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FINAL PROGRESS REPORT
UGC MINOR PROJECT IN SCIENCE
SCREENING OF HERBAL PLANTS AND ASCERTAINING THEIR EFFECT OF
THEIR EXTRACT ON THE INHIBITION OF MAMMALIAN AS WELL AS
HYALURONIDASE ISOLATED FROM PATHOGENIC BACTERIAL STRAINS
AND PROPOSING NOVEL POTENTIAL MICROBICIDES
REF: Approval letter : MRP(S)-540/09-10/KLMG 038/UGC-SWRO dated 30-11-2009
Sanction letter: MRP(S)-666/09-10/KLMG 038/UGC-SWRO dated 27-01-2010
Submitted by
DEPARTMENTS OF CHEMISTRY AND BIOCHEMISTRY
MAR ATHANASIUS COLLEGE, KOTHAMANGALAM
ERANAKULAM
KERALA
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To
Deputy Secretary and Regional Head
University Grants Commission
South Western Regional Office
P K Block, Palace Road
Gandhi Nagar, Bangalore
Sub: Minor Research Project in science entitled “Screening Of Herbal Plants And
Ascertaining Their Effect Of Their Extract On The Inhibition Of Mammalian As Well As
Hyaluronidase Isolated From Pathogenic Bacterial Strains And Proposing Novel
Potential Microbicides” (Approved letter: MRP(S)-540/09-10/KLMG 038/ UGC-SWRO
dated 30/11/ 2009; Sanctioned letter: MRP(S)-666/09-10/KLMG 038/ UGC-SWRO
dated 27/1/2010) sanctioned to Dr. Densely Jose, Associate Professor, Dept. of
Chemistry, M. A. College, Kothamangalam
Sir,
This is to inform you that the complete documents of the Final Project
Report, along with the Audited Utilization Certificate and Item-wise statement of
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expenditure of the Minor Research Project were forwarded to you, in original, vide letter
no: M2-UGC/034/13 dated 1/04/2013 by speed post at Kothamangalam post office
(686666) with receipt no.EL3249419731N. However a copy of the Final report along
with all the listed documents is again forwarded here with. Kindly do the needful as early
as possible.
Thanking you
Yours
faithfully,
Principal
Annexure -III
UNIVERSITY GRANTS COMMISSION
BAHADUR SHAH ZAFAR MARG
NEW DELHI – 110 002.
Annual Report of the work done on the Minor Research Project. (Report to be
submitted within 6 weeks after completion of each year).
1. Project report No. 1st
/2nd
/3rd
/Final : Final
2. UGC Reference No. :
Approval letter : MRP(S)-540/09-10/KLMG 038/UGC-SWRO dated 30-11-2009
Sanction letter: MRP(S)-666/09-10/KLMG 038/UGC-SWRO dated 27-01-2010
3. Period of report: from : 4/2/2010 to 04/08/2011
4. Title of research project : Screening Of Herbal plants And
Ascertaining The Effect Of Their Extracts
Of Mammalian Hyaluronidase As Well As
Hyaluronidase Isolated From Pathogenic
Bacterial Strains And Proposing Novel
Potential Microbicides
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5. (a) Name of the Principal Investigator : Dr. Densely Jose
(b) Dept. and University/College where work has progressed
: Department of Chemistry (Regional
research centre in Chemistry, MG
University) and Department of
Biochemistry, Mar Athanasius College,
Kothamangalam.
6. Effective date of starting of the project : 4 / 2 / 2010
7. Grant approved and expenditure incurred during the period of the report:
a. Total amount approved : Rs. 60,000/-
b. Total expenditure : Rs. 60,000/-
Report of the work done: (Please attach a separate sheet)
(i)Brief objective of the project : The objective of the study is to screen
potential herbal plants for their inhibitory
effect on Hyaluronidase activity and
assessing their potential antimicrobial
activity against pathogenic bacterial strains.
ii. Work done so far and results achieved and publications, if any, resulting from the work
(Give details of the papers and names of the journals in which it has been published or
accepted for publication) Results are enclosed along with this report.
(iii) Has the progress been according to original plan of work and towards achieving the
objective. if not, state reasons :yes
(iv)Please indicate the difficulties, if any, experienced in implementing the
project : Not applicable
(v)If project has not been completed, please indicate the approximate time by which it is
likely to be completed. A summary of the work done for the period (Annual basis) may
please be sent to The Commission on a separate sheet : Not applicable
(vi) If the project has been completed, please enclose a summary of the findings of the
study. Two bound copies of the final report of work done may also be sent to the
Commission : Attached
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(vii) Any other information which would help in evaluation of work done on the project.
At the completion of the project, the first report should indicate the output, such as (a)
Manpower trained (b) Ph. D. awarded (c) Publication of results (d) other impact, if any
: Publication of Results (Attached)
Signature of The Principal Investigator
Signature of The Co-
Investigators
Registrar/Principal
Annexure III
“Screening Of Herbal Plants And Ascertaining Their Effect Of Their Extract On
The Inhibition Of Mammalian As Well As Hyaluronidase Isolated From Pathogenic
Bacterial Strains And Proposing Novel Potential Microbicides”
Medicinal plants the world’s oldest health care products, play a key role in
traditional medicines. But these plants are not only used for primary health care;many
widely used pharmaceuticals are derived from plants and other natural sources. The plant
may be considered as a biosynthetic laboratory, not only for the chemical compounds
such as carbohydrates, proteins and lipids that are utilized as food by man but also for a
multitude of compounds like alkaloids, glycosides, volatile oils, resins etc. These
secondary metabolites are generally responsible for the therapeutic effects exhorted by
plant products. Plants produce a diverse range of bioactive molecules, making them a rich
source of different types of medicines. Higher plants, as sources of medicinal
compounds, have continued to play a dominant role in the maintenance of human health
since ancient times. Over 50% of all modern clinical drugs are of natural product origin
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and natural products play an important role in drug development programmes in the
pharmaceutical industry.
HA is the major constituent of the extra cellular matrix. In animals, HA is present
in every part of the body. HA present in the skin is about 50% of total HA, and that of
vitrous homour of eyes is found to be 0.1 to 0.4 mg/g. It is also present in synovial
fluid(4mg/dl)and in oocytes. Certain strains of bacteria such are streptococci and some
viruses also contain HA. HA plays a major role in physiological processes such as
fertilization, cell growth, and migration and in some pathophysiological processes such as
tumor spread and in certain eye diseases. Increased level of HA are seen in
morphogenesis, embryonic development, wound healing and in inflammation.
