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Rosemary extract

 

 

DEFINITION

Rosemary extract   is obtained from ground dried leaves of Rosmarinus officinalis L using   food-grade solvents, namely, acetone or ethanol. Solvent extraction is   followed by filtration, solvent evaporation, drying and sieving to obtain a   fine powder. Additional concentration and/or precipitation steps followed by   deodorisation, decolourisation and standardisation using diluents and   carriers of food grade quality maybe included to produce the final product.   Rosemary extract is characterised by its content of phenolic diterpenes,   carnosic acid and carnosol, the principal antioxidative agents. Other   antioxidant components present include triterpenes and triterpenic acids.   Rosemary extract is identified by the total content of carnosol and carnosic   acid as a ratio of reference volatile compounds which are responsible for   flavour.

Chemical names

Carnosic acid: 4a(2H)-Phenanthrenecarboxylic acid,   1,3,4,9,10,10a-hexahydro-5,6-dihydroxy-1,1-dimethyl-7-(1-methylethyl)-,   (4aR-trans)- 

Carnosol: 2H-9,4a-(Epoxymethano)phenanthren-12-one,   1,3,4,9,10,10a-hexahydro-5,6-dihydroxy-1,1-dimethyl-7(1-   methylethyl), (4aR- (4aα,9α,10aβ))-

C.A.S. numbers

Extract of   rosemary: 84604-14-8 ;

Carnosic Acid: 3650-09-7   ;

Carnosol: 5957-80-2

Chemical formula

Carnosic acid:   C20H28O4 ;

Carnosol: C20H26O4

Structural formula  

Formula weight

Carnosic acid: 332.43   ;

Carnosol: 330.42

DESCRIPTION

Beige to light brown   powder.

FUNCTIONAL USES

Antioxidant

CHARACTERISTICS


IDENTIFICATION


Solubility

Insoluble in   water; soluble in oil

Antioxidants/Reference   Volatiles Ratio

Total % of   carnosic acid and carnosol /Total % of reference volatiles: (-)-borneol,   (-)-bornyl acetate, (-)-camphor, 1,8-Cineole (eucalyptol) and verbenone: not   less than 15

PURITY

See description   under TESTS

Loss on drying

Not more than 5%   (80° under vacuum, 4 hours). Test 1 g   of sample

Residual solvents

Acetone: Not more than 50 mg/kg ;

Ethanol: Not more than   500 mg/kg

Arsenic

Not more than 3   mg/kg

Lead

Not more than 2   mg/kg

TESTS


IDENTITY TESTS

Antioxidant: Total % of carnosic acid and carnosol

Antioxidant/Reference   Volatiles Ratio

Reference Volatile   Ratio: Total % w/w of (-)-borneol, (-)-bornyl acetate, (-)-camphor,   1,8-Cineole (eucalyptol) and verbenone is determined using GC-MSD

Equipment and   Reagents

Equipment :GC/MS chromatograph with autosampler

Solvent:   Tetrahydrofuran (THF) from Carlo Erba, HPLC grade ref. 412452000 or   equivalent

Standards :

(-)-Borneol from   Fluka (Sigma-Aldrich) ref. 15598 or equivalent

(-)-Bornyl acetate   from Fluka (Sigma-Aldrich) ref. 45855 or equivalent

(-)-Camphor from   Fluka (Sigma-Aldrich) ref. 21293 or equivalent

1,8-Cineole   (Eucalyptol) (from Aldrich (Sigma-Aldrich) ref. C80601 or equivalent

Verbenone from   Fluka (Sigma-Aldrich) ref. 94882

Internal Standard:   Heptanon-4 from Fluka (Sigma-Aldrich) ref. 43570 or equivalent

Preparation of   Internal Standard Solution (ISS) :Accurately weigh 20 mg of 4-heptanon in a 50 ml   volumetric flask. Dilute to volume with THF and homogenise the solution. The   concentration of the Internal Standard Solution is approximately 400 μg/ml.

Preparation of   Sample Solution:   Accurately weigh 2.5 g   of the sample in a 10 ml volumetric flask. Add 500 μl of the Internal   Standard Solution, and dilute to volume with THF. Sonicate 5 min for liquid   samples or 10 min for powder extracts. Filter an aliquot through 0.45 μm   filter.

Preparation of   Standard Solutions (SS):   Accurately weigh 20 mg of each Standard into a 50 ml volumetric flask. Dilute   to volume with THF and homogenise the solution. The concentration of each   Standard in the Standard Solution is approximately 400 μg/ml.

Preparation of   Standard solutions for standard Curve (WSS):

 

Procedure: Load   the WSS and the Sample solution, onto the autosampler of the GC/MS using   following conditions. Inject in duplicate 1 μl of WSS.

GC conditions:

Column: FactorFour   Capillary column VF-5ms 30M x   0.25 mm Ft = 0.25.

