Controllable Transdermal Drug Delivery of Theobroma cacao Extract Based Polymeric Hydrogel against Dermal Microbial and Oxidative Damage

Human dermal layers are directly bared to the external environmental adver-sities like pollution, radiation, dust along with various chemical and mechan-ical stress conditions which constantly lead to the oxidative stress, eventually forming free radicals. These conditions also support the dermal microbial infections by invading the cutaneous layers. Therefore, the most efficient ap-proach accepted globally to combat these complications is to opt for transdermal application of exogenous antioxidants which helps in reducing the ill effects of oxidative stress and promotes the DNA repair. However, many scientific findings exhibited the potential role of Theobroma cacao for providing efficient skin protection. Theobroma cacao known for its vital procyanidin flavonoids, phenolic compounds, methylxanthines, catechin and epicatechin phytoconstituents with numerous health benefits, besides helping in smoothening and softening the damaged skin. These therapeutic benefits of cocoa phytocompounds are attributed to its anti-inflammatory and antioxidative characteristics. This study addresses the fabrication and optimiza-tions of Theobroma cacao extract loaded carbopol hydrogel system for increased antioxidative and antimicrobial effects. It was found in the studies that the hydrogel-based T. cacao extract (TCHG) has significantly improved the therapeutic index of the extract, fluorescens, Bacillus licheniformis, Micrococcus luteus, Aspergillus niger, Trichoderma harzianum, Rhizopus oryzae) infections. Also, the higher ROS quenching ability of TCHG as compared to T. cacao extract promises to be an effective transdermal formulation.


Introduction
Human skin is the most contacted and directly exposed organ known and thus, has multi-layered defense barrier systems for protection from external stress causes [1] [2] [3]. The dermal endogenous mechanism secures the cellular continuity, immunological balances, enzymatic defenses and cutaneous homeostasis, however, the exogenous compounds like ascorbic acid (AA), vitamin E, lycopene, cocoa etc. counter the quenching of ROS (Reactive Oxygen Species) besides, enhancing the antimicrobial properties [4] [5]. The flavonol and phenolic content of cocoa are also reported to contribute to enhancing the endogenous mechanism [6]. Cocoa also known as Theobroma cacao (T. cacao), sub-classified as Criollo, Forestero or Trinitario varieties has an essential phytoconstituents, theobromine (3,7-dihydro-3,7-dimethyl-1H-purine-2,6-dione) which belongs to the category of methylxanthines and affiliates to alkaloids (purine), affecting many physiological processes in body [7] [8] [9]. The pharmacological and toxicological properties of theobromine and other methylxanthines have been reviewed by many researchers and the chemical profile of roasted cocoa beans was found to be more complex [10]. But the primary compounds that induce its multiple beneficial functions are either naturally occurring or process-derived flavonoids, theobromine and magnesium [11]. It's been widely reported that the theobromine, an essential phytocompounds of T. cacao, scavenges reactive oxygen species (ROS) generated within the skin as a consequence of UV exposure, hormonal dysfunctioning, aging and other environmental factors which interfere with altered signaling pathways in the skin as a consequence [12] [13] [14]. Moreover, Cocoa's polyphenolic phytocompounds also help in delaying and reducing, the age-related brain impairments, including cognitive deficits in normal aging and perhaps neurodegenerative diseases too. It's also been reported to provide protection against nerve injuries, inflammation and persuade coronary vasodilatations and intensify endothelial nitric oxide content to prompt vascular slackening, expand vascular utility and also decline platelet linkage [15] [16] [17]. In the same way, polyphenols have anti-inflammatory action, over the inhibition of dissimilar transcription elements and cytokines [18] [19]. However, the current developments in pharmacological data has enhanced the competence of fragments to permit over the skin by refining the pharmacokinetics of medications; but then there is no sufficient and appropriate vehicle, that stayed de-Food and Nutrition Sciences veloped, to safeguard intense, stable and uninterrupted sustained drug delivery using non-invasive and patient compliant techniques [20] [21] [22]. Most of the dermal formulations developed are either cream or lotion based which doesn't permeate well through the skin and are less absorptive, therefore, other pharmaceutical intervention which can enhance the absorption and permeation through the multi-layered dermal structure is always preferred [22].
Hydrogels are unique materials that are elastic in nature and have a broad range of applications in cosmetics, medicine, biomaterials and food technologies [23]. They allow higher degree of dermal hydration, drug transportation and dissolution, proving to be a suitable candidate for transdermal application. Many therapeutic agents such as curcumin, turmeric, neem and sandalwood have been loaded in hydrogels for the purpose of efficient skin damage healing and treating infections [24]. It was suggested that the combination of bioactivity of cocoa encapsulated in hydrogel system may prove beneficial in treating skin infections, reducing skin oxidative stress and has a potential for cutaneous healing ability. Therefore, in the present study, we opted for developing T. cacao extract loaded hydrogel formulation that will be more patient compliant, would have higher impregnation and absorbability for transdermal application [24] [25] [26].

