Shenyang Pharmaceutical University LAB 16: INCLUSION PHARMACEUTICS III

Shenyang Pharmaceutical University LAB 16: INCLUSION PHARMACEUTICS III

LABORATORY 16:Preparation and characterization of inclusion complexes

1. LABORATORY OBJECTIVES

a) To learn the technique for preparing inclusion complexes by the co-precipitation method.

b) To learn how to identify inclusion complex formation.

1. INTRODUCTION

Inclusion compound (inclusion complex) is a complex in which one chemical compound (the "host") possesses a cavity in which molecules of a second compound (the "guest")can fit in. The definition of inclusion compounds is very broad, including long tunnels or channels formed between molecules in a crystal lattice in which the guest molecule can fit in. If the spaces in the host lattice are enclosed on all sides so that the guest molecule is ‘trapped’ as in a cage, such a compound is known as a cathrateor a ‘cage’ compound.There is no covalent bonding between the guest and the host molecule; the attraction is generally attributed to van der Waals forces.

Cyclodextrins are the most widely used molecules for the formation of host/guest-type inclusion complexes.Cyclodextrins (CDs) are cyclic oligosaccharides which are produced by enzymatic degradation of starch by glucosyltransferase most commonly derived from Bacillus maceransand have been recognized as a very useful pharmaceutical excipient. Chemically they are cyclic oligosaccharides containing at least 6 D-(+) glucopyranose units attached by α-(1, 4) glucosidic bonds. The 3 natural CDs, α-,β-, and γ-CDs (with 6, 7, or 8 glucose units respectively), differ in their ring size and aqueous solubility.

Cyclodextrins (CDs), with a lipophilic cavity and a hydrophilic outer surface, are capable of interacting with a large variety of guest molecules to form non-covalent inclusion complexes. Complexation with cyclodextrins has been reported to offer the following advantages: increased solubility; enhanced bioavailability; improved stability; the masking of bad taste or odor; reduced volatility; transformation of a liquid into a solid form; reduced side effects; and the possibility of providing sustained drug release.

In general, organic drug substances possessing one of the following requirements on structural features and physicochemical properties can form an inclusion complex with cyclodextrins. Theserequirements arethe number of atoms in the chemical structure is larger than 5 and the number of the fused-rings is less than 5; the molecular weight is in the range of 100~400; the solubility in water is less than 10 mg/mL, and the melting point is lower than 250℃. However, some drug substancesdo not form complex with cyclodextrinsalthough they meet these aforementioned requirements. This is mainly due to theirgeometric structure not matching the configuration of the cavity.In addition, inorganic drug substances can’t form complex with cyclodextrins. Different methods can be used to prepare inclusion compounds, including co-precipitation method (saturated aqueous solution method), kneading method, spray drying method, freeze drying method, and neutralization method. Among these methods, the most commonly used one is the co-precipitation method.

In this experiment, the guest molecule is zedoary turmeric oil, which is highly volatile and has been demonstrated to have strong anti-cancer effect in clinic. Curcumenol is the effective anti-cancer component of zedoary turmeric oil. Although zedoary turmeric oil injections and emulsions are available on the market, they are light-sensitive and showpoor stability. By complexing zedoary turmeric oil with cyclodextrin, it may provide the advantages of reduced volatility, converting the liquid into a solid form for the convenience of formulation design, and the possibility of sustained and controlled release.

1. METHODS

3.1 Preparation of zedoary turmeric oil(莪术挥发油)-βcyclodextrin inclusion complex

3.1.1 Preparation of zedoary turmeric oil

Mill thezedoary(莪术) into a medium sized powder. Weigh 200 g of the zedoary powder and add distilled water 10 times the volume of the powder. Extract for 3 h using a volatile oil distiller and a brown oily liquid is obtained. Dehydrate the liquid with anhydrous sodium sulfate to obtainthe zedoary turmeric oil.

3.1.2 Preparation of zedoary turmeric oil ethanol solution

Pipette accurately 1 mL of zedoary turmeric oil and dilute to 5 mL with anhydrous ethanol.

3.1.3 Preparation of saturatedβ-cyclodextrin aqueous solution

Weigh 8 g of β-cyclodextrin and transfer into a beaker, add 100 mL of distilled water and mix to form a saturated aqueous solution at 60±1℃.Keep the solution at this temperature for later use.

