INTRODUCTION

Pruritus manifests as an unpleasant sensation urging one to scratch. As one of the most common clinical manifestations in medicine, it is divided into acute and chronic stages, based on the duration of the clinical manifestation. Furthermore, it may manifest clinically as an adverse reaction to an administered drug. Its pathogenesis may involve several cytokines and inflammatory neuro-mediators and usually involves the central and peripheral nervous system [1].

Drugs may cause itching as a concomitant symptom of drug-induced skin reactions or in the form of pruritus without skin lesions [2]. Chloroquine-induced pruritus has been reported as being more predominant in black-skinned people and people of negroid descent while administration of the drug to whites rarely elicits itching , thus suggesting a genetic pre-dispositon to the symptom [3]. Furthermore, in vitro skin cell studies showed that chloroquine preferentially binds to melanocytes as opposed to keratinocytes [4]. The non-histaminergic nature of CQ itch makes it a good model for probing the mechanisms of chronic itch.

There are striking similarities between CQ itching pathways and other forms of chronic itch. These include the shared roles of skin, kappa opiate receptor, nitric oxide, serotonin via 5HT1B/D receptors, neural and spinal μ opiate receptors, cytokines (especially interleukins), and tumor necrosis factor [5].

Two varieties of coconut oil exist: virgin and refined oil. Virgin coconut oil (VCO) is made by cold-pressing the liquid from the fresh part of coconut meat. It has a milky appearance. This method of oil extraction preserves the contents of vitamin E, pro-vitamin A, and polyphenols. Various properties of this oil have been reported as anti-inflammatory, analgesic, and anti-cancer [6]. Natural plant oils, due to their easy availability, accessibility, and low cost are most commonly used globally for treating various skin ailments [7, 8]. Coconut oil contains compounds that exhibit antimicrobial, anti-inflammatory, antioxidant, and anti-pruritic actions making it an important, cost-effective, alternative, and complementary medicine to target various xerotic and inflammatory skin diseases in which the skin barrier is disrupted [9, 10].

CQ-induced pruritus has been an obstacle leading to poor compliance in patients [11]. Regardless of the route of administration, CQ is a major inducer of a very discomforting, generalized body itch within about 2 hours of administration. Chloroquine-induced itch is mostly independent of the route of administration as many patients are forced to stop further drug ingestion. This has led to poor patient compliance, CQ-induced resistance, increased cases of relapse, and an overall poor prognosis. Chloroquine-induced itching is often regarded as a characteristic ‘histamine-independent’ itch signaling pathway [12].

Several remedies for itching exist with varying degrees of efficacy [13]. Unfortunately, antihistamines are only effective if the itching (pruritus) is caused by histamine (as in urticaria). They only benefit non-histamine-mediated itch through their sedating tranquilizing properties [14].

Several substances have been shown to reduce the itching associated with chloroquine [15-18] while some other mechanisms have been discovered to ameliorate the itching associated with chloroquine use [19-21].

It has been reported that chlorpheniramine or hydroxyzine hydrochloride provided symptomatic relief [22].

Nwonu et al. [23] investigated the possibility of interaction upon concomitant administration of coconut fruit water and chloroquine sulfate in adult male rabbits. The results of the study demonstrated a strong interaction between coconut fruit water and chloroquine sulfate thus reducing its bio-availability. This oral administration is helpful for the management of drug toxicity. However, it can interfere with certain pharmacokinetic properties such as its volume of distribution, and plasma half-life in the process. There is a need for a dosage form that is less interfering with the drug’s pharmacokinetics. Choi et al. [12] reported that crotamiton inhibits both histamine- and chloroquine-dependent itch pathways and suppresses scratching behavior induced by both compounds in mice.

Coconut water and oil are already known to the population in their natural state as an age-long itch relief. There is a need for a natural pharmaceutical formulation that possesses: water solubility in external body fluids, cutaneous penetrability, cosmetic appeal (hair and skin), little or no pharmacokinetic interference, little or no adverse effects, ease of administration, sustained actions, safety, and physical stability.

Hydrogels have the ability to form three-dimensional polymer networks that are hydrophilic in nature and can absorb water to expand without dissolving. This makes them an attractive choice in drug delivery. Particularly, carboxymethyl cellulose hydrogels have attracted considerable research attention for the development of safe drug delivery carriers because of their non-toxicity, good biodegradability, good bio-compatibility, and low immunogenicity [24].

This study aims to formulate, characterize, and evaluate coconut water- and oil-loaded gels for their anti-pruritic activities on female Wister rats using the chloroquine-induced pruritus model in rats.