To provide and to further investigate the role of HA and Hyase in physiological
and pathological processes, selective and potent inhibitors are required. Heavy metals
like iron, copper and zinc salts, heparin, polyphenols and flavonoids are found to potent
inhibitors, but the inhibition was achieved only at concentrations much higher than that of
the physiological levels. Flavonoiods that inhibit sperm hyase also inhibit microbial
hyase.
OBJECTIVE OF THE STUDY
Collection and identification of different plants
Extraction of the plants by soxhlet extraction Method.
Phytochemical analysis of the plant extracts
Anti bacterial activity of the different plant extracts
Determination of % of inhibition of bovine testicular hyaluronidase by the
extracts
Materials and Methods
A) Plants selected:
1. Biophytum sensitivum
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Biophytum sensitivum is widespread in tropical Indo - Malaysia. The plant is
found throughout the hotter parts of India. It is very common in shady moist places,
roadsides, riverbanks and also on cultivated ground. The whole plant is reportedly
cooling, bitter, astringent, antipyretic and antiseptic. It is useful in fever, burning
sensation, hemorrhages, chronic cough, dysentery, urinary calculi and vaginal disorders.
It cures piles and hydrocele. The leaves are antiseptic and styptic and hence very useful in
healing wounds and ulcers. The leaves act as diuretic when given internally.
Whole plants of Biophytum sensitivum were collected from Kothamangalam area and
identified. Fresh plant materials were washed under running tap water and distilled water,
air dried and then ground into fine powder and stored in an airtight bottle. The extracts of
Biophytum sensitivum was prepared by soxhlet extraction in aqueous medium and in
methanolic medium. The methanolic and aqueous extracts of the plants of Biophytum
sensitivum were subjected to phytochemical screening, using the methods given in the
standard books. Different samples of pathogenic bacterial strains were collected from
Mar Baselious Medical Mission Hospital, Kothamangalam and were isolated and
identified by different methods.
2. Coleus amboinicus
According to Ayurveda, Coleous amboinicus, the plant under study is vatha and kapha
suppressant. It is a good pain killer. It is also very effective in convulsions paralysis and
has a small amount of necrotic effect. It is a good remedy in indigestion and is helpful in
avoiding tastelessness, diarrhoea, liver related problems and worm infestation. It is
effective in expelling out the extra amount of mucus present in the respiratory tract thus
preventing diseases like cough and asthma. It is also effective in treating kidney stones
and renal calculus as it is a diuretic.
3. Emilia songifolia
Distribution: Trough out India as a weed in cultivated fields and waste places
Morphology And Characters :A soft very variable annual herb, about 30 to 40 cm in
height ,variously branched, some times procumbent and root near the nodes. Simple
leaves both radical and cauline, lyrate pinnalifid with large terminal lobe. Flowers
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purplish in lax wrymbose heads,bracts almost equaling the corollar with scarious
margins, pappus hair white ,soft,nearly equaling the involucrate brads
4. Aloevera
Aloe vera plant has health, beauty, medical and skin care properties. The name
Aloevera derives from Arabic word “Alloeh” meaning “shining bitter substance” “Vera”
is Latin means “true”. The Egyptians called Aloevera is the plant of immortality. It is
widely used in the field of dermatology. Aloevera is used for medical purposes in
different cultures; India, Greece, Egypt, Mexico, Japan and China. Egyptians this as part
of regular beauty regimes. Alexander Great and Columbus used it tolerate solder’s
wounds .Aloevera was in use as a laxative in USA .In mid -1930’s, it was successfully
used to treat chronic and severe radiation dermitis.
I) COLLECTION OF PLANT STUDY MATERIAL
Fresh and dried leaves of Coleous amboinicus collected from Aromatic Medicinal Plants
Research station, Kothamangalam (Ernakulam district).The plants were freshly collected
and the leaves were separated from the stem. Then it was washed under running tap water
and with distilled water.After air drying, a portion of the samples were shade dried until
all the water molecules evaporated and the leaves get dry. Another portion of leaves were
dried in hot air oven at 40ºC. After drying, the plant leaves were ground well into fine
powder and then transferred into airtight containers. Another portion of washed fresh
leaves (30g) were ground well.
II) PREPARATION OF PLANT EXTRACTS (SOXHLET EXTRACTION)
10 g each of both shade dried and hot air oven dried leaves were taken and extracted with
ethanol and water as solvent for about 72 hours by soxhlet extractor. Similarly 30g of
ground fresh leaves were taken and extracted with ethanol and water as solvent for about
72 hours by soxhlet extractor.
III) PHYTOCHEMICAL ANALYSIS OF CRUDE EXTRACT
Ethanolic extracts of different plants were taken for phytochemical analysis of
carbohydrates, alkaloids, flavanoids, saponins, proteins, phenolic compounds,
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phlobotannins, cardiac glycosides, morphic alkaloid, oil and gums using the following
methods
1) Detection of carbohydrate and glycosides
a) Barfoed’s test: To 1 ml of the filtrate, 1ml of Barfoed’s reagent was added
and heated on a boiling water bath for 2 minutes. Red precipitate indicated
the presence of sugar.
b) Benedict’s test :To 0.5 ml of the filtrate, 1ml of Benedict’s reagent was
added and this mixture was heated on a boiling water bath for 2 minutes.
A characteristics coloured precipitate indicated the presence of sugar.
c) Molisch test: 1g of extract was dissolved in 1ml water.Then add two drops
of 1% alcoholic solution of alpha naphthol. 1ml concentrated sulphhuric
acid was added along the sides of the test tube. A deep violet colour at the
junction of two liquid indicated the presence of sugar.
d) Fehling test: 1ml of the filtrate is boiled in water bath with 1ml of fehlings
solution A and B .Red precipitate indicated the presence of sugar.
2) Detection of alkaloids
a) Mayers test: To a few ml of the filtrate, one or two drops of Mayers
reagent were added by the side of the tube. A white creamy precipitate
indicated the test as positive.
b) Wagners test : To a few ml of the filtrate, one or two drops of Wagners
reagent were added by the side of the tube. A reddish brown precipitate
confirmed the test as positive.
3) Detection of Phlobotannins : To 0.5 ml of sample mix 5ml of water and boil with
5ml 1% HCl . Red precipitate indicate the presence of phlobotannins
4) Detection of Flavanoids : 1ml of extract was dissolved in dilute NaOH solution.
