New Pharmaceutical Microemulsion System for Encapsulation and Delivery of Diospyrin, a Plant-Derived Bioactive Quinonoid Compound

Abstract

A new vegetable oil based oil-in-water microemulsion is developed and characterized as a prospective delivery system for in vivo application A particular weight percent composition 5/30/65 (clove oil/Tween-20/water) was selected (V1) from the clear oil-in-water zone of the pseudoternary phase diagram comprising clove oil, polyoxyethylene sorbitan monolaurate (Tween-20), and water. Two modifications of V1, (V2 and V3) were prepared by addition of dipalmitoyl phosphatidyl choline (DPPC), and a mixture of DPPC and cholesterol, respectively. A model drug diospyrin (a plantderived quinonoid compound) was encapsulated in the dispersed clove oil droplets of the three systems and designated as DV1, DV2, and DV3, respectively. The size of the dispersed clove oil droplets ranged between 9–20 nm as determined by dynamic light scattering. The stability of the vehicles, before and after encapsulation, was assessed under varying conditions of time and temperature and was found to be stable for 1 year and over a temperature range of 4-40°C. The ultraviolet-visible spectrum of diospyrin after encapsulation in the compartmentalized medium remained almost identical to that dissolved in chloroform. The single-dose acute toxicity of V1 and DV1 was assessed in vivo by carrying out survival study and enzyme assay in Swiss Albino mice.The vehicle was safe at a volume of 0.05 ml when injected intraperitoneally into the mice.

Keywords :

• Diospyrin
• Clove Oil
• O/W Microemulsion
• Quinonoid
• Tween-20

Development of microemulsions (Lawrence 1996; Paul and Moulik 1997; Tenjarala 1999; Trotta 1999; Moreno et al. 2000), liposomes (Gregoriadis 1993), niosomes (Uchegbu and Florence 1995; Sinha et al. 2002), organogels (Kantaria, Rees and Lawrence 1999), and nanocapsules (Watnasirichaikul et al. 2002) as drug delivery systems has been a major challenge for the pharmaceutical industry during the past decade. These organized systems used as carriers could achieve increased solubilization of lipophilic drugs with concomitant modification of their pharmacokinetic profiles, often leading to improvement in therapeutic efficacy (Trotta, Ugazio, and Gasco 1995; Corswant, Von Thorem, and Engstrom 1998; Cortesi and Nastruzzi 1999; Park and Kim 1999). Moreover, these systems are known to protect the encapsulated drugs against enzymatic degradation (Sariciaux, Alan, and Sado 1995) and to enhance membrane permeability induced by the surfactant and cosurfactant constituents (Swenson and Curatolo 1992).

It is desirable that delivery systems be biodegradable and nontoxic. In addition, tolerance toward additives, stability over a wide range of temperatures, low viscosity, small size, and ease of elimination from the body also are required for therapeutic application. Moreover, the size of the encapsulated particles needs to be controlled to avoid blockage of capillaries; hence, submicron-sized delivery systems are preferable for parenteral use (Kreuter 1994). Since microemulsions satisfy most of these criteria, and also because of their thermodynamic stability, optical clarity, and ease of preparation, considerable interest has been generated over the years to develop them as drug delivery systems. Thus, nontoxic and biocompatible amphiphiles are used to prepare “biological microemulsions,” in which vegetable oils (Acharya, Sanyal, and Moulik 2001a), fatty acid esters (Acharya, Sanyal, and Moulik 2001b), and triglycerides (Corswant et al. 1998) are used instead of aliphatic oils. Encapsulation of water insoluble drugs in the nanometer-sized oil droplets of the oil-in-water microemulsion might lead to its dissolution into aqueous phase in vivo resulting in better absorption.

