materials and methods - shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/6470/10/10_chapter...

CHAPTER 2

MATERIALS AND METHODS

2.1 MATERIALS

2.1.1 Plant materials

Garclnia cambogia dried fruits were collected from different areas of

Kerala like Angamaly, Edathua, Palaghat, Adimaly etc. The dried rinds of

fru~ts were also collected from Savanthvady (MIS Virnodkar Trading Co.),

Srl Lanka (Mls Mubarak Trading Co.) and Indonesia (Mk P.T Athan

Mitra Jaya). G. cambogia plant materials such as leaves, fresh fruits,

stem and rind were collected from Angamaly

2.1.2 Chemicals

Ethylene di-amine salt of HCA was purchased from Fluka Chemical

Company. Methanol, calcium chloride, sodium hydroxide, calcium

hydroxide, magnesium chloride, potassium hydroxide, standard buffer

Chapter 2 Materials and Methods

solutions, activated carbon, sulphuric acid, sodium meta vanadate, penta

bromo acetone, citric acid, tartaric acid, aspartic acid, ether, chloroform,

butanol, acetic acid, formic acid, anhydrous sodium sulphate, chloroform.

potassium permanganate, potassium bromide, ferrous sulphate,

hydroxylamine hydrochloride, ethyl alcohol, bromophenol, ferric chloride,

methyl orange, ammonium oxalate, silver nitrate, ferric ammonium

sulphate, ammonium thiocyanate etc. used were analytical grade.

HPLC grade methanol, acetonitrile (HPLC), water (HPLC) etc. were

purchased from E.Merck. Precoated HPTLC plate NH2 F254S was

purchased from E. Merck. HPTLC (Desaga), HPLC (Shimadzu), double

beam spectrophotometer (Shimadzu) etc. were used for the analysis.

HPLC C,, RP amide column was supplied by Shimadzu, cation exchange

resin - Zeocarb - 21 5 was purchased from Merk.

Materials and Methods

2.2 ANALYTICAL METHODS

2.2.1 Preparation of water extract

The water extract was prepared from dried rinds of Garcinia fruit by

extracting with water. Approximately weighed out 509 of dried sample into

a 500mL round bottomed flask. 300mL distilled water was added and

refluxed for 2 hours in a boiling water bath. Cooled, filtered and collected

the filterate. The residue was again refluxed with water. Repeated the

process and three washes were taken. All the washes were combined

and concentrated up to 50% of the total dissolved solvents (TDS) level.

Water extracts of Garcinia leaves, stem bark and fruits were also

prepared by the same procedure.

2.2.2 Determination of Moisture

Moisture content of the samples was determined by the method

described in Official and tentative method of Analysis. Association of

Analytical Chemists (1984).

Mater ia ls and Methods

a. Apparatus:

1 Moisture distillation apparatus made of glass including 500mL flat

bottom flask and condenser.

2 Heating mantle

3 Nylon brlstle burtle brush or wire loop long enough to extend through

the condenser.

b. Reagent:

Toluene commercial grade redistilled in lab

c. Procedure:

1 Weighed accurately 20-259 (sufficient to yield 1- 2mL H20) of the

powdered sample to the 500mL distilling flask.

2 Added about 100mL toluene sufficiently to cover the sample, added

3-4 boiling chips.

3 Assembled the apparatus and the trap was filled with toluene by

pouring it through the condenser until it just fills the trap. Inserted a

loose non-absorbing cotton wool into the tap of the condenser to


prevent condensation of atmospheric moisture in the condenser. Given

water connection to the condenser.

4. Heated to boil and reflux it about 2 drops per second for about 1 hour

until most of the water has been collected in the trap and then increase

the reflux rate to 4 drops per second.

5. Continued refluxing until 2 consecutive readings at 15 minutes intervals

show no change. Dislodge any water held in the sides of the condenser

carefully and trap by the brush or wire loop. Rinse the condenser

carefully using 5mL toluene.