A visible colour observed indicated flavanoids.
5) Detection of Proteins and amino acids
a) Biuret test : An aliquot of 2 ml of filtrate was treated with one drop of 2%
copper sulphate solution. To this 1 ml of ethanol was added, followed by
excess of potassium hydroxide pellets. Pink colour in the ethanolic layer
indicated the presence of proteins.
6) Detection of phenolic compounds and tannins
a) Ferric chloride test : The extract was dissolved in (50mg) 5ml of distilled water.
To this, few drops of neutral ferric chloride solution was added. Dark green
colours indicate the presence of phenolic compounds.
b) Lead acetate test
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The extract was dissolved in (50mg) 5ml of distilled water. To this, 3ml of
10% lead acetate solution was added. A bulky white precipitate indicated the
presence of phenolic compounds.
7) Detection of saponins
a) Frothing test: To 0.5 ml of sample added 5ml distilled water. Frothing
persistence indicated the presence of saponins
8) Detection of cardiac glycosides:
1ml glacial acetic acid containing trace of ferric chloride was added to 0.5g of
extract. Sulphuric acid was gently poured down the sides of the tube. Brown ring
at the interphase and violet ring beneath that layer and pale green upper layer was
an indicative of deoxy sugars.
9) Detection of morphine alkaloids
1ml of extract was evaporated to dryness and the residue was dissolved in 0.6ml
of 1% sulphuric acid. To this 2ml of distilled water and two drop sof 10% sodium
nitrite was added. The solution was then made alkaline with dilute ammonia
solution. Reddish brown precipitate indicated the presence of morphine alkaloids.
10) Detection of oil and fat
Small amount of extract was pressed between two filter paper. Oil stain on the
paper indicated the presence of oil.
11) Detection of gum and mucilage
100 mg of extract was dissolved in 10ml of distilled water and to this 25ml of
absolute alcohol was added with continues stirring .White or cloudy precipitate
indicates the presence of gum and mucilage.
IV) SCREENING OF PLANT EXTRACTS FOR ANTIMICROBIAL ACTIVITY
Disc diffusion method
Procedure:
1. Using a sterile loop,3-5 well isolated colonies of similar appearance were picked and
emulsified in 3-4ml of peptone water.
2. The turbidity of the suspension was matched with that of the McFarland’s Turbidity
Standard.
3. A sterile cotton swab was dipped into the bacterial suspension. Excess fluid was
removed by pressing and rotating the swab against the sides of the tube above the level of
the suspension.
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4. It was rubbed gently over the plate in several directions to obtain different planes, by
rotating the plate 600C each time. This was to obtain a confluent lawn of growth.
5. The plate was left aside with lid in place for 3-5 minutes, so as to allow the surface of
the agar to dry.
6. Sterile discs and wells were prepared by using sterile puncture.
7. The extracts were completely dissolved in suitable solvent and different concentrations
of plant extracts were added to the disc or wells using micropipette under sterile
conditions.
8. The plates were incubated overnight at 370C, under appropriate conditions suitable for
the organism.
9. Following incubation, the diameter of the zones of inhibition of growth including the
diameter of the disc was measured using a ruler. The diameters of the zones were
measured to the nearest millimeters and recorded.
V) DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION (MIC)
(Signleton,1999)
The MIC is regarded as the lowest concentration of antimicrobial agent which
completely inhibits the growth.
Procedure
b) Prepared stock solution of extracts as required. Labelled this concentration
as stock C.
c) A row of sterile test tubes was arranged and labels them C1-C10.
d) To all the test tubes 2ml nutrient broth was added.
e) 2ml of stock solution of extract was added to C1,after well mixing transfer
2ml to the C2.
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f) This procedure is continued upto 10th tube, using new pipette.
g) Inoculate one drop of an overnight broth culture of the test organism.
h) Incubate tubes for 18-24 hours at 37oC.
i) Inoculate a tube containing 2ml broth with organism and keep at 40c in the
refrigerator overnight, to be used as standard for the determination of
complete inhibition.
j) The minimum inhibition concentration (MIC) value was determined as the
lowest concentration of the crude extract in the broth medium that inhibited
the visible growth of the microorganism.
VI) DETERMINATION OF MINIMUM BACTERICIDAL CONCENTRATION
(MBC)
1. Sub cultured the test dilution on to a fresh drug-free solid nutrient agar
medium.
2. Incubated the plates for 18-24 hours at 37Oc.
3. The highest dilution that yielded no single bacterial colony on solid
medium was taken as MBC.
VI) ASSAY OF HYALURONIDASE (Spectrophotometric assay (Morgan-Elson
method)
For the investigation of potential enzyme inhibitors described in this thesis, the
hyaluronidase activity was determined by Morgan –Elson colorimetric assay. It is based
on the reaction between reducing N-acetyl-D-glucosamine residues of the substrate
(hyaluronic acid) with Ehrlich´s reagent (p- dimethylaminobenzaldehyde).In this
reaction, which forms a red colored product that can be detected colorimetrically at
600nm . hyaluronidase activity is determined by quantitation of the N-acetyl-D-
glucosamine residues at the reducing end of hyaluronic acid fragments produced by
enzymatic degradation. The mechanism of this reaction is, Hyaluronic acid is cleaved by
the hyaluronidases during incubation. Then the reducing N-acetyl-D-glucosamine
residues are cleaved under the basic assay conditions (pH 9, 100 °C) resulting in an
anomeric mixture of chromogen I (α-configuration) and chromogen II (β-configuration)
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that are transformed under acid catalysis to chromogen III via elimination of water. The
final red colored product is subsequently obtained after reaction with p-
dimethylaminobenzaldehyde.
VII) DETERMINATION OF THE EFFECT OF PLANT EXTRACTS ON THE
ACTIVITY OF BOVINE TESTICULAR HYALURONIDASE
Hyaluronidase inhibition was determined by ElsonMorgan assay by measuring the
amount of N-acetylglucosamine released from hyaluronic acid by the method
described by Abhijit Sahasrabudhe et al., 2010 with slight modifications
100 μl of Bovine Testicular Hyaluronidase (2mg/ml) dissolved in 0.2 M acetate buffer
(PH 3.6) was mixed with 100 μl of both aqueous and alcoholic extracts at different
concentrations in 5% DMSO. The control group was treated with 100 μl of 5% DMSO
instead of extracts and was incubated for 20 min at 370C . After 20 min 100 μl of calcium
chloride (12.5mM) was added to the reaction mixture and and again incubated for 20 min
at 370C .This Ca2+ activated hyaluronidase was treated with 100 μl Hyaluronic acid (8mg
mL-1) incubated at 370C for 40min.After incubation the enzyme reaction was stopped by
addition of 500 μl of alkaline borate solution and subsequent heating for 3 minutes in a
boiling water bath at 1000C. After cooling on tap water for 1 minute, added 100 μl
enzyme solutions into the control tubes. Then added 3ml of N,N-
dimethylaminobenzaldehyde to all the tubes and incubated in water bath at 370C for 20
minutes. A pink colour was developed it was measured at 586nm on Varian Cary 50 UV
spectrophotometer.
