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1/8www.pharmtech.com74 Pharmaceutical Technology OCTOBER 2004

Anitha Palamakula is a doctoral student inpharmaceutical sciences; Mahmoud

Soliman, PhD, is a postdoctoral research

associate; and Mansoor A. Khan, RPh,

PhD, is director at FDA/CDER/DPQR, White

Oak, LS Building 64, HFD-940, 5600 Fishers

Lane, Rockville, MD 20895, tel. 301.796.0016,

fax 301.796.9816, [email protected].

Indra K. Reddy is a professor and dean,

College of Pharmacy, Texas A&M

University–Kingsville (TAMU-K, Kingsville, TX).

Dr. Khan also serves as a distinguised visiting

professor at TAMU-K and is a member of

Pharmaceutical Technology ’s Editorial Advisory

Board.

*To whom all correspondence should be addressed.

Preparation and In Vitro Characterization of

Self-Nanoemulsified Drug

Delivery Systems of CoenzymeQ10 Using Chiral Essential Oil ComponentsAnitha Palamakula,Mahmoud Soliman, Indra K. Reddy,and Mansoor A. Khan*

P H O T O D I S C ,

I N C .

The authors developed

and characterized self-

nanoemulsified drug deliverysystems of coenzyme Q10 using

R and S enantiomers of limonene

and evaluated the progresss of

emulsification and drug-release

kinetics.

elf-emulsified systems are used extensively for improving

the formulation characteristics of poorly water-soluble

drugs (1). Conventional self-nanoemulsified drug deliv-ery (SNEDDS)-based formulations use mainly fixed oils

or essential oils to form fine oil-in-water emulsions of drugs in

aqueous solutions with the help of a surfactant and a cosurfac-

tant. Many of these formulations yield unstable products, how-

ever, because of the limited solubility of drugs in these oils aswell as rancidity problems (2).

Therefore, it was of interest to study natural substances such

as chiral components of essential oils for solubilizing drug com-

pounds with poor aqueous solubility. Chiral molecules such as

enantiomers of terpenes enhance the permeation of drugs

through skin (3). Limonenes, monocyclic monoterpenes ex-

tracted from citrus fruits, are used as flavoring agents. They existin two chiral conformations: R-()-limonene and S-()-

limonene. Limonene enantiomers have emulsion-forming and

solubilizing properties (4). Limonenes have been shown to pos-

sess chemopreventive activities in experimental animal mod-

els and hence have proved to be beneficial to health (5).The R-()-limonene isomeric form,also known as D-limonene ,

is more abundantly present than the racemic and S-()-limonene

forms. Limonenes are metabolized by cytochrome P450 enzymes

CYP2C9, CYP2C11, CYP2C19, and CYP2B1 to oxidized prod-

ucts such as carveols and perillyl alcohols (6). The hydroxylated

metabolites of R-()-limonene, sobrerol, carveol, and prillyl al-

cohol are more potent than the metabolites of S-()-limonene(6). R-()-limonene and its metabolites are weak inhibitors of

post-translational mediators of ras proteins, farnesyl transferase

(FTase), and geranylgeranyl transferase 1 (GGTase 1) and hence

shows potential tumor-growth inhibitory properties. Research

in the food industry has demonstrated R-()-limonene in mi-croemulsions as vehicles to enhance the solubilization of natu-

ral food supplements with nutritional and health benefits (7).

The available literature is scarce, however, about using these com-

ponents to prepare SNEDDS.

The study described in this article evaluated chiral molecules,

limonenes in SNEDDS. These studies were performed using

coenzyme Q10 (CoQ) as the model drug. CoQ is a neutral,highly lipophilic antioxidant with beneficial effects on cardio-

vascular and neurodegenerative diseases (8).

