Normative Instruction Nº 3, January 19th 2001
Annex VII - Regulation for Identity and Quality of Propolis Extract

1 - Reach:
1.1. Objective: To establish an identity and the minimum quality requests to be attended as a Propolis extract.
1.2. Ambit of Application: The present Regulation refers to Propolis Extract destined to national and international commerce’s.

2.Description:
2.1. Definition: Propolis Extract understands as a product that comes from soluble components of propolis extraction in neutral alcohol (alimentary grade) by ad equated technological process.
2.2. Designation (Selling Denomination): Propolis Extract.

3. References:
- ABNT (Brazilian Technical Norms Association).
- AOAC. Official Methods of Analysis of the Association of Analytical Chemists, Arlington, 1992.
- AOAC. Official Methods of Analysis of the Association of Analytical Chemists, 16^th Edition, Chapter 4.1.03, 1995.
- ASIS, M. Propoleo – El Oro Purpura de las Abejas. Cuba, 1989.
- BRAZIL. Ministerio da Agricultura e do Abastecimento. Portaria nº 368, dated 04.09.97 – Technical Regulation about Sanitarian and Hygienic Conditions.
- BRASIL. RIISPOA – Regulation of Industrial Inspection and Sanitarian of Products from Animal Origin. Decree nº 30.691 dated March 29^th, 1952.
- BRAZIL – Ministerio da Agricultura e do Abastecimento. Portaria SIPA nº 06/84. Hygienic-Sanitarian and Technological Norms for Honey, Bee wax, and Derivatives.
- FAO/OMS. Organización de las Naciones Unidas para la Agricultura y la Alimentación. Codees Alimentarius, CAC/vol. A, 1985.
- ICMSF – Microorganisms in Foods. 2. Sampling for microbiological analysis: Principles and specific applications. University of Toronto. Press, 1974.
- ICMSF. Compendium of Methods for Microbiological Examination of Foods, 1992.

4. Composition and Requisites:
4.1 Composition:
- Propolis Extract is composed by soluble elements from propolis in hydro alcoholic solution, alcohol and water.
4.2 Requisites:
4.2.1. Sensorial Characteristics:
4.2.1.1. Aroma: characteristic, depending on botanic origin (balsamic and resinous);
4.2.1.2. Color: it varies, depending on origin and concentration (amber, reedy and greeny tones);
4.2.1.3. Flavor: characteristic, from suave to strong, bitter and whet (piquant);
4.2.1.4. Aspect: limpid and homogeneous liquid.
4.2.2. Physical and Chemical Requisites;
4.2.2.1. Dry Extract: minimum of 11% (m/v);
4.2.2.2. Wax: maximum 1% from dry extract (m/m);
4.2.2.3. Flavonoids composition: minimum 0,25% (m/m);
4.2.2.4. Phenolic Composition: minimum 050% (m/m);
4.2.2.5. Activity of Oxidation: maximum 22 seg.;
4.2.2.6. Alcoholic contents; maximum 70º GL (v/v);
4.2.2.7. Methanol: maximum 0,40mg/l;
4.2.2.8. Spectrum of Absorption of Radiations UV visible; the propolis extract must be present characteristic peaks of principal flavonoids classes between 200nm and 400nm;
4.2.2.9. Lead Acetate: positive;
4.2.2.10. Sodium Hydroxide: positive;
4.2.3. Conditioning:
- Must be package in material bromatologicly adequate and it confers to the product an adequate protection.

5. Addictives:
It doesn’t admit.

6. Contaminators:
The organic and inorganic contaminators don’t have to be present in superior quantities to the to established limits by effective Regulation.
6.1. Other Contaminators:
Research of spurs (esporos) of Paenibacillus larvae, in 25ml of propolis extract (using the methodology described in the Portaria 248, from 12.30.1998). Accepted Result: absence of spurs (esporos) in 25ml. 

7. Hygiene:
7.1. General Considerations:
- The hygienic practices to elaborate the product have to be in accordance with the Technical Regulation on the Hygienic-Sanitarians Conditions and the Good Practices of Manufacturing for Elaborating/Industrialized Establishments of Foods.
7.2. Macroscopic Criterions:
- The product mustn’t contain strange matters, of any nature.
7.3. Microscopic Criterions:
- The product mustn’t contain strange matters, of any nature.

8. Weights and Measures:
It must be applied a specific Regulation.

9. Labeled:
It must be applied the specific Regulation with the following additional information – Dry Extract: minimum of ….%.

10. Analysis Methods:
- Official Analytical Methods for Quality Control;
- Validated Methodologies by The Ministry of Agriculture;
- Microbiologic Analysis Methods for Foods. Research of Paenibacillus larvae in honey and bee products. Portaria 248 date 12.30.1998. Ministério da Agricultura e do Abastecimento.

11. Sampling:
Following the procedures recommended by effective norm (ABNT).

Published in Official Diary of 01.23.2001, pages 18-20.
Ministério da Agricultura e do Abastecimento.

Required Analyses:

A) Dry Extract: minimum of 11% (m/v)
Weight 5,0g of propolis in a capsule of porcelain previous heating in stove at 105º C for three hours and “tarada”. Put the sample of propolis in the stove at 105º during three hours. Take it off from the stove and put it to desiccate for 1 hour, weight it and repeat the operation until the constant weight.
% resin = 100 x (A1 – A2)
Weight of sample
A1 – Weight of capsule + sample (before heating)
A2 – Weight of capsule + sample (after heating in stove)
P –   Weight of propolis sample.