Carrier gas: He;   flow rate 1 mL/min with constant flow

Split: 100/1

Temperature   Program:

Injector: 250°

Temperature:   Manifold: 150°, Transfer line: 240°, Quad: 230°

Auto sampler   specifications

Syringe: 10 μl

Injection volume:   1 μl

Rinse: pre-clean   solvent: 5 times, pre-clean sample: 5 times, post-clean solvent: 5 times

Washing solvent:   Tetrahydrofuran

MS Acquisition:

 

Calculation:

Calculate the   calibration curve by linear regression analysis for each individual volatile   standard using the equation

Area=a×(c×P)+b

where:

Area is Individual   Volatile Standard peak area in WSS chromatogram

c is Concentration   [μg/ml] of Individual Volatile Standard

a is Slope of the   regression line for Individual Volatile Standard

b is y-intercept   of the regression line for Individual Volatile Standard

P is Purity of   Individual Volatile Standard given by certificate of analysis from Supplier

Calculate the   concentration of the volatiles in the sample using the following formula:

where

AS is   Individual Volatile peak area in Sample Solution chromatogram

y is y-intercept   of Individual Volatile calibration curve

m is Slope of   Individual Volatile calibration curve

AIS is   Peak area of Internal standard in Sample Solution

chromatogram

V is Dilution   volume (ml)

W is Weight of   sample (g)

CIS is   Concentration of the Internal Standard Solution

With the software   Varian MS Workstation version 6.9 Service Pack 1, report the following   settings during the review:

Amount Standard is   1

Multiplier is   Dilution volume [ml]

Divisor is Weight   of sample [g]

The reported   result, Total Volatiles, is the sum of each Individual Volatile result.

The limit of   quantification (LOQ) is 20 ppm and the limit of detection (LOD) is 2 ppm.

A representative   GC-MS analysis of the volatile standards is shown below

 

METHOD OF ASSAY

Determine carnosic   acid and carnosol content by HPLC using the following conditions:

HPLC conditions:

Detector:   Ultraviolet (UV) 230 nm

Column: ZORBAX   SB-C18 (Agilent Technologies) or equivalent; 4.6-mm x 250-mm containing 5-μm   porous silica microparticles chemically bonded to octadecylsilane

Flow rate: 1.5   mL/min

Temperature: 25°

Injection size: 5   μl

Preparation of Mobile Phase:

Combine Acetonitrile with 0.5% phosphoric acid in water   (v/v) at a ratio of 65:35.

Preparation of   Solutions:

Preparation of   Phosphoric Acid Solution: Dissolve 0.5 ml of phosphoric acid, ACS grade, in   100 ml of Methanol, HPLC grade.

Reference Standard Solution: Dissolve 200-500 μg/ml of USP   Powdered Extract of Rosemary RS in Phosphoric Acid Solution. Sonicate for 5   min; filter through a 0.45-μm filter.

System Suitability   Standard Solution: Dissolve 100 μg/ml of USP Carnosic Acid RS in Phosphoric   Acid Solution. Sonicate for 5 min; filter through a 0.45-μm filter.

Sample Solution:   Dissolve 500 μg/ml of the sample in Phosphoric Acid Solution. Sonicate for 5   min; filter through a 0.45-μm filter

Procedure: Separately inject the System Suitability   Standard Solution, Reference Standard Solution and Sample Solution in   duplicate, and record the HPLC UV outputs. Identify the peaks present in the   chromatograms from the sample by comparison to the peaks from the Reference   Standard chromatograms.

Calculations:

System Suitability   Requirements:

Tailing Factor for   the Carnosic Acid Peak   in the chromatogram is 0.90 to 1.30

The RSD for the   Carnosic Acid peak response on replicate injections is not more than 2%

% Carnosic Acid or   Carnosol in sample:

where

Aanalyte   is peak area of the analyte of interest (carnosic Acid or Carnosol) obtained   from the chromatogram of the Sample Solution

AStd is peak area   of carnosic acid obtained from the chromatogram of System Suitability   Standard Solution

CCarnosic   Acid-SS is concentration of carnosic acid in the System Suitability   Standard Solution (μg/mL)

CCarnosic   Acid is concentration of carnosic acid in Sample Solution (μg/mL)

F is Relative   Response Factor of the analyte of interest (1.00 for carnosic acid; 0.92 for   carnosol)

MW1 is molar   weight of Carnosic acid (332.4 g/mol)  

MW2 is molar   weight of Carnosol (330.4 g/mol)  

Add the individual percentages of Carnosic acid and   Carnosol calculated using the above formula, and report the result as the   total content of Carnosic acid and Carnosol in the sample taken.



 

 

Reference:

1. Compendium of Food Additive Specifications. Joint FAO/WHO Expert Committee on Food Additives (JECFA), 82nd meeting 2016. FAO JECFA Monographs 19