S

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Materials and methodsMaterials. CoQ was a generous gift from Kyowa Hakko USA (New York, NY). R-limonene and S-limonene were purchased fromSigma Aldrich (Milwaukee, WI). Polyoxyl 35 castor oil (Cre-mophor EL) was obtained from BASF Corp. (Mount Olive, NJ).Capmul GMO-50 was obtained from Abitec Corp. (Janesville,WI). Hydroxypropyl methyl cellulose (HPMC) capsules weresupplied by Shionogi Qualicaps (Whitsett, NC). HPLC grademethanol and n-hexane were purchased from VWR Scientific(Minneapolis, MN). All chemicals were used as received.

Determination of CoQ solubility in R and S limonenes. Excess of CoQ(1 g) in 5 mL of limonenes were taken in amber,screw-cap, glass

vials, and the mixtures were stirred using magnetic stirrers. Sam-ples were taken after 24 and 48 h, filtered, and analyzed usinghigh-performance liquid chromatography (HPLC) (9).

Differential scanning calorimetry (DSC) of CoQ and binary systemsof CoQ with limonenes. CoQ and limonene (R or S) were mixedat various ratios between 90:10 and 10:90 (w/w). Approximately 5 mg of the mixture was sealed in an aluminum pan and ana-

lyzed using a differential scanning calorimeter (DSC 7,Perkin–Elmer, Norwalk, CT). Thermal analysis was carried outbetween 10 and 75 C under nitrogen-gas flow against an empty reference pan at a heating rate of 10 C/min. Ice was used tomaintain lower temperatures.

Fourier transform-infrared (FTIR) spectroscopy . FTIR spectroscopy was performed using a Nexus 470 FTIR unit (Thermo Nicolet,Madison,WI) attached to an attenuated total reflectance (ATR)accessory that was fitted with single-bounce diamond at 45 in-ternally reflected incident lights. The sampling area was 1 mmin diameter with a sample depth of several microns. Samplesfor CoQ and binary systems of CoQ with limonenes (90:10 to

10:90) were prepared using a small mortar and pestle. Analy-ses were performed by placing a small amount of the sampledirectly on the diamond disk and scanning for absorbance from4000 to 400 wave numbers (cm1) at a resolution of 1 cm1.

Stability of CoQ in limonenes. Stability studies were performedfor CoQ in either R or S limonene (500 mg/mL) taken in amberglass vials fitted with screw caps and subjected to various tem-perature and humidity conditions. Stability was evaluated at 4,25,37,and 25 C with 60% relative humidity (RH), 37 C with60% RH, and 45 C with 75% RH for as long as 3 months. Five-milliliter samples taken on days 1, 7, 15, 30, 45, 60, and 90 werediluted with methanol and analyzed using HPLC (9).

Formulation of the self-emulsified systems. A series of self-emulsifying systems were prepared with varying concentrationsof the limonenes (R or S), Cremophor EL,and Capmul GMO-50. The amount of CoQ was fixed at 25%. CoQ was accurately weighed into screw-capped glass vials and melted in a waterbath at 37 C. Cremophor EL and Capmul GMO-50 were addedto the mixture using a positive-displacement pipette and stirredwith a magnetic bar. While molten, formulations with variousconcentrations of surfactant, cosurfactant,and the limonene—each containing CoQ at a final loading of 30 mg—were filledinto size 3 HPMC capsules. Filled capsules were stored at roomtemperature until their use in subsequent studies.

Visual observations. To assess the self-emulsification proper-ties, formulation in a 120-mg capsule was introduced into 250mL of prewarmed water (37 C) in a glass Erlenmeyer flask at

Table I: CoQ SNEDDS ratios (% w/w).

R or S Cremophor Capmul

Formulation CoQ Limonene EL GMO-50

1 25 67.5 3.75 3.75

2 25 60 7.5 7.5

3 25 52.5 11.25 11.25

4 25 37.5 18.75 18.75

5 25 30 22.5 22.5

6 25 22.5 26.25 26.25

7 25 15 30 30

8 25 7.5 33.75 33.75

9 25 0 37.5 37.5

10 25 63.75 7.5 3.75

11 25 52.5 15 7.5

12 25 41.25 22.5 11.25

13 25 30 30 15

14 25 18.75 37.5 18.75

15 25 7.5 45 22.5

16 25 0 50 25

17 25 57 13.5 4.5

18 25 33 31.5 10.5

19 25 15 45 15

20 25 0 56.25 18.75

21 25 67.5 6 1.5

22 25 45 24 6

23 25 15 48 12

24 25 0 60 15

Temperature ( °C)

H e a t f l o w ( m W )

10.00.0

Temperature ( °C)


10.00.0

Temperature ( °C)


10.00.0

a b c

Figure 1: (a) DSC thermograms of CoQ; (b) binary mixture of CoQ with R-()-limonene; and (c) binary mixture of CoQ with S-()-limonene.

