**The Stability and Activity Changes of Apigenin and Luteolin in Human Cervical Cancer Hela Cells in Response to Heat Treatment and Fe2**+/**Cu2**<sup>+</sup> **Addition**

#### **Wan-Ning Liu 1, Jia Shi 1, Yu Fu <sup>2</sup> and Xin-Huai Zhao 1,\***


Received: 14 July 2019; Accepted: 12 August 2019; Published: 14 August 2019

**Abstract:** Flavonoids are natural polyphenolic compounds with desired bio-functions but with chemical instability and sensitivity to temperature, oxygen, and other factors. Apigenin and luteolin, two flavones of the flavonoid family in plant foods, were; thus, assessed and compared for their stability, especially the changes in anti-cancer activity in response to the conducted heat treatments and the addition of ferrous or cupric ions. The two flavones in aqueous solutions showed first-order degradation at 20 and 37 ◦C. The addition of ferrous or cupric ions (except for Cu2<sup>+</sup> at 37 ◦C) enhanced luteolin stability via forming the luteolin–metal complexes; however, Fe/Cu addition (especially at 37 ◦C) consistently impaired apigenin stability. Using the human cervical cancer Hela cells and two cell treatment times (24 and 48 h), it was evident that heat treatments (37 and 100 ◦C) or Fe/Cu addition could endow apigenin and luteolin with decreased activities in growth inhibition, DNA damage, intracellular reactive oxygen species (ROS) generation, and apoptosis induction. In general, higher temperature led to greater decrease in these activities, while Fe2<sup>+</sup> was more effective than Cu2<sup>+</sup> to decrease these activities. The correlation analysis also suggested that the decreased ROS generation of the two flavones in the Hela cells was positively correlated with their decreased apoptosis induction. It is; thus, concluded that the two treatments can influence the two flavones' stability and especially exert an adverse impact on their anti-cancer activities.

**Keywords:** apigenin; luteolin; degradation; ferrous ions; cupric ions; cervical cancer cells; growth inhibition; apoptosis

#### **1. Introduction**

Flavonoids are a class of secondary plant phenolic compounds existing in a wide range of human diets. Flavonoids are interesting target compounds to many researchers because they have anti-oxidative, anti-microbial, anti-inflammatory, and anti-cancer effects [1]. Flavonoids, as natural anti-oxidants, even can exert stronger anti-oxidant activity than that of anti-oxidative vitamins and synthetic phenols [2]. Flavonoid compounds, such as hesperetin, naringenin, poncirin, and diosmetin, are effective to inhibit harmful microorganisms; for example, they can inhibit the growth of *Helicobacter pylori* [3]. Furthermore, flavonoids have profound immune-regulatory and anti-inflammatory effects [4]. Cocoa flavonoids had immuno-regulation in the EL4.BU.OU6 cells by increasing the release of interleukin-4 [5]. Rutin, hesperidin, hesperetin, and quercetin were effective for both chronic and neurogenic inflammation [6]. Moreover, many researchers have paid special attention to the anti-cancer functions of flavonoids and flavonoid extracts. Quercetin, luteolin, chrysin, kaempferol, apigenin, and myricetin have cytotoxic effects on the human esophageal adenocarcinoma OE33 cells, resulting in growth inhibition, cell-cycle arrest, and apoptosis [7]. Baicalin could inhibit

the growth of several human prostate cancer cells, including DU145, PC-3, LNCaP, and CA-HPV-10 cells [8]. Naringenin from citrus fruits could inhibit the proliferation of human colon cancer HT29 cells [9]. All results suggest that dietary flavonoids are promising natural compounds with desired ability to reduce cancer risk. Subsequently, an inverse correlation between flavonoid intake and the incidence of laryngeal and esophageal cancers has been reported [10].

Fe and Cu are two essential trace elements in the body, and are widely found in human diets. Fe/Cu ions have active redox property and thus can easily react with dietary flavonoids, which might alter chemical structures, especially the bio-functions of flavonoids. When flavonoids interact with Fe/Cu ions, they are oxidized by the two ions with decreased absorbance at their maximum absorption peaks [11]. Flavonoids can chelate with the two ions and thus form complexes with changed properties. Flavonoid–Fe2<sup>+</sup> complexes showed enhanced stability, while flavonoid–Cu2<sup>+</sup> complexes had auto-oxidation [12,13]. Furthermore, flavonoid oxidation by Cu2<sup>+</sup> was irreversible [13]. However, superoxide scavenging capacities of rutin, taxifolin, epicatechin, and luteolin were weaker than their respective Fe/Cu complexes [14]. Overall, it is reasonable to believe that the anti-cancer potentials of flavonoids could be affected by these transition metal ions.

During food processing, Fe/Cu ions may easily enter food matrices, as food matrices have the opportunity to contact the surfaces of pipes and equipment made from the two metals. Furthermore, some treatments used in food processing might exert potential impacts on dietary flavonoids; for example, heat treatment is necessary or unavoidable. In general, flavonoids are sensitive to high temperature [15], because high temperature can promote their degradation. The higher temperature of elderberry anthocyanins gave rise to higher degradation rate constants [16], while flavonoids in cloudy apple juice at 80 to 145 ◦C also experienced increased degradation rates [17]. Dietary flavonoids at higher temperatures; therefore, might be endowed with changed bio-functions, mainly due to flavonoid degradation. Brazilian bean after boiling and draining had decreased flavonoid content and lower anti-oxidant capacity [18]. Thermal treatment of galangin, kaempferol, morin, and myricetin led to weakened growth inhibition on the human colon carcinoma HCT-116 cells [19,20]. Thus, the effects of heat treatment and metal entrance on anti-cancer functions of flavonoids in other cancer cells, like the human cervical cancer Hela cells, deserve further study.

The flavones are commonly found flavonoid compounds in natural foods, among those flavone members are apigenin and luteolin. Apigenin is rich in Chinese cabbage, bell pepper, garlic, bilimbi fruit, guava, wolfberry leaves, and local celery, while luteolin is rich in bird chili, onion leaves, and bilimbi fruit and its leaves [21]. Normally, flavones had been reported to have stronger anti-cancer activities due to their high lipophilicity [22]. Apigenin is a promising anti-cancer compound, because it could inhibit the growth of several cancer cells [23]. Luteolin also is served as a potential and emerging anti-cancer compound, due to its clear toxic effect on eukaryotic DNA topoisomerase I [24]. From a chemical point of view, apigenin and luteolin have several −OH groups in their molecules (Figure 1) and thus have different stability once they are heated or mixed with Fe/Cu ions. Whether apigenin and luteolin after these treatments still have good anti-cancer functions is important but unsolved at present. Such a study; thus, deserves consideration.

**Figure 1.** The chemical structures of flavone compounds apigenin and luteolin.

In this study, both apigenin and luteolin were measured for their stability under two temperatures (20 and 37 ◦C) or Fe2+/Cu2<sup>+</sup> addition. The two temperatures are regarded as room temperature of diet storage and average temperature of the body, respectively. Moreover, the latter is also the culture temperature of most cells. Afterwards, the two flavones were subjected to heat treatments at 37 and 100 ◦C or Fe/Cu addition, and then evaluated for their changes in anti-cancer activity using the human cervical cancer Hela cells as a cell model. Four indices including growth inhibition, cell morphology (or DNA damage), reactive oxygen species (ROS) generation, and apoptosis induction were used to clarify or compare activity changes. The study aimed to reveal whether the two treatments (heat treatment and Fe/Cu addition) could affect the stability of apigenin and luteolin as well as their anti-cancer effects in Hela cells.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

The apigenin and luteolin (purity >98%) were bought from Dalian Meilun Biological Technology Co. Ltd. (Dalian, Liaoning, China). The cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies Inc. (Kyushu, Japan). The reactive oxygen species (ROS) assay kit, Annexin V-FITC apoptosis detection kit, and Hoechst 33258 kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). 5-Fluorouracil (5-Fu) was bought from Tianjin Jinyao Pharmaceutical Co. Ltd. (Tianjin, China). All other chemicals used were of analytical grade. The water used in this study was ultrapure water generated with Milli-Q PLUS (Millipore Corporation, New York, NY, USA).

#### *2.2. Cell Line and Culture Conditions*

The Hela cells (STR: Amelogenin: X; CSF1PO: 9,10; D13S317: 12,13.3; D16S539: 9,10; D18S51: 16; D19S433: 13, 14; D21S11: 27,28; D2S1338: 17; D3S1358: 15, 18; D5S818: 11, 12; D7S820: 8,12; D8S1179: 12, 13; FGA: 18,21; TH01: 7; TPOX: 8,12; vWA: 16,18) used in this study were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). As recommended by the cell supplier, the cells were cultured in the Dulbecco's modified eagle's medium (DMEM) (Sigma-Aldrich, Co. St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin solution at 37 ◦C in a 5% CO2 atmosphere.

#### *2.3. Assays of Degradation Rates of the Two Flavones*

Both apigenin and luteolin were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions of 0.1 mol/L. The stock solutions were diluted with ethanol and then with 0.1 mol/L phosphate buffer solution (PBS, pH 7.3) to two final concentrations of 20 and 30 μmol/L, using respective dilution factors of 5000 and 3333. Otherwise, the stock solutions were diluted with ethanol and PBS similarly but with addition of CuCl2 or FeCl2, which resulted in a fixed molar ratio of flavones to Fe/Cu (3:1). All prepared solutions were incubated at two temperatures (20 and 37 ◦C) for 6 h, and measured for their absorbance values at various time points using two wavelengths (apigenin 354 nm; luteolin 360 nm) and a UV-visible spectrophotometer (UV-2401 PC, Shimadzu Co., Kyoto, Japan). PBS was used as blank in this assay. Residual levels of apigenin and luteolin were estimated using the respective standard curves generated from a serial of standard solutions.

Based on the established first-order reaction model of flavonoid degradation [25], the degradation rate constants (k, h<sup>−</sup>1) of apigenin and luteolin were calculated using a derived linear regression equation.

