19

MATERIALS AND METHODS

3.1 COLLECTION OF SEAWEEDS

Six species of seaweeds representing Chlorophyceae [Ulva lactuca Linnaeus

(VOCB 4097) and Caulerpa scalpelliformis (R.Brown ex Turner) C.Agardh

(VOCB 4093)], Phaeophyceae [Sargassum wightii Greville (VOCB 4096) and

Padina tetrastromatica Hauck (VOCB 4095)] and Rhodophyceae [Gracilaria

foliifera (Forsskal) Boergesen (VOCB 4094) and Acanthophora spicifera (Vahl)

Boergesen (VOCB 4092)] were collected in December 2007 during low tide period

in the early morning hours from the Hare Island, Tuticorin and Tiruchendur (Gulf

of Mannar region) southeast coast of Tamilnadu.

3.2 PHARMACOGNOSTIC STUDIES

The pharmacognostic studies, which include the study of the structural,

physical, chemical and sensory characters of crude substances of animals, plants

and mineral origin.

3.2.1 MACROSCOPIC STUDIES OF SEAWEEDS

Care was taken to select healthy plants and normal organs.

3.2.2 ANATOMICAL ANALYSIS OF SEAWEEDS

The required samples of different organs were cut and removed from the

plant and fixed in F.A.A (Formalin-5 ml + Acetic acid-5 ml + 70% Ethylalcohol-90

ml). After 24 hrs of fixing, the specimens were dehydrated with graded series of

tertiary butyl alcohol as per the schedule given by Sass, 1940. Infiltration of the

specimens was carried by gradual addition of paraffin wax (melting point-58°C-

60°C) until TBA solution attained supersaturation. The specimens were cast into

paraffin blocks.

20

SECTIONING

The paraffin embedded specimens were sectioned with the help of Rotary

Microtome. The thickness of the sections was 10-12 µm. Dewaxing of the sections

was by customary procedure (Johansen, 1940). The sections were stained with

toluidine blue as per the method published by O’Brien et al. (1964). Since toluidine

blue is a polychromatic stain, the staining results were remarkably good and some

cytochemical reactions were also obtained. The dye rendered pink colour to the

cellulose walls, blue to the lignified cells, dark green to suberin, violet to the

mucilage, blue to the protein bodies etc., wherever necessary sections were also

stained with Safranin and Fast green and IKI (for starch).

PHOTOMICROGRAPHS

Microscopic descriptions of tissues are supplemented with micrographs

wherever necessary. Photographs of different magnifications were taken with

Nikon labphoto 2 microscopic units. For normal observations bright field was used.

For the study of crystals, starch grains and lignified cells, polarized light was

employed. Since these structures have birefrigent property under polarized light,

they appear bright against dark background. Magnifications of the figures are

indicated by the scale-bars. Descriptive terms of the anatomical features are as

given in the standard Anatomy books (Esau, 1964).

3.2.3 DETERMINATION OF PHYSICAL CONSTANTS

The chemicals used in the determination of physico-chemical constants,

fluorescent study, phytochemical screening and quantification and the various

biochemical activities were of BDH (AR) or Sigma Chemical Company, St. Louis

M.O., U.S.A., throughout the study unless otherwise specified.

21

COLLECTION AND PROCESSING

Chosen species were brought to the laboratory. They were washed

thoroughly with tap water to remove the extraneous materials, shade dried

separately and powdered. The dried seaweed samples were packed in airtight

HDPE bags and stored at room temperature.

3.2.3.1 ASH VALUE

The ash content of a crude drug is generally taken to be the residue

remaining after incineration. It usually represents the inorganic salts naturally

occurring in the drug and adhering to it, but it may also include inorganic matter

added for the purpose of adulteration. Ash value varies within narrow limits in the

case of some individual drug. Hence, the ash value determination furnished a basis

for judging the identity and cleanliness of a drug and gives information related to its

adulteration with inorganic matter. Ash standards have been established for a

number of drugs. Usually these standards set a maximum limit on the total ash,

which is insoluble in dilute hydrochloric acid. The ash values of the phytodrugs are

helpful in detecting the quality and purity of the crude drugs in powder form.

Various ash types were determined as per standard procedures (Anonymous, 1996).

DETERMINATION OF TOTAL ASH

About 3 g of the powdered drug was accurately weighed in a silica crucible,

which was previously ignited and weighed. The powdered drug was spread as a

fine layer on the bottom of the crucible. The crucible was incinerated at a

temperature not exceeding 450°C until free from carbon. The crucible was cooled

and weighed. The procedure was repeated to the constant weight. The percentage

of total ash was calculated with reference to the air-dried drug.

22

DETERMINATION OF WATER-SOLUBLE ASH

The ash obtained in the determination of total ash was boiled for 5 minutes

with 25 ml of water. The insoluble matter was collected on an ashless filter paper

and washed with hot water. The insoluble ash was transferred into a tared silica

crucible and ignited for 15 minutes at a temperature not exceeding 450°C. The

procedure was repeated to get the constant weight. The weight of the insoluble

matter was subtracted from the weight of total ash. The difference in weight was

considered as the water-soluble ash. The percentage of water-soluble ash was

calculated with reference to the air-dried drug.