6 Continued refluxing for 5 more minutes. Stopped heating and allowed

the trap to cool room temp and noted the volume of the water

collected

Preparation of sample:

Taken about 509 of uniform sample and powdered to about 60 mesh

in a clean dry mixer grinder. Mixed the ground sample thoroughly.

d. Calculation

Moisture % = Volume of water (mL) x 100

Wt. of sample (g)

34


2.2.3 Determination of Acid value

Acid value of the extract was checked by Titration method as described in

Journal of AOAC (1969)

a. Apparatus:

1 Burette

2Erlenmeyer flask

b. Reagents:

1 Toluene - lsopropyl alcohol mixture (1 : I ratio)

2 0 1 N Alcoholic KOH

3 Phenolphthalein solution.

b. Procedure:

Weighed out 1-29 well mixed sample into 250 - 300mL Erlenmeyer flask.

Added 50 - 100mL toluene - isopropyl alcohol mixture and O.lmL

phenolphthalein solution. Titrated with 0.1N alcoholic KOH until

permanent faint pink appears and persists for 5 1 0 sec.

c. Calculation:

Acid value mL alc. KOH soln. X normality alc. KOH so1n.X 56.1

- - ..................................................................... Wt. of sample (g)


Difference between duplicate experiments should be

c 0. I mg KOHIg sample. -

2.2.4 Conversion of HCA into its Lactones

HCA and its lactone were prepared as described by

Krishnamoorthy et al. (1982 ).

As indicated, hydroxy citric acid present in G. cambogis is chiral and it is

easily converted into lactone.

Fruit rind of Garcinia cambogia (2009) was autoclaved with 600mL of

water at 115'C for 15 minutes. The cooled extract (2530%) was

decanted through several folds of cheesecloth and filtered on a Buchner

funnel (Whatman No.1 paper). The residue was washed with water. The

dark brown filtrate (volume 600mL) was concentrated to about lOOrnL on

a water bath and treated with 200mL of ethanol with stirring. The resulting

precipitate of pectinous material was removed either by centrifugation or

filtration. The acidic filtrate was neutralized (pH paper) by cautious

addition of 40% KOH, with careful stirring. The heavy oily liquid, which is

formed, was allowed to settle for a few minutes and the supernatant was


decanted and discarded. The oily liquid was washed with 60% ethanol

(five portions of 100mL). It was again washed with absolute alcohol

(two portions of IOOmL), the suspension being left to stand for

60 - 90 minutes each time. A further portion of IOOmL of absolute ethanol

was added and allowed to stand overnight. Ethanol was decanted, and

the yellow hygroscopic semisolid thus obtained was dried in vacuo at

80' C to remove traces of ethanol and stored in a desiccator. The yield

was about 409. A 10% solution of the salt was passed through a column

(20 x 3cm) of cation exchange resin (e.g., Zeocarb - 215). The column

was washed with water until free of acid and the effluent was evaporated

to dryness on a water bath. Instead of being dried in vacuo, the residue

(a thick syrup) can be seeded with a few crystals of the lactone to induce

crystallization. The crude crystalline material (light brown colour) was

further purified by extraction and recrystallization from ether.

2.2.5 Identification of HCA and lactone by paper Chromatography

The HCA and lactone were identified by ascending paper

chromatography as described by Krishnamurthy et a/. (1982)


Paper used : Whatmann No.1

Developing solvent: 1. n-Butanol : Acetic acid : water (BAW) 4:1:5 2. n-Butanol : Formic acid : water (BFW) 4:1:5

Spraying reagent : Bromophenol blue spray

2.2.6 Estimation of Citric acid

The citr~c acid present in the water extract and calcium salt of HCA were

determined by pentabrornoacetone method as described in methods in

Enzymology, Lowenstein (1981).

a. Apparatus:

1 Electronic balance

2 Welgh~ng Bottle

3 Cruclble

4 Volumetric flask - 500rnL & 250rnL 5 Measurtng cyllnder

6 P~pette - 25mL & 20mL

7 Desiccator

b. Reagents:

1 Dil HCI


3 Sulphur~c acid 1:1

4 Potassium bromide soln.- 40%

5 Ferrous sulphate soln. - 2509 of ferrous sulphate (FeS04.7H20) in a

mixture of lOmL of concentrated sulphuric acid and 700mL of water.

c. Procedure:

Weighed 2.89 of the anhydrous sample, dissolved in minimum quantity of

dilute hydrochloric acid transferred it into a 500mL volumetric flask, and

diluted to volume with water. Pipetted 50mL of this solution into a 250mL

glass-stoppered flask. Added 19mL of 1 : l sulphuric acid and lOmL of

40% aqueous potassium bromide solution. Warmed the solution to 55°C.