#### *2.4. Treatments of the Two Flavones for Cell Experiments*

Apigenin and luteolin were dissolved in DMSO to obtain 0.3 moL/L stock solutions, and diluted by the DMEM supplemented with 5% FBS to yield flavone concentrations of 20 to 80 μmoL/L using the dilution factors ranging from 15,000 to 3750. The stock solutions were also diluted by DMEM without FBS to a fixed flavone concentration of 42.1 μmoL/L (using dilution factor of 7126), and heated in the dark with a thermostatic water bath operated at 37 ◦C (or 100 ◦C) for 6 h (or 0.5 h). After heat treatment, the two solutions were immediately cooled in the ice water and added with the FBS to yield a final flavone concentration of 40 μmoL/L. The FBS was not involved in these thermal treatments. Or else, the stock solutions were diluted with DMEM supplemented with 5% FBS, and added with 100 mmoL/L CuCl2 or FeCl2 solution to yield a final flavone concentration (40 μmoL/L) together with a fixed molar ratio (3:1) of flavones to Fe/Cu.

#### *2.5. Assay of Growth Inhibition*

The cells (1 <sup>×</sup> 104 cells per 100 <sup>μ</sup>L per well) were seeded onto the 96-well plates and incubated for 12 h. After removal of cell medium, the cells were treated with 0.1% DMSO (negative control), 100 μmol/L 5-Fu (positive control), and the prepared flavone samples for 24 and 48 h, respectively, and then washed twice with the PBS. The CCK-8 solution of 100 μL (10 μL CCK-8 plus 90 μL DMEM containing 5% FBS) was added to each well, and the cells were further incubated at 37 ◦C for 1.5 h. A microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) was then used to measure the optical density values at 450 nm, which were used to calculate the percentages of growth inhibition as previously described [26].

#### *2.6. Hoechst 33258 Staining*

The cells in 6-well plates were grown to 70% confluence and incubated with the untreated or treated flavone samples (40 μmol/L) for 24 h. After discarding cell media, 4% methanol of 1 mL was added into each well to fix the cells at 4 ◦C for 10 min. After washing twice with the PBS buffer, the Hoechst 33258 (200 mg/mL) of 1 mL was added into each well to stain the cells for 10 min. The cells were then observed under a fluorescence microscope (Zeiss Axio Observer A1m, Carl Zeiss, Oberkochen, Germany), while cell images were taken at 350 nm using an objective of 40-fold.

#### *2.7. Assay of Apoptosis Induction*

The proportions of the apoptotic cells in different cell groups were detected using flow cytometry technique and Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining as previously described [27]. The cells were grown to 70% confluence in 6-well plates, incubated with the untreated and treated flavones at 40 μmoL/L for 24 and 48 h, harvested, washed with the cold PBS, and centrifuged at 110× *g* for 5 min to discard the supernatants. The pellets were re-suspended gently in the Annexin V-FITC binding buffer of 200 μL, and incubated with the Annexin V-FITC of 10 μL for 15 min in the dark at 20 ◦C. The binding buffer (300 μL) and PI (5 μL) were added into each well and mixed gently. The stained cells were assayed with a flow cytometer (FACS Calibur, Becton Dickson, San Jose, CA, USA), to detect the percentages of necrotic, late apoptotic, intact, and early apoptotic cells (Q1–Q4).

#### *2.8. Assay of Intracellular Reactive Oxygen Species*

In this assay, the cells were treated similarly as those in the assay of apoptosis induction. After cell harvesting and PBS washing, the cells were re-incubated with 20,70-dichlorofluorescein (DCF-DA, 5 mmoL/L) at 37 ◦C for 30 min in the dark, washed three times with the PBS, and re-suspended in the PBS of 1 mL. The cell suspension was seeded onto the 96-well plates and measured for their fluorescence intensities using a fluorescence microplate reader (Infinite 200, Tecan, Männedorf, Switzerland) and respective emission and excitation wavelengths of 488 and 525 nm. The relative ROS levels were expressed as the percentages of the control cells as previously described [28].

#### *2.9. Statistical Analysis*

All reported data collected from three independent experiments or assays were expressed as means or means ± standard deviations, and compared using the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). All obtained data meet the assumptions of normality and constant variance. Significant differences (*p* < 0.05) between the means of multiple groups were evaluated by the one-way analysis of

variance with Duncan's multiple range tests and two-way analysis of variance (ANOVA). The Pearson's correlation coefficient was also calculated using this software.

#### **3. Results**

#### *3.1. Instability of Apigenin and Luteolin at Two Temperatures or in the Presence of Fe2*+/*Cu2*<sup>+</sup>

Apigenin and luteolin showed typical UV-spectra with maximum absorption peaks around 354 and 360 nm, respectively. This study; thus, used two wavelengths to detect residual apigenin and luteolin, which were exposed to two temperatures or Fe2+/Cu2<sup>+</sup> for different time periods. The results indicated that both apigenin and luteolin were chemically instable in these cases, because their residual levels showed a decreasing trend (Figure 2). The calculated degradation rate constants (*k*) revealed how the higher temperature (37 ◦C) and the two ions affected the stability of apigenin and luteolin (Table 1). When their solutions were kept at 20 or 37 ◦C, apigenin and luteolin showed *k* values of 0.0207 and 0.0214 or 0.0226 and 0.0245 h−1, respectively. Higher temperature clearly led to higher *k* value (i.e., decreased stability). In the presence of Fe<sup>2</sup>+/Cu2<sup>+</sup>, apigenin gave greater degradation, because the measured *k* values were increased to 0.0395–0.0728 h<sup>−</sup>1. More importantly, higher temperature (37 ◦C) combined with Cu2<sup>+</sup> brought about more drastic apigenin degradation. As for luteolin, Fe2<sup>+</sup> resulted in lower *k* values (i.e., decreased degradation), while Cu2<sup>+</sup> at 37 ◦C led to enhanced degradation (i.e., larger *k* value). These results suggested that: (1) Both higher temperature and Fe2+/Cu2<sup>+</sup> caused structural instability for apigenin; and (2) only higher temperature and Cu2<sup>+</sup> could increase the instability of luteolin. Both heat treatments and Cu/Fe addition; therefore, might alter the anti-cancer activities of these two flavones.

**Figure 2.** Residual levels of apigenin and luteolin in the solutions incubated at 20 ◦C (**A**,**C**) and 37 ◦C (**B**,**D**) for different time periods.

Significance


None 0.0226 <sup>±</sup> 0.0001 <sup>E</sup> 0.0245 <sup>±</sup> 0.0006 <sup>b</sup> Fe2<sup>+</sup> 0.0520 <sup>±</sup> 0.0002 <sup>B</sup> 0.0203 <sup>±</sup> 0.0005 <sup>d</sup> Cu2<sup>+</sup> 0.0728 <sup>±</sup> 0.0010 <sup>A</sup> 0.0317 <sup>±</sup> 0.0004 <sup>a</sup>

**Table 1.** Degradation rate constants (*k*, h<sup>−</sup>1) of apigenin and luteolin in solutions treated with two temperatures or added with Fe2+/Cu2<sup>+</sup> s.

Different lowercase or capital letter superscripts after the values in the same column indicate that the means differ significantly according to one-way ANOVA (*p* < 0.05). The two asterisks indicate that the means differ significantly according to two-way ANOVA (*p* < 0.05).

Temperature \*\* \*\* Metals \*\* \*\* Temperature × Metals \*\* \*\*

#### *3.2. Growth Inhibition of the Flavone Samples on Hela Cells*

100 ◦C

The CCK-8 assaying results indicated that both apigenin and luteolin at 20–80 μmoL/L dose- and time-dependently had cytotoxic effects on the Hela cells (Figure 3), resulting in inhibition percentages of 30.6%–62.7% and 33.8%–70.6% (24 h) or 59.5%–76.4% and 62.3%–88.6% (48 h), respectively. Both apigenin and luteolin at 40 μmoL/L caused corresponding inhibition percentages of 52.0% and 57.9% (24 h) or 65.7% and 73.2% (48 h) in the cells. Thus, flavone concentration of 40 μmol/L was used in later study, because this concentration led to growth inhibition up to 50%–70%.

**Figure 3.** Growth inhibition of apigenin (**A**) and luteolin (**B**) of various concentrations on the Hela cells at treatment times of 24 and 48 h. 5-Fu, 5-fluorouracil as a positive control. Different capital or lowercase letters above the columns indicate that the means within the same group differ significantly according to one-way ANOVA (*p* < 0.05).

This flavone concentration was then used to compare different growth inhibition of these flavone samples with or without heat treatment or Fe/Cu addition in the Hela cells (Figure 4). Heat treatment at 37 ◦C decreased the inhibition percentages of apigenin and luteolin to 50.5% and 55.0% (24 h) or 63.2% and 67.5% (48 h), respectively. Heat treatment at 100 ◦C brought about much decreased growth inhibition, because the measured inhibition percentages of apigenin and luteolin were reduced to 48.4% and 51.1% (24 h) or 59.0% and 64.0% (48 h), respectively. Overall, heat treatment at 100 ◦C and Fe addition showed greater potential to decrease growth inhibition of the two flavones.

**Figure 4.** Growth inhibition of 40 μmoL/L flavonols (with or without thermal treatments and Fe/Cu addition) on the Hela cells with treatment times of 24 (**A**,**C**) and 48 h (**B**,**D**). Different capital or lowercase letters above the columns indicate that the means within the same group differ significantly according to one-way ANOVA (*p* < 0.05).

#### *3.3. Morphological Alteration of Hela Cells Treated by the Flavone Samples*

Morphological alteration of the treated cells can reflect potential apoptosis induction of the target substances. Morphological features of the treated Hela cells were; thus, observed using the Hoechst 33258 staining and fluorescence microscopy. In these results, the cell nuclei were dyed and observed in the fluorescent images. The apoptotic cells were observed as light blue while the viable cells were observed as dark blue. Moreover, the apoptotic cells often showed apoptotic morphology as the condensation and fragmentation of nuclei shrinkage as well as the formation of apoptotic bodies. In general, the untreated flavones were more effective than the treated ones to alter the morphological features of Hela cells, while 100 ◦C treatment and Fe addition brought about relatively higher cell density (Figure 5). Compared with the control cells without any treatment, the treated cells showed the typical apoptotic morphology and decreased cell density in the observation field. These results suggested that these assessed samples could damage DNA and thus had potential (but different) apoptosis induction towards the Hela cells.

**Figure 5.** Morphological features of the Hela cells treated with 0.1% DMSO, 100 μmol/L 5-fluorouracil (5-Fu), and 40 μmol/L flavone samples (with or without thermal treatment and Fe/Cu addition) for 24 h. A fluorescence microscope was used to photograph images (40×). The red and green arrows indicate the corresponding apoptotic and intact cells.