B) Wax (maximum of 1% of dry extract)
Weight 1 kg of propolis and put it in a “becker” (recipient) of 250ml. Add 100ml of distillated water and put it in water-bath (banho-maria) at 40º for 30 minutes. Filter it in a previous dry and hard paper filter, dry the filter paper (more than 80º C) until the constant weight.
Total insoluble solids % = 100 x (A2 – A1)
Weight
A1- Paper filter weight.
A2 - Paper filter weight + sample after filtered and dried in stove.
P – Weight of propolis sample.
% wax = % total insoluble solids.

C) Flavonoids composts
Reagents:
Quercetin PA
Ethanol PA
Aluminum Nitrate Solution 10%. Weight 17,6g of aluminum nitrate monohydrated – AL (NO3) 3 and 9H2O and dissolve it in distillated water, and complete the volume for 100ml.
Sample Preparing
Get 0,5ml of the sample and dilute it for 50ml with ethanol 80º C GL.

Preparing the standard curve:
Weight 50mg of quercetin, converting the weight on quercetin anhydrous. Dissolving in 100 ml of ethanol to 95% (stock solution). Prepare three samples from the stock solution that should contain quercetin anhydrous on proportions of 0,01 and 0,05 and 0,1mg/ml respectively. Measure the absorbance of each aliquot following the same procedure described for sample and makes the standard curve (Graphic of absorbance in relation to quercetin concentration).
Procedure:
Get 0,5ml of sample and add 4,3ml of ethanol at 80% 0,21ml of aluminum nitrate solution 10% and 0,1ml of acetate potassium solution 1M; let it resting at environment temperature during 40 minutes, measure the absorbance in 415nm, determine the content of quercetin in the sample, using a standard curve and considering the dilutions done in the sample.

D) Phenolic Composition  (minimum 0,50% (m/m)
Reagents
Reagent of Folin-Cicalteau
Saturated solution of sodium carbonate
Standard of phenol. Solution.
Dissolve 0,5000g of dry gallic acid in 100ml of water, inside a volumetric balloon of 100ml.

Procedure

C – Standard curve
Pipetting aliquot of 0 to 10ml of solution of tannic acid standard in volumetric balloon of 100ml and complete the volume with distilled water.
Pipetting 1 ml of each dissolution and transfer it for a balloon of 100ml.
Add 60ml of distilled water and agitate it.
Add 5ml of Folin Reagent and agitate again.
After 30 seconds and before 8 minutes add 10ml of sodium carbonate solution and complete the volume with distilled water and agitate it well.
Let it resting for 2 hours and do a reading at 765nm.

D- Sample
Dilute the sample in proportion of 1:25.
Transfer it for a balloon of 100ml, 1ml of the sample and add 60ml of distilled water.
Add 5ml of Folin Reagent and agitate it.
After 30 seconds and before 8 minutes add 10ml of sodium carbonate solution.
Complete the volume with distilled water and agitate it well.
Let it resting for 2 hours and the do a reading at 765nm.

E) Oxidation Activity:
Get 5ml of propolis extract and add 100ml of distilled water. After constant agitation, it has to be filtered in filter paper. Get 1ml of a filtered sample and put it in proof tube of 5ml. Add to this 1ml of distilled H20; after agitate it again, mix to this 1ml of sulfuric acid at 20%. This mix has to be agitated for 1 minute more, in which should be add 1 drop of permanganate potassium 0.1 N. With the help of a chronometer instrument, should be measured the time, in seconds, that corresponds to the time expensed for the coloring changing from yellow to pink. It is what determines oxidation indices. The determinations are done in duplicate (ASIS, 1989).

Spectrum of Absortions’Radiations UV visible.
(It presents characteristic peaks of the principal flavonoids classes between 200 and 400 nm).
An aliquot of 25ul of propolis extract is diluted in 30ml of ethanol at 80%. So, is analyzed the spectrum of absorption in the band of length wave of 200 to 400nm; (analyzed in spectrophotometer HP 8451A (PARK et al, 1995, 1997).  

F) Plumb Acetate and Sodium Hydroxide
Solutions:
Plumb Acetate at 10%
Putting in a volumetric balloon of 50ml, approximately 30ml of distilled water and 10g of plumb acetate. Agitate and complete the volume until reach 100ml of distilled water. The solution of plumb acetate at 10% remains stable during six months.
Sodium Hydroxide at 20%
Get a “becker” (recipient) and put inside 20g of sodium hydroxide and dissolve it with 50ml of distilled water. After that, transfer it to a balloon 100ml and complete the volume with distilled water, (with the solution in environment temperature).
Putting in a test glass 5ml of propolis extract, and take it to water-bath during 3 minutes. Filter it through paper filter with the solution in the state of environment temperature. Retire with a pipette, 1ml of filtered material and put it in “Erlenmeyer” of 50ml. Add to this 10ml of ethylic alcohol and agitate it carefully. Put 1ml of this solution in two glass tubes:

Tube 1- Add 0,5ml of sodium hydroxide solution at 20%;
Tube 2 – Add 0,5ml of Plumb Acetate solution at 10%.

Positive Reaction: The solution of the first glass tube should get color so fast with a coloring yellow/orange/ and / or darkness. The second glass tube solution will get a yellow greeny and precipitated, or, should present disturbance; (if it becomes disturbance, the precipitation will appear after 30 to 60 minutes.

Translation,
Tarcísio Martins / February 8^th