Added slowly 40mL of 5% potassium permanganate solution with

occasional shaking. Allowed it to stand 10 min. with occasional shaking.

Added 50mL of ferrous sulphate solution. Agitated gently until the

precipitate dissolved and the solution assumed a light yellow colour. The

solution was cooled under running water until crystals appeared. The

flask was closed and allowed to stand in an ice bath at 2-5'C for 2hr.

Filtered through a sintered glass crucible under suction employing 70mL

of ice water in the transferred and subsequent washing operation. Dried

the crucible and its contents overnight in a desicator containing

concentrated sulphuric acid and weighed. (Alternatively, the precipitate

39


may be dried by aspiration). Washed the pentabromoacetone from the

crucible under suction with anhydrous methanol. Dried the crucible at

100°C for 30 minute and re-weighed to obtain the tare weight of the

crucible^

d. Calculation:

O/O of citric acid = ON) (43.15)

W

Where W = Weight of pentabromoacetone

w = Weight of sample in aliquot taken

2.2.7 Estimation of Lactones

Lactones present in the crystals and different salts of HCA were estimated

spectrophotometrically as described by Lipmann and Tuttle (1945).

a. Apparatus:

1 UV Spectrophotometer

b. Reagents:

1 Hydroxylamine hydrochloride: Dissolved 349 of hydroxylamine

hydrochloride in 40mL of distilled water and made up to llitre by using

40


95% Ethyl alcohol; added 15mL of Bromophenol blue solution and

neutralized with 0.5 N alcoholic NaOH.

2 95% Ethyl alcohol

3 Bromophenol blue solution

4 5 N alcoholic NaOH

5 6MAq: NaOH

6 IMHCI

7 5% FeCI, solution.

c. Procedure:

Taken 0.29 of sample, added 4mL hydroxylamine reagent; added 0.8mL

6M NaOH and warmed gently in a water bath. Then it was cooled; added

8mL 1M HCI; added 1 drop of FeCI3 solution and filtered. Simultaneously

prepared a blank solution using the same method; 4mL Ethyl alcohol was

used instead of hydroxylamine reagent. Noted the absorbance at 495nm.

Comparing the absorbance of a sample of known lactone, percentage of

lactone in the sample can be calculated.

d. Calculation:

Lactone value of standard x Absorbance of sample

Absorbance of standard


2.2.8 Estimation of HCA by HPLC method

HCA was estimated by high-pressure liquid chromatography by the

method provided by industrial laboratory, USA. [Kucera et a/. 1981,

Robert 19941

Sample preparation

1. Weighed approximately 1.25g of sample and placed in a 500mL

volumetric flask

2 Added one to two (1-2) drops of 1:l concentrated H2SOdH20 to

the flask and made up to volume with distilled water.

3. Sonicated the flask for ten minutes or until all solids have

dissolved. Checked pH of solution to confirm that it was

approximately 5.5.

4. Quantitatively transfered a lOmL aliquot of the solution to a

25mL volumetric flask.

5 Dispensed 3mL internal standard solution (see below) into the

25mL Volumetric flask with sample, and brought the flask to

volume with distilled water. Filtered through a 0.45 micron disc filter.


HPLC Method for HCA Determination:

Hydroxycitric acid standards were prepared using secondary standard

Garcinia extract-50% (already standardised with 98% ED-HCA- Fluka).

0 08289 of standard extract (equivalent to 0.0414g of free acid) was

weighed accurately, dissolved in 25mL HPLC grade water containing

1mL 1: l H2SO4 IH20, filtered through 0.45micron disc filter and

transferred to a 50mL volumetric flask which was then brought to volume

w~th HPLC grade water. The resulting stock solution had a concentration

of 827pglmL Hydroxy citric acid.

An Internal Standard stock solution of L-Aspartic Acid (99%) was

prepared by dissolving 0.12609 of material in a 250mL volumetric flask

with HPLC grade water at pH 2.50 (see above). The internal stock

solut~on had a concentration of 504pglmL.


Standards for injection are prepared as follows:

7mL HCA stock plus 3mL internal Std. stock to yield 10mL of

579pglmL standard (A).