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25 C, and the contents were gently stirred manually. The ten-

dency to spontaneously form a transparent emulsion was judgedas good or bad on the basis of visual observation. Formulationsthat had poor or no emulsion formation were judged as bad.Phase diagrams were constructed that identified the good self-emulsifying region. All studies were repeated in triplicate withsimilar observations being made between repeats.

Emulsion droplet size analysis and turbidity measurements. For-mulation in one capsule (120 mg) was dissolved in 250 mL of water, pre-equilibrated at 37 °C, in an Erlenmeyer flask and gen-tly mixed manually. The resultant emulsions were evaluated fordroplet size and turbidity as follows.

Droplet size analysis. The droplet size distribution of the result-

ant emulsions was determined by taking the resultant emulsionin a small glass tube (60 50 mm). The particle size (volumediameter) was measured using a particle-sizing system (NiCompPSS ZW380, Santa Barbara, CA).All studies were repeated,andgood agreement was found among the measurements.

Turbidity measurement. Turbidity of the resultant emulsions (givenin nephlometric turbidity units [NTU)]) was measured usingan Orbeco-Hellige turbidimeter (model 966,Farmingdale, NY).The turbidimeter was carefully calibrated with formazin stan-dards. Accuracy of the instrument is essential, especially forsmall and diluted emulsions with high surfactant concentra-tions. The accuracy of the turbidimeter used was approximately

0.01 NTU with stray light less than or equal to 0.01 NTU.Zeta potential. The difference in potential between the surface

of the electro-neutral region of the solution and the surface of

tightly bound layer of ions on the parti-cle is the zeta potential . The zeta poten-tial was measured using a NiComp PSSZW380 instrument (Santa Barbara,CA).The nanoemulsions were taken in acuvvete, and the electrodes were attached

and placed in the ZW380 for measure-ment. Each sample was analyzed in trip-licate in automatic mode.

Dissolution studies. Flask method.Prelimi-nary dissolution experiments for capsulesfilled with the self-emulsified formula-tions of CoQ in R- or S-limonene weredetermined using an Erleynmeyer flask at 25 C. Capsules containing 30 mg of CoQ were introduced into 250 mL of pre-warmed distilled water. The amount of dissolved CoQ was analyzed by HPLC,

after the samples were filtered with a 10-mm VanKel filter. All of the experimentswere carried out in triplicate.

USP rotating-paddle method. Dissolutionprofiles of the capsules filled with the self-emulsified formulations of CoQ in R-limonene also were determined using aUSP2 rotating-paddle apparatus(VK7000, VanKel, Cary, NC).The disso-lution experiments in triplicate for eachformulation were carried out at 37 0.5

C, with a rotating speed of 50 rpm in 900 mL of water. Cap-

sules were held to the bottom of the vessel with aluminumsinkers. Three-milliliter samples were withdrawn after 5, 10,and 15 min, filtered with a 10-mm VanKel filter, and analyzedusing HPLC. (Details of this procedure is provided elsewhere[9]).

HPLC analysis. The HPLC analysis of aqueous CoQ samples waspreviously described by Nazzal et al. (9). In summary, CoQ wasanalyzed using a C18, 3.5 150 mm reverse-phase column(Nova-Pak; Waters, Milford, MA) at ambient temperatures. Amobile-phase composition of methanol:n-hexane (9:1) at a flow rate of 1.5 mL/min was used to elute CoQ at a wavelength of 275 nm. The samples were loaded into the autosampler (712

WISP) and then analyzed using the HPLC instrument attachedto a 510 pump and a 490E UV detector. The counts for areaunder the peak were determined using STAR 5.3 software (Var-ian, Walnut Creek, CA).

