18

Chapter – 2

Experimental Methodology

19

Chapter 2: EXPERIMENTAL METHODOLOGY

2.1 Phytochemical studies. 2.1.1. Selection of Medicinal plants. 2.1.2. Extraction, Fractionation and Isolation methods. 2.1.3. Fingerprinting of fractions and Isolated Compound by HPTLC,

HPLC & GC. 2.1.4. Characterization and Structure elucidation of isolated

compounds using NMR and GC-MS. 2.1.5. Selective extraction using aqueous solutions containing

surfactant. 2.1.6. A quantititative study on efficiency of extraction methods

2.2 Biological activity. 2.2.1. Bioassay guided isolation of bioactive molecules. 2.2.2. High throughput screening - Broth Micro Dilution – 96 wells titer

plate method. 2.2.3. Anti-fungal activity. 2.2.4. MDR Inhibitory activity. 2.2.5. Anti-dermatophytic activity. 2.2.6. Anti- bacterial activity. 2.2.7. Toxicity study – Cytotoxic activity - Brine shrimp Lethality

Bioassay.

2.1 Phytochemical studies: The recent interest in the plant kingdom as a potential source of new drugs, envisage alternate strategies for the fractionation of plant extracts rather than on a particular class of compound, since not all the chemical compounds elaborated by plants are of equal interest to the pharmacognosist. The so-called active principles are frequently alkaloids or glycosides and these, therefore, deserve special attention. Other groups such as carbohydrates, fats and proteins are of dietetic importance, and many such as starches and gums are used in pharmacy but lack any marked pharmacological action. Other substances, such as calcium oxalate, silica, lignin and coloring matters, may be assistance in the identification of drugs and the detection of adulteration. The present approach is based on bioactivity guided fractionation and isolation of active compounds. This phytochemical investigation of a plant involves the following;

20

Extraction of the plant material Separation and isolation of the constituents of interest Characterization of the isolated compounds and Quantitative evaluations

2.1.1. Selection of Medicinal plants 12 medicinal plants were selected based on the availability, chemotaxonomy and

Ethno medicinal applications. All the Plant materials were collected according to their

seasonal availability and shadow dried. Those materials were stored in airtight

containers for future use. Prof. Jeyaraman, Medicinal Plant Research Unit, West

Tambaram, Chennai – 45, authenticated all the plants. The herbarium sheets for each

plant are available in Asthagiri Herbal Research Foundation, Tambaram sanatorium,

Chennai, Tamilnadu. List of plants selected and their photocopies were given in Table

2.1 and Fig. 2.1.

Table 2.1. List of Plants used for this study

S.

No. Botanical Name Vernacular

name in Tamil Family Parts used

for the study

1 Wrightia tinctoria (Roxb.) R. Br. [WT]

Vet palai Apocyanaceae Leaves

2 Ocimum tenuiflorum Linn. [OCI] Krishna tulasi Labiatae Leaves 3 Coleus forskholii (Poir) Briq. [CF] ----- Labiatae Root 4 Phyllanthus amarus Schum &

Thonn. [PHY] Keela nelli Euphorbiacae Leaves

5 Urginea India kunth. [SQ] Nari venkayam Lilliaceae Bulb 6 Vitex negundo Linn. [VN] Vennochi Verbenaceae Leaves 7 Aloe barbadensis Mill. [ALOE] Kattalai Lilliaceae Leaves 8 Sesbania bispinosa (Jacq.) W.F.

Wight. [SB] Mullagathi Leguminosae Seed

9 Acyranthes aspera Linn. [ACY] Nayuruvi Amarantheceae Leaves 10 Azadirachta indica A. Juss. [AZA] Veppa Meliaceae Seeds 11 Begonia albo coccinea Hook. [BEG] Kaltamarai Begoniaceae Leaves &

Roots 12 Taxus baccata Linn. [TAX] Yew tree [Eng] Taxaceae Leaves

21

Fig. 2.1. Photos of the medicinal plants used in the present study

Wrightia tinctoria (Roxb.) R. Br.

Ocimum tenuiflorum Linn.

Coleus forskholii (Poir) Briq.

Phyllanthus amarus Schum & Thonn

Urginea India kunth.

Vitex negundo Linn.

Aloe barbadensis Mill.

