Carthamus red

TENTATIVE

Prepared at the 49th JECFA (1997)
superseding specifications prepared at the 39th JECFA (1992),
published in FNP 52 Addendum 1 (1992)

Information required on the levels of carthamin in carthamus red and on Method of Assay

SYNONYMS

Safflower red, carthamic acid, CI Natural Red 26 (No. 75140)

DEFINITION

Carthamus Red is obtained from the dried petals of Carthamus tinctorius L. To obtain carthamus red, carthamus yellow is extracted from the petals with water and the residue treated with aqueous sodium hydroxide or other alkali. Carthamus red is precipitated from the extract by addition of acid, separated by filtration and dried. The principal colouring matter is carthamin.

Class

Flavonoid

Chemical formula

C₄₃H₄₂O₂₂ (carthamin)

Formula weight

910.81 (carthamin)

Assay

Not less than 80% total colouring matters on a volatile matter-free basis

DESCRIPTION

Dark red to red-brown powder with a characteristic slight odour

FUNCTIONAL USES

Colour

CHARACTERISTICS

IDENTIFICATION

Solubility

Very slightly soluble in water
Very slightly soluble in ethanol
Practically insoluble in ether

Spectrophotometry

A solution of the sample in dimethyl formamide is red and shows an absorption maximum between 525-535 nm.

Thin layer chromatography

Carthamin appears as a red spot with an Rf value of about 0.40
See description under TESTS

Colour reaction I

Passes test
See description under TESTS

Colour reaction II

Passes test
See description under TESTS

PURITY

Lead

Not more than 10 mg/kg
Prepare a sample solution as directed for the organic compounds in the Limit Test, using 10 m g of lead ion (Pb) in the control.

Synthetic dyes

Passes test
See description under TESTS

TESTS

IDENTIFICATION TESTS

Thin layer chromatography

Activate some silica gel for 1 h at 110° and prepare a TLC plate. Prepare an 0.02% solution of the sample in dimethylformamide and apply 20 m l to the plate. Allow to dry and develop using a mixture of n-butanol, acetic acid and water (4:1:2 by volume) until the solvent front has ascended about 10 cm. Allow to dry. Carthamin appears as a red spot with an Rf value of about 0.40.

Colour reaction I

Dissolve 10 mg of the sample in 50 ml water. The colour of the solution is red. Add alkali to raise the pH to above 7. The colour changes to orange-yellow.

Colour reaction II

To 0.05 g of the sample add 2 ml of 5% phosphoric acid, heat for 1 h on a water bath. After cooling, filter and wash the residue with 3 ml of water. Combine the filtrate and the washings. Neutralize the combined solution with sodium hydroxide TS, add 5 ml of Fehling's TS and heat on a water bath for 10 min. A red precipitate is produced.

PURITY TESTS

Synthetic dyes

Basic dyes: To 1 g of the sample add 100 ml of 1% sodium hydroxide solution, and mix well. Extract 30 ml of this solution with 15 ml of ether. Then extract the ether layer twice with dilute acetic acid (5 ml); the dilute acetic acid layer does not contain any colour.
Acidic dyes: To 1 g of the sample add 1 ml of ammonia TS and 8 ml of water, and shake well. Discard an oily layer when separated. Proceed as directed (Ascending Chromatography) in the General Methods using 20 m l of the solution as the sample solution, and a mixture of pyridine and ammonia TS (2:1 by volume) as the developing solvent. Stop the development when the solvent front has advanced about 15 cm from the point of application. No spot is observed at the solvent front after drying under daylight. If any spot is observed, it should be decolourized when sprayed with a solution of stannous chloride in hydrochloric acid (2 in 5).

METHOD OF ASSAY

Place about 10 mg of the sample, previously dried and accurately weighed, in a 300-ml ground stoppered flask, add 150 ml of dimethylformamide, dissolve by shaking occasionally and allow stand for 2 hours. Filter this solution through a glass filter into a 200-ml volumetric flask. Wash the flask and filter with two 25-ml portions of dimethylformamide, combine the filtrate and the washings, add dimethylformamide to volume and mix. Determine the absorbance (A) at the maximum absorbance in the range of 525-535 nm using a 1-cm cell. Calculate the content using the absorptivity of carthamus red (information required).