#### *3.4. Pro-Oxidation of the Flavone Samples*

The Hela cells treated with or without these flavone samples were; thus, detected for their ROS levels (Table 2). The results indicated that all assessed samples had pro-oxidation in the cells, as the treated cells showed increased relative ROS levels (larger than 200%) than in the control cells (*p* < 0.05). The untreated apigenin and luteolin brought about relative ROS levels of 229% and 284% (24 h) or 263% and 281% (48 h), respectively. The apigenin and luteolin treated at 37 ◦C for 6 h resulted in lower ROS levels of 212% and 272% (24 h) or 260% and 263% (48 h), respectively. Apigenin and luteolin treated at 100 ◦C for 0.5 h showed much weaker ability to increase ROS generation than those heated at 37 ◦C for 6 h. For apigenin and luteolin, Fe addition led to the highest ROS reduction in the cells; however, Cu addition only decreased ROS levels to a slight extent, compared with Fe addition. Overall, both heat treatment and Fe/Cu addition consistently led to decreased ROS generation in the Hela cells.


**Table 2.** The measured reactive oxygen species (ROS) levels in the Hela cells treated with different samples for 24 and 48 h.

Different lowercase or capital letter superscripts after the values in the same column indicate that the means are significantly different according to one-way ANOVA (*p* < 0.05).

However, ROS generation of luteolin at 48 h was lower than that at 24 h (except 100 ◦C heat treatment) (Table 2). In these cases, the respective samples had stronger pro-oxidation, could enhance ROS to much higher levels and, thereby, caused greater cell apoptosis, which led to a lower number of viable cells. After a longer period, only a few viable cells continued to generate ROS. Finally, ROS generation with incubation time of 48 h was less than that with incubation time of 24 h.

#### *3.5. Apoptosis Induction of the Flavone Samples*

Apoptosis induction of the untreated and treated flavones were then assessed with the flow cytometry technique using the Annexin V-FITC/PI double staining and treatment times of 24 and 48 h (Figures 6 and 7).

**Figure 6.** Cell percentages of the Hela cells treated with 0.1% DMSO (control) and 40 μmoL/L flavone samples with or without thermal treatments and Fe/Cu addition for 24 h.

**Figure 7.** Cell percentages of the Hela cells treated with 0.1% DMSO (control) and 40 μmol/L flavone samples with or without thermal treatments and Fe/Cu addition for 48 h.

The control cells at 24 or 48 h only had total apoptotic cells (Q2 + Q4) of 3.4% or 3.7%. The cells treated with the untreated apigenin and luteolin for 24 (or 48) h led to increased total apoptotic cells about 12.8% and 16.1% (or 15.7% and 26.8%). If the cells were treated with the heated flavones, the total apoptotic cells were measured with the reduced percentages, especially using heat treatment at 100 ◦C. Subsequently, the total apoptotic cells were 7.3% and 10.2% (24 h) or 11.3% and 13.2% (48 h) with corresponding apigenin and luteolin treatments. When the two flavones were added with Fe2+, the respective apigenin and luteolin treatments resulted in the total apoptotic cells of 7.0% and 9.1% (24 h) or 8.2% and 10.1% (48 h). When the two flavones were added with Cu2+, the measured total apoptotic cells were 8.5% and 13.5% (24 h) or 10.7% and 21.2% (48 h) with respective apigenin and luteolin treatments. Data comparison further revealed how these treatments had positive or negative impacts on the apoptosis induction of the two flavones. Overall, the conducted heat treatment

(especially at 100 ◦C) caused decreased total apoptotic cell proportions, while Fe addition also resulted in much decreased total apoptotic cell proportions than Cu addition did.

Further data analysis revealed that the measured ROS levels (Table 2) in the cells with a treatment time of 24 h were positively and significantly correlated with the detected total apoptotic cell percentages (Figures 6 and 7), because the calculated Pearson's correlation coefficient (i.e., *r*-value) of the two indices was 0.854 (*p* < 0.05). This correlation meant that the decreased abilities in ROS generation of apigenin and luteolin possibly resulted in decreased apoptosis induction. In other words, the used treatments brought about flavone degradation and lower abilities to generate ROS in Hela cells, and thereby led to decreased apoptosis induction. However, this phenomenon was no longer observed when the cells were treated with a longer time of 48 h. The treatment time of 48 h led to too much cell death or the lower number of viable cells (Figure 4). Consequently, only fewer viable cells in the media were able to generate ROS. This fact meant that much higher extent of apoptosis induction of apigenin and luteolin led to lower ROS generation. Therefore, the calculated Pearson's correlation coefficient (*r*-value) of the two indices (i.e., ROS levels versus apoptotic cell percentages) decreased to 0.589 (*p* > 0.05). In this case, the measured apoptosis induction and ROS generation were positively but insignificantly correlated.

#### **4. Discussion**

Flavones, in general, have several −OH groups in their molecules, and; therefore, they as phenolic compounds are susceptible to oxidation. Heat treatment; thus, promotes flavone degradation, and is adverse to the stability and bio-activities of flavonoids. Polyphenols in the solid grape marc were degraded at 100–150 ◦C, leading to decreased anti-oxidation [29]. At the temperature of 250 ◦C, catechins might lose their DPPH radical scavenging ability completely due to the thermal degradation of catechins [30]. The anti-cancer activities of flavonoids (e.g., growth inhibition) are governed by their chemical structures [31,32]. Subsequently, structure changes of flavonoids will result in increased or decreased activity. It was found that heat treatment of cymaroside (i.e., luteolin-7-O-β-glucoside) led to the increased immuno-modulation by enhancing NK cells activity [33]. Additionally, the heated flavonoids showed decreased activities in the human colon carcinoma HCT-116 cells [19,20]. Thus, heat treatments (especially using 100 ◦C) in the present study caused greater degradation and decreased growth inhibition for both apigenin and luteolin.

It is well-known that Fe/Cu are capable of oxidizing flavonoids in solutions, resulting in flavonoid degradation [34]. However, flavonoids also can complex with multi-valent metal ions [35], resulting in changed stability. Thus, Fe/Cu added to apigenin and luteolin solutions might bring two major reactions: forming flavone–metal complexes and catalyzing flavone degradation [12,13]. From a chemical point of view, the redox cycling exists between transition metals and ligands [36]. Quercetin, rutin, and 3-hydroxyflavone in the presence of Fe<sup>2</sup>+/Cu2<sup>+</sup> exhibited a significant decomposition, yielding semiquinone compounds [36]. Both apigenin and luteolin; thus, could be oxidized by the added Fe/Cu, resulting in changed chemical stability. However, apigenin and luteolin are different in their chemical structures that; thus, govern their stability changes in the presence of Fe2+/Cu2<sup>+</sup>. Normally, one luteolin molecule can chelate 1.5 Fe<sup>2</sup>+/Cu2<sup>+</sup>, but apigenin without two adjacent <sup>−</sup>OH groups is almost unable to chelate the two ions [11]. Apigenin in the present study; thus, was instable in the presence of Fe2+/Cu2<sup>+</sup> (Table 1). On the contrary, luteolin has two adjacent <sup>−</sup>OH groups in its C-ring and thus can chelate the two ions; subsequently, it mainly showed enhanced stability in the presence of Fe<sup>2</sup>+/Cu2<sup>+</sup> (Table 1). Moreover, the Cu-added luteolin also showed decreased stability at 37 ◦C (but not at 20 ◦C), which was attributed to the stronger oxidation of Cu2<sup>+</sup> at this temperature. Consistent with the present finding, it was also found that quercetin bound with Fe2<sup>+</sup> had inhibited oxidation, while that bound with Cu2<sup>+</sup> received promoted oxidation [12]. In methanol medium, Cu2<sup>+</sup> also promoted quercetin oxidation [13]. It was reasonable in the present study that the two flavones showed worse stability in the presence of Cu2+, especially at the higher temperature.

*Foods* **2019**, *8*, 346

Hela cells have the potential to proliferate indefinitely and have been widely used for cancer research. It was reported that many flavonoids and their derivatives had the ability to inhibit Hela cells. Natural flavone eupatorine inhibited Hela cells through inducing cell-cycle arrest and apoptosis [37]. Wang and coauthors reported that quercetin could induce the apoptosis and autophagy of Hela cells [38]. Other researchers proved that quercetin had anti-cancer effects on HeLa cells via the adenosine 5'-monophosphate -activated protein kinase (AMPK)-induced HSP70 and down-regulation of epidermal growth factor receptor (EGFR) [39]. In this study, Fe2+/Cu2<sup>+</sup> showed different behaviors to affect the growth inhibition of apigenin and luteolin in the cells. Fe is one of the required nutritive elements for tumor growth [40], and is also reported to influence cell-cycle regulation at multiple sites [41]. Fe2<sup>+</sup> chelation of flavonoids is one of the important mechanisms in response to their growth inhibition in cancer cells. Fe addition thereby decreased luteolin's Fe-chelating activities, promoted apigenin oxidation, thus reasonably reduced its growth inhibition. Cu2<sup>+</sup> is capable of inducing cellular oxidative stress, bringing DNA damage, and then initiating cell apoptosis [42]. Cu addition for the two flavones; thus, gave rise to two chemical reactions: enhancing flavone degradation and increasing cellular Cu content. The enhanced flavone degradation led to decreased growth inhibition, whilst the increased Cu content brought about extra oxidative stress or higher cytotoxic effect on the Hela cells. Subsequently, Cu addition of the two flavones in this study was observed with less decreased growth inhibition than Fe addition. The bio-activity changes of flavonoids in the presence of transition metal ions had been observed in other studies; for example, the complexes of rutin and dihydroquercetin with Fe, Cu, and Zn had higher anti-oxidation than the free counterparts as the inhibitors of asbestos-induced cell injury [43]. Similarly, the free radical scavenging ability of quercetin–Cu complex was higher than free quercetin [44]. Metal ions such as Cu, Fe, and Zn also had been evidenced to impact anti-microbial, anti-viral, and anti-inflammatory activities of flavonoids [45]. The present results also provided another evidence to show different effects of Fe2+/Cu2<sup>+</sup> on anti-cancer activities of the two flavones.

