Examining the Naringin Content and Sensory Characteristics of Functional Chocolate Fortified with Grapefruit Peel Extract

Grapefruit peel contains a high concentration of naringin- a potent antioxidant with strong bioactive properties. In this study, a new type of functional chocolate fortified with grapefruit peel extract and different concentrations of aqueous methanol and ethanol were evaluated as extraction solvents. A new high-performance liquid chromatography (HPLC) method to analyze the naringin content of the fortified chocolates was developed with a recovery of 107% ± 3.1% and repeatability below 3.5%. A sensory evaluation was conducted to assess the preference for the chocolates among individuals who self-described a preference for bitter flavors. No significant preference was observed in the cases of astringency and aftertaste while the increased bitterness proved to be favorable. However, taste, flavor and overall acceptability were regarded somewhat less favorably. While chocolate proved to be a satisfactory carrier for naringin and had several enjoyable characteristics, further research may focus on improving the organoleptic properties of chocolates fortified by naringin. Supplementary Information The online version contains supplementary material available at 10.1007/s11130-023-01091-5.


Introduction
Naringin is a polyphenol and potent antioxidant abundant within the peels of grapefruit (Citrus paradisi), pomelo (Citrus grandis), and sour orange (Citrus aurantium). The molecule is a flavanone-7-O-glycoside between a naringenin aglycone and a neohesperidose moiety [1]. This compound has been shown to protect against neurodegenerative conditions such as Alzheimer's disease [2] cardiovascular diseases, and diabetes [3] while also exhibiting hepatoprotective, anti-obesity [4] nephroprotective [5,6] anti-inflammatory [7]and anti-carcinogenic characteristics [8,9]. Naringin is generally recognized as safe [1] and no significant toxic effects have been found [9]. Additionally, the NOAEL of naringin was found to be > 500 mg/kg of body weight when tested on Beagle dogs [10]. However, it is important to note that ingesting naringin-after being peel extract and to develop a novel HPLC method for analyzing the naringin content of the product.

Materials and Methods
The materials and methods used in this work are reported in detail in the Supplemental Material section.

HPLC-Method Validation
To validate our method, we evaluated the resolution, number of theoretical plates, retention time, and peak asymmetry of the method by injecting three replicate standards and chocolate extracts. The determination coefficient (R 2 ) of the calibration curve of naringin was 0.9999, which indicates the linearity of the HPLC method. The LOQ was found to be 4 µg/mL, while the LOD was estimated to be 1.2 µg/ mL. Recovery was determined to be 107% ± 3.1% while the repeatability and reproducibility of our method was evaluated by examining the relative standard deviation (RSD%) of naringin within the chocolate. The repeatabilities (intraday precisions) determined by triplicate measurements on four various days were 3.7, 0.4, 3.5, and 0.7%. The reproducibility (inter-day precision) calculated from 4 triplicate measurements (twelve replicates) was found to be 3.5%. Sensitivity was recorded as 6.586 mL/µg. Therefore, these results indicate that the used method was adequate for the quantitative and qualitative determination of naringin in chocolate.

Effect of the Solvent Composition on the Extraction Yield
To determine the most effective solvent for the extraction of naringin from grapefruit peel, we made triplicate measurements of wet grapefruit peel extracts prepared immediately before injection. While doing so, the solvent composition was varied. As seen in Fig. 1, solvent compositions of 25, 50, 75, and 100% aqueous ethanol and methanol were tested. A visualization of our results can be seen in Fig. 1.
Confidence intervals refer to standard deviation. Lettering shows which solvents produced statistically identical outcomes.
As shown in Fig. 1, pure methanol was proven to be the best extraction solvent while pure ethanol and deionized water yielded the lowest concentration of naringin. However, methanol is well-known to be highly toxic and the aqueous solvents were difficult to remove from the extract. Therefore, ethanol was chosen as an extraction solvent for the production of chocolates used for sensory evaluation. The observed superior solvency of methanol was in line with the findings of Feng et al. [22] where the extraction efficiency of methanol and ethanol for naringin from oranges were compared. However, ethanol is still commonly used due to its nature as a food-grade solvent and overall effectiveness [23].

