Propolis extraction protocol

Section 1

Protocol philosophy

This protocol synthesizes three established pharmaceutical approaches into a single, unified workflow:

1. The Sosnowski Cold-Purification Framework (US 4,382,886, 1983)

The foundational insight: propolis waxes and high-MW resins precipitate at low temperature (−20 °C) in ethanolic solution, while flavonoids and phenolic acids remain dissolved. This yields a clear, essentially colourless, non-staining filtrate free of emboligenic waxes. The Sosnowski protocol builds on Russian prior art (USSR Pat. 232470, 267014, 576115, 585846) and represents the only documented route to a parenteral-grade propolis starting material.

2. Optimized pH-Shift Two-Step Extraction

Different compound classes extract optimally under different conditions. Non-polar flavonoids (pinocembrin, chrysin, galangin) prefer high ethanol concentration (85%) with mild acidification (pH 3, citric acid). Polar phenolic acids (CAPE, caffeic acid, ferulic acid) prefer lower ethanol (23%) with mild alkalinity (pH 8, NaHCO₃). A sequential two-step extraction captures both fractions at their respective optima without thermal degradation.

3. The Legalon® SIL Prodrug Chemistry

Propolis flavonoids are phenolic acids — they possess no basic nitrogen for hydrochloride salt formation. Instead, succinic anhydride covalently esterifies phenolic –OH groups to hemisuccinates, appending free carboxylic acid handles. Neutralization with NaOH yields the water-soluble disodium salt. This is the only established chemistry for converting phenolic plant extracts into parenteral formulations.

Section 2

Equipment & machinery

Section 3 · Phase I

Raw material preparation

3.1. Starting material specification

• Source: Poplar-type propolis (European/North American origin)
• Form: Raw, unprocessed chunks (resinous, not powdered)
• Quality: HPLC-verified for pinocembrin/chrysin/CAPE markers
• Moisture: ≤8% (Karl Fischer)
• Wax content: ≤30% (gravimetric after cold precipitation)
• Total flavonoids: ≥10% w/w (quercetin equivalents)
• Batch: 500 g

3.2. Freezing & comminution

1. Place 500 g raw propolis in a sealed polyethylene bag inside a metal tray.
2. Transfer to ultra-low freezer at −80 °C for 24 hours.
3. Remove from freezer. Immediately transfer to a cryomill pre-cooled with liquid argon.
4. Pulverize at 25 Hz for 3 × 1-minute cycles with 30-second rests (to prevent heat buildup).
5. Sieve through 1 mm stainless steel mesh.
6. Return oversize particles to cryomill; repeat until all passes 1 mm mesh.

Yield: ~480–490 g fine propolis powder.  Storage: keep at −20 °C in amber glass container under argon until use (≤7 days).

Section 4 · Phase II

pH-shift two-step extraction

This is the critical yield-maximization phase. Two sequential extractions are performed on the SAME propolis powder — first for non-polar flavonoids (acidic, high ethanol), then for polar phenolic acids (mild alkaline, low ethanol). The combined extract is then processed as a single batch.

4.1. Chemicals & additives — exact quantities

Section 5 · Phase III

Sosnowski cold purification

Primary sourceReference: Sosnowski Z.M., Method for Extracting Propolis and Water Soluble Dry Propolis Powder, US Patent 4,382,886 (May 10, 1983). Building on Russian prior art: USSR Pat. 232470, 267014, 576115, 585846.

Principle: In ethanolic solution, propolis waxes (long-chain hydrocarbons, esters, fatty acids) and high-MW resins have inverse temperature solubility. At −20 °C, they precipitate as a viscous, opaque mass, while flavonoids and phenolic acids remain in solution. Cold filtration yields a clear, essentially colourless, non-staining filtrate free of emboligenic wax particles.

5.1. Procedure

1. Transfer the combined extract (~3.0 L) into a 5 L borosilicate flask with wide mouth.
2. Place flask in a deep-freeze at −20 °C ± 2 °C for 24 hours. Do not disturb during freezing. Agitation causes wax emulsification and prevents clean precipitation.
3. After 24 h, observe: the extract becomes viscous and opaque — waxes and resins have precipitated.
4. Pre-cool the following to −20 °C for 2 h:
   • Büchner funnel (glass or porcelain, ≥150 mm diameter)
   • Whatman No. 50 filter paper (hard-grade, quantitative, 2.7 µm)
   • 4 L vacuum filtration flask
   • 50 mL absolute ethanol (pre-cooled wash solvent)
5. Set up vacuum filtration in a cold room or with jacketed cooling maintained at −20 °C.
6. Pour the cold extract onto the pre-cooled filter. Apply gentle vacuum initially (≤100 mbar) to avoid pulling wax particles through the paper.
7. As filtration proceeds and the wax cake builds, gradually increase vacuum to ≤50 mbar.
8. When flow slows significantly, add 50 mL pre-cooled absolute ethanol to the filter cake to displace trapped filtrate. Apply gentle vacuum.
9. The filtrate must be very clear — light amber to straw-coloured, not cloudy or hazy. If hazy: repeat cold filtration with fresh Whatman No. 50 paper.
10. Bring clear filtrate to room temperature slowly (allow 2 h in ambient air; do not heat).