Flavonoids have both anti- and pro-oxidation in cells, depending on flavonoid concentrations and free radical sources [46]. The pro-oxidation of flavonoids plays an important role in their anti-cancer activities, via promoting the generation of intracellular ROS in cancer cells [47]. In general, a relative higher flavonoid level in cancer cells leads to pro-oxidation, promotes ROS generation, and, thereby, induces DNA damage [48]. Pro-oxidation of a tea polyphenolic compound, epigallocatechin-3-gallate, has been proved to govern its growth inhibition on colorectal HT29 cells, oral squamous carcinoma SCC-25 and SCC-9 cells, and premalignant leukoplakia MSK-Leuk1cells [49], while cytotoxic effects of quercetin, morin, and kaempferol on promyelocytic leukemia HL-60 cells were found to be caused by their pro-oxidation [22]. Both heat treatment and Fe/Cu addition of apigenin and luteolin led to oxidation and, thereby, altered the redox potential of the two flavones; the assessed samples; thus, had different abilities to generate intracellular ROS, and then showed different growth inhibition on Hela cells. Moreover, the enhanced ROS generation in cells suggests cell apoptosis, because this phenomenon is regarded as a classic way to trigger cell apoptosis [50]. Thus, flavonoids such as quercetin, luteolin, chrysin, kaempferol, apigenin, myricetin, and baicalin showed clear apoptosis induction to the human esophageal adenocarcinoma OE33 cells and three human prostate cancer cells, resulting in increased total apoptotic cells [7,8]. The conducted treatments in this study; thus, decreased ROS generation and apoptosis induction of the two flavones in the cells. It is reasonable that decreased ROS generation of the two flavones with treatment time of 24 h was positively and significantly consistent with their decreased apoptosis induction, as the correlation analysis results showed.

#### **5. Conclusions**

Two flavones, apigenin and luteolin, in aqueous solutions, had degradation to different extents, while Fe2+/Cu2<sup>+</sup> addition mainly resulted in stability (i.e., decreased degradation) for luteolin due to the formation of luteolin–metal complexes, but also led to instability (i.e., increased degradation) for apigenin. The flavone degradation was clearly enhanced at 37 ◦C (the classic temperature of cell culture) rather than 20 ◦C. The used heat treatments (37 and 100 ◦C) and Fe2+/Cu2<sup>+</sup> addition were adverse to

the anti-cancer activities of the two flavones against human cervical cancer Hela cells; subsequently, growth inhibition, DNA damage, and especially apoptosis induction (positively correlated with the intracellular ROS generation) of the two flavones were decreased. It is; thus, proposed that more attention should be paid to both heat treatment and some metal ions like Fe2+/Cu2<sup>+</sup> due to their negative effects when assessing the bio-activities of flavonoid compounds. However, this study only aimed to verify how the used heating treatments and two metal ions impacted flavone stability and anti-cancer activities in vitro. The related molecular mechanisms and an in vivo investigation will be carried out in a further study.

**Author Contributions:** W.-N.L. and J.S. performed the experiments; X.-H.Z. obtained the funding, designed the experiments, and analyzed the data; and Y.F. and X.-H.Z. wrote and revised the paper.

**Funding:** This research was funded by the Key Research Project in Science and Technology of the Education Department of Heilongjiang Province, grant number Project No. 11551z018.

**Acknowledgments:** The authors thank the anonymous reviewers for their valuable advice.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Ginger Water Reduces Body Weight Gain and Improves Energy Expenditure in Rats**

#### **Samy Sayed 1,2, Mohamed Ahmed 3, Ahmed El-Shehawi 1,4, Mohamed Alkafafy 1,3, Saqer Al-Otaibi 1, Hanan El-Sawy 5, Samy Farouk <sup>1</sup> and Samir El-Shazly 1,6,\***


Received: 19 November 2019; Accepted: 30 December 2019; Published: 2 January 2020

**Abstract:** Obesity is a serious global problem that causes predisposition to numerous serious diseases. The current study aims to investigate the effect of ginger water on body weight and energy expenditure through modulation of mRNA expression of carbohydrate and lipid metabolism. A white colored liquid obtained during freeze-drying of fresh rhizomes of *Zingiber o*ffi*cinal* was collected and named ginger water. It was used to treat rats, then blood and tissue samples were collected from the liver and white adipose at the end of the experiment. The serum was prepared and used for biochemical assays, while tissue samples were used for RNA isolation and gene expression analysis via Reverse transcription polymerase chain reaction (RT-PCR). Results of High Performance Liquid Chromatography (HPLC) analysis of ginger water revealed the presence of chrysin and galangin at concentrations of 0.24 μg/mL and 0.53 μg/mL, respectively. Average body weight gain decreased significantly in groups that received ginger water. In addition, both total cholesterol and serum triacylglycerol were reduced in the groups that received ginger water. Furthermore, mRNA expression of Sterol regulatory element-binding protein 1 (SREBP-1c) in the liver and leptin in adipose tissues were downregulated, while those of adiponectin, hepatic carnitine palmitoyltransferase1 (CPT-1), acyl-coA oxidase (ACO), Glucose transporter 2 (GLUT-2), and pyruvate kinase (PK) were upregulated in ginger water-treated groups. These results clearly revealed the lowering body weight gain effect of ginger water, which most likely occurs at the transcriptional level of energy metabolizing proteins.

**Keywords:** ginger water; obesity; energy homeostasis; gene expression; rat

#### **1. Introduction**

Obesity is a complex metabolic disorder that is currently a serious global problem. Obesity has been considered a fatal lifestyle disease during the past few decades because of increasingly high-fat and caloric-rich diets as well as genetic background [1,2]. The main reason for obesity is the energy imbalance in which the energy intake is higher than the energy expenditure. The main features of obesity include excessive fat mass and raised blood lipid concentration [3]. Obesity can lead to a wide range of diseases, such as type-2 diabetes, hypertension and hyperlipidemia, and cardiovascular diseases [4]. Therefore, prevention and treatment of obesity are a great health concern worldwide.

Although physical exercise and dieting are the preferred treatments for weight loss, in practice, this method is not effectively maintained, due to busy schedules. On the other hand, surgery is not preferable due to the risk factors and high cost. Therefore, there is a shift towards an increased use of medications to reduce weight with consideration of the side effects of these medications. Currently available antiobesity drugs attack body fat in various manners. They may promote metabolism and diminish appetite or they can affect fat digestion. Consequently, both health systems and researchers targeted the advancement of effective and safe therapies for obesity [5].

Natural products have been defined with different terms in various studies; functional food [6], food supplement [7], and the recently preferred definition "nutraceuticals" [8]. Although extensive research and patenting of nutraceuticals have been going for more than a decade, they do not have precise definition [9]. Nutraceuticals, when supported by clinical trials and known mode of action, have a major role in preventing as well as supporting the drug therapy of chronic diseases. In addition, the market of nutraceuticals is growing very fast with an expected market value of \$578.23 billion in 2025, although it faces challenges due to the absence of clear regulations and marking difference from food supplements. It is expected that nutraceuticals, in the future, will be approved and marketed side by side with the pharmaceuticals [10]. This indicates the need for an international consensus of regulatory framework for research, approval, safety, labelling, marketing, and use of nutraceuticals [9].

Natural plant compounds and their derivatives have been reported to treat obesity without mortality or obvious adverse impacts [11]. Plants that contain components with antiobesity activity have been used all over the world as alternative and complementary herbal therapies [12]. Herbal medicines are plant-derived raw or refined products that are used for the treatment of diseases. The antiobesity effects of many combinations of plant extracts were investigated. Most of these investigations indicated antiobesity effects, for example, decreasing body weight gain in both animals and humans. *Arachis hypogaea* decreased body weight gain, liver size, and liver triglyceride content, with an increase of fecal lipid excretion [13]. A reduction in food intake as a result of reducing appetite and an impacted hormonal status was shown with pomegranate [14].

Ginger (*Zingiber o*ffi*cinale* Roscoe, Zingiberaceae) is a well-known spice and flavoring material that has also been used in traditional medicine in many areas. Ethanolic extract of ginger had a reducing impact on the levels of blood glucose in rats fed on a high fat diet [15]. In addition, ginger ameliorates hyperlipidemia in diabetic rats by decreasing serum cholesterol and serum triglycerides [16,17]. Studies showed that ginger supplement improves fructose utilization-incited fatty liver [18] and adipose tissue insulin resistance in rats [19]. Ginger extract weakened the kidney injury induced by chronic fructose consumption. This was mediated by suppressing renal over-expression of proinflammatory cytokines [20]. The important active component of ginger root is the unpredictable oil and impactful phenol compounds, for example, gingerol, which is a very powerful anti-inflammatory compound [21]. Gingerol has appeared to stabilize adipocyte hormones, plasma, lipases, and lipid profiles in high fat diet induced obese rats [22].

Modern scientific research has revealed that ginger possesses various therapeutic properties, such as antioxidant effects and anti-inflammatory impacts [23]. Ginger water is obtained during the freeze-drying of ginger rhizomes as a byproduct. Its strong smell and milky color raised our attention to its potential similar biological effects to ginger extract. Most previous studies have focused on the effects of the main constituents of ginger extracts; however, there are no investigations that have specifically addressed the efficacy of the byproduct, ginger water. Therefore, this investigation aimed to study the lowering body weight gain effect of ginger water and to explore the molecular mechanisms underlying this impact through investigating the ability of ginger water to adjust mRNA expression of different genes related to carbohydrate and lipid metabolism.

#### **2. Materials and Methods**

#### *2.1. Experimental Design*

A total of fifteen ten weeks-old adult male Wistar rats were used in this study. Animals were obtained from the Experimental Animal Research Center, University of King Abdulaziz, Saudi Arabia. The animals were kept in polyethylene cages and held under laboratory conditions of 22 ◦C and 55% H in the animal house of Taif University, Saudi Arabia with a 12 h/12 h light/dark cycle. All animal groups were fed standard laboratory chow with free access to water. The Committee of Taif University for animal care and use has approved all procedures under the authorization number of 1-440-6145.

#### *2.2. Preparation of the Ginger Water*

Ginger water is not a ginger extract, but it is a byproduct obtained during lyophilization (freeze-drying) of ginger rhizomes. Fresh rhizomes of the ginger plant were washed, sliced, and freeze-dried at −60 ◦C. During the freeze-drying process, the condensed white colored liquid in the freeze-dryer was collected, named as ginger water, analyzed using High Performance Liquid Chromatography (HPLC), and used for the experiment.