The present work describes, for the first time, the preparation and physicochemical characterization of a stable o/w microemulsion composed of clove oil, Tween-20, and water. Clove oil, obtained from the spice plant Eugenia caryophyllata, and its main constituent, eugenol, both are used in pharmaceutical preparations, particularly to treat dental problems (Selvi and Niranjali 1998; Jadhav et al. 2004) Further, the application of this microemulsion also was explored to encapsulate a model drug, diospyrin, which is a highly lipophilic bioactive compound derived from an indigenous plant (Hazra et al. 1984). Presently, diospyrin, a bisnaphthoquinonoid, has emerged as a potential “lead molecule” for development of new drugs against cancer (Chakraborty et al. 2002), tuberculosis (Lall et al. 2003), and several parasitic diseases like visceral and cutaneous leishmaniasis (Ray et al. 1998; Hazra et al. 2002), which are causing major public health problems worldwide. Incidentally, the preparation of o/w microemulsion for delivery of another lipophilic drug, amphotericin B also used in the treatment of several types of leishmaniasis, recently has been reported (Moreno et al. 2001). In our present study, application of the clove oil-in-water microemulsion was examined as a delivery system in terms of its single-dose toxicity in vivo and compared with the dimethylsulfoxide formulation generally used for the delivery of lipophilic compounds in biological experiments.

MATERIALS AND METHODS

Clove oil was purchased from SD Fine Chemicals, India, with the following specifications (Gupta, Mukhopadhya, and Moulik 1995); acid value = 5.12 mg KOH/g; saponification value = 5.23 mg KOH/g; iodine value = 88.34% I₂; eugenol content 80%. The surfactant polyoxyethylene sorbitan monolaurate (Tween-20), cholesterol, dipalmitoyl phosphatidyl choline (DPPC), and dimethylsulfoxide (DMSO) were from Sigma Chemicals (USA).

Diospyrin was isolated from the stem-bark of Diospyros montana Roxb., and purified as described earlier (Hazra et al. 1984). Its chemical structure is presented in Figure 1). Spectroscopically pure chloroform, sodium pyruvate, 2, 4-dinitrophenyl hydrazine, α -ketoglutaric acid, and DL-alanine were purchased from SISCO Research Laboratories (India). p-Nitrophenylphosphate (PNPP) was a product of Loba Chemicals India, and diethanolamine was purchased from Merck, India. Doubly distilled water was used in all aqueous preparations.

FIG 1. Chemical structure of diospyrin.

Methods

Construction of Ternary Phase Diagram

To construct the phase diagram of the pseudoternary system of clove oil, Tween-20, and water, the amphiphile was dissolved in clove oil in different ratios in several stoppered test tubes and they were placed in a constant temperature water bath. To each of these samples, water was added from a micropipette under constant stirring condition until the solution became just turbid due to phase separation. The measurements were checked for reproducibility, and the compositions were expressed in weight percent to construct the phase diagram on triangular coordinates (Figure 2).

FIG 2. Phase diagram of the pseudoternary system of clove oil/Tween-20/water.

Selection of Vehicle Composition

For the encapsulation of diospyrin, a weight percent composition of clove oil/Tween-20/ water as 5/30/65 (V₁) was selected from the oil-in-water region of the pseudoternary phase diagram marked as S in Figure 2. Two other modifications of the delivery system were prepared: a DPPC (V₂) and a mixture of DPPC and cholesterol (V₃), respectively (Table 1a).

TABLE 1. Composition of drug delivery system before and after encapsulation of diospyrin

Composition	System
Clove oil/Tween 20/water (5/30/65) microemulsion	V1
V1 + 3.0 mgDPPC	V2
V1 + 1.5 mg DPPC + 0.4 mg cholesterol	V3
V1 containing 1.5 mg diospyrin	DV1A
V1 containing 1.0 mg diospyrin	DV1B
V2 containing 1.5 mg diospyrin	DV2
V3 containing 1.5 mg diospyrin	DV3

DPPC = dipalmitoyl phosphatidyl choline.

Encapsulation of Diospyrin in Microemulsion

First, 1.5 mg and 1.0 mg of diospyrin were dissolved separately in two portions of clove oil (0.25 ml each) and allowed to stand overnight to ensure complete dissolution. To each of the preparations, Tween-20 (1.5 ml) was added followed by water (3.25 ml). The compositions were thoroughly mixed in a vortex mixer to obtain the encapsulated samples (DV_1A and DV_1B, respectively). Similarly, diospyrin (1.5 mg each) also was incorporated in V₂ and V₃ to produce DV₂ and DV₃, respectively. The compositions of the delivery systems before and after incorporation of diospyrin are given in Table 1a. The final concentration of diospyrin in DV_1A and DV_1B amounts to 0.3 and 0.2 mg/ml, respectively.

