1

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

2

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

4

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)

13

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

+

+

+

+

+

-

+

-

-

+

15

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.

REFERENCES

Abhijit Sahasrabudhe and Manjushree Deodhar, (2010); Anti hyaluronidase and

Anti elastase activity Abinash C Bharati, Alakh N Sahu,(2012); Ethnobotany,

Phytochemistry and Pharmacology of Biophytum sensitivum DC; Phcog

Rev;Vol.6(11);68-73.

A. Garg, R. A. Anderson, L. J. D. Zaneveld, and S. Garg,(2005); Biological

Activity Assessment of a Novel Contraceptive Antimicrobial Agent; Journal of

Andrology;Vol.26(3);414-421.

Agarry O.O, Olaleye M.T and Bello-Michael, (2005); Comparative antimicrobial

activities of Aloe vera gel and leaf; African Journal of Biotechnology; Vol.4 (12);

1413-1414.

A.O. Adesuyi, O.A. Awosanya, F.B. Adaramola and A.I. Omeonu,(2011);

Nutritional and Phytochemical Screening of Aloe barbadensis ;Current Research

of Journal Biological Science;Vol.4(1);4-9.

Asma Bashir, Bushra Saeed, Talat .Y. Mujahid and Nayar Jehan,(2011);

Comparative Study of Antimicrobial Activities of Aloe vera Extracts and

21

Antibiotics against Isolates from Skin Infections; African Journal of

Biotechnology; Vol. 10(19); 3835-3840.

Atsushi Suzuki, Hidenao Toyoda, Toshihiko Toida and Toshio Imanari, (2001);

Preparation and Inhibitory Activity on Hyaluronidase of Fully O-Sulfated

Hyaluro- Oligosaccharides; Glycobiology; Vol .11(1); 57-64.

Berthold Rzany, MD, ScM, Petra Becker-Wegerich, MD, Frank Bachmann, MD,

Ricardo Erdmann, & Uwe Wollina, MD,(2009); Hyaluronidase in the Correction

of Hyaluronic acid-based Fillers: A Review and A Recommendation for Use;

Journal of Cosmetic Dermatology; Vol. 8; 317–323.of Garcinia indica;

International Journal of Botany;Vol.6(3);299-303.

Bishnu Joshi, Govind Prasad Sah, Buddha Bahadur Basnet, Megh Raj Bhatt,

Dinita Sharma, Krishna Subedi, Janardhan Pandey and Rajani Malla,(2010);

Phytochemical Extraction and Antimicrobial Properties of Different Medicinal

Plants; Journal of Microbiology and Antimicrobials; Vol. 3(1); 1-7.

B.Santoso,A.Kilmaskossu and P.Sambodo,(2007);Effect of Saponin from

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;

Cancer Therapies; Vol.7 (1); 42-50.

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 .

H.N. Krishna Kumar, E. Chandana, S.D.Preethi, Jyoti Bala Chauhan; In vitro

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.