Acyranthes aspera Linn.

Azadirachta indica A. Juss.

Begonia albo coccinea Hook

Taxus baccata Linn.

22

2.1.2. Extraction, Fractionation and Isolation methods Extraction method: The plant material was powdered by using cutter mill and mixer. The ground material

was soaked as cold maceration for 48 hrs with polar solvent (Methanol) and Non-polar

solvent (Hexane) separately. Aqueous solution containing Surfactant (1%w/v) and

Steam Distillation were used for some plants (Scheme 2.1). After effective maceration,

it was filtered in a Whatt-Mann filter paper; the filtrate was collected and concentrated

under reduced pressure using Rotovac – Flash Evaporator. This extraction process was

repeated two times and all the concentrated extracts were mixed together. The extracts

were characterized by the following parameters, such as

1. Percentage Yield,

2. Extractive values in various solvents and

3. TLC and HPTLC characterization.

PLANT MATERIAL

EXTRACT RESIDUE[ ST ]

PLANT MATERIAL

EXTRACT RESIDUE[ AE ]

PLANT MATERIAL

EXTRACT RESIDUE[ HE ]

PLANT MATERIAL

EXTRACT RESIDUE[ ME ]

4. STEAM DISTILLATION3. AQUEOUS EXTRACTION

2. NON POLAR EXTRACTION

1. POLAR EXTRACTION

METHANOL HEXANE AQUEOUS SURFACTANT

STEAM DISTILLATION

GENERAL EXTRACTION SCHEME

Scheme 2.1

23

Fractionation method:

The concentrated methanol extracts were further subjected to partial fractionation with

solvents of increasing polarity viz. Hexane: Chloroform: Ethyl acetate: Methanol

(Scheme 2.2) Methanol extracts were distributed in the non-polar solvent, Hexane.

Hexane dissolved portion, is called Hexane Fraction (HF), was filtered and

concentrated by removing the solvent under reduced pressure. The residue thus

obtained was fractionated subsequently with medium polar solvents such as

Chloroform (CLF), Ethyl acetate (EAF) and polar solvent, Methanol (MF).

All the fractions were also characterized by the following parameters.

1. Percentage Yield,

2. Extractive values in various solvents and

3. TLC and HPTLC Characterization.

Scheme 2.2.

General

Fractionation

Scheme

Using Solvents with increasing Polarity

METHANOLIC EXTRACT

[ME]

Hexane

FRACTION RESIDUE

[HF] CH Cl3

FRACTION RESIDUE

[CLF] Ethyl acetate

FRACTION RESIDUE

[EAF] Methanol

FRACTION RESIDUE

[MF] [AF]

24

Calculation of Percentage Yield

The Percentage yield was calculated for the extracts, fractions and major compounds with reference to the crude material taken using the formulas given below

Formula 1.

Percentage Yield Weight in grams of Extracts obtained with reference to = ------------------------------------------------- X 100 crude plant material Weight in grams of plant material taken Formula 2. Extractive Value Percentage yield in other solvent with reference to = ---------------------------------------------------- X100 Methanol extracts Percentage Yield in Methanol

Formula 3.

% Yield of fraction Weight in grams of fractions obtained with reference to = ---------------------------------------------------- x 100 methanol extracts Weight in grams of Methanol extract taken Formula 4.

% Yield of fraction % Yield of methanol % Yield of fraction with reference to x extracts with reference to

methanol extracts crude leaf powder with reference to = -------------------------------------------------------------- crude plant material 100

Isolation of compounds: Both bioactive extracts and fractions thus screened by biological activity study were subjected to simple column chromatography using silica gel, 70/325 meshes, as adsorbent and stationary phase. Solvent mixtures, Hexane – Ethyl acetate or Chloroform – Methanol were used as mobile phase. In some cases Preparative TLC was used for isolation of compounds.

25

Method 1- Column Chromatographic Isolation: Preparation of Admixture: 1 g of extract was admixed with 1 – 2 g Silica gel

(60/120 meshes) to get uniform mixing.

