Materials and Methods

Sample of Nigella sativa was collected from the commercial Gol market, Rail Bazar,

Faisalabad. The plant samples cleaned carefully and air dried under shade. The

phytochemical analysis was done and the phytochemical contents of the dried sample of

Nigella sativa were studied by using standard methods.

3.1 Instruments Used:

The following instruments were used for the phytochemical investigation of Nigella

sativa;

1. Electric muffle furnace

2. Refluxing apparatus

3. Distillation apparatus

4. Markham’s distillation apparatus

5. Electric shaker

6. Electrical oven

7. Analytical balance

8. Double beam spectrophotometer

3.2 Chemicals Used:

1. Concentrated H2SO4

2. NaOH

3. Sodium sulphate

4. Methanol

5. Ethanol
6. Chloroform

7. NaCl

8. Charcol

9. henolphthalein

10. 0.01N KOH solution

11. Iodine solution

12. Potassium Iodide

13. Sodium thiosulphate

14. Concentrated HCl

15. Mercuric chloride

16. Ammonium hydroxide

17. Sodium citrate

18. Sodium carbonate

19. Copper sulphate

20. Tartarate

21. Lead acetate

22. H2S saturated water

23. Ferric chloride solution

24. Carbon tetrachloride

25. Starch solution

3.3 Reagents Used:

3.3.1: Dragendroff’s Reagent:

This reagent was made according to Hartborn, (1973).

1. 0.6 grams Bismuth nitrate was dissolved in 3 ml concentrated HCl

and 10 ml distilled water was added.

2. Another solution was prepared by taking 6 grams KI in 10 ml distilled

water.

These two solutions were mixed together with 7 ml concentrated HCl and 15

ml water and diluted to 400 ml with distilled water.

3.3.2: Mayer’s Reagent:

This reagent was made according to Mayer, (1963).

1. 1.36 grams Mercuric chloride was dissolved in 60 ml distilled water.

2. Potassium Iodide 5.0 grams were dissolved in 20 ml distilled water.

These two solutions were mixed and the volume was made up to 100 ml with

distilled water.

3.3.3: Wagner’s Reagent:

This reagent was made according to Jenkin et al. (1967).

Potassium Iodide 2 grams were dissolved in 50 ml distilled water and 3.0 grams

of Iodine was added and the solution was made up to 100 ml with distilled water.
3.3.4: Benedict’s Solution:

This solution was prepared according to method of Adam et al. (1970).

1. 1. 2.0 grams sodium citrate and 11.5 sodium carbonate were dissolved in 10 ml

hot distilled water.

2. 2.0 grams of copper sulphate was dissolved separately in 20 ml hot distilled

water and added to sodium carbonate solution.

These two solutions were mixed and volume was made up to 100 ml.

3.3.5: Fehling Solution:

1. Fehling solution (A) was prepared by dissolving 3.5 grams CuSO4 in

100 ml distilled water.

2. Fehling solution (B) was prepared by dissolving tartarate in 20 ml

distilled water with gentle heating.

3. 5 grams of NaOH was dissolved in 20 ml water and these two

solutions were mixed and volume of 100 ml was prepared with

distilled water.

3.4: Preliminary Tests:

3.4.1: Moisture Contents:

Procedure:

To determine the moisture contents of the plant sample, 5 grams of

plant sample were taken in a pre-weighed crucible and placed in an oven at 100 oC

for 6 hours till constant weight. The percentage of moisture contents was calculated

by using the following formula;

Moisture contents (%) = Weight of sample after heating
_________________________ X 100
Weight of sample before heating

3.4.2: Ash Contents:

Procedure:

For the determination of ash contents of the plant sample 10 grams of

air-dried and ground plant sample was taken in a pre-weighed crucible and placed

in a muffle furnace at 600oC for n7 hours. Ash of plant material was weighed. This

procedure was repeated till a constant reading was achieved.