DETERMINATION OF ACID INSOLUBLE ASH

The ash obtained in the determination of total ash was boiled with 25 ml of

2N hydrochloric acid for 5 minutes. The insoluble ash was collected on an ashless

filter paper and washed with hot water. It was transferred into pre-weighed silica

crucible. The procedure was repeated to get constant weight. The percentage of

acid insoluble ash was calculated with reference to the air-dried drug.

3.2.3.2 EXTRACTIVE VALUES

Extractive values of crude drugs are useful for their evaluation especially

when the constituents of a drug cannot be readily estimated by any other means.

These values also indicate the value of the constituents present in a crude drug. The

extractive values were determined as per the standard procedures (Anonymous,

1996).

ETHANOL SOLUBLE EXTRACTIVE VALUE

5 g of the each air dried coarse powder of seaweed sample was macerated

with 100 ml of 90% of ethanol in a closed flask for 24 hours, shaking frequently

during the first 6 hours and allowing to stand for 18 hours. It was then filtered

rapidly taking precautions against loss of solvent. 25 ml of the filtrate was

23

evaporated to dryness in a tared flat-bottomed shallow dish dried at 105°C and

weighed. The percentage of ethanol soluble extractive value with reference to the

air-dried drug was calculated with reference to the air-dried drug.

WATER SOLUBLE EXTRACTIVE VALUE

5 g of shade dried coarsely powdered seaweed samples were macerated with

50 ml of water at 80˚C in a stoppered flask. It was shaken well and allowed to stand

for 10 minutes. It was cooled at 15˚C and 2g of Kieselghur was added and filtered.

5 ml of the filtrate was transferred to a tared evaporating dish, solvent was

evaporated on a water bath and the residue was weighed. The percentage of water

soluble extractive value with reference to the air-dried powder was calculated.

3.2.3.3 FLUORESCENT ANALYSIS

Many phytodrugs when suitably illuminated will emit light of a different

wavelength or colour from that which falls on them. This emitted light

(fluorescence) ceases when the existing light is removed. In fluorescence, the

fluorescent light is always of greater wavelength than the existing light. The

fluorescent analysis of drug extract helps to identify the drug with specific

fluorescent colour and also to find out the fluorescent impurities.

The drug powder was treated with acids like 1N HCl and 50% H2SO4 and

alkaline solutions like aqueous sodium hydroxide, alcoholic sodium hydroxide and

other solvents like picric acid, acetic acid, 5% ferric chloride, nitric acid with

ammonia and nitric acid. They were subjected to fluorescent analysis in daylight

and in UV light (254 nm and 365 nm) (Chase and Pratt, 1949).

3.2.4 PHYTOCHEMICAL SCREENING

The phytochemical screening involving various tests was carried out as per

the standard procedures (Harborne, 1979; Brinda et al., 1981; Lala, 1993).

24

Freshly collected plant material were dried in shade and then coarsely

powdered in a blender. 10 g of powdered samples were successively extracted in 40

ml of the solvent, BDH (AR) grade in the order of increasing polarity (hexane,

petroleum ether, benzene, chloroform, acetone and methanol) in a soxhlet apparatus

for 24 hours. All the extracts were filtered through Whatman No.41 filter paper.

All the extracts (hexane, petroleum ether, benzene, chloroform, acetone and

methanol) were subjected to qualitative tests for the identification of various

phytochemical constituents as per the standard procedures (Harborne, 1979; Brinda

et al., 1981; Anonymous, 1990; Lala, 1993). The methanol extracts of the seaweed

samples were used for GC-MS analysis and pharmacological studies.

TESTS FOR ALKALOIDS

MAYER’S TEST

To the powder, 2 ml of Mayer’s reagent was added; a dull white precipitate

reveals the presence of alkaloids.

TESTS FOR TERPENOIDS

NOLLER’S TEST

To 1 ml of extract with tin (one bit) and thionyl chloride (1 ml) were added.

Appearance of pink colour indicates the presence of Terpenoids.

TEST FOR STEROIDS

SALKOWSKI TEST

The powder was dissolved in chloroform and equal volumes of concentrated

sulphuric acid were dissolved. Formation of bluish red to cherry red colour in

chloroform layer and green fluorescence in the acid layer represents the steroid

components in the test samples.

25

TEST FOR COUMARIN

To 1ml of extract, 1 ml of 10% NaOH was added. The presence of

coumarin is indicated by the formation of yellow colour.

TEST FOR TANNIN

The sample powder was mixed with basic lead acetate solution. Formation

of white precipitate indicates the presence of tannins.

TEST FOR SAPONIN

To 1 ml of the extract, 5 ml of water was added and the tube was shaken

vigorously. Formation of honey comb, like froth indicates the presence of saponin.