% inhibition = (A – B) - (C – D) X 100
(A – B)
Where A is the control absorbance
B is the control blank absorbance
C is the sample absorbance
D is the sample blank absorbance
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RESULTS
1. Phytochemical Screening of plant extracts Preliminary phytochemical
screening of alcoholic extracts of different plants are shown in table 1. Phytochemical
analysis revealed the presence of carbohydrates and morphine alkaloids in aqueous
extracts but were absent in the alcoholic extract. Analysis of methanolic extract showed
the presence of most of the phytochemicals like carbohydrates, The proteins, alkaloids,
phenolic compounds, tannins, flavonoids and cardiac glycosides. phytochemical
screening indicated that methanol can extract more active principles of the plant than the
boiled water. Depending upon the phytochemical constituents three plants were selected ;
Aloe vera, Biophytum and leucas aspira were selected for further study.
Sl
no
Plant Aloe vera Biophytum Leucas
aspira
Coleus
ambonicus
Emilia
songifolia
Extract → Alc Aqu Alc Aqu Alc Aqu Alc Aqu Alc Aqu
1. Carbohydrate
a. Molisch’sTest
+
+
+
+
+
-
+
-
-
+
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b. Barfoed’s Test
c. Benedict’s Test
d. Fehling’s Test
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
+
+
+
-
-
-
-
-
-
+
+
+
2. Alkaloid Test
a. Wagner’s Test
b. Mayer’s Test
+
+
-
-
+
+
-
-
+
+
-
-
-
-
-
-
+
+
+
+
3. Flavanoids + + + + + + + - + -
4. Protein
a. Biuret Test
+
-
+
-
+
-
-
-
+
-
5. Phenolic
Compounds
a. Lead acetate
Test
b. Ferric chloride
Test
+
+
+
+
+
+
+
+
+
+
-
-
+
+
+
+
+
+
+
6. Oils + + + + + + + - + -
7. Steroids and
terpenoids
+ - + - + - + - - -
Table:1 Phytohemical analysis of plant extracts
2.Anti bacterial effect of plant extracts
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Ethanolic extract of Leucas aspera showed zone of inhibition for Klebsilla &, S.aureus
where as aqueous extract shows no zone of inhibition in disc diffusion method. Alcoholic
extract of aloevera showed antibacterial activities against Bacillus (Table:2) .The zone of
Plants▼ Solvent
Amou
nt of
extrac
t(mg)
Diameter of zone of inhibition obtained for
different organisms in mm
Bacterial
species►
Bacill
us
specie
s
E.c
oli
Klebsie
lla
species
S.ty
phi
S.aure
us MRSA
Leucas
aspera
Alcohol
50
100
150
- - 6±0.4 - 8±0.82 -
- - 18±0.6
0 -
15±1.8
2 -
- - 19±1.0
9 -
20±1.1
0 -
Aqueou
s
50
100
150
- - - - - -
- - - - - -
- - - - - -
Biophytu
m
enitivum
Alcohol
1.12 - - - 10 10
2.25 - - 12 11
4.5 - - 12 10
9.0 13 - - - 8 10
18.0 18 - - - 12 11
22.5 20 - - - 15 14
Aqeous
18.0 11 - - 11 13
36.0 19 - - - 16 16
45.0 21 - - - 17 17
18.0 11 - - - 11 13
Aloe vera
alcohol
50 - - - - - - -
100 7 - - - - - -
150 9 - - - - - -
aqeous
50 9 - 5 - - - -
100 11 - 5 - - -
150 13 - 7 - - - -
Table 2 : Anti bacterial assay of extracts
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inhibition increase with increase in concentration of extract.All aqueous extracts did not
possess antibacterial effect against any microorganism under study.Alcoholic extract of
Biophytum showed antibacterial activities against S aureus and Bacillus.
3) Determination of MBC& MIC values
Plant Leucas aspera Aloevera Biophytum sensitivum
Organism Aqueous Alcohol Aqueous Alcohol Aqueous Alcohol
MIC MBC MIC MIC MBC MIC MBC MIC MBC MIC MBC MIC
S.aureus - - 50 25 - - - - - - 110 55
Klebsiella - - 25 12.5 - - - - - - -
Bacillus - - - - - - 50 25 - - 110 55
Table :3 Determination of MBC/MIC of plant extracts
The ethanolic extract of Leucas aspera showed a MIC of 12.5mg in S.aureus &25mg in
Klebsilla and a MBC of 25mg in S.aureus & 50mg in Klebsilla.The MIC and MBC
studies showed that the extracts were bacteriostatic at lower concentrations and
bactericidal at higher concentrations. The MIC and MBC of alcoholic extract of
Biophytum was found to be 55 and 110 mg/ml respectively against both S.aureus and
Bacillus and that of Aloevera was 50 and 25 mg/ml against Bacillus.
4) Inhibition of Bovine Testicular Hyaluronidase by plant extracts
The aqueous and ethanolic extract of Biophytum sensitivum and Aloe vera showed
increase in percentage of inhibition towards Bovine Testicular Hyaluronidase with
increase in concentration of the plant extract (table 4).It can be found from the graph that
the alcoholic extract of Aloe vera showed more inhibition at 5, 10, 15 mg concentration
than the respective aqueous extracts. The percentage of inhibition of alcoholic extract of
A. vera at concentrations 5, 10, 15 mg was found to be 71.80%, 81.43%, 94.29% and that
of aqueous extract was 79.22%, 87.48% and 91.31% respectively. The aqueous extract of
B.sensitivum was found to have more inhibition than the aqueous at varying
concentration. The alcoholic and aqueous extract of B.sensitivum showed
38.205%,43.05%,59.45% inhibition and 73.51%,79% and 96.43% inhibition at 5,10,15
mg concentration respectively.The alcoholic extract of Leucas aspira showed more
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inhibition at 5, 10, 15 mg concentration than the respective aqueous extracts. The
percentage of inhibition of alcoholic extract of Leucas aspira at concentrations 5, 10, 15
mg was found to be 56.54%, 73.24%, 88.33 % and that of aqueous extract was 11.67%,
29.97% and 33.32% respectively.