MATERIALS AND METHODS

Materials

Plant materials

Mature coconuts (Cocos nucifera) used in this study were harvested in March, from a local herbalist farm in an open market in Awka, Anambra State, Nigeria. The plant samples were identified by a taxonomist and deposited at the Pharmacognosy and Traditional Medicine Department, Faculty of Pharmaceutical Sciences, Agulu, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.

Reagents

Ethanol (absolute), triethanolamine (JHD, Guangdong Guanghua Sci-Tech Co., China); chloroquine phosphate injection 40mg/ml (Labquin), Eurax cream (Novartis Corporation, Malaysia), Carbopol (Carbomer 934), carboxymethyl cellulose (CMC), potassium sorbate (Nantong Alchemy Biotech Development Co., China), Veet^® hair removal cream (Reckitt Benckiser Co., UK).

Methods

Extraction of cocos nucifera water

Coconuts were randomly selected for each experiment [25]. The coconuts were de-husked to obtain coconut water which was poured into clean plastic bottles and refrigerated until needed.

Aqueous extraction of coconut milk

Coconut milk preparation, filtration, and separation was carried out according to Jasman et al. [26]. The coconut meats were grated, weighed, and blended using an electric blender (Molgold, United Kingdom). The blended meat was soaked in warm water (50 °C) at a ratio of 1:2 (coconut meat: water). The mixture was kneaded, wrapped in a muslin cloth, and then squeezed to obtain coconut milk. The extracted coconut milk was poured into a clean beaker and allowed to stand.

Centrifugation of VCO (virgin coconut oil)

This was done using a Centrifuge (80-2B, China) at a speed of 1000 rpm for 20 min. The obtained oil was passed filtered through a Whatman no 1 filter paper. The volume of oil was determined.

 Pilot study of anti-pruritic effects of coconut water and coconut oil

This section of the pilot study aimed to determine the best route of administration of the pruritogen.

A total of 18 female albino rats ranging from 3-6 months old and weighing between 150-200 g were obtained from the Animal laboratory facility of the Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka. Animals were kept in clean cages and had free access to food and water. The rats were fed with pelletized feed (Vital Feeds, Nigeria). Also, they had 12-hour light and dark cycles. They were kept for one week to acclimatize to their new environment. The rats were divided into six (6) groups.

A pair of scissors was used to shave the napes off its fur to a diameter of about 1.5 cm. A small quantity of Veet^® hair removal cream (Reckitt Benckiser Co., UK) was applied to the shaved portions and left for a minute, after which, a water-whetted cotton wool was used to rub off the area till fur-less skin was seen. The shaved rats were used for the study on the next day to allow for acclimatization, as they had a tendency to scratch the shaved portions after shaving.

 The next day, the rats were given coconut fluids by oral gavage using a stainless-steel feeding needle (Harvard Apparatus, USA) inserted into a 1 ml syringe (BD, USA). The dose volume of the coconut milk, water, and oil was 20 ml/kg weight of the rat. A time interval of 15 min elapsed and then a 20 mg/kg dose of chloroquine was injected into rats through selected parenteral routes of drug administration, but the rats grouped in the oral route were given 158 mg/kg of chloroquine. Evaluation of pruritus was done by video recording of the animals, followed by visualization and counting of scratching bouts. The number of scratching bouts and distance run for each animal were registered over 30 min after the CQ challenge and the results were analyzed [27].

Determination of the anti-pruritic effects of coconut oil and water on CQ-induced itch

The was done to determine the anti-pruritic efficacy of the coconut extracts, upon topical administration. The dorsal views of the neck region of the rats were shaved as previously described.  The next day, eight female Wistar rats were placed in two groups of four animals. Afterward, 0.5 ml each of coconut oil and coconut water was topically administered to the rats on the shaved portions, according to their respective groups. The rats were left for a 15-minute interval and then administered 2 mg (50 µl of 40 mg/ml) chloroquine, injected subcutaneously into the shaved portions. Evaluation of pruritus was done by video recording of the animals, followed by visualization and counting of scratching bouts as previously described [27].