	Information required on the levels of carthamin in carthamus red and on Method of Assay
SYNONYMS	Safflower red, carthamic acid, CI Natural Red 26 (No. 75140)
DEFINITION	Carthamus Red is obtained from the dried petals of Carthamus tinctorius L. To obtain carthamus red, carthamus yellow is extracted from the petals with water and the residue treated with aqueous sodium hydroxide or other alkali. Carthamus red is precipitated from the extract by addition of acid, separated by filtration and dried. The principal colouring matter is carthamin.
Class	Flavonoid
Chemical formula	C₄₃H₄₂O₂₂ (carthamin)
Formula weight	910.81 (carthamin)
Assay	Not less than 80% total colouring matters on a volatile matter-free basis
DESCRIPTION	Dark red to red-brown powder with a characteristic slight odour
FUNCTIONAL USES	Colour
CHARACTERISTICS
IDENTIFICATION
Solubility	Very slightly soluble in water Very slightly soluble in ethanol Practically insoluble in ether
Spectrophotometry	A solution of the sample in dimethyl formamide is red and shows an absorption maximum between 525-535 nm.
Thin layer chromatography
	Carthamin appears as a red spot with an Rf value of about 0.40 See description under TESTS
Colour reaction I	Passes test See description under TESTS
Colour reaction II	Passes test See description under TESTS
PURITY
Lead	Not more than 10 mg/kg Prepare a sample solution as directed for the organic compounds in the Limit Test, using 10 m g of lead ion (Pb) in the control.
Synthetic dyes	Passes test See description under TESTS
TESTS
IDENTIFICATION TESTS
Thin layer chromatography
	Activate some silica gel for 1 h at 110° and prepare a TLC plate. Prepare an 0.02% solution of the sample in dimethylformamide and apply 20 m l to the plate. Allow to dry and develop using a mixture of n-butanol, acetic acid and water (4:1:2 by volume) until the solvent front has ascended about 10 cm. Allow to dry. Carthamin appears as a red spot with an Rf value of about 0.40.
Colour reaction I	Dissolve 10 mg of the sample in 50 ml water. The colour of the solution is red. Add alkali to raise the pH to above 7. The colour changes to orange-yellow.
Colour reaction II	To 0.05 g of the sample add 2 ml of 5% phosphoric acid, heat for 1 h on a water bath. After cooling, filter and wash the residue with 3 ml of water. Combine the filtrate and the washings. Neutralize the combined solution with sodium hydroxide TS, add 5 ml of Fehling's TS and heat on a water bath for 10 min. A red precipitate is produced.
PURITY TESTS
Synthetic dyes	Basic dyes: To 1 g of the sample add 100 ml of 1% sodium hydroxide solution, and mix well. Extract 30 ml of this solution with 15 ml of ether. Then extract the ether layer twice with dilute acetic acid (5 ml); the dilute acetic acid layer does not contain any colour. Acidic dyes: To 1 g of the sample add 1 ml of ammonia TS and 8 ml of water, and shake well. Discard an oily layer when separated. Proceed as directed (Ascending Chromatography) in the General Methods using 20 m l of the solution as the sample solution, and a mixture of pyridine and ammonia TS (2:1 by volume) as the developing solvent. Stop the development when the solvent front has advanced about 15 cm from the point of application. No spot is observed at the solvent front after drying under daylight. If any spot is observed, it should be decolourized when sprayed with a solution of stannous chloride in hydrochloric acid (2 in 5).
METHOD OF ASSAY	Place about 10 mg of the sample, previously dried and accurately weighed, in a 300-ml ground stoppered flask, add 150 ml of dimethylformamide, dissolve by shaking occasionally and allow stand for 2 hours. Filter this solution through a glass filter into a 200-ml volumetric flask. Wash the flask and filter with two 25-ml portions of dimethylformamide, combine the filtrate and the washings, add dimethylformamide to volume and mix. Determine the absorbance (A) at the maximum absorbance in the range of 525-535 nm using a 1-cm cell. Calculate the content using the absorptivity of carthamus red (information required).