Ash contents were calculated by using the following formula;

Ash contents (%) = Weight of ash

____________ X 100
Weight of plant

3.4.3: Determination of Crude Fibers:

Procedure:

5 grams of plant sample was taken in a 250 ml beaker and boiled in

200 ml of 1.25% v/v concentrated H2SO4 solution for half an hour. It was filtered

and the residue was boiled in 200 ml of 1.25% of w/v NaOH solution for half an

hour. It was filtered, dried and weighed and estimated the percentage of crude fibers

by the following formula;

Crude Fibers (%) = Loss in weight

_______________ X 100
Weight of sample
Loss in weight= Weight of residue-Weight of ash contents.

3.4.4: Determination of Crude Lipids:

Procedure:

5 grams of air dried and ground plant sample and 2 grams of sodium

sulphate were taken in pestle and mortar and macerated. This macerated mixture

was taken in an Iodine flask and fat solvent (methanol + chloroform with ratio 2:1)

was added in it and shaked for one hour. This process was repeated until total lipid

contents were extracted out with fat solvent. The whole extracted material was

distillated until small amount left behind. The extracted material was taken in

separating funnel and 10-20 ml fat solvent and 2-5 ml of 1% NaCl were added in it.

It was shaked very well and kept horizontally for 2-3 minutes. The organic layer

(lower layer containing lipids) was separated. The whole lipids with fat solvent

were taken in a pre-weighed beaker, solvent was evaporated in a water bath and the

crude lipids were weighed. The percentage of lipids was calculated by using

formula;

Crude lipids (%) = Weight of crude lipids

__________________ X 100
Weight of the plant

3.4.5: Determination of Acid Value:

10-20 ml of fat solvent of methanol and

chloroform (2:1) was added in a beaker, contained re-weighed lipids extracted from

5 grams of plant material. 0.5 grams Charcol was added in it and left it for a night.

Next day it was filtered. Clear solution containing lipids was obtained. This clear
solution was taken in a titration flask, 2-3 drops of phenolphthalein were added in it

and titrated it by 0.01N KOH solution. End point was faint pink colour that

remained for 20-30 seconds. Same procedure was repeated with blank. Acid value

was estimated by using the following formula;

Acid value = (Y-X) x (Normality of OKH) x 56.1

_____________________________
Weight of sample

Where,

Y= Amount of 0.01 N KOH used for sample.

X= Amount of 0.01 N KOH used for blank.

3.4.6: Determination of Iodine Value:

Pre-weighed lipid extracted from the plant

sample was dissolved in 10 ml of fat solvent. This material was taken in an Iodine

flask, 80 ml iodine solution was added in it. Flask was closed and allowed to stand

for 30 minutes with constant shaking then 10 ml of 15% w/v potassium iodide

solution was added in it, shaked well and 100 ml water was added. This solution

was titrated against thiosulphate solution till yellow colour just disappeared; starch

solution was added to this solution and again titrated against sodium thiosulphate

solution till blue colour was bleached. The same procedure was repeated with blank

(with no oil or fat). Iodine value was calculated by using the following formula;

Iodine value = (X-Y) x 6.35

_______________________ X 100
Weight of sample X 1000
Where,

X= Volume of 0.05 N Na2SO3.5H2O used for blank.

Y= Volume of 0.05 N Na2SO3.5H2O used for sample.

3.5: Phytochemical screening:

Appropriate portions of the powdered plant sample were subjected to

phytochemical screening for the presence of the alkaloids, tannins, saponins,

flavonoids, anthraquinone, unbound anthraquinones, glycosides, and cardiac

glycosides. The results were recorded in Table 2.

3.5.1: Detection of Alkaloids:

For the detection of alkaloids in plant samples, the methods described by Brain and

Turner, (1975) were used. 10 grams of finely powdered plant sample was boiled

with 100 ml acidified ethyl alcohol (97ml ethanol+ 3ml conc. H2SO4) in a flask for

five minutes. The contents were mixed and centrifuged at 500 rpm for 20 minutes.

One ml supernatant was taken and tested with Dragendroff’s, Mayer’s, and

Wagner’s reagent. If there is no precipitation it is not worth continuing the test. A

precipitation at this stage is due to the alkaloid for protein. In case of positive result

at this stage the rest of the extract is made alkaline by the addition of 50%

ammonium hydroxide and extracted with 50 ml of chloroform. The contents were

transferred to a separating funnel and gently mixed for 10-20 times. Vigorous

shaking was avoided to prevent the formation of emulsion. The lower chloroform

layer was extracted with 30 ml glacial acetic acid or some quantity of 50% HCl.
The mixture was shaken vigorously, the chloroform layer was discarded and acetic

acid layer was divided into two equal portions.