Results and discussionDetermination of CoQ solubility in limonenes. The solubility of CoQ in R- and S-limonenes was determined to assess the useof these solvents to prepare CoQ SNEDDS. Results indicatedthat CoQ is fairly soluble in monoterpenes,R-limonene (571.6 5.03 mg/mL), and S-limonene (543 3.6 mg/mL). As a re-sult of stability problems posed by fixed oils and essential oils

used in SNEDDS (2), using monoterpenes as an alternative tooils in the preparation of SNEDDS might provide formula-tions with increased stability. Microemulsions consisting of

Table II: CoQ SNEDDS with R-()-Limonene, Cremophor EL, and Capmul GMO-50.

Particle-size

Appearance Spontaneity of Turbidity volume weighing

Formulation Ratio of SEDDS emulsification NTUs SD (nm) SD

1 1:1 Good, clear Good 80 3 43.3 2

2 1:1 Good, clear Good 209 7 911.7 2

3 1:1 Good, clear Good 27 1 911.8 80

4 1:1 Good, clear Good 84 5 70.2 5.1

5 1:1 Turbid Bad 405 8 43 0.3

6 1:1 Turbid Bad 62 4 69.2 3.9

7 1:1 Turbid Bad 73 6 52.6 3.9

8 1:1 Turbid Bad 69 5 1000

9 1:1 Turbid Bad 97 1 41.1 3.7

10 2:1 Good, clear Good 40 1 25.5 1.6

11 2:1 Good, clear Good 303 7 911 80

12 2:1 Good, clear Bad 247 8 911.7 80

13 2:1 Good, clear Bad 240 7 33.7 2.2

14 2:1 Turbid Bad 431 6 51.6 1.7

15 2:1 Turbid Good 91 2 32.7 3.4

16 2:1 Turbid Bad 277 7 10.9 0.8

17 3:1 Good, clear Bad 214 8 911.3 0.8

18 3:1 Good, clear Good 217 3 148.6 0.8

19 3:1 Turbid Good 103 1 40.5 5.4

20 3:1 Turbid Good 103 2 12.9 0.8

21 4:1 Good, clear Good 207 3 99.6 24.8

22 4:1 Good, clear Bad 520 8 82.6 11

23 4:1 Turbid Good 135 8 29.2 3.5

24 4:1 Turbid Bad 272 4 30.8 3.4

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ethoxylated sorbitan ester (Tween 60), water, R-()-limonene,

ethanol, and propylene glycol were prepared and used as food-grade vehicles for enhancing the phytosterols solubilization(7). Microemulsions that are vehicles for enhancing solubi-lization of natural-food supplements increase the solubiliza-tion of lipophilic antioxidant, lycopene (10), thereby indicat-ing the effectiveness of R-()-limonene in emulsion formations.

DSC of CoQ and binary systems of CoQ with limonenes. Figure 1shows thermographs for CoQ and binary systems of CoQ withlimonene (R or S) mixed at a ratio of 90:10 w/w. The meltingpoint of CoQ and a binary system of CoQ with R-()-limonenewas 49 C. A binary mixture of CoQ with S-()-limoneneexhibited a broad endotherm with a melting point of 44 C.

These results could be attributed to the physicochemical differ-ences owing to the isomerism of these two enantiomers. Fur-thermore, lemon oil—volatile oil consisting of limonenes—reduces the melting point of CoQ by forming eutectic semi-solid product. Thus, the reduction in peak time for the binary mixture of CoQ with S-()-limonene could be a result of eu-tectic formation, whereas the R-()-limonene did not effectthe CoQ melting point.

FTIR spectroscopy. CoQ compatibility with limonenes wastested using FTIR spectrometry in conjugation with ATR. Bi-nary mixtures of CoQ with limonenes (ratios from 90:10 to10:90) were scanned for absorbance over a range of 4000 to 400

wave numbers (cm1

) at a cm1

resolution. Figures 2a–c show the absorption spectra. Yang and Her demonstrated using FTIR in conjugation with ATR to handle liquids and semisolids (11).