5mL HCA stock plus 3mL internal Std. stock to yield IOmL of

413.71~glmL standard (B).

3mL HCA stock plus 3mL internal Std. Stock to yield 10mL of

248.1 pglmL standard (C).

2mL HCA stock plus 3mL internal std. stock to yield 10mL of

165.5~glmL standard (D).

ImL HCA stock plus 3mL internal std. stock to yield lOmL of

827pglmL standard (E).

Standards and samples were analyzed using a varian 5500 HPLC

system, a varian 9065 diode array ultraviolet absorbance detector, and

varlan LC Star software.


Instrument conditions are as follows

Column :DuPond Zorbax (or Alltech Altima) C18, 25cm x

4.6mm, 5pm with C18 guard column attached.

Mobile phase : 0.1M Na2S04 pH adjusted to 2.10 with H2S04

filtered and degassed, isocratic.

Flow rate : 0.5mUmin

Detector Wavelength : 203nm

Injection Volume :50pL

Standards were injected manually and a regression curve was

generated using the amount of material injected and the ratio of the

HCA peak area to that of the Internal Standard for each sample. The

peak area ratio of HCA to Standard was used to determine the amount of

HCA injected with each sample from the linear regression. The amount

of HCA detected is multiplied by a dilution factor then divided by the total

sample weight to give the percentage of free acid in the sample.


Sample calculation of % HCA content

Area of HCA peak of sample x conc. of standard (~glmL)

Area of HCA peak of standard .......................................................................................

cone. of sample (1glmL)

2.2.9 Estimation of Magnesium

The amount of magnesium present in magnesium hydroxy citrate was

estimated as given in Official method of analysis, AOAC (1984).

Weighed out approximately 209 sample, transferred to 400mL beaker,

and dissolved in 200mL HCI (1+3). Covered beaker, heated to boiling

point and continued boiling for 10 min. Filtered through paper and washed

residue with small amounts of hot water until filtrate was chloride free.

Combined filtrate and washings, cooled and diluted to 500mL (soln. X)

Placed 250mL solution X in 400mL beaker, acidified with HCI (1+1), and

added 2-39 (NH4),HP04 and enough HCI (1+1) to produce clear solution

when all (NH4),HPO4 was dissolved. When cold, made slightly alkaline

with NH40H, stirring constantly. Added 2mL excess of NH40H and let


stand for 12 hours. Filtered supernate and washed 4 times by decantation

with NH40H (1+10). Dissolved the precipitate in HCI (1+1), diluted to

about 150mL, added little (NH,)2HP04 , and precipitated with NH40H as

before. Kept for stand for 12 hours., filtered, washed till the solution

became chlorine free with NH40H (I+lO).Placed the precipitate in

porcelain crucible, ignited, heated over blast lamp, and weighed as

MgPz07

Calculation :- Wt of Magnesium = Wt. of Mg2P207 X 0.21842.

2.2.10 Estimation of HCA by HPLC using Cqe RP arnide column

Estimation of HCA was carried out by HPLC using a new method

developed by Benny Antony et a/. (under publication). In this C ~ B RP

amide column was used and the procedure is described in detail in

Chapter 5

2.2.11 Eatitnation of HCA by HPTLC method

The HCA was estimated by High Performance Thin Layer

Chromatography (HPTLC). The detailed procedure is given in Chapter 5.

Chapter 2

Method


Ascending, one dimensional development in the

HPTLC developing chamber, without chamber

saturation.

Application : 2 - 3 microliter using the applicator AS 30 as

5mm bands

Stationary phase : Precoated HPTLC plates, NH, F, S

(Merck), prewashed with methanol

Mobile phase Methanol : Water - 6:2 (vlv)

D~stance run 7cm.

Runn~ng t~me 30 minute

Detection After development the plate was heated

10 minutes at 1 50°C on the thermo plate and the

resultant fluorescent zones were examined

under the UV lamp at 366nm.


2.2.12 Estimation of HCA by Spectrophotometric method

The HCA was estimated spedrophotometrically by the method by

Benny Antony et a/. (1999).