TEST FOR FLAVONOIDS

SHINODA’S TEST

To few mg of the powder, magnesium turnings and 1-2 drops of

concentrated hydrochloric acid were added. Formation of red colour shows the

presence of flavonoids.

TESTS FOR QUINONES

To 1ml of the extract 1 ml of Conc.H2SO4 was added. Formation of red

colour shows the presence of Quinone.

TEST FOR ANTHRAQUINONES

BORNTRAGER’S TEST

The powder / extract were macerated with ether and after filtration; aqueous

ammonia or caustic soda was added. Pink red or violet colour in the aqueous layer

after shaking indicates the presence of anthraquinones.

TEST FOR PHENOLS

To 1 ml of the extract, 2 ml of distilled water was added followed by few

drops of 10% aqueous ferric chloride. Appearance of blue or green colour indicates

the presence of phenols.

26

TEST FOR PROTEIN (BIURET TEST)

NINHYDRIN TEST

Two drops of freshly prepared 0.2% ninhydrin reagent was added to the

powder and heated. Development of blue colour reveals the presence of proteins,

peptides or amino acids.

TEST FOR CARBOHYDRATES (SUGAR)

FEHLING’S TEST

To the powder, equal quantities of Fehling’s solution A and B were added

and on heating, formation of a brick red precipitate indicates the presence of

carbohydrates.

TEST FOR GLYCOSIDES

The extract was mixed with a little anthrone on a watch glass. One drop of

concentrated sulphuric acid was added and made into a paste and warmed gently

over the water bath. The presence of glycosides was identified by dark green

colouration.

TEST FOR CATACHIN

A test solution of drug and Ehrlich’s reagent followed by a few drops of

Conc.HCl give pink colour indicates the presence of catachin.

3.2 QUANTIFICATION OF PHYTOCHEMICALS

3.2.1 ESTIMATION OF PROTEIN (Lowry et al., 1951)

PROCEDURE

500 mg of dry seaweed powdered sample was ground well with 15 ml of

phosphate buffer (0.2M, pH7.2). The extract was centrifuged for 10 minutes at 3000

rpm. The lower layer was discarded and the supernatant was taken. To the

supernatant, an equal amount of cold 5% TCA was added. It was left for 30

minutes in an ice bath. The precipitated protein was taken and again centrifuged.

27

The supernatant was discarded and the pellet was dissolved in 25 ml of 0.2N

NaOH. From this 1 ml was taken and mixed with 4 ml of alkaline copper reagent.

It was shaken well and was allowed to stand for 10 minutes at room temperature.

Then 0.1 ml of diluted Folin phenol reagent was added and mixed well. After 20

minutes, the O.D was read at 650 nm using UV-VIS Spectrophotometer SL 150.

Bovine Serum Albumin was used as the standard.

3.2.2 ESTIMATION OF CARBOHYDRATE (Sheifter et al., 1950 )

PROCEDURE

100 mg of the dry seaweed powder was ground with 5 ml of 80% methanol.

The extract was centrifuged at 5000 rpm for 15 minutes. The supernatant was

collected and made up to 10 ml with 80% of methanol. To this 10 ml of petroleum

ether was added and mixed well. The lower layer was taken for carbohydrate

estimation.

To 0.1 ml of the extract, 4.9 ml of anthrone reagent (0.2% w/v in

Conc.H2SO4) freshly prepared was added and kept in a boiling water bath for 10

minutes. After cooling, the O.D was read at 625 nm using UV-VIS

Spectrophotometer SL 150. Glucose was used as the standard.

3.3.4 ESTIMATION OF LIPID (Bligh and Dyer, 1959)

PROCEDURE

1 g of dry seaweed powder was ground with 10 ml of distilled water. The

pulp was transferred to a conical flask and 30 ml of Chloroform-methanol mixture

(2:1v/v) was added and mixed well. It was kept in the dark at room temperature

overnight. After this period, a further addition of 20 ml chloroform and 20 ml of

water was made. The resulting solution was subjected to centrifugation, when 3

layers were seen. A clear lower layer of chloroform containing all the lipids,

28

coloured aqueous layer of methanol with all water soluble materials and a thick

pasty interface were seen.

The lower layer was carefully collected in a pre weighed beaker and

evaporated. When the solution was free of organic solvents, the weight was

determined again. The difference in weight gave the weight of the lipid.

3.3.4 ESTIMATION OF CARBON (Walkley and Black, 1943)

PROCEDURE

100 mg of dried sample powder was taken in a conical flask. To this was

added 4 ml of 1N potassium dichromate solution and 2 ml of concentrated sulphuric

acid. The flask was kept undisturbed for about 30 minutes.

After the reaction, the content was diluted with 20 ml of distilled water. To

this 2 ml of phosphoric acid (85%v/v) was added followed by 1 ml of

diphenylamine indicator. This was then titrated against 0.4N ferrous ammonium

sulphate. The end point was a change into brilliant green. The amount of carbon

was calculated using the following formula:

Organic carbon (%) =

S

T -1

g

951.3

Where,

g = weight of the sample in gram

S = ml of ferrous solution consumed in blank titration

T = ml of ferrous solution consumed in sample titration.