Several characteristic sharp peaks were ob-tained in the absorption spectrum of CoQ.CoQ bands at 1608 cm1 corresponds tothe benzoquinone ring and another char-acteristic peak for monosubstituted iso-prenoid units was observed at 1643 cm1

(see Figure 2a). No significant change inthese peaks was observed for the binary mixtures of CoQ with R-limonene (see Fig-ure 2b). This resemblance of characteris-tic peaks indicates the intactness of CoQmolecular structure in binary mixtureswith R-limonene. These two characteris-tic peaks of CoQ, however, decreased asthe ratio of S-limonene increased in the bi-nary mixture with CoQ (see Figure 2c).This disappearance of characteristic peaksat higher S-limonene concentration (70%

S-limonene) in CoQ binary mixture indi-cates possible interactions or degradation.The FTIR spectrum for all the ratios of R-limonene indicated no chemical interac-tion in these binary mixtures, thus suggest-ing the compatibility of CoQ withlimonenes.

Stability of CoQ in limonenes. The stabil-ity of CoQ in R or S limonene was eval-uated at 4, 25, and 37 C with 60% RH;37 C with 60% RH; and 45 C with 75%

RH for as long as 3 months. Figures 3a and 3b depict that the

amount of CoQ remained stable when subjected to the abovetemperature and humidity conditions. CoQ was found to bestable with less than 5% degradation in both forms of limonene.Results of the solubility, DSC, and FTIR studies clearly show that these essential oil components can be used with CoQ with-out causing significant chemical interactions or degradation.

Formulation of the self-nanoemulsified systems. A series of self-emulsifying systems were prepared by using a constant amountof CoQ (25%) with varying concentrations of the limonenes(R or S), Cremophor EL, and Capmul GMO-50. The compo-sitions of these formulations included 1:1, 2:1, 3:1, and 4:1 ra-tios of surfactant to cosurfactant. Twenty-four formulations for

each R- or S-limonene were designed with various ratios (seeTable I). Because the drug and cosurfactant are fixed to a cer-tain percentage, the only variables in these formulations werethe surfactant and limonene. The amount of cosurfactant wasfixed to one part for 1,2, and 3 parts of surfactant and screenedfor a formulation with greater emulsification properties. Thesurfactant is limited in its use because of restrictions in theamount of oils it can emulsify. Incorporating cosurfactant sta-bilizes the diffusional degradation of the emulsions by form-ing miniemulsions (12). For this study, cosurfactant, a mono-diglyceride of food-grade oleic acid Capmul GMO-50 (glycerolmono-oleate),was selected because of its multifunctional emul-

sifying capabilities. Capmul GMO-50 complies with the FoodChemical Codex for glycerol mono-oleate and is generally rec-ognized as safe according to 21 CFR 184.1505.

Table III: CoQ SNEDDS with S-limonene, Cremophor EL, and Capmul GMO-50.