Column Packing: 30 g of Silica gel, 70/325, (The ratio of admixture: adsorbent is 1:

30) was taken in a suitable column (80 cm length, 5 cm diameter) and packed very

carefully without air bubbles using Hexane as filling solvent. The column was kept

aside for 1 hr and allowed for close packing. Then admixture was added at the top of

the stationary phase and started separation of compounds by the eluting with various

solvent mixtures with increasing order of polarity i.e., 0, 1, 5, 10, 15, 20, 25, 30, 40

and 50 % V/V of Ethyl acetate in Hexane. All the column fractions were collected

separately and concentrated under reduced pressure. Finally the column was washed

with ethyl acetate and methanol. If needed further column separation was carried out

for some column fractions. Those column fractions obtained were studied for

bioactivity and characterized.

Method 2 – Preparative TLC separation:

Preparation of preparative TLC plate: Plates were prepared by using Silica Gel – G

as adsorbent and 20 x 20 glass plates. 100 g Silica gel-G was mixed with sufficient

quantity of distilled water to make slurry. The slurry was immediately poured into a

spreader and plates were prepared by spreading the slurry on the glass plates. The

thickness of the layer was fixed as 1.5 mm. Plates were allowed to air dry for 1 hour

and layer was fixed by drying at 105° C for 1 hour.

Plate development: Using a syringe and needle about 10 ml of 1% w/v solution of

extracts or fractions were loaded gradually over the plate. The loaded plates were

eluted by suitable eluent mixture in separation tank. Before elution the tank was

allowed 30 min. for saturation with eluent mixture.

Collection and Purification of isolated compound: After successful separation, the

marked compounds were scraped by using a spatula, collected separately and extracted

26

in a suitable solvent. After evaporating the solvent dry weight and % yield was

calculated for the pure compound.

2.1.3. Fingerprinting of extracts, fractions and Isolated Compounds using

HPTLC, HPLC & GC

All the extracts, fractions and isolated compounds were characterized by the

chromatographic techniques such as HPTLC, HPLC and GC. Chromatographic

profiles were prepared for the extracts and fractions. Compounds were identified by

HPTLC using marker compounds. Presence major compounds were identified and

their percentage yield was calculated (Formula. 5) using HPTLC densitogram and

HPLC chromatogram.

Formula 5. % Yield of the major % Area x % yield with reference to crude material compound with reference = -------------------------------------------------------------------- to crude plant material 100 2.1.4. Characterization and Structure elucidation of isolated compounds

using NMR & MS

Isolated compounds and bioactive compounds were characterized and identified by

Mass and NMR spectrophotometric method. Chemical structures of the compounds

were elucidated.

NMR spectra were recorded on a Bruker 200 MHz instrument using TMS as the

internal standard. CDCl3, D2O and DMSO were used as the solvents.

LRMS were recorded on a Shimadzu QP 1000A and QP 5000 mass spectrometer.

FAB-HRMS was recorded using Autospec FAB Mass spectrometer.

27

2.1.5. Selective Extraction using aqueous solutions of surfactant:

Isolation of compounds from the plant species is one of the challenging, tedious,

complicated and expensive jobs. Another draw back in conventional methods of

extraction is using organic solvents such as Hexane, Toluene, Chloroform etc., and

also these solvents are more poisonous.

We have taken an attempt to develop a simple and safer method for isolation of

compound by using a suitable solvent mixture. Amphiphilic character of surfactants

had attracted and directed us to use them in extraction process. We had chosen

surfactants based on their biodegradability and pharmaceutical grade. Linear Alkyl

Benzyl Sulfonic acid (LABSA), an anionic surfactant and Poly oxyethylene sorbitan

mono oleate (Tween 80), a non-ionic surfactant, were selected for this study. They

were pharmaceutical grade and biodegradable additive used for pharmaceutical

preparations. 1 %w/v aqueous solution of these surfactants was used for this study.

Method: The dried and powdered plant material (Ocimum sanctum) was soaked in 1%

w/v of aqueous solution of surfactant, Linear Alkyl Benzyl Sulfonic acid (LABSA)

and Poly oxyethylene sorbitan mono oleate (Tween 80), for 48 hours. The extract was

collected by filtration and subjected to the following treatment. The aqueous extract

was neutralized (pH 7.4 – 8.0) with sodium bicarbonate; during this neutralization

process magnetic stirrer kept the extract under continuous stirring. Further the extract

was partitioned with Di-chloro methane (DCM), DCM layer was collected and the

extract was concentrated by vacuum evaporation. (If further purification needed,

Vacuum Liquid chromatographic method could be adopted) The extract was

characterized by the Instrument, Gas chromatography and Mass Spectrometry

(Scheme 2.3).