Yield: ~2.8 L clear, wax-free propolis extract.  Mass of dissolved solids: ~150–180 g (wax fraction removed: ~30–50 g).

5.2. Purity stress test (−80 °C checkpoint)

1. Take a 50 mL sample of the clear filtrate in a borosilicate test tube.
2. Place in ultra-low freezer at −80 °C for 24 hours.
3. After 24 h, examine:
   • Crystal-clear = PASS — wax removal was complete at −20 °C.
   • Cloudy, hazy, or precipitate = FAIL — residual waxes remain. The entire batch must be re-subjected to cold precipitation (return to −20 °C for additional 12 h, then re-filter).
4. This stress test confirms that the −20 °C filtration was sufficient for parenteral-grade purity.

Section 6 · Phase IV

Solvent removal

Objective: remove bulk ethanol and water to obtain a purified propolis resin substrate for esterification. Constraint: temperature must not exceed 40 °C to prevent thermal degradation of CAPE and flavonoids.

6.1. Option A — Thin-film evaporator (preferred)

1. Transfer clear filtrate (~2.8 L) to thin-film evaporator.
2. Set jacket temperature: 35–38 °C.
3. Set vacuum: ≤30 mbar.
4. Feed rate: 200 mL/min.
5. Collect distillate (ethanol/water) for solvent recovery.
6. Continue until a yellowish bed remains in the receiver (~150–180 g). It is not a dark resin: the Sosnowski cold purification has already removed the wax and high-molecular-weight resin fraction, so what the ethanol leaves behind is light.
7. Verify residual ethanol by GC-Headspace: target ≤0.5% w/w.

Time: ~2–3 hours.

6.2. Option B — Lyochrysalis vacuum evaporation

1. Transfer filtrate to Lyochrysalis chamber trays.
2. Enable Cryolapse condenser at −110 °C.
3. Pull vacuum to ≤50 mbar.
4. DO NOT enable TgShift or LyoLevit — this is evaporation, not lyophilization.
5. Maintain at ambient shelf temperature (no heating).
6. Evaporate until the yellowish bed remains.

Time: ~6–8 hours.

6.3. Azeotropic cleanup (if residual ethanol >0.5%)

1. Dissolve the bed in 200 mL ethyl acetate.
2. Evaporate under vacuum (rotovap, ≤35 °C).
3. Ethyl acetate forms an azeotrope with residual ethanol, removing it completely.
4. Repeat if necessary.

Result: the purified yellowish bed, ethanol-free, ready for esterification.

Section 7 · Phase V

Succinic anhydride esterification

Primary sourceReference: Legalon® SIL (silibinin-C-2′,3-dihydrogen succinate, disodium salt) — Rottapharm/Madaus. Approved IV formulation for Amanita phalloides poisoning.

Chemistry: propolis flavonoids are phenolic acids. They possess no basic nitrogen for HCl salt formation. Succinic anhydride covalently esterifies phenolic –OH groups to hemisuccinates, appending free carboxylic acid handles. The flavonoid chromone ring system remains intact.

7.1. Stoichiometric basis

Section 8 · Phase VI

Sodium salt formation (water solubilization)

The hemisuccinate product carries free terminal carboxylic acid groups (–COOH). Neutralization with NaOH deprotonates these to carboxylate anions (–COO⁻Na⁺), conferring water solubility. This is the same mechanism as Legalon® SIL.

8.1. Reagents & quantities

Section 9 · Phase VII

Sterile filtration

Objective: remove particulates and bioburden. Note: 0.22 µm filtration removes bacteria and particles but does NOT remove endotoxins (LPS, 1–10 nm). Endotoxin levels must be verified separately by LAL test (USP <85>).

9.1. Procedure

1. Perform all steps in ISO Class 5 laminar flow hood or isolator.
2. Pre-filter the 1.5 L solution through 0.45 µm PES (47 mm, sterile) to protect final sterile filter.
3. Immediately pass through 0.22 µm PES (47 mm, sterile, pharma grade) into a sterile 2 L borosilicate receiving vessel.
4. Use peristaltic pump or gentle argon pressure (≤0.2 bar) to drive filtration. Do not exceed 2 bar differential pressure.
5. If filtration rate drops >50%, replace filter — do not force.
6. Retain 20 mL sample for:
   • Sterility testing (USP <71>)
   • Endotoxin testing (LAL, USP <85>)
   • pH verification

Acceptance: sterile (no growth); endotoxins <5 EU/kg body weight; pH 7.2–7.6.