Plant Solvent Concentration(mg/
ml) % of inhibition
Leucas aspira
Ethanol
5 56.54
10 73.24
15 88.33
Aqueous
5 11.67
10 29.97
15 33.32
Biophytum
sensitivum
Aqueous
5 38.21%
10 43.05%
15 59.45%
Ethanol
5 73.51%
10 79.00%
15 96.43%
Aloevera
Ethanol
5 71.80%
10 81.43%
15 94.29%
Aqueous
5 79.22%
10 87.48%
15 91.31%
Table:4 Inhibitory Effect of Alcoholic and Aqueous Extracts on the activity of Bovine
Testicular Hyaluronidase
DISCUSSIONS
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Plants have provided a source of inspiration for novel drug contributions towards human
health. About 64% of the total population remains dependent on traditional medicine &
medicinal plants for provision of their health care needs. Increase in plants with
compounds as plant derived medicines have made significant antimicrobial properties has
revived as a consequence for current problem associated with the use of antibiotics.
Even though pharmacological industries have produced a number of new antibiotics in
the last three decades, resistance to these drugs by microorganisms has increased. In
general, bacteria have the genetic ability to transmit & acquire resistance to drugs, which
are utilized as therapeutic agents. Hence, more studies pertaining to the use of plants ass
therapeutic agents should be emphasized, especially those related to the control of
antibiotic resistant bacteria. The purpose of this study was to assess the antibacterial
potential of Leucas aspera.
Hyase is known to play a major role in fertilization (Rogers 1982). Hyase inhibitors can
be used as contraceptives, because they can arrest the penetration of the sperm (Pincus
1948). Hyaluronidase plays major role in cancer metastasis & in angiogenesis (Dacailiu,
1996). So that it is used in cancer therapy. For many years, Hyaluronidases especially
Bovinine Testicular Hyaluronidase preparations, are widely used in many fields like
Orthopaedia, Surgery, Ophthalmology, Oncology, Dermatology & gynaecology (Borrel,
2000).
The phytochemical analysis indicated that ethanol can extract more active principles of
the plant than the water. On qualitative examination the aqueous extract of the plant was
shown to contain phytoconstituents like alkaloids, flavonoids, oil, phenolic compounds
and tannins while the alcoholic extracts was shown to contain alkaloids, carbohydrates,
flavonoids, proteins, cardiac glycosides, oils, phenolic compounds and tannins.
In the present study the aqueous and alcoholic extracts of Leucas aspera showed that
with the increase in the concentration it showed considerable inhibition towards Bovine
Testicular Hyaluronidase. The alcoholic extract of Aloe vera exhibited much inhibition
towards BTH than aqueous extracts. The % of inhibition was found to be increased as
concentration of the extracts increased. The alcoholic extract of Aloe vera was reported to
possess anti-ulcer, anti-inflammatory, analgesic and antipyretic effect and these
properties could be attributed to the presence of alkaloids and flavanoids (Metowogo et al
20
2008).Any of these secondary metabolites may be responsible for the antihyaluronidase
effect of the plant.
Now the present study showed the anti-hyaluronidase effect of the plant, which further
supported its therapeutic potential. The results of the study suggests that these three
medicinal plants can be used clinically as contraceptives, anti-inflammatory, anti-tumor,
anti-allergic, anti aging & anti arthritic agents.
CONCLUSION
Aloe vera, Leucas aspira and Biophytum sensitivum are medicinally important plants. At
higher concentration (15 mg) Aloe vera and Biophytum sensitivum exhibited about 95%
inhibition on BTH. As the plants were found to have antimicrobial properties, the plant
extracts could be used as microbicide to prevent sexually transmitted disese ; in
cosmetics as anti-wrinkling agents, or as anti-tumor agents. The study thus confirmed the
potential of Leucas aspira, A.vera and B.sensitivum as natural antioxidants,
contraceptives, anticancer, anti-inflammatory and anti-arthritis agent.
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Agarry O.O, Olaleye M.T and Bello-Michael, (2005); Comparative antimicrobial
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Ricardo Erdmann, & Uwe Wollina, MD,(2009); Hyaluronidase in the Correction
of Hyaluronic acid-based Fillers: A Review and A Recommendation for Use;
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Biophytum petersianum Klotzsch on Ruminal Fermentation,Microbial Protein
Synthesis and Nitrogen Utilization in Goats;Animal Feed Science and
Technology;Vol.137(1&2);58-68.
C.Guruvayyorappan, Girija Kuttan, (2008); Protective Effect of Biophytum
sensitivum (L.) DC on Radiation Induced Damage in Mice;
Immunopharmacology and Immunotoxicology; Vol.30 (4); 815-835.
C.Guruvayoorappan,Girija Kuttan,(2007);Apoptotic Effect of Biophytum
sensitivum on B16F-10 cells and its Regulatory Effects on Nitric Oxide and
Cytokine Production on Tumor Associated Macrophages; Integrative Cancer
Therapies;Vol.6(4);373-380.
C.Guruvayoorappan, Girija Kuttan, (2008); Biophytum sensitivum (L.) DC
Inhibits Tumor Cell Invasion and Metastasis through a Mechanism Involving
Regulation of MMPs, ProlylHydroxylase, LysylOxidase, ERK-1, ERK-2, STAT-
1 and Proinflammatory Cytokine Gene Expression in Metastatic Lung Tissue;
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22
Damintoti Karou, Mamoudou H. Dicko, Jacques Simpore, and Alfred S.
Traore,(2005);Antioxidant and Antibacterial activities of Polyphenols from
Ethnomedicinal Plants of Burkina Faso; African Journal of Biotechnology ;Vol. 4
(8);823-828 .
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Antimicrobial Activity and Phytochemical Screening of Aloe vera Linn.;
International Journal of Current Pharmaceutical Research;Vol.4(3);45-47.
Hunnicutt G R, Myles D.G, (1996); Sperm Surface Protein PH-20 is Bifunctional:
One Activity is a Hyaluronidase and a Second, Distinct Activity is required in
Secondary Sperm-Zona Binding; Biol Reprod; Vol.55 (1); 80-86.