Diospyrin Dispersion in Dimethyl Sulphoxide

A dispersion (D_p) of diospyrin in DMSO was prepared by maceration of the compound with a droplet of Tween-20. A calculated volume of DMSO was used to prepare D_p to deliver the requisite dose of diospyrin in vivo to compare its effect with the same amount of the drug delivered through its microemulsion formulation (DV₁). The vehicle (DMSO + Tween-20) also was used as a “control” in the biological studies presented in Table 2 and Table 3.

TABLE 2. Single dose acute toxicity, in Swiss mice, of a microemulsion vehicle (V₁), diospyrin encapsulated in the same vehicle (DV_1A and DV_1B), and diospyrin dissolved in DMSO with the help of Tween−20™(D_p)

Group of mice	Diospyrin dosage (mg kg^−1)	Injection volume (ml)	Mortality (%) within 1 day	Survival (%) after 30 days
DMSO + Tween 20
	—	0.10	0	100
V1	—	0.10	33.3	66.7
V1	—	0.05	3.3	96.7
Dp	0.75	0.05	6.7	93.3
DV1A	0.75	0.05	3.3	96.7
DV1B	0.50	0.05	0	100

DMSO = dimethylsulfoxide. The standard deviations in mortality and survival of the animals were within ±8%.

TABLE 3. Estimation of the activity of alkaline phosphatase (AP) and glutamate pyruvate transaminase (GPT) in blood serum of Swiss A mice collected 24 hr after treatment

Groups of mice	Dose of Dp^a (ml/kg)	AP activity unit^b	GPT activity unit^c
Untreated normal	Nil	8.9 ± 1.7	24.4 ± 2.3
DMSO^d	Nil	8.9 ± 0.9	21.1 ± 3.4
V1	Nil	7.7 ± 0.5	23.0 ± 2.1
Dp	0.75	8.7 ± 0.5	20.3 ± 1.5
DV1A	0.75	8.6 ± 0.5	29.8 ± 3.2
DV1B	0.50	7.2 ± 2.4	21.7 ± 1.4

^aVolume of vehicle used for intraperitoneal treatment = 0.05 ml.

^bUnit for AP activity: μ mol of p-nitrophenylphosphate hydrolyzed/min/dl of serum.

^cUnit for GPT activity: μ mol of pyruvate formed/min/dl of serum.

^dDMSO = dimethylsulfoxide.

Characterization of the Delivery System

The empty as well as the drug-encapsulated microemulsions—V₁, V₂, V₃, DV_1A, DV_1B, DV₂, and DV₃—were characterized in terms of macroscopic appearance, particle size, surface tension, and pH. Their color, isotropy, and homogeneity were examined visually, as well as by cross-polarized light at room temperature. The droplet size distribution of the oil phase and the polydispersity of empty and drug-loaded vehicles, as well as their modifications, were determined by dynamic light scattering (DLS). Ultraviolet-visible spectra of diospyrin dissolved in a chloroform solution, as well as preparations DV_1A, DV₂, and DV₃, were recorded.

The pH of the delivery system with and without diospyrin was determined in triplicate at room temperature using a global digital pH meter. The pH was maintained at 7.0 by dilute NaOH. The surface tension of the drug delivery vehicle as well as the drug-loaded vehicle was determined with a calibrated du Nouy Tensiometer (Kruss, Germany) by the platinum ring detachment technique. The results were accurate within ± 0.1 mN m⁻¹. The surface tension of V₁ and DV_1A was 29.5 mNm⁻¹.