3.1.4 Preparation of zedoary turmeric oil-β cyclodextrin inclusion complex

Accurately pipette5 mL of zedoary turmeric oil ethanol solution prepared in 3.1.2 and adddrop wise into the saturated β-cyclodextrin aqueous solution prepared in 3.1.3 under constant stirring at 60±1℃. Rinse the pipette with an additional5 mL of anhydrous ethanol and add to the solution.After the appearance of white precipitatesin the solution, continue stirring for an additional 4 h.Stop heating the mixture and stirto cool down to room temperature. Placethe sample inthe refrigerator for 12 h (ice bath can also be used in the experiment). After the precipitation is completed, filtrate the mixture, wash the precipitates with a 5 mL of ethanol for 3 times on the filter. Dry the precipitates at50℃. Weigh the precipitates and calculate the yield.

3.2 Identification of the formation of inclusion complex

3.2.1 Thin layer chromatography (TLC)

a) Preparation of silica gel G plate: Triturate one part of the stationary phase (silica gel G) with 3 parts of 0.5% CMC-Na aqueous solution in a mortar following the same direction. After the air bubbles are dissipated, pour the mixture on the spreader and prepare a thin layer with a thickness of 0.2~0.3 mm on glass plates.Dry the plates at room temperature. Before use, the plates may be activated by heating in an oven at 110°C for 30 minutes (or 120°Cfor 20 minutes).Visually examine the plate for uniform thickness before use.

b) Preparation of sample solutions

Preparation of zedoary turmeric oil test solution (A): Accurately pipette 0.5 mL of zedoary turmeric oil and dilute to 10 mL with anhydrous ethanol at a final concentration of 0.05μl oil/μl ethanol.

Preparation of zedoary turmeric oil-β cyclodextrin inclusion complex test solution (B): Weigh accurately the predetermined amount of inclusion complex (corresponding to 0.5 mL of zedoary turmeric oil) and dilute to 10 mL with anhydrous ethanol. The supernatant is used for the test.

Pipette 10 μl of test solution A and B respectively using a microsyringe. Apply the solutions on a line parallel to the lower edge of the plate with aninterval of at least 10 mm between the center of each applied solution spot. Continue to apply the prescribed volume of the test solution to the same spot to obtain an area of 2 to 5 mm in diameter. Allow the solvent to evaporate at room temperature. Usea mixture of petroleum (石油醚) and ethyl acetate(乙酸乙酯)（9：1）as the developing agent. Before developing, the plate is placed in the chromatographic chamber saturated with the solvent vapor for 10 min. The chamber is sealed with the lid closed to allow the equilibrium between the solvent and its vapor. Unless otherwise indicated, the chromatographic separation is performed in thechamber using ascendingor horizontal development technique.

For ascending development, the plate is placed in the chamber and before sealing the chamber with the lide, ensures that the plate is in a prefectvertical position and that the sample spots or bands are above the surface of the mobile phase.Remove the plate when the mobile phase has moved more than15 cm. Dry the plate. For horizontal development, the chamber is provided with a reservoir to hold the mobile phase and it also contains a device for directing the movement of the mobile phase on the stationary phase. The distance traveled by the front of the mobile phase should be about 15 cm. Examine the plates after being sprayedwith a 1% vanillin-sulphuric acid (香草醛浓硫酸液) as the color developing agent. The visual comparison ofthe size or intensity of the spots or zones may serve as a semi-quantitative estimation.

3.2.2 Differential thermal analysis（DTA）

a) Samples preparation: Sample A is zedoary turmeric oil, sample B is β cyclodextrin, sample C is the prepared inclusion complex, and sample D is a physical mixture of β cyclodextrin and zedoary turmeric oil with the same ratio as that for the inclusion complex.

b) DTA conditions：The nitrogen (N2) flow rate is 40mL/min，with the range of ±100uV, the scanning rate is 10C/min, speed of the paper is 600mm/h.Same amounts of sample and reference should be used for the measurement.

3.3. Measurement of oil content in the zedoary turmeric oil-β cyclodextrin inclusion complex

3.3.1Accurately pipette 1mL of zedoary turmeric oil and transfer into a round-bottom flask, and add 100 mL of distilled water. Extract zedoary turmeric oil in the sample according to the method described in Part 3.1.1 and measure the volume.