28

Scheme 2. 3.

Powdered Plant materialSoaked in aqueous solution of Surfactant (1 % w/w) for 48 hrs.

Aqueous extract

- Neutralization with NaHCO3- Partition with Dichloro

methane

DCM Layer Aqueous Layer

Marc.

Extract and characterization by GC-MS

- Vacuum Evaporation- Further Purification

by VLC

Selective

Extraction

Scheme

Using Aqueous solution containing Surfactant

2.1.6. A quantitative study on Efficiency of Extraction methods:

Efficiency of extraction methods for Coleous forskhollii using various solvents was

studied. Dried Roots were collected and powdered by cutter mill. 100 g of powdered

plant was taken separately and were soaked in solvents of Hexane, Methanol, Aqueous

solution containing 1% w/v of LABSA and 1%w/v aqueous solution of Tween-20

(Poly oxyethylene sorbiton mono laurate). The maceration period was 48 hrs. (Scheme

2.1.) The extracts were collected separately after filtration and vacuum evaporation of

solvent. Percentage of Forskolin (a Major chemical constituent of Coleous forskhollii)

content of each extract was calculated by using the HPLC report and the following

formula.

29

Formula 6. Percentage of Area of Test Concentration of Standard Forskolin content = --------------------- X -------------------------------- X 100 In Extract (%Fe) Area of Standard Concentration of Test Formula 7 Percentage of Forskolin % Fe x % Yield of crude extract content in Crude plant = -- ------------------------------------------------ material (%Fm) 100 2.2. Biological activity Bioactive fractions and molecules with anti-infective properties were screened from

the plants by following two systematic approaches such as, bioassay guided isolation

and high throughput screening methods.

2.2.1. Bioassay guided Isolation

Bioassay-guided isolation method integrates the processes of separation of compounds

in a mixture, using various analytical methods, with results obtained from biological

testing. The process begins with testing an extract to confirm its activity, followed by

crude separation of the compounds in the matrix and testing the crude fractions

(Scheme 2.4). Further fractionation is carried out on the fractions that are determined

to be active, at a certain concentration threshold, whereas the inactive fractions are set

aside or discarded. The process of fractionation and biological testing is repeated until

pure compound(s) are obtained. Structural identification of the pure compound then

follows. This methodology precludes overlooking novel compounds that are often

missed in studies that only identify those compounds with which the investigator is

familiar. Moreover, the possibility of discovering an unknown molecular site of action

is maximized (Agnes et al., 2001).

30

Extracts

Extraction Various solvents with varying polarity

Bioassay

No activity Active No activity

Fractionation using solvents with increasing polarity or VLC methodBioassay

NO NO NO YES NO NO NO NO

Isolation and Structure ElucidationUsing Chromatographic technique and Instrumental analysis

Bioassay

Guided

Isolation of

Bioactive

molecules

PLANTS

Scheme 2.4. General scheme for bioassay-guided isolation

2.2.2. High throughput Screening – Broth Micro-dilution Method (NCCLS,

USA-M27A) The method described here is intended for testing yeasts that cause invasive infections.

The broth micro-dilution was performed by using sterile, disposable, U-shaped, 96

wells micro-titre plates (Scheme 5).

Step 1: Inoculum Preparation Medium - PYD Broth: Saboroud Dextrose Broth (Peptone 0.2%w/v and Dextrose

0.5%w/v) with 0.2 % w/v Yeast extract was used for this study. Saboroud Dextrose

Broth and Yeast extract were weighed accurately and dissolved in distilled water,

filtered, sterilized by autoclave method and stored at 4° C.

31

High throughput Screening

1 2 3 4 5 6 7 8 9 10 11 12

ABCDEFGH

Row A contains the highest and Row F contains the lowest concentration of compound.