3.3.5 ESTIMATION OF CRUDE FIBRE (AOAC, 1970; Sadhasivam and

Manickam, 1992)

PROCEDURE

Dried sample powder of 1g was defatted by using petroleum ether in a

soxhlet apparatus for 16 hours or till the petroleum ether extractant was colourless.

29

Petroleum ether was evaporated and this defatted residue was used for crude fibre

estimation.

The residue was taken in a 50 ml beaker and successively digested with 25

ml of 0.255N H2SO4 followed by 25 ml of 0.313N NaOH solution till all the liquid

got evaporated. The digested sample was transferred to a cheese cloth placed in

funnel inserted in a 250 ml flask. The sample was washed with boiling water

followed by absolute ethanol, till the washing appeared clear. Then the digested

washed residue was transferred to an ashing dish (preweighed dish W1). The

residue was dried for 2 hours at 130 ± 2˚C. The dish was cooled in a desiccator and

weighed (W2). Then it was ignited for 30 minutes at 600 ± 15˚C. Once again it

was cooled in a desiccator and reweighed (W3). The crude fibre was calculated as

follows:

Crude fibre (%) = 100 sample theof Weight

)1W3W()1W2W(ignition on weight in Loss×

−−−

3.3.6 ESTIMATION OF CALORIFIC VALUE (Karzinkin and

Tarkovskaya, 1964)

PROCEDURE

Sample weighing 20 mg was placed in a round bottomed flask. To the flask

was added 3 ml of 5% (w/v) potassium iodate solution and 20 ml of concentrated

sulphuric acid. The control flask contained 3 ml of potassium iodate (5%w/v) and

20 ml of concentrated sulphuric acid. The flasks were connected to the reflex

condenser without shaking and heated on heating mantles. Boiling was continued

for an hour.

After combustion, the experimental and control flasks were cooled and 50

ml of distilled water was added separately. The liquid was mixed well and the

flasks were heated (not boiled) until the complete disappearance of its pink colour

30

and the smell of iodine. The contents were cooled and each flask was diluted with

250 ml of distilled water and 10 ml of potassium iodide was added after transferring

them into a separate 500 ml conical flask.

Then the flasks were titrated against 0.1N sodium thiosulphate using starch

(1%w/v) as indicator. The end point was the disapperance of blue colour. The

energy content was calculated using the following formula:

Energy value (KJg-1dry wt)

= weightSample

3.38 4.184 0.6667 valuealExperiment - value titrecontrol ×××

3.3.7. ESTIMATION OF PHENOLICS (Maxon and Rooney, 1972)

EXTRACTION

500 mg of air-dried powdered sample was taken in a 100 ml flask, to which

50 ml of 1% (v/v) HCl in methanol was added. The samples were shaken on a

reciprocating shaker for 24 hours, at room temperature. The contents were

centrifuged at 10000×g for 5 minutes. The supernatant was collected separately and

used for further analysis.

3.3.7.1 ESTIMATION OF TOTAL FREE PHENOLS (Sadasivam and

Manickam, 1992)

1 ml aliquot of the above extract were pipetted into different test

tubes to which 1 ml of Folin-Ciocalteu’s reagent followed by 2 ml of 20%(w/v)

Na2CO3 solution were added and the tubes were shaken and placed in a boiling

water bath for exactly 1minute. The test tubes were cooled under running tap water.

The resulting blue solution was diluted to 25 ml with distilled water and the

absorbance was measured at 650 nm with a help of a Spectrophotometer. If

precipitation occured, it was removed by centrifugation at 5000×g for 10 minutes

31

before measuring the absorbance. The amount of phenolics present in the sample

was determined from a standard curve prepared with catechol. A blank containing

all the reagents minus plant extract was used to adjust the absorbance to zero.

Average value of triplicate estimations was expressed as g100gˉ1 of powdered

sample on dry weight basis.

3.3.7.2 ESTIMATION OF TANNINS (Burns, 1971)

From suitable aliquots of the above extract tannin content was quantified by

the Vanillin-HCl method of Burns (1971) using Phloroglucinol as a standard at 500

nm with VU-VIS Spectrophotometer SL 150. The average values of triplicate

estimates of all samples were expressed as g100gˉ1 of powdered samples on dry

weight basis.

3.3.8 ESTIMATION OF ANTHOCYANINS (Vivekanandan, 1991)

1g fresh seaweed thallus was cut into pieces and incubated in the extraction

medium (25 ml) for 48 hours. The extraction medium was prepared by mixing 80

ml of methanol, 20 ml of distilled water and 1 ml of concentrated hydrochloric acid.