Ratio of Particle-size

Surfactant: Appearance Spontaneity of Turbidity volume weighting

Formulation Cosurfactant of SNEDDS emulsification NTUs SD (nm) SD

1 1:1 Good, clear Good 37 4 156 22.6

2 1:1 Good, clear Good 211 7 18.6 1.3

3 1:1 Good, clear Good 68 4 12 0.5

4 1:1 Good, clear Good 524 3 92.2 12

5 1:1 Turbid Bad 122 7 911.0 80

6 1:1 Turbid Bad 135 2 36.9 2.1

7 1:1 Turbid Bad 204 8 911 79

8 1:1 Turbid Bad 152 4 911.9 80

9 1:1 Turbid Bad 149 1 35.8 4.8

10 2:1 Good, clear v. Good 49 2 124 17.7

11 2:1 Good, clear Good 25 5 911.8 80

12 2:1 Good, clear Good 105 2 136.6 14

13 2:1 Good, clear Good 147 3 98.0 10.5

14 2:1 Turbid Good 282 7 11.9 0.5

15 2:1 Turbid Bad 300 8 12.1 0.4

16 2:1 Turbid Bad 717 6 50.3 4.9

17 3:1 Good, clear Good 138 7 73.4 9.2

18 3:1 Good, clear Good 167 4 91.6 13.1

19 3:1 Turbid Good 79 1 17.5 1.8

20 3:1 Turbid Bad 350 5 10.9 0.8

21 4:1 Good, clear v. Good 99 3 10.4 0.7

22 4:1 Good, clear Bad 354 8 21.5 2.2

23 4:1 Turbid Bad 144 4 38.3 4.1

24 4:1 Turbid Good 522 6 17.2 2

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Because the hydrophile–lipophile balance (HLB) of the mix-ture is an important factor for obtaining the maximum emul-

sification rate, the surfactant and cosurfactant should be se-lected to yield a mixture with optimum HLB (13,14). CapmulGMO-50 has a calculated HLB value of 3–4 and thus can beused with high HLB-value surfactants such as Cremophor ELto obtain optimum HLB of the mixture. Cremophor EL is apolyethoxylated castor oil having an HLB value of 12–14. Itfunctions as nonhemolytic, and it is a solvent-compatible emul-sifier and solubilizer. In this study, surfactant:cosurfactant ra-tios of 3:1 and 4:1 formed more stable emulsions with smallerglobule size. The spontaneity and efficacy of the emulsificationshowed that stable emulsions formed with a low amount of oilsand low surfactant concentrations. This explains the advantage

of using a cosurfactant to avoid the toxic effects related to usinghigher surfactant concentrations.

Visual observations. The self-emulsification properties of for-mulation in a capsule (containing 30 mg of CoQ) were assessedby measuring the tendency to spontaneously form a transpar-ent emulsion. Tables II and III show the results for spontaneity of emulsification and appearance of formulations for R- andS-limonenes, respectively. These results indicated the forma-tion of stable emulsions at higher oil concentrations with allratios of surfactant and cosurfactant, which could be a resultof the solubility of CoQ in limonenes. Further, the presence of surfactant and cosurfactant helps in the formation of oil in

water emulsions.A phase diagram for the R-()-limonene formulations iden-

tifies the good self-emulsifying region (see Figure 4). Among

the screened formulations, 3:1 and 4:1 ratios of surfactant tocosurfactant showed maximum oil loading as high as 90% andthus the solubilization of CoQ. These results are in accordancewith the solubilizing capacity of oil and surfactants.

Turbidity and particle-size were measured for the resultantemulsions (see Tables II and III). Turbidity, a linear measure-ment inversely proportional to the solubility of the compound,has been used to predict the dissolution characteristics of com-pounds (15). In our studies, the turbidity of the CoQ formula-tions in R-()-limonene increased at higher surfactant andco-surfactant levels; that is, when the oil concentration was low.The turbidity of CoQ formulations in S-()-limonenes, how-

ever, showed comparatively higher values, except for formula-tions 3,10, 19, and 21. This result could be attributed to the for-mation of mesomorphic phases at the surfactant–water interface.When SNEDDS are diluted with water, various mesomorphicphases are formed (16). No particular trend was observed forturbidity of these formulations to correlate with dissolution.

Particle-size measurements of these formulations revealedthe formation of nanoemulsions (see Tables II and III). Themean diameters for the volume-weighed particle-size determi-nations of most formulations were below 100 nm, and someformulations also showed bimodal distributions having anotherpeak at 911 2 nm. The secondary peak obtained at 911 nm

was constant for all formulations and was present for blank (i.e.,empty) shells of HPMC capsules dissolved in water. Hence,weconcluded that the formulations obtained from using chiral

Table IV: Zeta potential.