Section 10 · Phase VIII

Lyophilization

Objective: convert sterile aqueous solution into a stable, solid cake for long-term storage and IV reconstitution. The Lyochrysalis platform is fully engaged here — TgShift, LyoLevit, and Cryolapse are used for an aqueous peptide-like phenolic salt matrix.

10.1. Procedure

1. Aseptic filling: in ISO Class 5 hood, dispense 10 mL of sterile filtrate into the product chamber of each Lyoprester™ dual-chamber cartridge. Set the plungers to the drying position so the chamber stays open to vapour. Number of cartridges: ~150.
2. Loading: transfer the cartridges to the Lyochrysalis shelf, pre-cooled to 5 °C.
3. Freezing (TgShift-enabled):
   • Ramp shelf to −45 °C at 1 °C/min.
   • Hold 6 hours to ensure complete solidification.
   • TgShift protocol: apply RF modulation at −35 °C for 30 min to raise Tg′ and prevent amorphous collapse during primary drying.
4. Primary drying (Cryolapse + LyoLevit):
   • Pull vacuum to 0.1 mbar.
   • Ramp shelf to −20 °C over 24 h.
   • LyoLevit: engage orbital RF levitation at 0.5 mm amplitude to maximize sublimation surface.
   • Cryolapse: program intentional pressure-collapse cycles (0.3 → 0.1 mbar) every 4 h.
5. Secondary drying:
   • Ramp shelf to +25 °C at 0.2 °C/min.
   • Hold under vacuum (0.05 mbar) for 18 hours.
   • Target residual moisture: ≤2% (Karl Fischer).
6. Backfill & sealing:
   • Backfill chamber with sterile argon to 800 mbar.
   • Drive the plungers home under vacuum so each cartridge seals with the argon headspace inside it.
   • The dried cake and its reconstitution liquid are now closed in the same cartridge, in separate chambers, and stay apart until use.

Yield: ~150 sealed Lyoprester cartridges. Each carries ~1.0 g of sterile lyophilised cake (equivalent to ~600 mg propolis flavonoid succinate sodium salt) with its reconstitution volume already loaded in the adjacent chamber.

Section 11 · Phase IX

Reconstitution & administration

11.1. Reconstitution

Nothing is drawn up, and nothing is injected into a vial. The cartridge already holds both phases; reconstitution is an actuation of the cartridge, performed in the closed system.

1. Bring the cartridge to room temperature before actuating. A cold cake dissolves slowly and a cold liquid carries more dissolved gas.
2. Actuate the cartridge. Advancing the plunger drives the reconstitution volume from its chamber through the bypass into the cake chamber. The path is closed throughout — no needle, no open vial, no air ingress.
3. Because the liquid enters through the bypass rather than being aimed at the cake surface, the cake wets from the base upward instead of being struck from above. This is the point of the architecture: a phenolic salt solution is surface-active and foams readily when a stream is fired into it, and the dual chamber removes the operation that causes it.
4. Invert gently two or three times until dissolution is complete. Do not shake. Foam is not merely cosmetic here — it is air brought to a large interface with a surface-active solute.
5. Resulting solution: ~60 mg/mL propolis succinate sodium salt, pH 7.4, clear to light amber.
6. For infusion, transfer to a compatible carrier (5% dextrose or 0.9% NaCl) at the target concentration.
7. Inspect visually before administration. Reject if hazy or particulate.

11.2. Storage conditions

• Sealed cartridge: 2–8 °C, protected from light; the cake sits under its argon headspace inside the cartridge. Stability: ≥24 months (accelerated data required).
• Reconstituted solution: use within 4 hours at 20–25 °C; within 24 hours if refrigerated at 2–8 °C.
• Transport: ship sealed cartridges at 2–8 °C with cold packs. Do not freeze.

Section 12

Master chemicals & materials — shopping list

12.1. Raw materials & solvents

Section 13

Molar ratios & stoichiometry summary

All quantities expressed in molar (M), millimolar (mM), micromolar (µM), and nanomolar (nM) for standardization and scale-up calculations.

Section 14

Stability, degradation risks & mitigation

This section details every thermal, chemical, and oxidative risk point in the protocol, with exact temperature ceilings, pH boundaries, and mitigation strategies.

Section 15

Critical quality attributes (CQA)