Sample Preparation (T. cacao Extract)
The T. cacao extract was prepared by defatting the cocoa butter from the extract completely with no loss of polyphenolic content as reported by P. Camasca, meeting the current regulation and ordinance standards regarding cocoa processing [27]. Then the cocoa extract (2 grams) was mixed with 10 ml of n-hexane solution and kept in a tightly capped reagent bottle on shaker for 24 hours. Thereafter, the defatted extract solution was subjected for ultra-sonication (25 kHz) for 30 minutes followed by a filtration and drying of filtrate in a vacuum-dryer (DZF-6050) at 37˚C and stored at −20˚C for further use [28] [29] [30].

Total Phenolic Estimation
Phenols react with phosphomolybdic acid in Folin-Ciocalteu chemical in an al-

Total Flavonoid Estimation
For quantifying the total flavonoid content in the given TCE sample, they were subjected for the aluminium chloride (AlCl 3 ) colorimetric method as reported by

Preparation of T. cacao Extract Loaded Hydrogel (TCHG)
The hydrogels are prepared and molded through various biochemical or physical crosslinks that construct the system and physical steadiness. These physical cross linkages include van der Waals interfaces or hydrogen attachment. Here in the present study, we have prepared the T. cacao defatted extract solution (TCE) loaded polymeric hydrogel by dispersion method [36] [37]. To prepare the gel carbopol-940 (10 µg/ml) was dispersed in water for two hours to get swollen up gelling mixture under constant mild stirring [38]. Further, the prescribed amount of TCE (10 µg/ml) was added in the gelling mixture along with triethanolamine (5 µg/ml) and glycerin (30 µg/ml) to maintain the pH and improve viscosity of the prepared hydrogel (TCHG

Determination of Spreadability
The spreadability analysis of optimized hydrogel formulation (TCHG) was done by determining range of the expanded diameter of TCHG, when 500 mg of the same was placed between the two glass plates. The glass plates were left for 1 minute so that the gel can spread over maximally within a circle. Thereafter, the attained diameter of the spread gel on glass plates was measured. The shorter the time interval, the better will be the spreading coefficients [42].

Scanning Electron Microscopy Analysis (SEM)
The surface morphology and shape of the optimized TCHG was analyzed by SEM (Scanning electron microscopy

DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Assay
DPPH is widely used antioxidant assay for analyzing the scavenging ability of a compound against free radicals [45]. The antioxidative effect in this assay is measured on the basis of fading away of DPPH (free radical) in test samples which turns purple to yellow color as soon as the DPPH formed by absorption of hydrogen from the antioxidant reduces. Similarly, here in the experiment, 0.5 ml of DPPH solution was added in 500 μl of the test samples (AA, TCE and TCHG) prepared in different concentrations (10 -50 μl) and incubated for 30 minutes at 37˚C followed by recording the λ maxima at 517 nm [46] [47] [48].
The scavenging ability (%) of each test samples were calculated by below mentioned formula: where, A (OC) and A (OS) refer to the absorbance of the control and sample at 517 nm.

Hydrogen Peroxide Scavenging Ability (H2O2 Assay)
Hydrogen peroxides (H 2 O 2 ) are the free radical reactive oxygen species (ROS) which are produced as the by-products of normal aerobic metabolism and increases during a stressed condition, infections, exercise, radiation etc. H 2 O 2 is not toxic itself but eventually, under the mentioned conditions may get converted into even more toxic radicals (hydroxyl radicals) and thus, its removal is important [51].

Antimicrobial Activity by Agar Well-Diffusion Assay
The TCE and TCHG were screened for their antimicrobial potential using agar well diffusion assay. without extract (HG) and distilled water (DW)). Streptomycin or amphotericin B was selected as a positive control for bacterial and fungal plates (200 µl) respectively [61]. The plates were incubated at 37˚C for 24 -48 h. Thereafter, antimicrobial activity was determined by measuring the diameter of zones of inhibition in mm [62]. The means and standard deviation (±SD) of the diameter of zones of growth inhibitions for the treatments are shown in results [63].

Phytocompounds Estimation
The presence of the total phenolic content in T. cacao extract was determined by the Folin-Ciocalteu method, the total phenolic content in the extract was found to be 97.58 ± 1.38 mg/ml. Similarly, the presence of total flavonoids content in TCE was estimated by AlCl 3 method and was found to be 68.02 ± 0.59 mg/ml, after plotting the standard graph with gallic acid.