Katsunari lppoushi, Yuichi Yamaguchi, Hidekazu Itou, Keiko Azuma and Hisao
Higash,(1999); Evaluation of Inhibitory Effects of Vegetables and Herbs on
Hyaluronidase and ldentification of Rosmarinic Acid as a Hyaluronidase Inhibitor
in Lemon Balm(Melissa officinalis L. ); FoodSci. Technol. Res;Vol.6(1);74-77.
Mehdi Rahmanian and Paraskevi Heldin,(2002); Testicular Hyaluronidase
Induces Tubular Structure of Endothelial Cells Grown In Three-Dimensional
Colagen Gel Through A CD-44 Mediated Mechanism; Int. J. Cancer: Vol.97,
601–607
Natarajan, M.S. Shivakumar and R. Srinivasan, (2010); Antibacterial activity of
leaf extracts of Biophytum sensitivum (L.) DC; J.Pharm Sci. & Res; Vol.2 (11);
717-720
S. Arunkumar and M. Muthuselvam, (2009); Analysis of Phytochemical
Constituents and Antimicrobial Activities of Aloe vera L. against Clinical
Pathogens; World Journal of Agricultural Science, Vol. 5 (5); 572-576.
Simenou Titrikou,Kwashie Eklu-Gadebeku,Aklesso Mouzou,Kodjo
Aklikokou,Messanvi Gbeassor,(2007);Calcium Antagonistic Activity of
Biophytum petersianum on Vascular Smooth Muscles of Wistar Rat, Iranian
Journal of Pharmacology and Therapeutics;Vol.6(2);185-189.
Sivaraman Padavattan,(2006); Crystal Structure Determination of Hyaluronidase,
a Major Bee Venom Allergen, in Complex with an IgG Fab Fragment and
23
Purification and Biophysical Characterization of Bovine Testes Hyaluronidase.
JMB;Vol.368;745-752.
Siveen KS, Kuttan G;Effect of Amentoflavone,(2011);A Phenolic Compound
from Biophytum sensitivum on Cell Cycling and Apoptosis of B16F-10 Melanoma
Cells; Environ Pathol Toxicol Oncol;Vol.30(4);301-309.
Urvashi Nandal and Raju Lal Bhardwaj,(2012); Aloe vera for Human Nutrition,
Health and Cosmetic Use-A review; International Research Journal of Plant
Science; Vol. 3(3); 38-46.
V.H.Bhaskar and V.Rajalakshmi,(2010); Anti tumor Activity of Aqueous Extract
of Biophytum sensitivum Linn; Annals of Biological Research; Vol.1 (3); 76-80.
Vinata B. Lokeshwar and Melanie A. Simpson, (2009); Hyaluronan and
hyaluronidase in genitourinary tumors; Front Biosci; Vol.13; 5664-5680.
Yebpella G. G, Adeyemi Hassan M. M., Hammuel C, Magomya A. M, Agbaji A.
S and Okonkwo E M,(2011); Phtyochemical Screening and Comparative Study of
Antimicrobial activity of Aloe vera Various Extracts; African Journal of
Microbiology Research;Vol.5(10);1182-1187.
Yun Hu, Juan Xu and Qiuhui Hu (2003); Evaluation of Antioxidant Potential of
Aloe vera (Aloe barbendis miller) Extracts; J.Agri.Food.Chem; 51; 7788-7791.
Annexure - V
24
UNIVERSITY GRANTS COMMISSION
Bahadur Shah Zafar Marg
New Delhi – 110 002.
STATEMENT OF EXPENDITURE IN RESPECT OF MAJOR/MINOR
RESEARCH PROJECT
1. Name of Principal Investigator : Dr. Densely Jose
2. Deptt. of University/College : Dept. of Chemistry, M A College,
Kothamangalam
3. UGC approval No. and Date : MRP(S)–666/0910/KLMG038/UGC-
SWRO dated 04-02-2010
4. Title of the Research Project : “Screening Of Herbal Plants And
Ascertaining Their Effect Of Their Extract On The Inhibition Of Mammalian As Well As
Hyaluronidase Isolated From Pathogenic Bacterial Strains And Proposing Novel
Potential Microbicides”
5. Effective date of starting the project : 04-02-2010
6. (a) Period of Expenditure: From 04-02-2010 to 04-08-2011
7. Details of Expenditure
S.No. Item Amount
Approved Rs. Expenditure Incurred Rs.
i. Books & Journals 10,000 10,000
ii. Equipment 10,000 10,000
iii. Contingency 15,000 15000
iv. Field Work/Travel 5000 5000
v. Chemicals & Glassware 20,000 20,000
It is certified that the grant of Rs.60,000 sanctioned (Amount received till date Rs 56,000
(Rupees fifty six thousand only ) from the University Grants Commission under the
scheme of support for Major Research Project entitled “Screening Of Herbal Plants And
Ascertaining Their Effect Of Their Extract On The Inhibition Of Mammalian As Well As
Hyaluronidase Isolated From Pathogenic Bacterial Strains And Proposing Novel
Potential Microbicides” vide UGC letter No. F. MRP(S)–666/0910/KLMG038/UGC-
25
SWRO dated 04-02-2010 has been fully utilized for the purpose for which it was
sanctioned and in accordance with the terms and conditions laid down by the University
Grants Commission.
SIGNATURE OF
PRINCIPAL INVESTIGATOR REGISTRAR/PRINCIPAL
26
Annexure – VIII
UNIVERSITY GRANTS COMMISSION
BAHADUR SHAH ZAFAR MARG
NEW DELHI – 110 002
PROFORMA FOR SUBMISSION OF INFORMATION AT THE TIME OF
SENDING THE FINAL REPORT OF THE WORK DONE ON THE PROJECT
1. NAME AND ADDRESS OF THE PRINCIPAL INVESTIGATOR : Dr. Densely Jose,
Selection Grade Lecturer, Department of Chemistry, Mar Athanasius College
,Kothamangalam,Eranakulam,Kerala.
2.NAME AND ADDRESS OF THE INSTITUTION : Mar Athanasius
College,Kothamangalam, Eranakulam, Kerala.