Concentration

Wells to perform sterility test, growth medium aloneWells for growth control, medium + inoculum ( with out compound)

A

F

Broth micro dilution –96 wells titre plate bioassay(NCCLS, USA-M 27- A)

Scheme 2. 5. High throughput screening method – 96 wells plate assay Method: The inoculum was prepared by picking five colonies of > 1 mm in

diameter from 24 hr old culture of fungi. The colonies should be suspended in 5 ml of

sterile saline (0.145 mol / lit). The resulting suspension was vortexed for 15 sec and

the cell density was adjusted with a spectrophotometer by adding sufficient saline to

increase the transmittance to that produced by a 0.5 McFarland standard (Table 2) at

530 nm wavelength. This procedure yields a stock suspension of 1 x 10 6 to 5 x 106 cfu

/ ml. A working suspension was made by a 1: 100 dilution followed by a 1: 20

dilution of the stock suspension with PYD broth medium which resulted in 5 x 10 2 to

2.5 x 10 3 cfu / ml.

Step2 : Preparation of Anti-fungal agents (Extracts / Fractions / Compounds)

Preparation of stock solution: Stock solution of the extract, to be tested, was

prepared by dissolving 20.48 mg of extract in 1 ml of Dimethyl sulphoxide (DMSO) in

an Eppendorff’s centrifuge tube. All those stock solutions were stored at - 20° C.

32

Dilution of anti-fungal agent: Sterile Eppendorff centrifuge tubes, 1.5 ml, were

used for dilution. Two-fold dilutions of extracts were prepared according to the Table

2.3. A single pipette was used for measuring all diluents and then for adding the stock

anti-fungal solution to the first tube.

Table 2. 2. Preparation of Mc. Farland standards

Standard Tube No

BaCl 2 solution, 1% w/v

(ml)

Cold Solution of H2SO4, 1% w/v

(ml)

Corresponding Bacterial

concentrations. (Millions / ml)

%Transmittanceat 530 nm

(%)

0.5 0.05 9.95 150 72.00 1 0.1 9.9 300 54.67 2 0.2 9.8 600 34.80 3 0.3 9.7 900 23.70 4 0.4 9.6 1200 19.70 5 0.5 9.5 1500 14.00

Table 2. 3: Scheme for preparing dilutions of Anti-fungal extracts to be used in

Broth Micro-Dilution Susceptibility Tests Step Concent-

ration

(μg/ml)

Source Volume

(μl)

Medium

+(μl)

Intermediate Concentration

(μg/ml) (Working solution)

Final Concentration

at 1: 1 dilution (μg/ml)

Code for Concentr

-ation

1. 20480 Stock 100 700 2560 1280 A 2. 2560 Step 1 200 200 1280 640 B 3. 2560 Step 1 200 600 640 320 C 4. 640 Step 3 200 200 320 160 D 5. 640 Step 3 100 300 160 80 E 6. 640 Step 3 100 700 80 40 F

Step 3: Experimental Procedure: The Broth micro dilution method was performed by using sterile, disposable, U-

bottom micro-titer plate. The diluted compound solutions were dispensed into the

wells of columns A to F of the micro dilution plates in 100 μl. volumes with a

micropipette. Column A contained the highest and column F contained the lowest

concentration of compound.

33

Each well of a micro dilution plate is inoculated with 100 micro liters of the

corresponding diluted inoculums suspensions. Column G may be used to perform

sterility testing with compound and inoculums free medium only. Column H may be

used as growth control well containing culture medium without compound for each

organism tested (Scheme 2.5). The plates are incubated at 37 degree C for 24 hrs and

absorbance measured in Elisa Reader (ANTHOS 2020, version 1.2) at 630 nm.

Percentage killing of microorganism corresponding to the control and the Inhibitory

concentrations are calculated and tabulated.

Formula 8.

A Test - A Blank % Killing = 1 - ----------------- X 100

A Control - A Blank 2.2.3. Anti-fungal activity Materials Fungal strains (obtained from National Chemical Laboratory, Pune)

1. Candida albicans [ATCC No. 10231]

2. Candida krusi [ATCC No. 3219]

3. Candida tropicalis [ATCC No. 1380] and

4. Saccharomyces cerevisiae [ATCC.No. 2366]

Culture Medium used: PYD Broth (Saboroud Dextrose Broth (Peptone 0.2%w/v

and Dextrose 0.5%w/v) with 0.2 % w/v Yeast extract)

Standard Anti fungal agent: Fluconazole.

Cell density of Inoculum (i.e., working suspension of fungi):

5 x 10 2 to 2.5 x 10 3 cfu / ml.