S-Limonene R-Limonene

Formulation formulations formulations

1 30.31 0.91 297.01 8.89

2 2393.20 40.7 425.97 4.28

3 42.83 6.88 66.77 4.03

4 52.78 4.21 9.55 3.94

5 280.97 34.4 28.09 5.40

6 45.26 4.14 123.61 14.7

7 815.75 27.25 84.24 3.91

8 820.47 47.10 268.38 1.75

9 1.98 0.12 84.24 5.12

10 548.61 6.54 26.78 6.91

11 65.61 3.53 160.89 6.6

12 62.52 8.39 4.58 1.18

13 50.58 9.23 67.35 0.69

14 64.76 2.26 15.11 1.30

15 7.00 0.68 4.96 2.10

16 160.15 8.16 67.58 1.64

17 7.08 0.91 34.32 5.90

18 0.86 9.12 72.14 1.39

19 61.99 5.18 26.41 1.82

20 10.46 3.11 80.26 4.37

21 49.96 0.75 7.04 0.98

22 70.66 7.25 197.18 8.41

23 76.34 3.43 109.98 9.04

24 5.49 1.78 508.12 10.74

Table V: Dissolution studies (% released after 15 min).

R-Limonene

S-limonene R-limonene formulations

Formulation formulation formulation (VanKel method)

1 64.60 0.69 62.87 2.68 9.76 5.24

2 47.87 0.44 34.34 2.22 2.48 0.79

3 42.98 0.90 24.03 2.12 2.08 0.48

4 28.34 1.04 30.94 3.33 0.94 0.02

5 28.19 1.14 56.22 15.44 0.00 0.00

6 19.99 0.25 51.47 12.70 0.57 0.81

7 9.95 0.10 40.92 5.97 0.21 0.30

8 1.66 0.01 22.64 2.74 0

9 1.56 0.14 1.51 0.23 1.06 0.51

10 51.99 0.40 69.1 14.24 1.01 0.78

11 64.15 0.34 66.1 1.99 3.66 3.36

12 49.74 0.13 34.8 0.80 15.05 4.35

13 41.19 0.22 67.35 0.69 16.28 7.79

14 51.93 0.26 61.50 0.30 36.07 6.80

15 7.00 1.28 33.62 2.12 1.46 2.07

16 5.49 1.90 9.06 0.54 0

17 0.86 1.22 64.58 0.45 40.64 5.56

18 40.68 3.58 53.24 0.31 62.59 20.05

19 17.07 1.47 42.76 0.88 112.91 1.11

20 6.62 0.11 9.78 0.34 20.83 10.52

21 0.75 0.21 69.31 1.90 58.38 13.24

22 17.25 3.32 44.99 0.40 98.65 0.85

23 25.29 3.31 55.53 1.04 117.64 25.36

24 4.44 0.73 23.98 5.74 6.06 0.42

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molecules, limonenes, are nanoemulsions. Because R-()-limonene is studied more extensively for its use in microemul-sions of food supplements compared with S-()-limonene andit shows better compatibility in DSC studies, R-()-limoneneis of interest for further studies.

Zeta potential. Table IV lists the zeta potential values obtained

for the 24 formulations prepared with R-()-limonene and the24 formulations prepared with S-()-limonene. The values ob-tained for limonene formulations are strongly negative. An ab-

solute zeta potential25 mV indicates a deflocculated systemin which repulsive forces exceed the attractive forces, thereby keeping the particles dispersed. In contrast, a zeta potential25mV indicates a flocculated system in which particles come

together as a result of attractive forces exceeding the repulsiveforces. Table IV shows that R-limonene formulations resultedin 16 deflocculated systems, S-limonene formulations resultedin 4 deflocculated systems, and all other formulations showedhighly negative zeta-potential values. The zeta potential allowsprediction of emulsion stability (17). This study showed thatusing R-limonene formed more stable formulations than usingS-limonene.