3.3.2Weigh the amount of inclusion complex containing 1 mL of zedoary turmeric oil, tranferit into a round-bottom flask, and add 100 mL of distilled water. Extract zedoary turmeric oil according to the method described in Part 3.1.1and measure the volume.

Use the following equations to calculate the oil content in the inclusion complex, encapsulation ratio, and the yield.

（1）

（2）

（3）

3.4 Determination of Curcumenol content by UV spectrophotometry

3.4.1 Preparation of the standard solution

Weigh accurately 250 mg of curcumenol (莪术醇) and transfer it into a 25 mL of volumetric flask, add anhydrous ethanol to volume, and shake to mix. Pipette accurately 1 mL of the above solution and transfer it into a 50 mL of volumetric flask, add 1 mL of 10% Tween 80, and add anhydrous ethanol to volume (0.2 mg/mL).

3.4.2 Preparation of sample solutions

a) Preparation of the zedoary turmeric oil control solution: Pipette accurately 0.1 mL of zedoary turmeric oil and transfer it into a 25 mL of volumetric flask, add 0.5 mL of 10% Tween 80, and add anhydrous ethanol to volume.

b) Preparation of the zedoary turmeric oil-β cyclodextrin inclusion complextest solution: Weigh a specific amount of the inclusion complex containing 0.1 mL of zedoary turmeric oil,tranfer it into a beaker,and add 20 mL of anhydrous ethanol. Shake for 10 min and set it aside for 1 h. Filter and transfer the filtrate into a 25 mL volumetric flask, add 0.5 mL of 10% Tween 80, and add anhydrous ethanol to volume.

c) Preparation of the blank solution: Accurately pipette 0.5 mL of 10% Tween 80 and transfer it into a 25 mL volumetric flask, and add anhydrous ethanol to volume.

3.4.3Method of measurement

Accurately pipette 0.5 mL of the standard solution and the test solution respectively and transfer them into two 100 mL volumetric flasksseparately, add 0.2% vanillin-sulphuric acid solution to volume, and mix by shaking. Set aside for 30 min and measure the absorption at the wavelength of 510 nm. Calculate the content of curcumenol using the following equation:

Cx=（Ax/Ar）Cr

where Cx is the concentration of the test solution, Ax is the absorption of the test solution, Cr is the concentration of the control solution, Ar is the absorption of the standard solution.

1. RESULTS AND DISCUSSION

4.1 List the oil content, encapsulation ratio(EP), and yield of the inclusion complex in Table 1.

Table 1. Oil content, encapsulation ratio and yield of the inclusion complex

Sample / Oil content % / EP% / Yield %
Inclusion complex

4.2 Identification of the inclusion complex

4.2.1Draw the TLCplate after applying the developing agent, describe the final location of the spots and Rf valuesof the guest substance before and after inclusioncomplex formation, indicating the formation of the inclusion complex.

4.2.2 Based on the DTA theromograms, describe the shape of the peak and the temperature corresponding to the peaks before and after inclusioncomplex formation, indicating the formation of the inclusion complex.

4.2.3 List the curcumenol content results in Table 2.

Table 2. Curcumenol content in different samples

Samples / Ax / Ar / Cx / Cr / %
Volatile oil
Inclusion complex

1. QUESTIONS

a) What are thekey parameters for the preparation of inclusion complexes? How to control these parameters?

b) What are the structural requirementsfor the guest molecule with relation to the host molecule for the preparation of inclusion complexes?

c) How many methods can be used to confirm the formation of an inclusion complex?

References

[1] H. C. Ansel, N. G. Popovich and L. V.Allen, Jr. Pharmaceutical Dosage Forms and Drug Delivery Systems, , 8th ed., Williams and Wilkins, Baltimore, 2005.

[2] M.E. Aulton. Pharmaceutics: The science of dosage form design. 1988.

[3] R. Challa, A. Ahuja, J. Ali, and R.K. Khar. Cyclodextrins in Drug Delivery: An Updated Review. AAPS PharmSciTech 2005; 6 (2), E329-E357.

(Shirui MAO）

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