The extract was centrifuged after 48 hours. O.D was measured at 530 and 657 nm

with UV-VIS Spectrophotometer SL 150. The amount of anthocyanin (mggˉ1) was

calculated as below:

Amount of anthocyanin = OD 530 – 0.3 × OD 657

3.3.9 ESTIMATION OF NITROGEN (Humphries, 1956)

PROCEDURE

STEP I (DIGESTION PROCESS)

100 mg of the dry powdered seaweed sample was heated in a digestion flask

having 2 ml salycylic acid (5%w/v in Conc.H2SO4), 300 mg powdered sodium

thiosulphate, a pinch of the catalyst (CuSO4, K2SO4, SiO2, 1:8:1) and 1 ml of

32

concentrated H2SO4 at 100˚C till the appearance of apple green colour of the digest.

After cooling, the digest was made up to 50 ml with distilled water.

STEP II (DISTILLATION PROCESS)

10 ml aliquot was transferred to a micro Kjeldahl distillation flask. To this

was added 10 ml NaOH (40%w/v) solution and 2 ml glass distilled water and the

flask was heated. The liberated ammonia was collected in 10 ml of 2% (w/v) boric

acid solution containing a drop of the double indicator, a mixture of bromocresol

green solution and methyl red solution. Distillation was stopped when the reddish

colour changed into peacock blue. This solution was then titrated against 0.02N

(v/v) H2SO4. The end point was the reappearance of the reddish colour. The

volume of H2SO4 consumed was noted (Titre value).

Amount of Nitrogen (mg/g) = Titre value×2.8×10

3.3.10 ESTIMATION OF MINERALS (K, Ca, Mg, and Na) (AOAC, 1970)

PROCEDURE (TRIPLE ACID DIGESTION)

500 mg of dry seaweed powder was mixed with 10 ml of concentrated nitric

acid, 4 ml of 60% (v/v) perchloric acid and 1ml of concentrated sulphuric acid . The

mixture was left undisturbed overnight. After that it was heated on a hot plate

containing concentrated sulphuric acid in a beaker until brown fumes ceased to

come out and then was allowed to cool. After cooling, it was filtered through

Whatmann No.42 filter paper. The filtrate was made up to 100 ml with glass

distilled water.

The filtrate was used for the estimation of K, Ca, Mg and Na using a Flame

photometer with KCl, CaCl2, MgCl2 and NaCl employed as standards for

calibration.

33

3.3.11 ESTIMATION OF VITAMINS

3.3.11.1 ESTIMATION OF VITAMIN A (Eitenmiller and Landen Jr. 1998)

2.5 g of the sample was dissolved with an accuracy of 0.1% in 5 ml of

pentane R and dilute with 2-propanol R1 to a presumed concentration of 10 µ/ml to

15 µ/ml. O.D was read at 225 nm using UV-VIS Spectrometer SL 150 at the

absorption maximum of 326 nm. The amount of vitamin A was calculated as

below:

Amount of Vitamin A = A225 × V ×1900

100 × m

A225 = absorbance at 326nm

m = mass of the substance to be examined in grams.

V = total volume to which the substance to be examined is diluted to

give 10 µ/ml to 15 µ/ml.

1900 = factor to convert the specific absorbance of esters of retinol into

International units per gram.

3.3.11.2 ESTIMATION OF VITAMIN B3 (Sadasivam and Manickam, 1992)

EXTRACTION

5 g of air-dried powdered sample was extracted with 30 ml of 4N sulphuric

acid and steamed for 30 minutes. It was allowed to cool and made up to 50 ml with

distilled water. This suspension was filtered through Whatmann No.1 filter paper

and to 25 ml of the filtrate; 5 ml of 60% basic Lead acetate was added. The pH of

the above suspension was adjusted to 9.5 and centrifuged. 2 ml of Conc.H2SO4 was

added to the supernatant. The supernatant with Conc.H2SO4 was allowed to stand

for 1hour and centrifuged again. 5 ml of 40% ZnSO4 was added to the supernatant

and the pH was adjusted to 8.4. The supernatant was collected by centrifugation and

the pH was adjusted to 7. This supernatant was used as the source material.

34

ESTIMATION

1 ml of aliquot of the above extract was pipetted into different test tubes and

the volume was made upto 6 ml with distilled water. 3 ml of cyanogen bromide

was added and the contents of the tubes were shaken. 1 ml of 4% aniline was added

to each test tube after 10 minutes. The yellow colour developed after 5 minutes was

read at 420 nm against reagent blank. The niacin content present in the samples

was calculated by referring to a standard graph of niacin using niacin as standard

and pressed as mg100gˉ1

of powdered samples.

3.3.11.3 ESTIMATION OF VITAMIN C (Sadasivam and Manickam, 1992)

EXTRACTION

3 g of air-dried powdered sample was ground with 25 ml 4% oxalic acid

and filtered. 10 ml of the filtrate was taken in a conical flask. Bromine water was

added drop by drop with constant stirring to remove the enolic hydrogen atoms in

the ascorbic acid filtrate (till the filtrate turned orange yellow). The excess of

bromine was expelled by blowing in air. The final volume was made up to 25 ml

with 4% oxalic acid solution and it was used as the source material.