Preparation of standard

Working standards were prepared by ethylene diamine salt of HCA

(98% ED - HCA). The salt equivalent to 0.0429 of the free acid was

weighed accurately and dissolved in 5mL of I N H2S04 and approximately

25mL of distilled water was added. It was filtered and transferred into a

50mL volumetric flask and was made up to the volume using distilled

water [Conc 828 pglmL]. 1mL of this solution was pipetted into a 50mL

standard flask and made upto the volume using distilled water.

Preparation of sample

About 0.29 of calcium salt was accurately weighed and dissolved in 5mL

of I N H2S0, and the solution was diluted to 25mL with distilled water. The

solution was decolourised with activated charcoal, filtered and washed

with small portions of distilled water and then made upto 50mL.


Procedure

About 0.9mL of 5% sodium meta vanadate solution was added to the

sample solution and to the standard solutions. The yellow colour slightly

changed to reddish orange after 20 minutes. The absorbance at 467nrn

was measured against a blank which was prepared without sample

solution. The experiment was repeated using different volumes

(1 5, 2, 2 5 and 3mL) of the standard solution. A calibration graph was

plotted w~th the concentration of HCA against absorbance and the

percentage of HCA in the sample was calculated.

2.2.13 Determination of pH

The pH of water extract of HCA and other solutions were checked using a

pH meter. The calibrations and determinations were carried out as

described in Practical Biochemistry by Plummer (1978).

a. Apparatus

DH meter

b. Reagents

Buffer solut~ons having pH 7, 4.2 and 9

50


c. Procedure:

Standardisation of pH meter:

The pH meter was calibrated before use by means of a standard solution.

Potassium hydrogen phthalate was the recommended standard. At 15'C,

005mollL solution has pH4.00. The pH at other temperatures (0 to 60°C)

can be obtained from the following equation:

pH = 4.00 + % ((t - 15)/100)~

Other standards used for the calibration of a pH meter

1. 0.025mollI. Potassium dihydrogen phosphate1 0.025molIL. Disodiurn

hydrogen phosphate-pH6.86at25°Cand pH 6.84 at 37'C

2. 0.01 mollL Sodium tetra borate - pH 9.18 at 25OC and 9.06 at 37'C

One percent solution was prepared using the given sample. pH meter

was dipped in the solution. Adjusted the pH to the cover. The steady

reading was noted.


2.2.14 Estimation of Calcium

The amount of calcium present in the calcium salt of HCA was estimated

by the procedure given in Analytical chemistry of foods by James (1999).

Calcium is precipitated at about pH 4 as the oxalate and the oxalate is

then dissolved in sulphuric acid liberating oxalic acid which is titrated with

standard potassium permanganate solution

a. Reagents.

1 0.01 M Potassium permanganate solution

2. Dilute ammonium hydroxide solution

3. Dilute acetic acid

4. Solid ammonium oxalate

5 Dilute sulphuric acid

b. Procedure

Prepared an ash solution of the sample and pipetted out 50mL of the

solution in a 250mL beaker and neutralised with dilute ammonium

hydroxide solution until it was just alkaline to litmus then acidified with

dilute acetic acid. Brought to boiling and excess of ammonium oxalate

(about 0.89) was added and boiled vigorously for thirty minutes.

52


Poured the supernatant liquid through a 12.5cm Whatman No.1 filter

paper in a funnel and washed the precipitate twice with hot water by

decantation through the same filter. Finally transferred the precipitate to

the filter paper and the washed the residue on the filter paper a number of

times with small quantities of distilled water. The washings were

continued until the filtrate was free from oxalate. This was shown by

collecting a few drops of filtrate at intervals in a test tube and adding

calcium chloride solution. A white precipitate indicated the presence of

oxalate. Discarded the filtrate and washing. Transferred the filter paper

carrying the precipitate used for the precipitation and added 60 ml of

warm dilute sulphuric acid. Stirred the contents of the beaker macerating

the filter paper. Warmed to 70°C and titrated with 0.01M potassium

permanganate solution to a persistent pink colour

c. Calculation

Calculated the percentage of the calcium in the sample given that;

Tx0 .1 Percentage of calcium of sample = ----------

W

Where T= titere of 0.01M potassium permanganate, and W = weight of

the sample


2.2.15 Estimation of Salt content by Mohr's method

The salt content was estimated by Mohr's method as described by

James C. S (1999) in Analytical food chemistry.