DLS Measurements

The dimensions of the nano-dispersions were measured in a DLS spectrophotometer (Model DLS 700, Otsuka Electronics, Japan), using a He-Ne laser of wavelength 632.8 nm (Moulik et al. 1999). The sample tube was placed in the thermostated chamber of the goniometer, and the scattered intensity was monitored at 90°. The autocorrelation function of the scattered intensity was obtained from a 1024-channel photon correlator. The experimental solutions were filtered (three to four times) through Millipore filters (0.45 μ m) to remove the extraneous particles. The results of measurements for each sample were processed using the instrument's software to obtain the hydrodynamic diameter (d_h), the polydispersity index (PDI), and the diffusion coefficient (D) of the dispersed droplets. The measurements were repeated five times (Figure 3, Table 1b).

FIG 3. Particle size distribution of the drug delivery system (V_1A) of weight percent composition clove oil/Tween-20/water (5/30/65).

TABLE 1b. Dynamic light scattering data for empty and drug-loaded microemulsion of clove oil/Tween-20™/water (5/30/65)

System	Diffusion coefficient 10^7 D/cm^2 sec^−1	Polydispersity index	Hydrodynamic diameter, dh/nm
V1	2.95	0.369 ± 0.038	17.0
V2	5.91	0.453 ± 0.040	8.9
V3	5.66	0.559 ± 0.045	9.3
DV1A	6.02	0.540 ± 0.042	20
DV1B	6.02	0.540 ± 0.042	20
DV2	4.33	0.472 ± 0.042	12.2
DV3	5.17	0.491 ± 0.043	10.2

Spectroscopic Measurement

The individual spectra of clove oil, 0.25M Tween-20™ (concentration same as that in the drug delivery vehicle), and the compositions DV_1A, DV₂, and DV₃ (concentration of diospyrin 0.16 mM) were recorded in a Shimadzu (model 1601A) ultraviolet-visible spectrophotometer (Figure 4 and Figure 5). The measurements were repeated three times to check reproducibility. The biochemical studies were undertaken using Ultrospec 2000 ultraviolet-visible spectrophotometer (Pharmacia Biotech, Sweden).

FIG 4. Ultraviolet-visible spectra of diospyrin in CHCl₃.

FIG 5. Visible spectra of clove oil, Tween-20, and diospyrin encapsulated in different delivery systems. (Insets; a = clove oil; b = 0.25 mM Tween-20, c = DV_1A, d = DV₂, e = DV₃.) Concentration of diospyrin for all the spectral studies were 0.16 mM. The error in measurement is ± 1.5% that falls within size of the symbols used.

Stability Studies

The drug delivery vehicles, with and without diospyrin, were found to be physically very stable; no phase separation, appearance of haziness or turbidity, and change of color were observed even after 1 year. The stability was examined in the temperature range of 4–40°C.

Water Tolerance

All the empty and drug-loaded delivery systems were tested for dilution effect. The formulations were found to remain stable on dilution up to 60% with water.

Biological Studies

Single-Dose Acute Toxicity Studies

The study was started with two groups of Swiss A mice (body weight 20 g; 10 per group) that were administered intraperitoneally (i.p.) by a single bolus injection of 0.05 ml and 0.1 ml of V₁, respectively. As the lower dose was found to be well tolerated, the injection volume was fixed at 0.05 ml for all the treatments carried out with D_p, DV_1A, and DV_1B. Control experiments with vehicles, DMSO and V₁, also were performed similarly. The mortality occurring within 24 hr was recorded as a measure of acute toxicity of the treatment protocols used, and the survival was followed up to 30 days. All the experiments were repeated three times for confirmation, and the results (mean of three observations ± standard deviation) are presented in Table 2 as percentages of the mortality and survival of experimental mice.

Biochemical Study

Another set of experiments was set up as above with 5 mice in each group and treated in the same way. The mice were sacrificed after 24 hr to collect blood serum from each mouse for estimation of the following liver function enzymes (Bergmeyer and Gawehn 1974).

Serum Alkaline Phosphatase (AP)

A buffered substrate solution of PNPP (1.25 mM; pH 9.8) was prepared by mixing with diethanolamine (0.1M). 0.4 ml of this solution and incubated with 10 μl of serum sample at 25°C for 30 min. Sodium hydroxide solution (0.05 N; 2 ml) was added and the absorbance was measured at 420 nm. The activity was determined from the standard curve of p-nitrophenol in terms of PNPP hydrolyzed per minute.