#### *2.3. HPLC Analysis of Ginger Water*

The obtained ginger water was subjected to analysis using HPLC. Briefly, ginger water was filtered through syringe filters and used for HPLC analysis against nine flavonoid standards (Cyanidine chloride, Myrecitine, Quercetine, Chrysine, Malvidine chloride, Delphinidine chloride, Naringenine, Caffeic acid, and Galangin). The HPLC conditions were similar to those mentioned previously by the authors in [24]. Samples were assayed on an HPLC Hewlett-Packard Phenomenex Luna C18 column (4.6 × 250 mm, 10 μm particle size, Hewlett-Packard, Palo Alto, CA, USA). Separation was done at 12 min linear gradient from 100% of 100 mM ammonium acetate (pH 5.5) to 100% methanol. The flow rate was 1.5 mL/min and oven temperature of 35 ◦C with injection volume of 20 μL. Sample components were monitored at 260 nm. For calibration, standard compounds were dissolved in ethyl alcohol. Then, each peak area was converted to micrograms per mL.

#### *2.4. Animal Treatment*

The animals were randomly distributed into three groups of five animals each. The first group received tap water and feed ad libitum throughout the experimental period and considered as a control group. The second and third groups received ginger water at a rate of 25% and 50% (*v*/*v*) in their drinking water, respectively. Treatment proceeded for approximately a month. Body weight and the average of daily food consumption were measured weekly until the experimental period ended.

#### *2.5. Sampling*

By the end of treatment and before animal sacrifice, animals were fasted for 10 h. Blood samples were directly collected from retro-orbital puncture after diethyl ether anesthesia. Serum samples were arranged and stored at −80 ◦C until use in subsequent analysis. Then, rats were killed by decapitation. Specimens for RNA isolation were collected from liver and white adipose tissue. Samples were kept in QiaZol (Qiagen Inc., Valencia, CA, USA) and stored at −80 ◦C for using in gene expression analysis.

#### *2.6. Biochemical Assays*

Total cholesterol (TC) and serum triacylglycerol (TAG) were measured cholorametrically using commercial kits (HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Wiesbaden, Germany) according to the manufacturer's instructions.

#### *2.7. Gene Expression Analysis*

#### 2.7.1. RNA Extraction and cDNA Synthesis

Tissue, 100 mg, was used for isolation of total RNA using QIAzol reagent (QIAGEN Inc., Valencia, CA, USA) as explained previously [14]. RNA quality was tested by agarose gel electrophoresis. Concentration and purity of RNA were evaluated at 260 nm and by determination of OD260/<sup>280</sup> ratio, respectively. For cDNA synthesis, 4 μg of RNA were used with oligo-dT primer and M-MuLV reverse transcriptase (GoScript™ Reverse Transcriptase Promega, Fitchburg, WI, USA) as described previously [25] in the PeX 0.5 Thermal Cycler (Thermo Electronic Corporation, Milford, MA, USA). The obtained cDNA was directly used for Reverse transcription polymerase chain reaction (RT-PCR) or kept at −20 ◦C for future use.

#### 2.7.2. Semi-Quantitative-PCR

Expression of different genes related to energy metabolism was estimated by semi-quantitative PCR using their corresponding primers (Table 1). The tested genes included pyruvate kinase (PK), sterol regulatory element-binding protein-1c (SREBP-1c), glucose transporter-2 (GLUT-2), carnitine palmitoyl transferase-1 (CPT-1), acyl-CoA oxidase (ACO), and hormone sensitive lipase (HSL). The expression of leptin as well as adiponectin was also tested. Primers were designed using the Oligo-4 computer program and nucleotide sequence published in GeneBank (Table 1). PCR was conducted in 25 μL volume using PCR GoTaq®Master Mix (Promega Co., Fitchburg, WI, USA) as detailed previously [14]. The number of cycles and annealing temperatures of primers are summarized in Table 1. Expression of GAPDH mRNA was used as a reference (Table 1). PCR products were subjected to 1% agarose electrophoresis with ethidium bromide staining. PCR product bands were photographed under UV light. The intensities of the bands were densitometerically quantified using the NIH imageJ program (https://imagej.nih.gov/ij/).


**Table 1.** Primer sequence and PCR conditions used in this study.

#### *2.8. Statistical Analysis*

Results were analyzed statistically using one-way ANOVA and Scheffe's protected least significant difference test, by using SPSS software (SPSS version 13.0, IBM, Chicago, IL, USA) with *p* < 0.05. Results were expressed as means ± standard errors (SE).

#### **3. Results**

#### *3.1. Chemical Composition of Ginger Water*

HPLC analysis of the obtained ginger water revealed that, among the nine standards used in the HPLC analysis, only chrysin and galangin were detected in the ginger water, at concentrations of 0.24 μg/mL and 0.53 μg/mL, respectively (Figure 1).

**Figure 1.** HPLC chromatograms of ginger water and reference standards. (**A**) Standard mix1, (**B**) ginger water, (**C**) standard mix2, and (**D**) ginger water.

#### *3.2. E*ff*ect of Ginger Water on Food Consumption and Average Change of Body Weight*

The obtained results indicated that there was no significant decrease in neither the food consumption nor the water intake in the groups that received ginger water compared to the control. On the other hand, the weekly average body weight exhibited significant differences in the groups that received 25% and 50% ginger water compared to the control group starting from the second week (Figure 2A). The difference was indicated in the lowering body weight gain in the 25% and 50% groups compared to the control. Meanwhile, there are no significant differences among groups that received ginger water at different dose rates.

**Figure 2.** The effect of ginger water on body weight. Values are mean ± standard errors (SE) (*n* = 5). (**A**) Control, control group; 25%, 25% (*v*/*v*) ginger water-treated group; 50%, 50% (*v*/*v*) ginger water-treated group. The effect ginger water on serum level of (**B**) cholesterol and (**C**) triacylglycerol. Values are mean ± SE (*n* = 5). Cont: control, 25%:25% (*v*/*v*) ginger water-treated group, 50%:50% (*v*/*v*) ginger water-treated group. \* *p* < 0.05 vs. the control.

#### *3.3. E*ff*ect of Ginger Water on Serum Total Cholesterol and Triacylglycerol*

Administration of ginger water significantly decreased both serum total cholesterol and serum triacylglycerol compared to the control group. Meanwhile, the difference between groups that received 25% and 50% ginger water is not significant (Figure 2B,C).

#### *3.4. E*ff*ect of Ginger Water Treatment on HSL and SREBP-1c mRNA Expression*

The obtained results showed that the ginger water-receiving groups did not show significant differences with the control group in hormone sensitive lipase (HSL) mRNA expression. On the other hand, ginger water treatment at 25% and 50% induced 50% and 60% downregulation in SREBP-1c mRNA expression, respectively (Figure 3A,B).

**Figure 3.** Effect of ginger water on HSL (**A**) SREBP-1c, (**B**) mRNA expressions in hepatic tissue of rats. Results of densitometric analyses and demonstrative blots of at least five independent experiments are displayed. Values are expressed as means ± SE. Cont: control, 25%:25% (*v*/*v*) ginger water-treated group, 50%:50% (*v*/*v*) ginger water-treated group. \* *p* < 0.05 vs. the control.

#### *3.5. E*ff*ect of Ginger Water Treatment on the Leptin, Adiponectine, and Resistin mRNA Expression in White Adipose Tissue*

The expression of leptin mRNA was significantly downregulated (more than 3-fold) in response to receiving ginger water in both treated groups compared to the control one. Leptin mRNA expression did not show significant differences between 25% and 50% ginger water receiving groups (Figure 4A). Concerning adiponectin mRNA expression, the results showed a significant upregulation (about 2.5-fold) in groups that received ginger water compared to the control group, without significant differences between the two treated groups (Figure 4B). In the same context, ginger water treatment significantly suppressed resistin mRNA expression (Figure 4C).

**Figure 4.** Effect of ginger water on leptin (**A**), adiponectin (**B**), and resistin (**C**), and expression of mRNA in white adipose tissue of rat. Results of densitometric analyses and demonstrative blots of at least five independent experiments are displayed. Values are expressed as means ± SE. Cont: control, 25%:25% (*v*/*v*) ginger water-treated group, 50%:50% (*v*/*v*) ginger water-treated group. \* *p* < 0.05 vs. the control.

#### *3.6. E*ff*ect of Ginger Water Treatment on the GLUT-2 and PK mRNA Expression*

The expression of GLUT-2 mRNA showed a significant upregulation in groups that received 25% and 50% ginger water compared to the control group (Figure 5A). In a parallel manner to GLUT-2, PK showed upregulation in groups that received ginger water that reached a significant degree in the group treated with 50% ginger water compared to the control group (Figure 5B).

**Figure 5.** Effect of ginger water on GLUT-2 (**A**) and PK (**B**) mRNA expressions in the hepatic tissue of rats. Results of densitometric analyses and demonstrative blots of at least five independent experiments are displayed. Values are expressed as means ± SE. Cont: control, 25%:25% (*v*/*v*) ginger water-treated group, 50%:50% (*v*/*v*) ginger water-treated group. \* *p* < 0.05 vs. the control.

#### *3.7. E*ff*ect of Ginger Water Treatment on the CPT-1 and ACO mRNA Expression*

The expression of CPT-1 mRNA showed upregulation in the groups that received ginger water with a significant degree in the 50% group compared to the control group (Figure 6A). Similarly, ACO mRNA showed significant upregulation in groups that received 25% and 50% ginger water compared to the control group (Figure 6B).

**Figure 6.** Effect of ginger water on CPT-1(**A**) and ACO (**B**) expression of mRNA in rat hepatic tissue. Results of densitometric analyses and demonstrative blots of at least five independent experiments are displayed. Values are expressed as means ± SE. Cont: control, 25%:25% (*v*/*v*) ginger water-treated group, 50%:50% (*v*/*v*) ginger water-treated group. \* *p* < 0.05 vs. the control.

#### **4. Discussion**

The use of herbal medicines has increased over the last few years for treatment of obesity. This is due to the rise in population, high cost of medicinal treatment for common disorders, side effects of different current therapeutic drugs, and the appearance of drug resistance. Ginger is considered one of the most commonly used species worldwide [26]. It belongs to the plant family that includes turmeric and cardamom. It has a strong aroma due to its high content of the pungent ketones, including gingerol, which is used in research studies as an extract [27]. Beneficial effects of ginger on obesity and its associated metabolic disorders have been shown [28,29]. It was reported that ginger extract decreases aortic atherosclerotic lesion areas, plasma cholesterol, triacylglycerol, and low-density lipoprotein [30]. In addition, ginger powder strongly decreased serum lipid levels in volunteers [31]. Moreover, ginger meal (1%) significantly lowered cholesterol levels [32]. Our obtained results showed that administration of ginger water significantly reduces the serum triacylglycerol and total cholesterol compared to those of the control group, indicating the hypolipidemic effects of ginger water. Although gingerols constitute the main portion of fresh and dry ginger, many constituents have been detected using different analytical methods [33]. In the present study, two compounds (Chrysin, Galangin) were detected in the ginger water at concentrations of 0.24 μg/mL and 0.53 μg/mL, respectively, using HPLC analysis.