34

Concentration of Extracts used:

A - 1280 μg / ml. B - 640 μg / ml. C - 32O μg / ml.

D - 160 μg / ml. E - 80 μg / ml. F - 40 μg / ml.

Experimental method: Screening of anti-fungal fractions and MIC testing was performed with PYD medium

against yeast culture in micro-titer plate by the protocols provided by NCCLS, as

described in Chapter 2 -Section 2.2.2. The compounds tested were diluted and tested at

different concentrations in order to determine more precise MIC (Minimum Inhibitory

concentration). MICs were recorded as the lowest concentration of compound that

inhibited 80% growth after 24 h of incubation at 35°C. Each test was performed at

least in triplicate.

2.2.4. MDR Inhibitory activity

Measurement of R6G uptake:

Rhodamine 6G and Fluconazole uptake by the Candida albicans, both susceptible and

resistant strains, were studied by following the method given by Shigefumi Maesaki

et al., 1999. Yeast cells, 5 colonies of 24 h culture, were grown in 50 ml of PYD broth

at 37° C for 14 h. 1x 108 yeast cells per ml were transferred to 50 ml of fresh PYD

broth and incubated at 37° C for 4 h. The cells were harvested in 50 ml Falcon tubes

and centrifuge at 5000 g for 5 min. The pellets were washed thrice with 20 ml of

Phosphate buffered saline (PBS). They were then suspended in a Glucose – free PBS

buffer, at a concentration of 1x 108 cells / ml. and incubated at 37° C for 1 h. in

reciprocating shaker. A stock solution of R6G was prepared by dissolving the dye in

DMSO at a concentration of 10 mM. A final concentration of 10 μM of R6G was

added to the cell suspension and incubated at 37° C in a reciprocating shaker. After

incubation for 5, 10, 15, 20, 25 min, 1 ml samples were withdrawn and centrifuged at

35

9000 g for 2 min. The supernatants (750 μl) were collected and absorption was

measured at 527 nm. (Scheme 2.6)

Candida albicans, 5 colonies, 24 hrs cultures

Transfer 10 8 cfu / ml culture Into 50 ml of fresh SDB medium

Dispersed the pellets in PBS – 10ml and transfer into 20 ml buffer, the final concentration is 10 8 cfu / ml

Collect the samples of 2 ml at 5 minutes intervals i.e., 5, 10, 15 & 20 minutes

Calculate the extra cellular concentration of Fluconazole and compare with Standard, Verapamil

Scheme 6. Flow chart for determination of MDR efflux mechanism in

Candida albicans, a Fluconazole resistant culture.

- Inoculated in 50 ml PYD medium - Incubated for 14 hrs at 37° C

- Incubated for 4 hrs in shaker at room temp. - Centrifuged at 5000 rpm for 5 minutes and collect the pellets. - Washed the pellets with Phosphate Buffer Saline [PBS] with 20 ml for two times

- Kept it in the shaker for 1 hr. - Added the inhibitors and shake for 20 minutes - Added Fluconazole; the final concentration is 10 μM

- Centrifuged the collected samples at 9000 rpm - Took Absorbance the supernatant liquid containing Fluconazole at 212 nm

36

2.2.5. Anti-dermatophytic activity

Materials.

Fungal strains (obtained from SPIC Science Foundation, Chennai):

1. Trichophyton rubrum

2. Trichophyton mentagrophytes

3. Epidermaphyton floccosum

Culture Medium used: PYD Broth (Saboroud Dextrose Broth (Peptone 0.2%w/v

and Dextrose 0.5%w/v) with 0.2 % w/v Yeast extract)

Preparation of compounds:

As described in section 2.2.2., extracts and fractions were dissolved in dimethyl

sulfoxide and diluted in PYD broth to a final concentration ranges between 1280 and

40 µg/ml.

Concentration of Extracts and fractions

A - 1280 μg / ml. B - 640 μg / ml. C - 32O μg / ml.

D - 160 μg / ml. E - 80 μg / ml. F - 40 μg / ml.