Serum Glutamate Pyruvate Transaminase (GPT)

A buffered substrate solution (pH 7.4) was prepared by dissolving 2 mM α -ketoglutaric acid and 0.2 M DL-alanine in 0.1 M phosphate buffer. A chromogen solution was prepared with 2,4-dinitrophenyl hydrazine (1 mM dissolved in 1N HCl) and sodium pyruvate (2 mM). The serum sample was added to buffered substrate solution (1 ml) and incubated at 37°C for 30 min. The chromogen solution (1 ml) was added to this mixture and allowed to stand at room temperature for 20 min, followed by addition of sodium hydroxide (0.4 N; 10 ml). The absorbance at 520 nm was noted after 5 min, and the activity was calculated from the standard curve of pyruvate solution and expressed in terms of pyruvate formed per minute.

RESULTS AND DISCUSSION

Phase Behavior of the Ternary System

In the present work, clove oil was used as the internal phase because diospyrin was soluble in clove oil. A nearly symmetrical phase boundary has been observed with 43% amphiphile at its maximum (Figure 2). The clear “pseudomonophasic” microemulsion zone covered 48% of the total area of the triangle. The compositions falling under the curve were found to be macroscopically biphasic. To obtain a good oil-in-water zone for microemulsion, amphiphiles with high hydrophilic–lipohilic balance (HLB) values were chosen (the HLB value of Tween-20 is 16.7). A large clear zone was obtained without the addition of a cosurfactant, making the composition simple. Presence of a cosurfactant is undesirable for pharmaceutical application (Baroli et al. 2000). Enhanced solubility of steroids in three component microemulsion systems of soyabean oil, polyethylene ether surfactants, and water also is reported (Malcomson and Lawrence 1993, 1995). The majority of such pharmaceutical microemulsions use a large number of constituents and hence suffer from the complexity of their compositions (Trotta et al. 1995, 1999).

Droplet Size

In addition to composition, bioacceptability of the delivery system also is influenced by the particle size. The size of the dispersed clove oil droplets ranges between 9–20 nm (Table 1b). The nano-droplets with drug were nearly 2-fold larger than those without drug. A representative size distribution observed by the DLS method for the sample V_1A is illustrated in Figure 3. The broadness of the distribution speaks in favor of polydispersity (PDI = 0.369). Their diffusion coefficients were inversely proportional to the droplet size. Because the diameter of the dispersed clove oil droplets of the present microemulsion system is much smaller than the smallest blood capillary (400 nm), the chances of capillary blockage during transport of the vehicle should be minimum (Kreuter 1994). A small size distribution of the vehicle is favorable for higher circulation time (T_1/2) after in vivo application. Park and Kim (1999) reported micro-emulsion system of ethyl oleate, Tween-20, and water with droplet diameter of ∼100 nm. The mean droplet diameter of betamethasone encapsulated microemulsion composed of isopropyl myristate, aerosol OT, butanol, and water ranged between 20–30 nm (Trotta et al. 1990).

Diospyrin Encapsulation and Stability

Microemulsions prepared with nonionic surfactants are characteristically sensitive to temperature variations (Oh et al. 1995), whereas the present system was quite stable in the temperature range of 4–40°C. An added benefit of microemulsions with nonionic surfactants is their low susceptibility to phase separation with variations in ionic strength and pH (Constantindes 1995) which are likely to be encountered in the biological environment. Therefore, the chances of precipitation of the drug due to phase separation would be less during transport through the blood vessels.

The present systems of V₁, DV_1A, and DV_1B have been observed to tolerate 60% of water uptake. The droplet structure of o/w microemulsion is often retained on dilution (Constantinides 1995). However, the process of dilution results in gradual desorption of surfactant located at the droplet interface. The process is thermodynamically driven by the requirement of surfactant to maintain an aqueous phase concentration equivalent to its critical micelle concentration (cmc) under the prevailing condition of temperature, pH, and ionic strength. Because nonionic surfactants typically have lower cmcs, the present o/w microemulsion formulation is likely to offer superior in vivo stability. As phosphatidylcholine and cholesterol exist in the body in major amounts, a good acceptability may be expected when these substances are used as excipients for the delivery system (as in liposomes).