Galangin is a member of the flavonol class of flavonoids and chemically known as 3,5,7-trihydroxyflavone. It is the active constituent of the rhizome of the *Alpinia galanga* plant, which belongs to the Zingiberaceae family [34]. Galangin has been proven to have various pharmacological effects [35], such as antimicrobial activity [36], anticancer [37], anti-inflammatory [38], antioxidative [39], metabolic enzyme modulation [40], and antiobesity [41] effects. Moreover, galangin was found to produce a significant decrease in serum lipids [42]. Other recent studies have revealed that galangin significantly contributed to the protection against acetaminophen-induced acute injury in liver and kidney [43]. An earlier study has shown that galangin has antioxidant activity in vitro and in vivo, free radical scavenging activity, tweaks enzymatic activity, and suppresses genotoxicity of chemicals [39].

Chrysin (C15H10O4) has been shown to be a very active flavonoid exerting some pharmacological properties, such as anti-inflammatory activity through blocking histamine release and expression of proinflammatory cytokines [44,45]. Antiasthmatic activity occurs via suppressing the nuclear factor-kB (NF-kB) and inducible nitric oxide synthase (iNOS) [46]. The anticancer activity of chrysin was also reported [47,48], as well antihypercholesterolemic and cardioprotective activities [49,50].

The antiobesity effect of plant preparations may act through inducing thermogenesis [51], stimulating lipolysis and decreasing lipogenesis [52], suppressing appetite [53], or decreasing lipid absorption [54]. In the current study, the administration of ginger water at a concentration of 25% and 50% showed a marked decrease in the lipogenesis process that was demonstrated by the inhibition of SEREP1c mRNA expression. The obtained data of body weights are parallel with that of leptin levels where nontreated controls showed higher body weights and leptin levels compared to the ginger water-treated groups. These results agree with that of previous studies [55].

On the contrary to leptin, adiponectin mRNA expression was higher in ginger water treated groups compared to the control group. Plasma adiponectin concentration and mRNA expression are decreased in obesity and insulin resistance [56]. Gingerol is well known to decrease serum adiponectin [57]. Therefore, this upregulated adiponectin expression could clarify the lowered blood glucose level. This might be caused by the reduced hepatic gluconeogesis and elevated insulin sensitivity [58].

Administration of ginger water apparently upregulated the hepatic mRNA expression of the lipid degradation gene, HSL, contrasted with the control. This suggested that the ginger water effects are partially caused by the downregulation of the mRNA expression of genes engaged with lipogenesis and upregulation of those concerned with lipolysis.

The lipogenic transcription factor, SREBP1c, regulates lipid metabolism via controlling the gene expression of enzymes for fatty acid synthesis, uptake, and triacylglycerol synthesis [59]. The obtained results showed a significant reduction in the mRNA expression of SREBP1c in groups that received ginger water compared to the control group. The ginger oil effectively suppressed the expression of PPARγ (Peroxisome proliferator-activated gamma), and SREBP1c [60]. Ethanolic extract of ginger reduces the levels of blood glucose in high fat diet-fed rats [15]. It has been also shown to have hypoglycemic and hypolipidemic effects in diabetic rats [16] and mice [61]. The current results showed that ginger water upregulated the expression of GLUT-2 mRNA, which plays a central role in glucose transportation from blood to liver. Moreover, Hepatic PK mRNA expression was upregulated by ginger water. PK is a key player in the glycolytic pathway. Thus, ginger water improves energy metabolism through enhancing glucose uptake via GLUT-2 mRNA expression upregulation, enhancing glucose oxidation via PK mRNA expression upregulation, and enhancing lipolysis and inhibiting lipogenesis via upregulating HSL and downregulating SREP1-c mRNA expressions, respectively. These findings could explain the obtained lipid profile in ginger water-treated groups. Moreover, ginger water could improve energy metabolism through enhancing insulin sensitivity via upregulation of adiponectin and/or downregulating both leptin and resistin expression [62,63]. These findings are in agreement with those of the previous study on the effect of vitamins A and E on lipid and carbohydrate metabolism in diet-induced obese rats [64].

The rate limiting enzyme, Acyl-CoA oxidase (ACO), catalyzes the first step in the peroxisomal β-oxidation [65]. The obtained results revealed that both 25% and 50% of ginger water resulted in upregulation of hepatic tissue ACO mRNA expression. These findings are in line with the previous work, which showed that the treatment with ginger extract led to upregulation of ACO mRNA expression, suggesting its ability to reduce liver fat accumulation through motivation of peroxisomal β-oxidation [66].

Carnitine palmitoyl transferase-I (CPT-I) is a regulatory enzyme of mitochondrial β-oxidation through controlling fatty acid transport to the mitochondrial matrix [18]. Our results revealed that ginger water administration led to upregulation of CPT-1 mRNA expression in hepatic tissue. Upregulation of cellular CPT-I expression motivated fatty acid oxidation and considerably decreased the hepatic triacylglycerol content in both high-fat diet or standard diet [67].

In conclusion, ginger water has a lowering body weight gain effect. It seems to show such activities by regulating the lipid metabolism through stimulation of lipolytic pathways and downregulation of lipogenic pathways. Additionally, ginger water may be helpful in insulin sensitization and facilitating glucose transportation to liver cells as well as improving glucose metabolism. Moreover, ginger water could have nutraceutical potential for controlling body weight, preventing obesity and obesity-associated diseases through its incorporation as food flavor, and in dietary supplements, especially for those going on a diet to lower body weight gain.

**Author Contributions:** Formal analysis, S.S.; Investigation, A.E.-S., H.E.-S. and S.E.-S.; Methodology, M.A. (Mohamed Alkafafy); Resources, S.A.-O., S.F.; Writing—original draft, M.A. (Mohamed Ahmed). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Taif University, Grant Number 1-440-6145.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

## *Arthrospira Platensis* **(Spirulina) Supplementation on Laying Hens' Performance: Eggs Physical, Chemical, and Sensorial Qualities**

**Besma Omri 1,\*, Marwen Amraoui 1, Arbi Tarek 1, Massimo Lucarini 2, Alessandra Durazzo 2, Nicola Cicero 3, Antonello Santini 4,\* and Mounir Kamoun <sup>1</sup>**


Received: 10 July 2019; Accepted: 28 August 2019; Published: 2 September 2019

**Abstract:** The present study evaluated the effects of dietary supplementation of spirulina on laying hens' performances: Eggs' physical, chemical, and sensorial qualities. A total of 45 Lohman White hens, 44 weeks of age, were randomized into 3 groups of 15 birds. Hens were given 120 g/d of a basal diet containing 0% (control), 1.5%, and 2.5% of spirulina for 6 weeks. Albumen height and consequently Haugh unit were significantly affected by dietary supplementation of spirulina (*p* < 0.05) and by weeks on diet (*p* < 0.05). This supplement did not affect (*p* > 0.05) egg yolk weight or height. However, spirulina increased egg yolk redness (a\*) from 1.33 (C) to 12.67 (D1) and 16.19 (D2) and reduced (*p* < 0.05) the yellowness (b\*) parameter from 62.1(C) to 58.17 (D1) and 55.87 (D2). Egg yolks from hens fed spirulina were darker, more red, and less yellow in color than egg yolks from hens fed the control-diet (*p* < 0.0001). However, spirulina did not affect (*p* > 0.05) egg yolks' total cholesterol concentration. In conclusion, a significant enhancement of egg yolk color was found in response to spirulina supplementation. Further investigations are needed to evaluate the impact of spirulina on egg yolks' fatty acids profile.

**Keywords:** cholesterol; egg quality; Haugh unit; spirulina; yolk color

#### **1. Introduction**

*Arthrospira platensis* (spirulina) is a filamentous spiral-shaped blue-green algae [1,2]. It has been recognized as a genus of photosynthetic bacteria (*Arthrospira*). This microorganism belongs to the class of Cyanophyta/Cyanobacteria that grow naturally in warm and alkaline aquatic media. From the perspective of a nutraceutical view [3–8], spirulina is considered as a functional food due to its high protein content (65% to 70% dry matter), high amount of vitamin and mineral content, and wide variety of natural carotene and xanthophyll phytopigments [9,10], and it is generally regarded as safe (GRAS) by the European Food Safety Authority (EFSA) [11]. Spirulina is a source of other nutritionally beneficial organic molecules, such as gamma linoleic acid, phenolic acids, and chlorophyll [12,13]. Deng et al. [14] and Bashandy et al. [15] reported that spirulina has many health benefits, including antioxidant properties, hypolipidemic action, and immunostimulating or anti- inflammatory effects [16,17]. These properties have been verified using laboratory animals [18,19]. Spirulina was used as alternative dietary sources in poultry diets [20]. In these diets, spirulina can be used up to 10% as a partial replacement of

conventional proteins without any adverse effects [21]. Dietary vitamin and mineral premixes can be omitted when spirulina algae are included in chicken rations [22], due to its nutrient-rich composition. Zeweil et al. [23] reported that dietary supplementation of spirulina in chickens under heat stress conditions could decrease adverse effects of chronic heat stress on growth performance and immunity of a Gimmizah local strain of chicken. Spirulina could also be used as an effective way to improve the poultry product quality to meet consumer preferences [24], owing to its high concentration of carotenoids [25]. Zahroojian et al. [26] and Mariey et al. [12] found that dietary inclusion of spirulina at a concentration of 2% to 2.5% in laying hens' feed intensified egg yolk color to make it more aesthetically pleasing for consumers. This coloration intensification is thought to be due to spirulina's high concentration of β-carotene [27]. Dietary incorporation of spirulina can also reduce egg yolk total cholesterol and saturated fatty acid content and increase its omega-3 polyunsaturated fatty acids levels [28,29]. According to Zahroojian et al. [26], it has been shown that addition of spirulina at a level of 2% to 2.5% in the laying hen diet was associated with a significant increase of egg yolk color determined by comparison with the BASF Ovo-Color Fan, an Ovo-Color Yolk Fan supplied by BASF (Florham Park, NJ, USA). However, egg yolk color estimation using the color measuring device, Chroma Meter, was not reported. Therefore, the objective of the present study was to evaluate the effect of dietary incorporation of spirulina on laying hens' performances, egg physical characteristics, egg yolk color, and total cholesterol concentration.