Sliver nitrate reacts with sodium chloride to produce insoluble silver

chlor~de. At the end point silver irons reacts with the potassium chromate

indicator to produce a reddish brown precipitate.

a. Reagents

1 0 1M Silver nitrate

2 Potasslum chromate indicator

b. Procedure

We~ghed out l g of the sample into 250mL in a volumetric flask. Then

pipetted out 25mL of the diluted brine into a 250mL conical flask. Added

ImL of the potassium chromate indicator and titrated with 0.1M silver

nitrate solution until a distinct reddish- brown colour appeared and persist

on brisk shaking. Repeated the analysis to obtain concordant results


c. Calculation

Percentage of salt content in sample = 5EixQl x T

5

Where T + mean titre of 0.1M silver nitrate in mL

2.2 16 Estimation of Sodium

The sod~um present in the extract was estimated by Volhard's method as

described by James C. S (1999) in Analytical chemistry of Foods.

a. Reagents:

1 0 05M silver nitrate solution

2 Concentrated Nitric acid

3 005M potassium thiocyanate solution

4 Iron alum indicator (ammonium iron (Ill) sulphate), saturated solution

b. Procedure:

Weighed accurately 2-39 of sample into a conical flask. Added 10mL

distilled water and 25mL 0.05M silver nitrate solution from a dispenser.

Warmed the contents to 75-80°C to facilitate dispersion of the sample on

sw~rling

55


Added IOmL concentrated Nitric acid and boil gently for about 10

rnlnutes Cool and add a small amount of iron alum indicator and about

50mL dlstllled water and titrated the unused silver nitrate with 0.05M

potasslum thiocyanate solution to persistent reddish-brown end-point.

In the same manner carried out a blank titration using 25mL of silver

nltrate solution and the same volumes of reagents and water. The

d~fference between this blank titration and the sample titration is the

volume of potassium thiocyanate equivalent to the chloride in the sample

c. Calculation:

The percentage of sodium content of the sample was calculated by the

following formula,

Percentage of sodium = (6 - S)mL x 0.05x0.0585 xlOO Weight of the sample

Where B = blank titre and S = sample titre


2.2.17 Determination of Bulk density

The bulk density of the hydroxy citrates products were checked by the

procedure given in ASTA Analytical methods and Guenther (1947).

a. Procedure:

1 Filled the sample to be studied in the measuring cylinder by

gentle tapping up to 100mL.

2 The weight of the sample was found out

b. Calculation:

Bulk density (glmL) = Wt. of the substance 1 100

2.2.18 Estimation of Potassium

The potassium content of Garcinia extract - potassium salt (potassium hydroxy citrate) was checked by the procedure described in Official

Method of Analysis, AOAC (1984).

a. Procedure:

Weighed approximately 59 of the sample in a petridish and heated up to

redness in a furnace. Cooled and added 50% HCI and boiled. Filtered the

solut~on Into a lOOmL volumetric flask. Washed with water and collected


all wash~ng and filtrate. Evaporated the aliquot to remove excess HCI.

While the solution was boiling, 10% Barium chloride solution was added

as long as precipitate forms, and then enough saturated Barium

hydroxide solution to make liquid strongly alkaline. After precipitate

settled, filtered and washed with hot water, heated filtrate to boiling point,

added enough (NH4),C03 solution (1 part (NH4),C03 in 5 parts NH40H

solution) to precipitate all the barium. Filtered and wash with HzO.

Evaporated filtrate to dryness and ignited residue below redness to remo

ammonium salts. Added little water and few drops of ammonium

carbonate solution to residue. Filtered into weighed petridish, evaporated,

~gnited below redness and weigh.

D~gested residue with hot water, filtered through small filter and diluted

filtrate to provide 220mL liquid for each 100mg K,O. Acidified with few

drops HCI and added excess platinum solution (10.5 g H2Pt CI, 1100 mL).

Evaporated on water bath to thick paste, treated residue repeatedly with

80% alcohol, decanted through weighed gooch or other filter,

transferred precipitate to filter and washed thoroughly with 80%

alcohol. Dried 30 minutes at 100°C , weighed and estimated the amount

of Dotasslum


b. Calculation

Percentage of potassium = Weight of the precipitate x 2.615

2.2.19 Estimation of Heavy metals

The Heavy metals like Lead, Zinc, Copper, Arsenic, Cadmium. Cobalt etc.

were estimated by Atomic Absorption Mass Spectrophotometer (AAMS).