Phospholipids are used in a number of microemulsions meant for pharmaceutical applications, but in all such preparations, addition of cosurfactant is needed (Trotta et al. 1990, 1995). Cortesi et al. (1997) observed enhanced solubility of camptothecin in microemulsion (comprising Labrasol®, plurol isostearate, isostearyl isostearate and water) over that in water. The drug encapsulated formulation was stable for 30 days whereas diospyrin encapsulated microemulsion was stable over one year. A pseudoternary system of ethyl oleate, Tween-20, and isotonic saline was reported as a parenteral drug carrier for the poorly soluble drug flurbiprofen (Park and Kim 1999).

Spectral Features

The ultraviolet-visible spectra of diospyrin in CHCl₃ produced a sharp peak at 250 nm and a broad peak at 425 nm (Figure 4). The spectra of both clove oil and Tween-20 in water (inset Figure 5) produced intense and sharply increasing absorbance at wavelength <400 nm. The sharp peak of diospyrin at 250 nm was thus masked in the o/w microemulsion by the contributions from the oil and the amphiphile. The absorption spectrum of the drug in DV₁, DV₂, and DV₃ (depicted in Figure 5) has the same electronic transition with the broad maximum at 430 nm as has been observed for diospyrin in CHCl₃. In the compartmentalized condition, the spectral patterns of the drug with and without the additives (cholesterol and DPPC) were the same indicating that the additives did not alter the spectral feature of the encapsulated drug.

Single Dose Acute Toxicity Studies

The survival data of mice treated i.p. with a single dose of diospyrin delivered either through DMSO (D_p) or the microemulsion (DV_1A and DV_1B), along with the appropriate “vehicle control” groups, are presented in Table 2. The vehicle V₁ was found to be safe for i.p. injection at a volume of 0.05 ml. The doses of diospyrin in D_p and DV₁ were chosen to be in the same range (0.5–0.75 mg/kg) as done previously for treatment of tumor-bearing mice (Hazra et al. 1984). V_1A and V_1B were found to be reasonably well tolerated in i.p. treatment at the dose used in the present study. Performing enzyme assays on the aforesaid groups of mice also supported this. As shown in Table 3, the treatments did not produce any deleterious effect on the liver function enzymes.

CONCLUSION

Clove oil/Tween-20/water could form stable o/w microemulsions with droplet dimensions of 9–20 nm that could favorably encapsulate the oil-soluble quinonoid compound, diospyrin. From the study of the single dose acute toxicity of the microemulsion vehicle, we found that it was reasonably tolerated by Swiss mice when delivered i.p. at a dose of 0.05 ml, without and with incorporation of diospyrin. The vehicle did not impart any hepatotoxicity at this dose as we found from the assay of liver function enzymes carried out on the blood sera collected from the experimental mice. Eugenol, which constitutes ∼ 80% of clove oil, is regarded as a safe food–flavoring agent, and the acceptable limit for human intake given by the Food and Drug Administration is 2.5 mg/kg (IARC 1985, FAO/WHO 1982). In the microemulsion composition presented here, the clove oil delivered per dose in vivo was 0.125 mg/kg. The biological acceptability of nonionic surfactants has been reported (Attwood 1994). The amount of Tween-20 used in the treatment regimen (0.8 mg/kg) also was considerably less than the limit of 25 mg/kg approved by FAO and WHO (FAO/WHO 1974). Hence, the feasibility of the above microemulsion formulations as a drug delivery system for compounds that are insoluble in water but soluble in clove oil appears to be promising. Further, the prospect of its application through routes other than i.p. remains to be investigated.

S. Gupta, S. K. Sanyal, and S. Datta thank the Department of Science and Technology, Government of India, for financial support (research grant no. SP/S₁/H-31/99) and S. P. Moulik thanks the University Grants Commission for an Emeritus Fellowship. B. Hazra is grateful for a Research Grant [No.58/4/2000/BMS] from the Indian Council of Medical Research, New Delhi.