#### **2. Materials and Methods**

#### *2.1. Diet Preparation*

Two kilos of spirulina (*Spirulina platensis*) were purchased from a regional producer located in the region of Gabes (Tunisia). A standard diet (control diet) (C) for laying hens based on corn and soybean meal and 2 supplemented diets, designated as follows: 1.5% of spirulina-supplemented diet (D1) and 2.5% of spirulina-supplemented diet (D2) were individually prepared by mixing the control diet (C) thoroughly with the designated supplements at the required incorporation levels as shown in Table 1.



αControl (C) provided following nutrients per 100 g: Ca, 4.3 g; P, 0.6 g; Na, 0.14 g; Cl, 0.23 g; Fe, 4 mg; Zn, 40 mg; Mn, 7 mg; Cu, 0.3 mg; I, 0.08 mg; Se, 0.01 mg; Co, 0.02; methionine, 0.39 g; methionine + cysteine, 0.69 g; lysine, 0.89 g; Retinol, 800 IU; Cholecalciferol, 220 IU; α-tocopherol, 1.1 IU; Thiamin, 0.33 IU; Nicotinic acid, 909 IU. ¥ Metabolizable Energy = 2707.71 + 58.63 \* EE−16.06 \* NDF [30].

#### *2.2. Ethical Considerations*

All procedures related to animals' care, handling, and sampling were conducted under the approval of the Official Animal Care and Use Committee of the Higher School of Agriculture of Mateur (protocol N◦05/15) before the initiation of the study and followed the Tunisian guidelines.

#### *2.3. Experimental Design*

Forty-five 44-week-old *Lohman White* laying hens were divided randomly into 3 groups of 15 birds. Each group was allocated to one of the three following dietary treatments: (1) Control diet (C), (2) 1.5% of spirulina-supplemented diet (D1), and (3) 2.5% of spirulina-supplemented diet (D2). Each hen was fed daily 120 g of a basal diet containing 0% (control), basal diet plus 1.5% g of spirulina, or basal diet plus 2.5% g of spirulina. The ingredients and chemical composition of the diets are shown in Table 1. Hens were housed in cages with individual feed troughs and common water troughs in a room with ambient temperature of about 20 ◦C and a photoperiod of 16 h light:8 h darkness cycle. Water was provided ad libitum throughout the trial period, which lasted 42 days.

#### *2.4. Data Collection*

All the birds were weighed individually at the beginning and the end of the experiment to determine the live weight changes. Feed was offered once daily at 7:30 a.m. and refusal was measured weekly. Egg production and weight were recorded daily. Daily feed consumption, laying rate (number of laid eggs × 100/number of feeding days), and feed conversion ratio (feed consumption/number of eggs × egg weight) were calculated per week.

Eggs laid during the 1st, 14th, 21st, 28th, 35th, and 42nd days were used for egg physical characteristics measurements (egg albumen, yolk and shell weights, albumen and yolk heights, Haugh unit, yolk diameter, yolk index, and shell thickness) and egg yolk color using the color measuring device Konica Minolta Chroma Meter CR- 400/410 (Minolta Corp.) according to the CIE (Commission Internationale de L'Eclairage) L \* (lightness: negative towards black, positive towards white), a \* (redness: negative towards green, positive towards red), and b \* (yellowness: negative towards blue, positive towards yellow) color system and colorimetric interval, dL \* (Lightness interval), da \* (Red/Green interval), and db \* (Yellow/Blue interval), between the spirulina-supplemented diet (D1 and D2) and control diet (C). The instrument was set perpendicular to the egg yolk surface in a Petri dish. The parameters, L \*, a \*, and b \*, were measured three times and the final values were calculated as the averages of the three corresponding values measured.

Haugh unit (UH) was calculated according to the formula [31]:

$$\text{Haugh unit} = 100 \times \log \text{ (HA} - 1.7 \times \text{W0.37} + 7.6),$$

where: HA = albumen height (mm) and W = egg weight (g).

Yolk index was calculated according to the formula:

Yolk index = Yolk height (mm)/Yolk diameter (mm).

Shell thickness, albumen and yolk heights, and yolk diameter were measured using a caliper. Eggs laid during days 1 and 28 of the experimental period were pooled per hen and used for egg yolk total cholesterol determination.

#### *2.5. Chemical Analysis*

The dry matter of diets (DM) was determined at 105 ◦C for 24 h. All other analyses were done on samples dried at 65 ◦C and ground in a mill to pass through a 0.5-mm screen. Ash content was determined by igniting the ground sample at 550 ◦C in a muffle furnace for 10 h. The Association of Official Analytical Chemists method [32] was used for crude protein (CP) determination.

Egg yolk samples pooled per hen were solubilized in 2% (w/v) NaCl solution [33] and used for cholesterol determination using standard enzymatic-colorimetric methods (cholesterol enzymatic colorimetric test, CHOD-PAP Biomaghreb, Tunisia).

#### *2.6. Statistical Analysis*

Data of repeated measurements (feed refusal and intake, laying rate, egg mass, feed conversion ratio, egg physical characteristics, yolk total cholesterol, and yolk color traits) were tested for diet, week on diet effects, and their interaction using mixed models with compound symmetry covariance structures of SAS (Statistical Analysis System) [34].

Data of the hens' live weight change were tested for diet effect using the general linear model (GLM) procedure of the Statistical Analysis System (SAS) [34] according to the following model:

Yij=u+Ai+eij,

where:

Yij= represents the *j*th observation on the *i*th treatment;

μ = overall mean;

Ai= main effect of the *i*th treatment;

eij= random error present in the *j*th observation on the *i*th treatment.

#### **3. Results**

#### *3.1. Laying Performance*

Laying hens' performances are shown in Table 2. All hens showed a loss of body weight at the end of the experimental period, but this weight loss was not affected (*p* > 0.05) by dietary treatment. Feed refusal and consumption was not affected (*p* > 0.05) by dietary addition of spirulina and did not change (*p* > 0.05) over the weeks on the diet. In parallel, the laying rate and consequently daily egg mass production were not affected (*p* > 0.05) by dietary treatment and weeks on the diet and their interaction. Only egg weight was affected (*p* < 0.05) by dietary treatment. Supplementation of 2.5% spirulina (D2) increased (*p* < 0.05) egg weight from 62.76 ± 1.53 g to 64.33 ± 1.83 g. The feed conversion ratio (FCR) was not affected (*p* > 0.05) by dietary treatment, weeks on diet, and their interaction.


**Table 2.** Effect of spirulina on hens' live weight changes and laying performances.

<sup>α</sup> **C** = control diet with 0% of spirulina; <sup>α</sup> D1 = control diet supplemented with 1.5% of spirulina; <sup>α</sup> D2 = control diet supplemented with 2.5% of spirulina; and SEM = standard error of the mean; <sup>β</sup> trt = treatment; <sup>β</sup> W = week; <sup>β</sup> trt \* W <sup>=</sup> treatment – week interaction; \* = *p* < 0.05; NS = *p* <sup>≥</sup> 0.05; ab: Mean in the same row with different superscripts are significantly different (*p* < 0.05).

#### *3.2. Egg Physical Characteristics*

The determined physical characteristics of eggs (egg, yolk, albumen and shell weights, yolk and albumen heights, yolk diameter, UH (Haugh Unit), and yolk index) are shown in Table 3.

Ours results showed that egg and albumen weights were affected (*p* < 0.05) by dietary inclusion of spirulina. Mean egg weight varied from 62.22 ± 2.98 g (C) to 64.43 ± 3.04 g (D2). Albumen weight of hens fed on 2.5% spirulina was the highest, with mean values of 36.20 ± 2.1 g vs. 35.08 ± 2.5 g (D1) and 34.41 ± 1.81 g (C). Shell thickness was affected by dietary treatment (*p* < 0.05), weeks on diet (*p* < 0.0001), and their interaction (*p* < 0.0001).

Albumen height and consequently Haugh unit were significantly affected by dietary supplementation of spirulina (*p* < 0.05) and by weeks on diet (*p* < 0.05). Dietary incorporation of spirulina did not affect (*p* > 0.05) egg yolk weight and height. However, these parameters were influenced (*p* < 0.05) by weeks on diet. Egg yolk diameter and index were affected by weeks on diet (*p* < 0.0001) and the interaction, treatment\*week on diet.

Concerning albumen height, UH, and shell thickness, for each diet, differences between parameters at week 1 and their average mean at week 3 and week 6, as well as differences between means at week 3 and week 6, were compared (Table 4). Tested differences of albumen height were significant (*p* < 0.05) for the control diet, indicating an increase of albumen height at week 1. For each of the D1- and D2-diets, only differences between heights at week 3 and week 6 were significant (*p* < 0.05), indicating an increase of albumen height at week 6. UH did not change (*p* < 0.05) over time for the control, decreased (*p* > 0.05) at week 3 for the D1-diet, and increased (*p* > 0.05) for the D1-diet and D2-diet at week 6. Shell thickness increased (*p* > 0.05) for the D2-diet at week 1, week 3, and week 2 and for the D2-diet at week 6.


**Table 3.** Effect of spirulina on egg physical characteristics.

<sup>α</sup> **C** = control diet with 0% of spirulina; <sup>α</sup> **D1** = control diet supplemented with 1.5% spirulina; <sup>α</sup> **D2** = control diet supplemented with 2.5% spirulina; **SEM** & = standard error of the mean; <sup>β</sup> trt = treatment; <sup>β</sup> W = week; <sup>β</sup> trt \* W <sup>=</sup> treatment–week interaction; \*\*\* = *p* < 0.0001; \*\* = *p* < 0.001; \* = *p* < 0.05; NS = *p* <sup>≥</sup> 0.05; ab: Mean in the same row with different superscripts are significantly different (*p* < 0.05).


**Table 4.** Week effect of the diet's distribution on albumen height, UH (Haugh Unit), and shell thickness.

<sup>α</sup> **C** = control diet with 0% spirulina; <sup>α</sup> **D1** = control diet supplemented with 1.5% spirulina; <sup>α</sup> **D2** = control diet supplemented with 2.5% spirulina; \*\*\* = *p* < 0.0001; \*\* = *p* < 0.001; \* = *p* < 0.05; NS = *p* ≥ 0.05.