3. UGC APPROVAL NO. AND DATE . MRP(S)–540/09-10/KLMG038/UGC-SWR
dated 30-11-2009
4. DATE OF IMPLEMENTATION : 04-02-2010
5. TENURE OF THE PROJECT :
6. TOTAL GRANT ALLOCATED : 60,000
7. TOTAL GRANT RECEIVED : 56,000
8. FINAL EXPENDITURE : 60,000
9. TITLE OF THE PROJECT : “Screening Of Herbal Plants And Ascertaining Their
Effect Of Their Extract On The Inhibition Of Mammalian As Well As Hyaluronidase
Isolated From Pathogenic Bacterial Strains And Proposing Novel Potential Microbicides”
10. OBJECTIVES OF THE PROJECT: To prepare plant extract and study their
antibacterial effect and evaluate their effect on bovine testicular hyalunidase
11. WHETHER OBJECTIVES WERE ACHIEVED: yes
12. ACHIEVEMENTS FROM THE PROJECT: Five plants selected for the study were
evaluated their phyto chemical analysis. Three plants with highest number of
phytochemicals were selected for antibacterial assay and hyaluronidase inhibition assay.
All the three plant extracts were found to have anti bacterial effect and have very high
inhibitory effect on bovine testicular hyaluronidase.
13. SUMMARY OF THE FINDINGS : Attached as a separate sheet
( IN 500 WORDS )
27
14. CONTRIBUTION TO THE SOCIETY : . Hyaluronidase inhibitors can be used as
anti-ageing and anti-wrinkling agents in cosmetics; anti-allergic or anti-inflammatory
cases; as prophylactic measure to prevent spreading of HIV and other sexually
transmitted diseases; as anti-tumor agents etc. (Satardekar K.V et al, 2010).The medicinal
plants studied in this project were potent hyaluronidase inhibitors. So the plants can be
useful in many fields where hyaluronidase inhibitors are employed
15. WHETHER ANY PH.D. ENROLLED/PRODUCED : No
OUT OF THE PROJECT
16. NO. OF PUBLICATIONS OUT OF THE PROJECT: 3
a) Asha Gangadharan, Elizabeth Jacob, and Densely Jose 2014; Phytochemical
Analysis, Antibacterial And Antihyaluronidase Activity Of Three Indigenous
Medicinal Plants; World Journal Of Pharmacy And Pharmaceutical Sciences;
3(6), 751-761
b) Asha Gangadharan, Elizabeth Jacob, Aswathy Vijay, Chithira M J and
Densely Jose; Phytochemical analysis and anti hyaluronidase activity of three
indigenous medicinal plants; a poster presentation in a UGC Sponsored
National Conference cum workshop on Neutraceuticals - Perspective,
Prospects and Challenges; organized by Department of Chemistry and Botany
Mar Athanasius College , Kothamangalam on 12 and 13 March 2013.
c) Veena Ravindran K, Deepa V Ravindran, Asha Gangadharan, Densely Jose ;
Phytochemical analysis Anti bacterial activity and the effect of Leucas aspira
on Bovine Testicular hyaluronidase ; a poster presentation in a UGC
Sponsored National seminar on Clinical applications of Molecular Biology,
Organized by Department of Biotecchnology, Mar Athanasius college
,Kothamangalm, on 3 and 4 February,2012.
( PRINCIPAL INVESTIGATOR ) (REGISTRAR/PRINCIPAL)
(CO-INVESTIGATORS)
28
SUMMARY
The dry plants were extracted using water and ethanol via soxhlet extraction method.
The crude extract obtained was evaporated to dryness and the yield was determined.
Phytochemical analysis of the alcoholic extracts of plant extracts revealed the
presence of carbohydrate, alkaloids, flavonoids, proteins, phenolic compounds, oils,
steroids & terpenoids. The aqueous extracts of the plants were shown to contain
carbohydrate, flavonoids, phenolic compounds and oils.
The percentage of inhibition of alcoholic and aqueous extract of A. vera at
concentrations 5, 10, 15 mg/ml on the activity of Bovine Testicular Hyaluronidase
was found to be 71.80%, 81.43%, 94.29% and 79.22%, 87.48% and 91.31%
respectively. The alcoholic and aqueous extract of B sensitivum showed
38.205%,43.05%, 59.45% inhibition and 73.51%,79% and 96.43% inhibition at
5,10,15 mg/ml concentration respectively.
The percentage of inhibition of alcoholic and aqueous extract of Leucas aspira at
concentrations 5, 10, 15 mg/ml on the activity of Bovine Testicular Hyaluronidase
was found to be 56.54,73.24, 88.33 and 11.67, 29.97and 33.32% respectively.
29
Poster presented on UGC sponsored National Seminar On Clinical Applications Of
Molecular Biology At Mar Athanasius College, Kothamangalam
Phytochemical Analysis of Leucas aspera and its Effect on Bovine
Testicular Hyaluronidase and Multidrug Resistant Bacteria Asha Gangadharan1, Elizabeth Jacob2 and Densely Jose3
1& 2 Dept. of Biochemistry, Mar Athanasious College , Kothamangalam, Ernakulam, Kerala. 3Dept. of Chemistry, Mar Athanasious College , Kothamangalam, Ernakulam, Kerala
ABSTRACT
Plants have always played a major role in the treatment of human diseases. Worldwide
interest in the use of medicinal and aromatic plants is increasing nowadays. Leucas
aspera belonging to the family Labiatea, was used for treatment of various diseases in
traditional system of medicine. In this study phytochemical analysis of Leucas aspera
and its effects on Bovine Testicular Hyaluronidase and multi drug resistant bacteria was
evaluated. The effect of alcoholic and aqueous extracts of Leucas aspera on the specific
activity of bovine testicular hyaluronidase was tested by Elson - Morgan assay and was
found to be decreased for both of the extracts with increasing concentration of extracts.
The effect of the plant extracts on the kinetics of bovine testicular hyaluronidase was
also evaluated and showed a mixed non competitive type of reversible inhibition on the
activity of the enzyme. The extracts affect kinetic parameters Vmax and Km. The
apparent Km increases and apparent Vmax decreases with increasing concentration of
both extracts. The extracts of Leucas aspera act as an inhibitor which affects both the
velocity and the affinity of the enzyme. The aqueous and alcoholic extract was tested for
antibacterial activity against Multidrug Resistant organisms. Both extracts showed
antibacterial activity against Klebsiella and S.aureus with MIC of 25 mg and 12.5 mg
and MBC of 50 mg and 25 mg respectively. This work has revealed the potential of
Leucas aspera plant exract in the area of pharmacology as an antibacterial agent and as a
contraceptive. A further study involving isolation and identification of bioactive
compounds from Leucas aspera extracts should be carried out as it will help us to
understand which constituent is responsible for the effect. Ancient knowledge coupled
with scientific principle, can come to the forefront and provide us with powerful
remedies to eradicate disease.