ESTIMATION

2 ml aliquots of the above extract were pipetted into each of the different

test tubes and the volume was made upto 3 ml with distilled water. 1 ml of 2%

DNPH (2, 4–dinitro phenyl hydrazine) reagent and 1 or 2 drops of 10% thiourea

were added to each test tube. The contents of the test tubes were mixed thoroughly

and incubated at 37˚C for 3 hours. After incubation, 7 ml of 80% sulphuric acid

was added to each test tube to dissolve the orange red osazone crystals and the

absorbance was measured at 540 nm in UV-VIS Spectrophotometer SL 150 against

a reagent blank. The ascorbic acid content present in the sample was calculated by

35

referring to a standard graph of ascorbic acid. The average value of triplicate

determination was expressed as mg100gˉ1of powdered samples.

3.3.11.4 ESTIMATION OF VITAMIN E (Eitenmiller and Landen Jr. 1998)

MOBILE PHASE

10 ml of phosphoric acid was diluted with water to 1000 ml to obtain

solution A. Filtered and degassed mixture of methanol and solution A (95.5) was

prepared.

STANDARD PREPARATION

An accurately weighed quantity of USP Alpha Tocopherol RS was

dissolved in methanol and diluted quantitatively with methanol to obtain a solution

having a known concentration of about 2 mg/ml.

SYSTEM SUITABILITY PREPARATION

An accurately weighed quantity of USP Ergocalciferol RS was dissolved in

methanol and diluted quantitatively with methanol to obtain a solution having a

concentration of 0.65 mg/ml. 1.0 ml of this solution was transferred to a 100 ml

volumetric flask containing accurately weighed 100 mg of USP Alpha Tocopheryl

Acetate RS. This was dissolved in 30 ml of methanol with the aid of sonication and

mixed. This solution was stored in a refrigerator.

ASSAY PREPARATION

20 ml of the solution retained as specified in the dilution for assay

preparation in the assay for Vitamin A was transferred to a suitable container and

evaporated in vacuum at room temperature to dryness. The residue with the aid of

methanol was transferred to a suitable volumetric flask and diluted with methanol to

volume to obtain a solution having a concentration of about 2 mg of alpha

tocopherol.

36

CHROMATOGRAPHIC SYSTEM

The liquid chromatograph was equipped with a 254 nm detector and an 8

mm × 10 cm column that contained 5 µm packing L1. The flow rate was about 2

ml/minute. The relative retention times were about 0.5 for ergocalciferol and 1.0

for alpha tocopheryl acetate: the resolution R between ergocalciferol and alpha

tocopheryl acetate was not less than 12 and the tailing factor was inbetween 0.8

to 1.2.

The relative standard deviation for replicate injections was not more

than 3.0 %.

PROCEDURE

Equal volumes (about 100 µl) of the standard preparation and the assay

preparation were separately injected into the chromatograph, the chromatograms

were recorded and the peak areas were measured. The quantity, in mg, of alpha

tocopherol (C29H50O2) alpha tocopheryl acetate (C31H52O3) was calculated taken by

the formula:

CD = (rU/rS)

In which,

C = concentration in mg/ml, of the corresponding USP reference

standard in the standard preparation.

D = dilution factor, in ml for the assay preparation.

rU & rS = peak responses for the relevant Vitamin E form obtained from the assay

preparation and the standard preparation respectively.

It was estimated using HPLC (LD 044, Hitachi, Japan).

3.3.12 ESTIMATION OF β–CAROTENE (AACC, 1995)

2 g of sample was taken in 100 ml glass stopper flask and 10 ml water

saturated butanol (WSB) was added. The contents of the flasks were mixed

37

vigorously for 1 minute and kept overnight (16–18hours) at room temperature under

dark for complete extraction of β-carotene. The contents were shaken again and

filtered completely through the Whatmann no.1 filter paper into a 100 ml

volumetric flask. The O.D was measured at 440 nm.

3.4 GC-MS STUDIES

GC-MS was performed with GC Clarus 500 Perkin Elmer equipment.

Compounds were separated on Elite-I capillary column (100%

Dimethylpolysiloxane). Samples were injected with a split ratio of 10:1 with a flow

rate of helium 1ml/minute (carrier gas). Mass detector-Turbo Mass gold-Perkin

Elmer Software-Turbomass 5.1 was used as a detector. Other conditions are oven

temperature up to 110°-2 minute hold; upto 280° at the rate of 5 deg / min-9 minutes

hold. Injector temperature was maintained at 250°C.

The constituents were identified after comparison with those available in the

Computer Library (NIST ver.2.1) attached to the GC-MS instrument and reported.

3.5 ANTIOXIDANT ACTIVITY

EVALUATION OF ANTIOXIDANT ACTIVITY

EXTRACTION

10 g of the six powdered samples representing the aforesaid six marine plant

species were subjected to extraction with methanol (40 ml) (British Drug Houses

(AR) grade solvent (E.Merck India Ltd., Mumbai, India) in a soxhlet extractor

(Borosilicate Brand, The Science House, Chennai, India) for six hours and the

extraction was repeated twice. Similar extracts were also prepared using distilled

water. The extracts were then concentrated to dryness under reduced pressure and

controlled temperature (40˚-50˚C). The resultant residues were stored in a

refrigerator till further use.