These experiments were carried out at Quality control laboratory of

Sp~ces Board. Cochin.

2.2.20 Estimation of Aflatoxin

Aflatoxtn content in garcinia rind and calcium hydroxy citrate were

esttmated at Shiva Analytical Laboratory, Bangalore. The estimations

were carr~ed out using GCMS.

2.2.21 Estimation of Crop contaminants like saffrole, agaric acid and hydrocyanic acid.

The common crop contaminants present are saffrole, agaric acid,

hydrocyanic acid. These were estimated by HPLC at Shiva Analytical

Laboratory Bangalore.

2.2.22 Determination of Microbial parameters

The common microbial contaminants associated with herbal products are

TPC. Yeast and Mould, E. Coli, Staphylococcus, Salmonella etc. These

59


were est~rnated in Garcinia rind and its salts. The experiments were

carried out at Sterling Farm R&D Centre, Tripunithura.

2.2.23 Determination of Optical rotation

Optical rotation was found out by using the method described by

Gunther (1947).

a. Apparatus:

1 Polarimeter

2 100rnm polarimeter tube

b. Procedure:

1. Optical rotation of liquids :

The oil or i ~ q u ~ d should be free from suspended material. Placed the

100rnm polarirneter tube containing the oil or liquid under examination in

the trough of the instrument between the polarizer and analyzer. Slowly

turned the analyzer until both halves of the field, viewed through the

telescope, showed equal intensities of illumination. At the proper setting,

a small rotation to the right or to the left will immediately caused a

pronounced mequality in the intensities of illumination of the two halves of

the field


Determined the direction of rotation. If the analyzer was turned

counterclockwise from the zero position to obtain the final reading, the

rotation was laevo (-); if clockwise, dextro (+).

After the direction of rotation has been established, carefully readjusted

the analyzer until equal illumination of the two halves of the field were

obtained. Adjusted the eyepiece of the telescope to give a clear, sharp

l~ne between the two halves of the field. Determined the rotation by

means of the protractor; read the degrees directly, and the minutes with

the aid of either of the two fixed verniers. Experiment was repeated to get

concordant values.

2. Optical rotation of solids :

The optical activity of a solid was best determined in solution and

expressed as specific rotation.

Specific rotation = [a] ~ t ' C= a

Ic

Where

[a] C = Specific rotation at temperature t°C, using sodium light

tr =Observed rotation in degrees of the solution at temperature to

I = Length of polarimeter tube in decimeters

Chapter ? Materials and Methods

c = Concentration of solution expressed as the number of grams

of active substance in 100 cc of solution.

2.2.24 Estimation of Refractive index

The refractive index was measured by adopting procedure described in

Essential oils by Guenther (1947)

a. Apparatus:

1 Refractometer

b. Reagents:

1 Sample solution (oils)

2. Methanol

3. Distilled water

c. Procedure:

Placed the ~nstrument in such a position that diffused daylight or some

form of artificial light can readily be obtained for illumination. Circulated

through the prisms a stream of water at 2OoC. Carefully cleaned the

prisms of the instrument with alcohol and then with ether. To charge the

instrument, opened the double prism by means of the screw head and

placed a few drops of the sample on the prism, opened the prisms slightly

by turning the screw head and poured a few drops of sample into the


funnel-shaped aperture between the prisms. Closed the prisms firmly by

t~ghtening the screw head. Allowed the instrument to stand for a few

minutes before the reading was made so that the sample and instrument

will be at the same temperature. Moved the slide backward or forward

until the field of vision was divided into a light and dark portion. The line

d~viding these portions was the " border line", and as a rule, will not be a

sharp line but a band of color. The colors were eliminated by rotating the

screw head of the compensator until a sharp, colorless line was obtained.

Adjusted the borderline so that it falls on the point of intersection of the

cross hairs. Read the refractive index of the substance directly on the

scale of the sector. A second reading was taken a few minutes later to

assure that temperature equilibrium had been attained.

2.2.25 Statistical analysis

Stat~stlcal analysis was carried out by students 't' test [Bannet and

Franklin, 19671

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