Dissolution studies. Dissolution of S- and R- limonene formu-lations was carried out to evaluate the release of drug from theformulations and to determine the emulsifying capacities of SNEDDS to solubilize the drug in aqueous medium. Cumula-

tive percent released after 5, 10, and 15 min was analyzed, andthe dissolution rate within the first 15 min appeared to be fastenough to release the drug up to 100% in few formulations. Ini-tial screening of the formulations was performed using a flask method to get a rough estimate of the dissolution characteris-tics. The results, shown in Table V, indicate that the maximumrelease of drug from these formulations was as high as 60%,which could be attributed to the delayed solubility of the HPMCcapsule shells and thus incomplete release of the drug. HPMCcapsule shells dissolve within 3–4 min in aqueous media (18).The release of CoQ, further analyzed using USP VanKel disso-lution apparatus, indicated that formulations 19, 22, and 23

showed faster and complete drug release above 90%. The re-maining formulations, however, did not release the drug, owingto the encapsulation of drug in the capsule-shell material. The

0.05

0.000.04

0.02

0.00

0.04

0.02

0.000.06

0.04

0.02

0.02

0.01

4000 3000 2000 1000

Co-Q

R-limonene

S-limonene

R-limo 50

S-limo 50

Wavenumbers (cm-1)

A b s o r b a n c e

1 4 4 4

. 3 2

1 6 1 9

2 9 6

1 2 6 2

. 7 0

1 0 9 4

. 0 5

8 8 6

. 4 9

0.05

0.05

0.00

0.05

0.000.04

0.060.040.02

0.05

0.05

4000 3000 2000 1000

R-limo 10

Wavenumbers (cm-1)

A b s o r b a n c

e

2 9 5R-limo 20

R-limo 30

R-limo 40

R-limo 50

R-limo 60

R-limo 70

1 4 5 0

. 8 0

1 1 0 7

. 0 0

8 8 0

. 0 0

7 8 2

. 7 0

0.00

0.01

0.00

0.01

0.00

0.02

0.02

0.01

0.02

0.04

4000 3000 2000 1000

S limo 10

Wavenumbers (cm-1)

A b s o r b a n c e

S limo 20

S limo 30

S limo 40

S limo 50

S limo 60

S limo 70

0.01

0.01

0.02

a

b

c

Figure 2: (a) FTIR spectra of CoQ, R-()-limonene, S-()-limonene,

and mixtures of 50%

50% CoQ:R-limonene and CoQ:S-limonene; (b)FTIR spectra of mixtures of CoQ with increasing ratio of R-()-

limonene (from 10% to 70% w/w); (c) FTIR spectra of mixtures of CoQ

with increasing ratio of S-()-limonene (from 10% to 70% w/w).

-5

20

45

70

95

120

0 20 40 60 80

4 deg

25 deg

25 deg 60% RH37 deg

37 deg 60% RH

45 deg 75% RH

100

-5

20

40

60

80

120

0 20 40 60 80

4 deg

25 deg

25 deg 60% RH

37 deg

37 deg 60% RH

45 deg 75% RH

100

100

a

b

Figure 3: (a) Stability of CoQ in R-()-limonene; (b) stability of CoQ in

S-()-limonene.

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range of oil, Cremophor EL, and Capmul GMO-50 for theseformulations was 15–67.5%, 6–48%, and 1.5–12%, respectively.Cremophor EL has been used as a surfactant in microemul-sions. It increases the dissolution rate as well as the bioavail-ability of numerous lipophilic drugs and provides clinically ben-eficial effects (19). In these studies, limonenes minimized the

use of higher concentrations of Cremophor EL, which wouldotherwise be toxic at high concentrations.

ConclusionThe present investigation illustrated the potential use of essentialoil pure components for the preparation of self-nanoemulsified

drug delivery systems. The SNEDDS prepared from chiral mole-cules of limonene showed good stability and enhanced solubility for formulation development. DSC and FTIR studies revealed nochemical interactions of these excipients with CoQ. The dissolu-tions profiles indicated immediate-release liquid dosage forms of CoQ using R-()-limonene with less than 50% of CremophorEL. Limonenes provide an attractive alternative to using essentialoils in SNEDDS of lipophilic drugs.

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0.00

0.25

0.50

0.75

1.00

1.00

0.75

0.50

0.25

0.001.000.00 0.25 0.50 0.75

R limonene

C

a p m u l

G M O

C r e m o p

h o r E

L

Figure 4: Pseudo-ternary phase diagram indicating the efficient self-

emulsification region for formulations with R-()-limonene.

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