38

PREPARATION OF THE EXTRACTS AND STANDARD

For each of the six stock solutions; 20 mg methanol/water residues were

dissolved in the respective solvents. The stock solutions were serially diluted with

respective solvents to get lower concentrations (1000, 750, 500, 250, 100 µgmlˉ1).

Each concentration was prepared in triplicate. These were subjected to the

following in vitro assays to assess the antioxidant potential. Vitamin E standard

(US Pharmacopoeia alpha tocopherol, USP-India Pvt. Ltd., Hyderabad) was

dissolved in methanol and diluted quantitatively with methanol to obtain a

concentration of 100µg mlˉ1. All data about antioxidant activities are the average

of triplicate analyses.

NITRIC OXIDE (NO) RADICAL SCAVENGING ACTIVITY

Sodium nitroprusside in aqueous solution at physiological pH spontaneously

produce nitric oxide, which reacts with oxygen to produce nitrite ions, which can be

determined by the use of the Griess Illosvoy reaction (Garrat, 1964). Griess

Illosvoy reagent was slightly modified using napthylethylene diamine

dihydrochloride (0.1%) instead of 1-napthylamine (5%) (Senevirathne et al., 2006).

Scavengers of nitric oxide compete with oxygen and reduce the production of nitric

oxide (Marcocci et al., 1994). The reaction mixture (3 ml) containing Sodium

nitroprusside (10mM, 2 ml), phosphate buffer saline (pH 7.4, 0.01M, 0.5 ml) and

extract (methanol or water, 100-1000µg mlˉ1) or standard solution (0.5 ml) was

incubated at 25˚C for 150 minutes. After incubation, 0.5 ml of the reaction mixture

containing nitrite was pipetted and mixed with 1ml of sulphanil acid reagent (0.33%

in 20% glacial acetic acid) and allowed to stand for 5minures for completing

diazotization. Then, 1 ml of napthylethylene diamine dihydrochloride (0.1%) was

added, mixed and allowed to stand for 30 minutes in diffused light. The absorbance

of the pink coloured chromophore was measured at 540 nm against the

39

corresponding blank solutions in an Elico double beam UV-VIS

Spectrophotometer. The percentage of NO scavenging by the different

concentrations of the six seaweed samples and standard was calculated using the

following formula:

NO scavenged (%) = A0 – A1 × 100

A0

Where,

A0 = the absorbance of the control

A1 = the absorbance of the test samples or standard (Govindarajan et al., 2003;

Badami et al., 2005).

H2O2 SCAVENGING METHOD

The ability of the methanol and water extracts of the six marine organism

samples to scavenge hydrogen peroxide was determined according to the method of

Ruch et al. (1989).

Extract (1.0 ml) was added to a hydrogen peroxide solution prepared in

0.1M phosphate buffer saline (pH7.4, 40mM and 0.6 ml). Absorbance of hydrogen

peroxide at 230 nm was determined after ten minute against a blank solution

containing phosphate buffer without hydrogen peroxide. The percentage of

scavenging of hydrogen peroxide of water and methanol extracts of different

concentrations was calculated using the formula:

Percentage scavenged (H2O2) = A0 – A1 × 100

A0

Where,

A0 = absorbance of the control

A1 = absorbance of the test samples or standard (Gulcin et al., 2003).

40

TOTAL ANTIOXIDANT ACTIVITY

The total antioxidant activity was evaluated by the method of Prieto et al.

(1999). An aliquot of the extract (100-1000 µg mlˉ1, 0.1 ml) was combined in

eppendorf tube with 1 ml of reagent solution (0.6M sulphuric acid, 28mM sodium

phosphate and 4mM ammonium molybdate). In case of blank, 0.1 ml of methanol

or distilled water was used in place of sample. The tubes were capped and

incubated in a boiling waterbath at 95˚C for 90 minutes. After cooling to room

temperature, the absorbance of each solution was measured at 695 nm using UV-

VIS Spectrophotometer SL 150. Vitamin E (USP alpha tocopherol) was used as the

standard and the total antioxidant activity expressed as equivalents of vitamin E

(µggˉ1).

3.6 ANTIMICROBIAL ACTIVITY

5 g of each algal sample was extracted in 100 ml of acetone, methanol and

water. The extraction solvent was evaporated under vacuum and used for

antimicrobial assay by Disc Diffusion Technique (Bauer et al., 1966) and confirmed

by Minimum Bactericidal Concentration (MBC) study (NCCLS, 1997a, b).

3.6.1 DISC DIFFUSION TECHNIQUE (DDT)

Test organisms were cultivated on Mueller Hinton Broth by lawn culture

technique at 37˚C for overnight before inoculation for assay. Standardised bacterial

suspension which contained 106 numbers of bacteria/ml was added to the medium

and poured to sterile petridishes. After medium solidified, the discs impregnated

with extracts were placed onto the surface.