1. To serve as a blank (control)

2. To second portion added few drops of Dragendroff’s reagent.

3. To third portion added Mayer’s reagent.

4. To fourth portion added few drops of Wagner’s reagent.

The presence of alkaloids was indicated by the precipitation of orange (in case of

Dragendroff’s reagent, greenish white (in case of Mayer’s reagent), and reddish

brown (in case of Wagner’s reagent) colours. The experiment was repeated twice to

have constant result.

3.5.1: Culvenor and Fitzeralz’d Method for the Detection of Alkaloids:

This method of Culvenor and Fitzerald, (1963) was used to extract and detect the

alkaloids in plant samples. 5 grams powdered plant sample was grinded with 1

gram sand and 30 ml chloroform. The slurry formed was made basic by the addition

of 10 ml of 50% NH4OH and filtered. The filtrate was acidified with 10 ml of 2N

H2SO4. The acid layer was separated and few drops of layer were tested with

Dragendroff’s reagent and Mayer’s reagent. Turbidity confirmed the presence of

alkaloids.
 3.5.2: Detection of Glycosides and Cardiac Glycosides:

1. Glycosides:

5 grams of powdered plant sample were boiled; 30 ml of 70%

ethanol was added and then filtered. 5-8 ml of 4% lead acetate was added to it till

no further precipitation of chlorophyll and other materials like phenol, sugars and

acids occurred. The contents were filtered; 0.5 ml lead acetate was added to 2 ml

filtrate to check re-precipitation until no precipitates were obtained. Excess of lead

acetate was removed by adding 15 ml water saturated with H2S gas. Black

precipitates of lead sulphide were removed by filtration. The PbS free filtrate was

concentrated by heating on a boiling water bath. Benedict’s or Fehling’s solutions

were added to few drops of concentrated solution separately, tom form red

precipitates which indicated the presence of glycosides.

3.5.3: Cardiac Glycosides:

The PbS free filtrate obtained earlier was extracted with 50 ml chloroform. The

organic layer was separated and evaporated to dryness. The residue was dissolved in

3 ml of 3.5% ferric chloride in glacial acetic acid followed by the addition of 1.5 ml

concentrated H2SO4 along the walls of the test tube. On standing brown layer formed

at the interface was due to the presence of deoxy sugars and pale green colour is

formed in the upper layer due to the presence of steroid nucleus, indicating the

presence of cardiac glycosides.

3.5.4: Detection of Unbound Anthraquinones:

The method of Brain and Turner, (1975) is used. Fo0r the detection of anthraquinones

in the plant sample, 10g of powdered plant sample is boiled in 50 ml distilled water

for 10 minutes, filtrate is extracted with 10 ml distilled water followed by the addition

of 0.5% NH4OH. Pink colouration or precipitation indicated the presence of unbound

anthraquinones.

3.5.5: Detection of Bound Anthraquinones:

For the detection of bound anthraquinones in plant sample 10g air dried and

powdered plant sample is mixed with 30 ml of 10% Ferric Chloride and 1 ml of

concentrated HCl, contents are refluxed for 10 minutes on water bath. Filter while

sample is hot., cool and is extracted with 20 ml CCl4. The separated organic layer is

washed with water and shaked with 0.5% NH4OH. Occurrence of pink colour

indicates the presence of bound anthraquinones.

3.5.6: Detection of Saponins:

To detect the presence of saponins in the plant sample, 2.5g powdered plant sample is

extracted with 12.5 ml hot water and the contents are filtered 0.8% saline are taken in

test tubes followed by the addition of 5 ml distilled water in one test tube and 5 ml of

each sample extract in other test tube separately. Few drops of fresh blood are added

to each test tube. The contents are centrifuged for 15 minutes. Hemolysed red blood

cells in the extract will indicate the presence of saponins in the plant sample.
3.5.7: Detection of Flavonoids:

The method of Willstalter, (1966) is used to detect the presence of flavonoids in the

plant samples. 5g of air dried and ground plant sample is boiled in 80% ethanol for 10

minute, filtered and followed by the addition of magnesium metal. After cooling light

green colour indicated the presence of flavonoids in the sample.