QUILLAIA EXTRACTS

Prepared at the 55th JECFA (2000) and published in FNP 52 Add 8 (2000), superseding tentative specifications prepared at the 29th JECFA (1985) and published in FNP 34 (1986) and in FNP 52 (1992). An ADI of 0-5mg/kg bw was established at the 29th JECFA (1985).

SYNONYMS

Quillaja extracts, Soapbark extracts, Quillay bark extracts, Bois de Panama, Panama bark extracts, Quillai extracts; INS No. 999

DEFINITION

Obtained by aqueous extraction of the milled inner bark of Quillaja saponaria Molina (family Rosaceae) or of the wood, including stems and branches. Quillaia extracts (QE) contain a number of triterpenoid saponins (QS) consisting of glycosides of quillaic acid, some sugars (including glucose, galactose, arabinose, xylose and rhamnose) along with polyphenols (including tannins), calcium oxalate and other minor components.

The QS contains sugars linked to the triterpene at carbon 3 (glucuronic acid and two of the following three sugars: galactose, rhamnose or xylose) and at carbon 28 (rhamnose, fucose, xylose, glucose and arabinose). The principal saponin component of commercial QE is designated as QS-18.

Quillaia extracts are available commercially as “non-refined” and “semi-refined” extracts, each of which may be sold as aqueous solutions or as dried powders. The extracts are usually preserved with sodium benzoate or ethanol. Fresh non-refined QE contains an average value of 190 g of QS/kg. (“Highly refined” quillaia extract, which is not specified by this monograph, is typically used in human and animal vaccines)

Formula weight

Monomeric saponins from about 1800 up to about 2000

Assay

QS as specified by the vendor.

DESCRIPTION

Non-refined liquid extract has a red-brownish colour; the powder form is light brown with a pink tinge. The liquid or powder semi-refined QE has a light colour.

FUNCTIONAL USES

Emulsifier, foaming agent.

CHARACTERISTICS

IDENTIFICATION

Solubility (FNP 5)

Very soluble in water, insoluble in ethanol, acetone, methanol and butanol

Foam

Dissolve 0.5 g in 9.5 g of water. Add 1 ml of this mixture to 350 ml of water in a 1000 ml graduated cylinder. Cover the cylinder and vigorously shake 30 times and allow to settle. Record the foam level in ml after 30 min. Typical values are 150 ml of foam.

QS-18 saponin

Determine as in Method of Assay. The major peak of the sample is compared to the QS-18 peak of the standard.

Colour and Turbidity

Powdered form only: Dissolve 0.5 g in 9.5 g of water. The solution must be free of any visible aggregates. Determine the solution absorbance against water, at 520 nm. Maximum acceptable levels are 1.2 absorbance units.

PURITY

Water (FNP 5)

Powdered form: not more than 6% (Karl Fischer Method)

Loss on drying (FNP 5)

Liquid form: 50 to 70% (2g, 105°, 5 h).

pH (FNP 5)

4.2 - 5.5 (4 % solution).

Ash (FNP 5)

Not more than 12% on dry basis (use 1.0 g of the powder-form sample; for liquid sample use residue from loss on drying).

Tannins

Not more than 7 % on dry basis.

See description under TESTS

Lead

Not more 2 mg/kg.

Determine using atomic absorption technique appropriate to the specified level. The selection of the sample size and method of sample preparation may be based on the principles of the method described in FNP 5, “Instrumental Methods”.

TESTS

PURITY TESTS

Tannins

Weigh 3 g of the powder form or equivalent amount of liquid sample, based on solids content determined from loss on drying. Dissolve in 250 ml of water. Adjust the pH to 3.5 with acetic acid. Dry 25 ml of this solution at 105 ° for 5h to determine the dry weight in g solids/l (S initial). 50 ml of the solution are mixed with 360 mg of polyvinyl polypyrrolidone (PVPP). Stir the solution for 30 min at room temperature, followed by centrifugation at 3000 rpm. Recover the supernatant and determine dry weight after drying at 105 °, 5 h (S final). The amount of tannins in the sample is estimated as:

% tannins on dry basis= 100 x (S initial - S final) / S initial

S initial: g solids / l before treatment with PVPP

S final: g solids / l after treatment with PVPP

METHOD OF ASSAY

Sample Preparation

For powder sample dissolve 0.5 g in 9.5 g of water and filter through 0.2 μm filter before injection, into HPLC. For aqueous extracts (~ 550 g solids/l) dilute 1 g in 9 g of water and filter through 0.2 μm filter before injection.

Standard preparation

Dissolve 1.5 g of purified saponins ( SuperSap, Natural Response, Chile; Quil-A, Superfos., Denmark or similar, contain at least 90% saponin by weight) in 100 ml of water and filter through 0.2 micron filter before injection.

Reverse phase HPLC conditions:

Column: Vydac 214TP54 or similar, 4.6 x 250 mm length, 5μm pore

Column temperature: room temperature

Pump: Linear gradient

A: 0.15% TFA in HPLC grade water.

B: 0.15% TFA in HPLC grade acetonitrile.