Fabrication and Optimization of T. Cacao Hydrogel
The T. cacao extract loaded hydrogel (TCHG) was fabricated by using Carbopol-940 along with the hydro-dispersion of cocoa extract and glycerin as shown in Table 1 (Fabrication and optimization of hydrogel using Carbopol 940 at various concentrations). The drug and polymer ratio were optimized at different concentrations in variable batches (F1, F2, F3, and F4) followed by-physical parameters like pH, viscosity, appearance, homogeneity and dug release content analysis.

In-Vitro Drug Release Kinetics
The in-vitro drug release kinetics of all the batches of TCHG were further assessed for cumulative amount of phenolic content released in 12 hours. It was found that the in-vitro drug release was best explained by Hixon's equation, as the plots showed the highest linearity (R 2 = 0.9931), followed by Higuchi's equation (R 2 = 0.9912). As it is clear from Table 3 below that the maximum release of drug was obtained at 12 th hour in F2 formulation. The linear profile (approx.) as shown in Figure 1 was seen in all the formulations. The expected characteristics of hydro particles of sustained release were verified. The compound release from the hydrogel were clearly observed to attain 90.94% in 12 hours and on the order of release rate obtained for all the formulations (Table 4) we can rank them in the following descending order F2 > F3 > F4 > F1. Table 3 illustrates the profile of in-vitro release of T. cacao extract from various formulations of TCHG hydrogel. It is clearly observed that over 90% of T. Cacao was released from F2 formulation within 12 h, whereas F1, F3 and F4 re-

Scanning Electron Microscopy (SEM)
The SEM analysis of morphology of TCHG (F2) depicted the uneven and apparently flaky surface appearance with average porosity, giving avenue for decent extract loading capacity followed by controlled release characteristics. The balanced viscosity in the TCHG (F2) formulation might have led to an efficient inter micellar continuities in the hydrogel composites resulting in increased storage coefficients and long hydrophilic chain as shown in Figure 2.

Antioxidant (AO) and Antiradical Analysis
The standard graph of Ascorbic acid (AA) at different concentration (10 -50 µg/ml), and was analyzed 517 nm. After plotting the values on the standard graph the R 2 value was found out to be 0.996. Thereafter the same the antioxidant activity was evaluated for cocoa gel as well as the extract.
The antioxidant activity of gel was found out to be maximum at the concentration of 50 µg/ml which was 92.15% ± 1.42% whereas; the extract shows the % antioxidant activity of 40.31% ± 1.13% at the same concentration as shown in Figure 3. Therefore, it was well reflected from the results that cocoa gel (TCHG) has a maximum antioxidant activity followed by the standard (AA) as shown in Table 5.

2,2-Azinobis (3-Ethylbenzothiazoline-6-Sulfonic Acid) Radical Assay (ABTS)
The antioxidant activity was plotted against various concentration (10 -50  Figure 4. Therefore, it was well reflected from the results that cocoa gel (TCHG) has a maximum antioxidant activity followed by extract and the standard AA as shown in Table 6.

Hydrogen Peroxide Scavenging Activity-H₂O₂ Assay
The scavenging behavior of the test samples T. cacao extract (TCE) as well as cocoa gel (TCHG) was dose-dependent as it increases with the increase in concentration. The antioxidant activity was plotted against various concentration (10 -50 µg/ml) of test samples Theobroma cacao extract (TCE) as well as cocoa gel (TCHG). The antioxidant activity of gel was found out to be maximum at the concentration of 30 µg/ml which was 98.12% ± 0.42% whereas; the extract shows the % antioxidant activity of 57.13% ± 1.13% at the same concentration as shown in Figure 5. Therefore, it was well reflected from the results that cocoa gel (TCHG) has a maximum antioxidant activity followed by extract (TCE) and the standard (AA) as shown in Table 7.

Nitric Oxide Scavenging Activity-NO Assay
The antioxidant activity was plotted against various concentration (10 -50 µg/ml) of test samples Theobroma cacao extract (TCE) as well as cocoa gel (TCHG). The antioxidant activity of gel was found out to be maximum at the concentration of 50 µg/ml which was 92.54% ± 0.69% whereas; the extract shows the % antioxidant activity of 61.5% ± 1.43% at the same concentration as shown in Figure 6. Therefore, it was well reflected from the results that cocoa gel (TCHG) has a maximum antioxidant activity followed by extract (TCE) and the standard (AA) as shown in Table 8.

Antimicrobial Activity
When the samples were loaded in the agar plates, there occurs the diffusion of the sample into the well which leads to the inhibition of growth of microbes in and around the wells loaded with samples. These inhibitions are clearly seen in the plates after incubation of 24 hours to 72 hours for anti-bacterial and anti-fungal activity. These zones are marked and then measured in mm which determines the inhibition of the microbes by the test samples as shown in Figure 7.
Out of the 3 bacterial strains used, maximum inhibition zones were found in the species of Bacillus licheniformis. These zones were measured as shown in Table   9 and were further analyzed for MIC calculation as shown in Table 10.
where a = diameter of the inhibition zones, b = diameter of the well.
Then plotting the relation between X 2 and log (concentration) and from the curve the MIC is calculated. The antilog of the value of the intercept falling on the X-axis determines the MIC of the sample [65].