Disc diffusion technique was studied by incorporating 0.01 ml of the

varying concentration of the extract in sterilised discs of 5 mm diameter punched

out from Whatmann No., 1 filter paper. Sterilised discs were incorporated with

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varying concentrations (20 µg/ml, 10 µg/ml, 5 µg/ml, 2.5 µg/ml, 1.25 µg/ml, 0.63

µg/ml, 0.31 µg/ml, 0.16 µg/ml and 0.08 µg/ml) of the different extracts.

The antimicrobial characteristics of acetone, methanol and extracts of

seaweeds were tested. The test organisms used were,

Gram positive organism

1. Staphylococcus aureus (ATCC 923)

Gram negative organisms

2. Escherichia coli (ATCC 25922)

3. Klebsiella pneumoniae (ATCC 700693)

4. Pseudomonas aeruginosa (ATCC 25583)

The fungi used were,

5. Candida albicans (ATCC 2091)

6. Aspergillus niger (ATCC 9029)

The acetone, methanol and water extracts of six seaweed samples were

placed on the culture plates and the plates were incubated for overnight. After

overnight incubation the zone size inhibition was measured with the help of a scale

and recorded.

Standard antimicrobial discs (Gatifloxin for bacteria and Nystatin for fungi)

were used as a control.

3.6.2 TUBE DILUTION TECHNIQUE (TDT) FOR MBC STUDY

Minimum Bactericidal Concentration of different extracts was studied by

making subcultures from tubes showing no visible growth on Muller Hinton agar

media. The mixture was incubated at 37˚C for overnight. After overnight

incubation subcultures were made on Mueller Hinton Agar and the plates were

incubated for overnight. The plates were observed for growth.

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The end point dilution showing inhibition by DDT was taken as the criteria

and MBC for each extract was tested for three dilutions–the end point dilution, the

previous and the next dilution.

3.7 PHARMACOLOGICAL STUDIES

ACUTE TOXICITY STUDY

For toxicity studies, a set of six Albino rats of either sex were administered

orally with the test substance in the range of doses 200-2000 mg/kg and the

mortality rates were observed after 72hours. The methanol extract of A. spicifera

showed no mortality at 2000 mg/Kg. Therefore 2000 mg/Kg dose was considered as

LD50 cut off dose (safe dose). So 1/20 of that was selected (100 mg/Kg) for the

experiment as administrative dose.

ANTI-ARTHRITIC ACTIVITY (FREUND’S ADJUVANT-INDUCED

ARTHRITIS)

PREPARATION OF FREUND’S ADJUVANT

25 mg of steam killed Mycobacterium tuberculosis cells were finely

grounded using mortar and pestle with sufficient amount of liquid paraffin is

referred as complete Freund’s adjuvant. The liquid paraffin referred in the study as

incomplete Freund’s adjuvant.

INDUCTION OF ADJUVANT - INDUCED ARTHRITIS

Albino rats weighing about 200-250 g were divided into 4 groups of 6

animals each. The dosages of the drugs administered to the different groups were

group I-control (normal saline 0.5 ml/Kg), group II–A. spicifera (100 mg/kg).

Group III-Indomethacin (10 mg/Kg). All the drugs were administered orally.

Adjuvant arthritis was induced by subcutaneous injection of complete Freund’s

adjuvant (0.1 ml) into the plantar tissue of the left hind paw of each rat. Rats in this

group were known as inflamed control and test groups. Group IV- served as the

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non-inflamed control group consisted of rats injected with 0.1 ml liquid paraffin

(Incomplete Freund’s adjuvant). The test groups consisted of complete Freund’s

adjuvant-injected rats challenged with doses of the test drugs administered orally 18

hours and 2 hours before induction of arthritis. The drugs administered were

continued daily at the same time of the day for 19 more days. Development of

adjuvant induced swelling in the paws of both the injected and non-injected limbs

of each rat were monitored daily as the percentage increase in paw volume.

3.8 STATISTICAL ANALYSIS

All the biochemical parameters were estimated on triplicate determinations.

Standard deviation and standard error were calculated for the various quantified

biochemical parameters, antioxidant activity and antiarthritic activity. In the case of

antiarthritic activity, statistical difference were compared by student’s‘t’ test.

Correlation and student’s‘t’ test analysis were performed with regard to antioxidant

activity.

(i). Standard deviation

SD = 1N

d2

−

∑

Where, d = deviation of each score from mean

N = total number of observations

(ii). Student’s‘t’ test

Student’s‘t’ test was used to compare two means by applying the formula:

t = 2

2

2

1

21

SESE

xx

+

+

Where,

1x and 2x = represent the means compared and

SE1 and SE2 = respective standard errors

44

(iii).Standard error

SE = 1n

SD

−

The level of significance for the‘t’ at corresponding degrees of freedom

(Df= N-2) was read from the probability table given in Zar (1974), where ‘N’ is the

number of scores in both the experiments.

(iv). Simple correlation coefficient (r)

The simple correlation coefficient ‘r’ was determined from the formula.

r = ∑ ∑

∑

yX

xy22