Inoculum preparation:

Scraping the spores of the fungi from the agar plates with a loop and suspending them

in sterile saline solution prepared the inocula. The fungal spores suspensions were

filtered once through sterile gauze to remove the hyphae. For the preparation of

ascospores suspensions we used 0.05% Tween 20 in sterile saline solution (Panreac

Química S.A.). Inocula of T. mentagrophytes, T. rubrum and E. floccosum were

prepared using hemocytometer. The final concentration of inocula in the microtiter

plates was 5 x 10 2 to 2.5 x 10 3 spores/ml.

Experimental method

Screening of anti-dermatophytic fractions and MIC testing was performed with PYD

medium against dermatophytes in micro-titer plates by broth micro dilution method as

37

described in Chapter 2-Section 2.2.2. The compounds tested were diluted and tested at

several different concentrations in order to determine more precise MICs. MICs were

recorded as the lowest concentration of compound that 80% inhibited growth after 24 h

of incubation (72 h incubation in the case of T. rubrum) at 35°C. Each test was

performed at least in triplicate.

2.2.6. Anti-bacterial activity Materials Anti-bacterial strains (obtained from Christian Medical College, Vellore) 1. Staphylococcus aureus [ATCC No. 25923]

2. Pseudomonas aeruginosa [ATCC No. 25853]

3. Escherichia coli [ATCC No. 25922] and

4. Klebsiella pneumoniae [ATCC No. 700603]

Culture Medium used: Muller Hinton broth Standard Anti-bacterial agent: Ciprofloxacin Cell density of Inoculum: 5 x 10 2 to 2.5 x 10 3 cfu / ml. Concentration of Extracts and fractions

A - 1250 μg / ml. B - 625 μg / ml. C - 32O μg / ml.

D - 160 μg / ml. E - 80 μg / ml. F - 40 μg / ml.

Experimental method: Screening of anti-bacterial fractions and MIC testing was performed with Muller-

Hinton medium against both Gram positive and Gram negative bacterial culture in

micro-titer plates by the protocols provided by NCCLS, as described in section 2.2.2.

The compounds tested were diluted and tested at several different concentrations in

order to determine more precise MICs. MICs were recorded as the lowest

concentration of compound that 80% inhibited growth after 20 h of incubation at 35°C.

Each test was performed at least in triplicate.

38

2.2.7. Toxicity Study and Cytotoxic activity Brine shrimp Lethality assay: A rapid general Bioassay

Materials:

Artemia salina Cyst (complementary sample from Sr. Suganthi Dason Marine

Institute, Thuthukudi)

Experimental procedure;

1. Seawater was collected from the Marina beach, Chennai and sterilized by moist

heat sterilization, filtered and used for this study. (Artificial seawater could be

prepared by dissolving 38g sea salt in one liter of water, filtered and used for

this study)

2. Put seawater and shrimp eggs in small tank, one side of the divided tank. Lamp

was lighted the other side to attract hatched shrimp through perforation in the

dam.

3. Allowed two days for the shrimp to hatch and mature as nauplii.

4. Prepared vials for testing; for each fraction, tested initially at 100, 100 and 10

μg/ml; prepared three vials at each concentration for a total of 9 vials; weighed

20mg of sample and added 2 ml of solvent, DMSO (20 mg/ 2ml); from this

solution transferred 500, 250 or 50 μl to vials corresponding to 1000, 500 or

100 μg/ml, respectively.

5. After 2 days (when the shrimp larvae are ready), sterile seawater was added to

each vial, 10 shrimp per vial were transfered (30 shrimp per dilution), and the

volume was adjusted with seawater to 5 ml per vial. (Shrimp can be used 48 –

72 hours after the initiation of hatching. After 72 hours they should be

discarded).

6. 24 hours later counted and recorded the number of survivors.

39

200 mg of cyst of Artemia Salina Nauplii stage Artemia 10 Nauplii Each vial contained 5 ml of Seawater and various

concentrations of testing substance

Inhibitory concentration at which 50 % nauplii were killed (IC50) was calculated

Scheme 7. Brine Shrimp Lethality Bioassay Methodology

- Transfer into 400 ml of Sea water - Set the Aerator and the 100 W bulb to

give enough heat for hatching. - Continue this process for 48 to 72 hours

Until hatching occurs

Transfer each 10 nauplii into the reaction bottle, in which different concentration of testing substance is present - After 24 hours the live naupli can be counted

100 500 1000 Control

40

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