Discussion
In the quantitative analysis of the cocoa extract which is done to confirm the presence of certain phyto-constituents as well quantify the amount of these phyto-chemicals in the plant. The quantification of metabolites helps determine the impact of presence and amount of heavy metal present in the sample on the over-all well-being of the plants [66]. Moreover, since these metabolites are responsible for imparting health benefits to the host upon consumption, thus it is essential to understand the extent of impact of these metals on plants to correlate their effect on humans [67]. rapeutic effects and has much more value in the same way the results obtained in this study was approximately near to the values as described above [69] [70].
Hence, the study of Theobroma cacao is validated to the best of knowledge.
Hydrogel formulation containing cocoa hydrogel was successfully prepared and showed effective as well as better carrier for the transdermal drug delivery.
The formulated hydrogel was optimized for pH, viscosity, drug release, extrudability, homogeneity [71]. As per Ashwini Jaybhave et al., administration of this through dermal route bypasses the disadvantages of oral route and maintain the consistent plasma levels for the therapy of single dose [72]. Therefore, as proved  [79]. Moreover, the potential value of antioxidants is often low due to their low levels in cells and tissue, thus limiting their possible use as therapeutic drugs [80]. For example, in the therapeutic strategies to prevent progressive neuronal loss based on antioxidant activity, the antioxidant must be able to cross the blood brain barrier and be present at the respective brain region for neuroprotection or the transdermal layer for skin cancers (Aruoma, 2003) [81]. That being said, food, nutraceuticals, and complementary medicines facilitate the administration or ingestion of high quantities of antioxidant compounds, which can produce high or moderate levels of antioxidant agents in the blood and tissue [82] [83]. This protects the natural antioxidants from destruction and consequently reduces cell and tissue damage. The use of T. cacao as an anti-microbial agent in many traditional medicinal system has indicated that T. cacao may be useful for specific infectious diseases [89]. Hence, there is no need to scientifically evaluate the plant so that it can be used as a novel anti-microbial agent [90]. There are various publications which report the anti-microbial property of T. cacao comparison of the present findings with previous publications is difficult. Plants are known to vary in their composition with changes in their habitat and climatic conditions [91]. The methods used to check anti-microbial activity of various plant extracts and selection of the test organisms vary between publications. Moreover, a comprehensive report on the activity of T. cacao on skin infections causing pathogens is not available [92]. Therefore, the present study indicates that T. cacao extracts and gel both have a potent anti-microbial activity against various skin pathogens [93] [94] [95]. However, MIC values (Table 10) indicates that it has higher action against fungal strains than that of the bacterial strains [96]. Moreover, gel extract has a more effective action towards the microbial strains as compared to pure extract which was evident from the MIC values [97]. This indicates that the polyphenolic components of T. cacao extract, which are responsible for its anti-microbial activity, are more effective as gel than that of the powder [98].
However, the study reported by Chou et al., P. fluorescens has been found to be more sensitive toward the cocoa extract which confirms the result obtained for the gel extract but the same was not found for the powder extract [99].
Cocoa hydrogel is being prepared using 1% carbopol-940 as a gelling agent, as it shows better drug release as compared to other formulations, i.e. 90.94% and the formulation follows Hixon-Crowell model of drug release kinetics. Appearance of the gel was found to be homogenous with no grittiness and brown in colour. The pH was found to be 6.68 ± 0.54 and viscosity 35.965 ± 0.56.

Conclusions
T. cacao has been proved to be very effective in managing many disorders and infections which are associated with skin. Phytochemical studies of T. cacao have shown the presence of various versatile and important phytoconstituents such as flavonoids, phenols, alkaloids and saponins. Out of which maximum quantity was estimated for phenolics which was 97.58% ± 1.51%. The hydrogel prepared showed a good release of drug and had increased bioavailability. The ex-vivo studies of cocoa hydrogel performed showed the maximum drug release of cocoa extract after 12 consecutive hours which proves the effectiveness of the hydrogel. Therefore, the hydrogel containing cocoa proved for in-vitro anti-microbial activity and anti-cancer activity.
On evaluating the antioxidant and antiradical properties of cocoa hydrogel by DPPH, ABTS, NO, H 2 O 2 , it showed that cocoa hydrogel has better radical scavenging effect than the standard of ascorbic acid (AA).
Further results affirmed our study for the activity of cocoa gel against some sensitive bacterial and fungal strains. The anti-bacterial study of cocoa was es-