Table 1. Formula for different polymer-based gels

Key

A-Coconut water gel (CMC- based); B-Coconut water gel (Carbopol-based); C-Coconut water gel (CMC + Carbopol-based); D-Coconut oil gel (CMC-based); E-Coconut oil gel (Carbopol-based); F-Coconut oil gel (CMC + Carbopol-based); G-Plain gel

Preparation of polymer-based gels of coconut water and coconut oil

Twenty grams of seven gel formulations were prepared, (labeled A-G) according to the formula presented in Table 1. Briefly, the required quantities of CMC, glycerol, ethanol, and potassium sorbate were weighed out and placed in a mortar. Water was added and the mixture was stirred gently until the CMC dissolved. Then two drops of triethanolamine were added to enhance the gelation process of the CMC. Gentle stirring continued till a uniform mixture was obtained. The mixture was transferred to a clean and dry container and made up to the required weight. The entire procedure was repeated using carbopol and then CMC/carbopol co-polymers and also with coconut oil in place of coconut water, while a plain gel was formed without the addition of the coconut oil or water.

Characterization and stability evaluation of formulated gels

The gels formed were characterized accordingly at intervals of 7, 30, and 90 days post formulation to evaluate their stability at a temperature of (25 ± 1°C).

Organoleptic characterization

The gels were physically observed for color, appearance, clarity, consistency, and phase separation.

Determination of pH

The Digital pH meter (AVI-65, India) was used. Calibration was done using buffer solutions (pH 4 and 7). Triplicate readings were taken.

Determination of viscosity

The viscosity of the gel formulations was measured with a rotational viscometer (IIDJ-1B, China) using spindle no 5.

Comparative evaluation of the anti-pruritic activity of the formulated gels

The animals (27 Wister rats) were shaved as previously described. Subsequently, the rats were divided into nine groups of three rats per group (Groups A-I).

The rats in groups A-H were administered with the formulations above and after 15 min, 2 mg (50 ul of 40 mg/ml) of chloroquine injection was injected subcutaneously to all rats in each group. Evaluation of pruritus was done as previously described [27].

Statistical analysis

The recorded data were subjected to paired t-tests to check for statistical differences in the mean number of scratching bouts across different groups. Analysis of variance (ANOVA) was carried out to assess the relationship between the anti-pruritic reductions in the different treatment classes being tested. All the statistical analyses were computed with IBM SPSS statistics software version 23. A P value < 0.05 was considered the statistically significant cut-off.

RESULTS AND DISCUSSION

Yield of coconut oil

The percentage (extraction) yield of coconut oil was 15.5 % v/w.

Pilot study on the anti-pruritic effects of coconut extracts

The results of the administration of the various extracts to animals were recorded in Table 2. 

Table 2. Pilot study on coconut extracts

Key

One scratch is indicated with asterisks (*).

A - SC (Subcutaneous); B - IM (Intramuscular); C - IP (Intraperitoneal); D - O (Oral); E – IV (Intramuscular); F – Control

Evaluation of anti-pruritic effects of coconut extracts
Figure 1 shows the anti-pruritic effects of topically-administered coconut water, and coconut oil against chloroquine-induced itching over a 30-min period.

Physical properties of coconut gel formulations

Table 3 shows the physical properties of the formulated gels. From the results, the formulations were emollient, non-greasiness, and ease removed.

Table 3. Organoleptic evaluation

Key

A-Coconut water gel (CMC- based); B-Coconut water gel (Carbopol-based); C-Coconut water gel (CMC + Carbopol-based); D-Coconut oil gel (CMC-based); E-Coconut oil gel (Carbopol-based); F-Coconut oil gel (CMC + Carbopol-based); G-Plain gel; H-Eurax.

Characterization of gels

pH

The mean pH readings of the coconut-based gels over 90 days are presented in Figure 2a. The pH values obtained ranged from (7.00 ± 0.49) to (9.5 ± 0.03).

Mean viscosity

The mean viscosity values of the coconut-based gels over 90 days are presented in Figure 2b. The values obtained ranged from (201 ± 19.56) mPaS to (1890 ± 56.3) mPaS. The values of the formulations were constant, with a slight increase between days 1 and 7.

Stability studies of gels

The stability evaluation of the gels is presented in Table 4.

From the results of the stability test, degradation of the gel formulations occurred as changes in coloration, loss of pleasant coconut aroma (Coconut water and oil gel), and separation of the oil from the formulation (Coconut oil gel). Degradation occurred in all of the gel formulations except in the coconut oil gel containing CMC and Carbopol alongside the plain gel formulations.

Table 4. Stability studies of gels

Key: +++ = Good             ++ = moderate               + = poor

A-Coconut water gel (CMC-based); B-Coconut water gel (Carbopol-based); C-Coconut water gel (CMC+ Carbopol)-based; D-Coconut oil gel (CMC-based); E-Coconut oil gel (Carbopol-based); F-Coconut oil gel (CMC + Carbopol)-based; G-Plain gel    

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