30
A Poster Presented In A UGC Sponsored National Conference Cum Workshop On
Neutraceuticals - Perspective, Prospects And Challenges;
Organized By Department Of Chemistry and Botany,
Mar Athanasius College , Kothamangalam
On 12 and 13 March 2013
“PHYTOCHEMICAL ANALYSIS AND ANTI-HYALURONIDASE ACTIVITY
OF THREE INDIGENOUS MEDICINAL PLANTS”
Dr. Densely Jose*, Mrs. Asha Gangadharan#, Mrs. Elizabeth Jacob#,
Abstract Medicinal plants the world’s oldest health care products, play a key role in traditional medicines.
Plants produce a diverse range of bioactive molecules, making them a rich source of different
types of medicines. Aloe vera, Leucas aspira and Biophytum sensitivum are medicinally
important plants. .In this study phytochemical analysis of Aloe vera, Leucas aspira and
Biophytum sensitivum and their antihyaluronidase effect was studied by evaluating inhibition of
extracts on Bovine Testicular Hyaluronidase. The plants were extracted using water and ethanol
via soxhlet extraction method. The effect of alcoholic and aqueous extracts of extracts on the
activity of bovine testicular hyaluronidase was tested by Elson – Morgan assay. Phytochemical
analysis of the alcoholic extracts of plant extracts revealed the presence of carbohydrate,
alkaloids, flavonoids, proteins, phenolic compounds, oils, steroids & terpenoids. The aqueous
extracts of the plants were shown to contain carbohydrate, flavonoids, phenolic compounds and
oils. The percentage of inhibition of alcoholic and aqueous extract of A. vera (at concentrations 5,
10, 15 mg/ml) on the activity of Bovine Testicular Hyaluronidase was found to be 71.80%,
81.43%, 94.29% and 79.22%, 87.48% and 91.31% respectively. The alcoholic and aqueous
extract of B sensitivum showed 38.205%, 43.05%, 59.45% inhibition and 73.51%,79% and
96.43% inhibition at 5,10,15 mg/ml concentration respectively. The percentage of inhibition of
alcoholic and aqueous extract of Leucas aspira at concentrations 5, 10, 15 mg/ml on the activity
of Bovine Testicular Hyaluronidase was found to be 56.54, 73.24, 88.33 and 11.67, 29.97 and
33.32% respectively. The study of potent inhibition on hyaluronidase enzyme confirmed the
potential of Leucas aspira, A.vera and B.sensitivum as anti-allergic, contraceptives, anticancer,
anti-inflammatory and anti-arthritis agent.
*Department of Chemistry, M A College, Kothamangalam
#Department of Biochemistry, M A College, Kothamangalam
31
CERTIFICATE
This is to certify that one copy of the report of the Minor Research Project titled
“Screening of Herbal Plants and Ascertaining their Effect of their Extract on the
Inhibition of Mammalian as well as Hyaluronidase Isolated from Pathogenic Bacterial
Strains and Proposing Novel Potential Microbicides” granted (Approved letter: MRP(S)-
540/09-10/KLMG 038/ UGC-SWRO dated 30/11/ 2009 and Sanctioned letter: MRP(S)-
666/09-10/KLMG 038/ UGC-SWRO dated 27/1/2010) to Dr. Densely Jose, Dept. of
Chemistry, Mar Athanasius College, Kothamangalam is submitted in the department
library and the summary of the project is published on our college website
(www.macollege.in) in accordance with the terms and conditions laid down by the
U.G.C.
Thanking you .
Yours faithfully
Principal
32
UGC MINOR PROJECT
“Screening Of Herbal Plants And Ascertaining Their Effect Of Their Extract On The Inhibition Of
Mammalian As Well As Hyaluronidase Isolated From Pathogenic Bacterial Strains And Proposing
Novel Potential Microbicides”
MRP(S)–666/0910/KLMG038/UGC-SWRO
Dr. Densely Jose, Dept. of Chemistry, M A College, Kothamangalam
Mrs. Asha Gangadharan, Department of Biochemistry, M A College, Kothamangalam
Mrs. Elizabeth Jacob, Department of Biochemistry, M A College, Kothamangalam
SUMMARY
Medicinal plants the world’s oldest health care products, play a key role in traditional medicines. Plants
produce a diverse range of bioactive molecules, making them a rich source of different types of medicines.
Aloe vera, Leucas aspira and Biophytum sensitivum are medicinally important plants. As the plants were
found to have antimicrobial properties, the plant extracts could be used as microbicide to prevent sexually
transmitted disese ; in cosmetics as anti-wrinkling agents, or as anti-tumor agents.In this study
phytochemical analysis of Aloe vera, Leucas aspira and Biophytum sensitivum and anti bacterial effect and
its effects on Bovine Testicular Hyaluronidase were evaluated. The plants were extracted using water and
ethanol via soxhlet extraction method. The effect of alcoholic and aqueous extracts of extracts on the
specific activity of bovine testicular hyaluronidase was tested by Elson - Morgan assay. Phytochemical
analysis of the alcoholic extracts of plant extracts revealed the presence of carbohydrate, alkaloids,
flavonoids, proteins, phenolic compounds, oils, steroids & terpenoids. The aqueous extracts of the plants
were shown to contain carbohydrate, flavonoids, phenolic compounds and oils.The percentage of inhibition
of alcoholic and aqueous extract of A. vera at concentrations 5, 10, 15 mg/ml on the activity of Bovine
Testicular Hyaluronidase was found to be 71.80%, 81.43%, 94.29% and 79.22%, 87.48% and 91.31%
respectively. The alcoholic and aqueous extract of B sensitivum showed 38.205%,43.05%, 59.45%
inhibition and 73.51%,79% and 96.43% inhibition at 5,10,15 mg/ml concentration respectively.The
percentage of inhibition of alcoholic and aqueous extract of Leucas aspira at concentrations 5, 10, 15
mg/ml on the activity of Bovine Testicular Hyaluronidase was found to be 56.54,73.24, 88.33 and 11.67,
29.97and 33.32% respectively. The study thus confirmed the potential of Leucas aspira, A.vera and
B.sensitivum as natural antioxidants, contraceptives, anticancer, anti-inflammatory and anti-arthritis agent.