Characterization of Grapefruit peel Extract
The grapefruit peel had a dry matter content of 22.3% which was determined gravimetrically. The grapefruit peel extract was a dull, red-orange powder comprising 61.9%, 57.9%, and 42.95% of dry peel mass when extracted with Fig. 1 Effect of solvent composition on naringin yield from grapefruit methanol, ethanol, and 75% ethanol respectively. The measured naringin content of the dry extract was 130 ± 2.3 mg/g when using ethanol, 111.9 ± 1.6 mg/g when using methanol, and 96.2 ± 1.4 mg/g when using 75% ethanol. The extraction yield from the dry peel was 54.5 mg/g with methanol, 45.6 mg/g with ethanol, and 53.6 mg/g with 75% ethanol. This was in line with the findings of Li et al.
[24] (52.03 mg/g when using methanol) showing that our extraction method provided sufficient yield. As highlighted by Sharma et al. [9], and Li et al. [25], naringin poses no health risks below 2 g per person or in a dose of less than 200 mg/kg in body weight.

HPLC Analysis of Fortified Chocolate
The protocol for chocolate production and analysis was based on the method used by Sik et al. [26]. Naringin and naringenin were both detected at 280 nm during the procedure. Our experiments showed that pure methanolic extraction resulted in the highest naringin yield (54.5 mg/g) from grapefruit peel so methanol was chosen for the extraction of naringin from the fortified chocolate. Methanolic solvents are commonly used to extract polyphenols from chocolate matrices as seen in studies by Belščak-Cvitanović et al. [27], Poliński et al. [28], and Carvalho et al. [29]. Fig. 2 shows the chromatogram measured for chocolates and the standard solution.
As seen from the chromatogram, naringin was successfully separated from the other minor components of the fortified chocolates. In addition, the measured concentration was 1.04 mg/g with 101 ± 3.4% recovery. This data highlights that dark chocolate provides a suitable matrix for fortification with naringin. Other authors have also examined the potential for chocolates to be fortified with fruit extracts offering proof of the suitability of chocolate to serve as a medium for functional ingredients. For example, Kaur et al. [30] produced a meat chocolate fortified with blueberry and raspberry extract and observed improved microbiological quality and boosted antioxidant activity. Polinski et al. [31] also measured an increase in phenolic content and antioxidant capacities when making dark chocolates fortified with chokeberry, elder flower, and elderberry extracts.

Effect of Extraction time on Naringin Yield
The amount of naringin that could be extracted from the chocolates as a function of time was also investigated. We also investigated the factor of extraction time during the extraction of naringin from chocolate. As seen in Fig. 3, extractions of 15, 30, 45, and 60 min provided a statistically identical result. and 100 ?g/mL naringin standard solution (c). Naringin is absent from control chocolate research may focus on improving the sensory characteristics of fortified chocolates. A possible solution to this issue may be using encapsulation technologies, which may decrease the changes in flavor while maintaining or increasing the antioxidant activity and bioavailability of naringin [32,33]. Alternatively, naringin may be hydrolyzed to its less-bitter aglycone moiety naringenin through the use of naringinase, or a combination of α-L-rhamnosidase and β-glucosidase [34].

Conclusion
Our study evaluated chocolate as a potential oral delivery system of naringin. Ethanol was chosen as to extract naringin from grapefruit peel due to its volatility and lesser toxicity when compared to methanol. A simple, rapid, accurate and precise HPLC-DAD was developed to quantify naringin in the fortified chocolate. While increase in bitterness was found to be appealing to some, further studies may seek to minimize organoleptic changes through the addition of sweeteners, nanoencapsulation, or the enzymatic hydrolysis of naringin.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing Interests
The authors declare no competing interests.

Conflict of Interest
The authors declare that they have no known conflicting financial or relational interest that may have influenced the contents or methodology within this work.
Ethics Approval Not applicable.

Results of Organoleptic Evaluation
Due to the strong change in the organoleptic properties caused by the fortification with grapefruit peel extract, the sensory evaluation was focused towards individuals who enjoy bitter flavors. However, the evaluators showed no significant preference for either chocolate in the categories of aftertaste, or astringency. In fact, a clear preference was shown for the enhanced bitterness of the chocolate. However, taste, flavor and overall acceptability was greater in the case of the control chocolates. A summary of the organoleptic trials can be seen in Fig. 4.
There was no significant difference found within the evaluation of the visible traits of the chocolate. Even though the increase in bitterness may be welcome for some, further   Exploring the rosmarinic acid profile of dark chocolate fortified with freeze-dried lemon balm extract using conventional and Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.