evaluate the
watery fluid and its medicinal properties.
Received on 17.03.2011 Modified on 22.03.2011
Accepted on 11.04.2011 © RJPT All right reserved
Asian J. Pharm. Ana. 1(1): Jan.-Mar. 2011; Page 19-21
MATERIALS AND METHODS:
Authentication and collection of flowers:
Plant was identified at by Dr. Madhukar Bachulkar, Botany
department Vivekanand College Kolhapur. Voucher
specimen (SGK/ BV/Cog.-6) has been deposited at
Pharmacognosy dept., Bharati Vidyapeeth College of
Pharmacy, Kolhapur. Fresh flowers were collected from
local plants early in the morning in the month of Dec. 2009.
Collection and evaluation of watery fluid for stability:
Freshly collected flowers were sorted for other impurities
and washed with water and drained. Floral base of each
flower was punctured at base with pointed needle and fluid
was collected in cleaned screw cap glass bottles (500ml).
The collected fluid was then subjected for stability studies
using 5ml of sample in screw cap glass bottles kept in
stability chamber at normal temp. (280
C±20
C, relative
humidity 65%) and in refrigerator at 40
C. It was further
analyzed for sedimentation, color change, microbial growth
at regular intervals.

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Determination of viscosity, density, pH and
fluorescence:
Fresh watery fluid was analyzed for viscosity, density and
pH using standard official methods4
. Fresh fluid was
collected, filtered and subjected for physical analysis. pH
was analyzed using digital pH meter, viscosity by oswald
viscometer and density by gravity bottle. Undiluted sample
of watery fluid was then observed in UV cabinet for
determination of florescence.
Fig.No.1: Mineral content of watery fluid at floral base of
Spathodea campanulata
Determination of total dissolved solids, sedimentation
and miscibility:
Total dissolved solids were determined by withdrawing
10ml of fresh fluid in tarred porcelain dish and evaporated
on digital thermostatic water bath at 400
C.the dried residue
was weighed and total dissolved solids was determined. The
process was repeated thrice and average was calculated.
Fresh watery fluid (10ml) was kept in three different screw
cap bottles under constant observation for any
sedimentation. Further the watery fluid was tested for
miscibility with different polar and non polar solvents
including neutral acidic and basic aqueous solutions.
Determination of mineral content5
:
1g of dried residue of fluid was subjected to acid digestion
using 10 ml of equal amount of perchloric, sulphuric and
nitric acids on water bath until all fumes were ceased and
filtered through Whatman no-1 filter paper. Concentrate
was dissolved in double glass distilled water. The sample
was subjected for atomic absorption and mineral content
was determined.
Phytochemical screening and TLC studied 6,7
:
Watery fluid was studied for secondary metabolites and
found to contain sugars, minerals and steroids. TLC studies
were carried out with different solvent systems showed
presence of steroids with blue fluorescence at short
wavelength (254nm) using n-hexane: methanol: water
(3:2:1)
Antimicrobial activity,8
:
Dried residue of watery fluid was tested for antimicrobial
activity using standard MTCC cultures by agar well
diffusion method. Bacterial cultures were subculture on
nutrient agar and fungal cultures on potato dextrose agar.
Only fresh cultures were used for testing the activity.
Different concentrations were tried along with standard
ciprofloxacin, Doxycyclin and Fluconazole. Bacterial and
fungal cultures were incubated at (370
C±20
C) and at room
temperature respectively. Zone of inhibitions (mm) were
determined in triplicate.
Fig.No.2: Antimicrobial activity of watery fluid at floral base of
Spathodea campanulata
Table No.1 Determination of viscosity, density, pH and fluorescence of watery fluid
Sample Viscosity* pH Density * Fluorescence
Short Long Day
Watery fluid 0.8645cp 6.4 0.9426g/ml Milky white green yellowish green
*Average of five determinations
Table No.2: Determination of mineral content of watery fluid at floral base
Sr. No. Part used
Mineral content mg -1
100g
Ca Mg Fe Mn Zn Pb Co
01 Watery fluid 1.294 1.272 1.986 0.873 0.215 0.082 0.008
* Average of three determinations
Table No. 3: Phytochemical screening of watery fluid at floral base
Sample Sugars Alk. Tannins Glycosides steroids proteins Org. acids
R NR HT CT a c s f co ST TT C O T
Watery
fluid
+ + - - - - - - - - + - + + + -

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Table No. 4: Antimicrobial activity of watery fluid at floral base of Spathodea campanulata
Sample Conc.
used
Zone of inhibition in mm.*
E.coli P.aeruginosa M.luteus B.subtilus S.aureus C.albicans A.niger P.notatum
2%acacia
suspension
0.2ml -- -- -- -- -- -- -- --
Doxycyclin 01µg 22 18 23 25 23 NT NT NT
Ciprofloxacin 01µg 25 21 27 25 26 NT NT NT
Fluconazone 05 µg NT NT NT NT NT 16 17 17
Watery fluid 500 µg 13 12 15 14 13 11 12 13
1000µg 17 15 19 18 17 13 15 15
3000µg 20 18 22 21 19 15 17 17
*Average of triplicate
RESULTS AND DISCUSSION:
The watery fluid was stable for more than 48h in
atmospheric condition and more than six months at
refrigeration. Mineral contents found were Ca, Mg, Fe, Cr,
Zn, Mn, Ni etc. The fluid showed presence of maximum
(Iron 1.986 mg -1
100g) followed by calcium (1.294mg -1
100g ) and minimum of cobalt (0.008 mg -1
100g).
Phytochemical studies showed presence of Steroids,
minerals, proteins, organic acids, sugars and absence of
alkaloids, tannins and terpenes. Dried residue has showed
antimicrobial activity at minimum 500µ against tested
micro-organisms and significant at 3mg per well compare
to standards. The antimicrobial activity was found
concentration dependent. The viscosity, density and pH
were found to be 0.8645cp, 0.9426g/ml and 6.4
respectively. Undiluted fluid has shown milky, greenish and
yellowish green fluorescence at short, long and day
wavelengths respectively. Total dissolved solids were found
to be 3.15% w/v, sedimentation time was 48h. Fluid was
miscible with aqueous ethanol, methanol, acetone and ethyl
acetate and immiscible in non polar solvents. It was found
more stable at neutral pH while turbid at alkaline pH. The
watery fluid was stable for more than 48h in atmospheric
condition and more than six months at refrigeration.
CONCLUSION:
From the above study it was confirmed that the watery fluid
showed antimicrobial activity possibly due to presence of
minerals or steroids. Fresh juice needs to be preserved in
refrigerator for long storage since sedimentation occurred
after 48h. Further studies are in process to find out the
chemical moieties responsible for antimicrobial activity.
ACKNOWLEDGEMENT:
Authors are thankful to Dr. Madhukar Bachulkar, Botany
department Vivekananda College Kolhapur for
authentication of plant. We are also thankful to Mr. Kalas,
Common Facility Center (CFC) Shivaji University
Kolhapur for helping in atomic absorption studies and Dr.
H. N. More, Principal, BVCOPK for providing the facilities
to carry out this work.
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