#### *3.3. Egg Yolk Color*

Egg yolk color traits determined by a Konica Minolta Chroma Meter CR-410 were affected by dietary treatment (*p* < 0.0001), weeks on diet (*p* < 0.0001), and their interaction (*p* < 0.0001) (Table 5). Hens fed the control diet had the highest (*p* < 0.0001) lightness L\*, with a mean value of 70.55 vs. 65.98 and 63.74 corresponding to hens fed with 1.5% and 2.5% spirulina, respectively. These values showed that the egg yolk of the control group was characterized by an intense yellow color.

Dietary supplementation of spirulina increased egg yolk redness, a\*, from 1.33 ± 2.34 (C) to 12.67 ± 8.94 (D1) and 16.19 ± 9.85 (D2). The redness mean value (a\*) corresponding to the control group indicated that this group had a weak red hue. Concerning the yellowness (b\*), hens fed the diet without spirulina supplementation had the highest mean value (62.1 ± 2.66), which indicated that egg yolk color was sufficiently intense. Dietary inclusion of spirulina resulted in a significant decrease (*p* < 0.0001) of the egg yolk yellowness (b\*). In fact, hens fed 2.5% and 1.5% spirulina had a yellowness mean value of 55.87 ± 3.93 and 58.17 ± 3.41, respectively.

The colorimetric interval between the spirulina-supplemented diet and control diet are represented in Table 6. These colorimetric intervals, dL\*(lightness interval), da\*(red/green interval), and db\*(yellow/blue interval), between the spirulina-supplemented diet and control diet (C), determined by a Chroma Meter, showed that egg yolks from hens fed spirulina were darker, more red and, less yellow in color than egg yolks from hens fed the C-diet (*p* < 0.0001). It was found that egg yolk color parameters (L\*, a \*, and b\*) changed over time for all diets (Table 7). L\* decreased (*p* < 0.0001) in the C-diet, D1-diet, and D2-diet after the first week and increased (*p* < 0.0001) at week 3 and 6 for the D1-diet and D2-diet. However, egg yolk lightness decreased (*p* < 0.001) at the sixth week for the control diet. Concerning egg yolk redness (a\*), a significant increase (*p* < 0.0001) was found at week 1 for the three diets. This increase was recorded during the experimental period for the D1 and D2 differences between the mean a\* values at week 3 and were significant (*p* < 0.0001), indicating a decrease of egg yolk redness. Egg yolk yellowness increased (*p* < 0.0001) at week 1, week 3, and week 6 for C-, D1-, and D2-diets. By contrast, differences between the mean b\* values for D2 at week 3 and for C at week 6 were negative (*p* < 0.0001), indicating a decrease of egg yolk yellowness.


**Table 5.** Effect of spirulina on egg yolk color.

<sup>α</sup> C = control diet with 0% spirulina; <sup>α</sup> D1 = control diet supplemented with 1.5% spirulina; <sup>α</sup> D2 = control diet supplemented with 2.5% spirulina; SEM & <sup>=</sup> standard error of the mean; <sup>β</sup> trt <sup>=</sup> treatment; <sup>β</sup> <sup>W</sup> <sup>=</sup> week; <sup>β</sup> trt \* W <sup>=</sup> treatment–week interaction; \*\*\* <sup>=</sup> *<sup>p</sup>* <sup>&</sup>lt; 0.0001; a,b,c: Means in the same row with different superscripts are significantly different (*p* < 0.05); L\*: lightness, a\*: redness and b\*: yellowness.

**Table 6.** Colorimetric interval between the spirulina-supplemented diet and control diet.


<sup>α</sup> C = control diet with 0% spirulina; <sup>α</sup> D1 = control diet supplemented with 1.5% spirulina; <sup>α</sup> D2 = control diet supplemented with 2.5% spirulina; SEM **&** = standard error of the mean; <sup>β</sup> trt = treatment; <sup>β</sup> W = week; <sup>β</sup> trt\*W = treatment–week interaction; \*\*\* = *p* < 0.0001; \* = *p* < 0.05; a,b: Means in the same row with different superscripts are significantly different (*p* < 0.05).


**Table 7.** Week effect of the diet´s distribution on egg yolk color.

<sup>α</sup> C = control diet with 0% spirulina; <sup>α</sup> D1 = control diet supplemented with 1.5% spirulina; <sup>α</sup> D2 = control diet supplemented with 2.5% spirulina; \*\*\* = *p* < 0.0001.

#### *3.4. Egg Yolk Cholesterol Concentration*

The effect of dietary supplementation of 1.5% and 2.5% spirulina on egg yolk total cholesterol concentration is represented in Table 8.

Our data showed that egg yolk concentration of total cholesterol was only affected (*p* < 0.05) by the weeks on diet. However, egg yolk concentration of total cholesterol was 14.35 ± 0.88 mg/g for hens fed the C-diet vs. 13.89 ± 1.21 and 14.39 ± 1.23 mg/g for hens fed 1.5% and 2.5% spirulina, respectively.


**Table 8.** Effect of dietary incorporation of spirulina on egg yolk cholesterol concentration.

C = control diet with 0% spirulina; D1 = control diet supplemented with 1.5% spirulina; D2 = control diet supplemented with 2.5% spirulina; SEM = standard error of the mean; Trt = treatment; W = week; Trt \* W = treatment–week interaction; \* = *p* < 0.05; NS = *p* ≥ 0.05; a: Means in the same row with the same superscripts are not significantly different (*p* ≥ 0.05).

#### **4. Discussion**

#### *4.1. Laying Performances*

Spirulina inclusion was without impact on feed consumption. Our results are in agreement with those reported by Dogan et al. [35], who reported that dietary addition of 0.5%, 1%, and 2% of spirulina did not affect feed consumption of laying quails.

In the present study, hens' live body weight losses were 56.00 vs. 19.33 g/42 d and laying rates were high (92.19% vs. 96.38%) and not affected by spirulina inclusion. Hens were fed ad libitum before our experimental study and then feed was restricted to 120 g/d so that animals showed this loss of body weight throughout the experimental period. Furthermore, only egg weight was significantly increased from 62.76 (C) to 63.18 g (1.5% spirulina) and 64.33g (2.5% spirulina). This increase of egg weight could be attributed to the high protein content in spirulina. These results are partially in agreement with those reported by Mariey et al. [12], who found that hens' live weight of Sinai (S) and Gimmizah (G) was not affected by dietary supplementation of 0.1%, 0.15%, and 0.2% spirulina. However, this supplementation decreased the feed conversion ratio. The lowest value was attributed to hens fed 0.2% spirulina; 3.46 versus 4.54 for the control group. Dogan et al. [35] also reported that dietary inclusion of 0.5%, 1%, and 2% spirulina did not affect the laying rate, feed conversion ratio, and egg weight. By contrast, Selim et al. [36] found that inclusion of 0%, 0.1%, 0.2%, and 0.3% spirulina increased hens' final weight from 1222 g (0%) to 1227 (0.1%), 1238 (0.2%), and 1253 g (0.3%). However, Mariey et al. [12] reported that dietary incorporation of 0.1%, 0.15%, and 0.2% spirulina increased the laying rate, egg weight, and egg mass. Zahroojian et al. [26] showed that addition of 1.5%, 2%, and 2.5% spirulina in the diet of 128 Hy-line White hens did not affect the laying rate and egg weight. This absence of changes in laying hens' performances associated with the use of spirulina might be attributed to the rate of inclusion of this alga, the variety, cultural practices, and climate.

#### *4.2. Egg Physical Characteristics*

With the exception of egg and albumen weights, albumen height, UH, and shell thickness, egg characteristics were not affected (*p* > 0.05) by dietary treatment and observed changes over time were numerically small and, therefore, of little physiological significance. Selim et al. [36] reported that dietary addition of 0.1%, 0.2%, and 0.3% spirulina did not affect egg physical characteristics (albumen index, albumen, yolk and shell weights, yolk index, and Haugh unit) determined at the end of the fourth week of the experiment trial. However, hens fed 0.1%, 0.2%, and 0.3% spirulina had a thicker shell of 0.356, 0.401, and 0.423 mm compared to those fed the control diet (0.314 mm). This finding could be attributed to the high calcium content of spirulina. Concerning the Haugh unit, Parisse [37] reported that eggs with a Haugh unit higher than 70 are considered excellent eggs, eggs with 70 to 60 Haugh units are acceptable, while eggs with Haugh units below 60 are of poor quality. Our results showed that hens fed 1.5% spirulina had the highest Haugh unit, with a mean value of 97.28 versus 95.99 (2.5%) and 93.33 (0%). Mean values of the present study were higher than 70, thus our eggs may be considered as excellent eggs. This high Haugh unit could be attributed to the fact that this parameter was determined on each egg laid on the same day and not on stored eggs. The pigment content of the supplemented spirulina could be responsible for the observed difference between treatments.

Mariey et al. [12] reported that dietary supplementation of spirulina at 0.1%, 0.15%, and 0.2% did not affect shell weight, albumen percentage and index, and Haugh's unit. Our data showed that dietary inclusion of spirulina did not affect egg yolk weight, height, diameter, and index. These results were not in agreement with those reported by Dogan et al. [35], who found that incorporation of 2% spirulina increased the egg yolk index of laying quails from 47.48 to 48.45 mm, shell weight from 1.55 to 1.68 g, and shell thickness from 0.199 to 0.207 mm. Mariey et al. [12] also reported that dietary supplementation of 0.15% spirulina increased egg yolk weight from 31.10 to 32.90 g.

By contrast, Zahroojian et al. [26] reported that incorporation of 1.5%, 2%, and 2.5% spirulina did not affect eggs' physical qualities (yolk index, Haugh unit, shell thickness, shell weight, and specific gravity)

#### *4.3. Egg Yolk Color*

Egg choice by consumers is no longer only based on yolk cholesterol content or fatty acids profile but also on its color [38]. The required degree of pigmentation varies among and within countries, but golden to yellow colors are usually considered more attractive [39]. Egg yolk color intensification can be achieved by dietary supplementation of carotenoids. However, laying hens are unable to synthesize these pigments. They need to be provided in their diet's ingredients [40]. Carotenoids are synthesized by algae, plants, fungi, and some bacteria. In the present study, egg yolk color intensification was achieved with dietary incorporation of spirulina. Concerning egg yolk color evaluation, it was estimated by colorimetric determination of lightness (L \*), redness (a \*), and yellowness (b\*) indexes and colorimetric intervals (ΔL \*, Δa \*, Δb \*).

Our results showed dietary supplementation of spirulina increased egg yolk redness and reduced yolk yellowness. Studies on the effect of spirulina on egg yolk color traits measured by a Chroma Meter are lacking. However, Mariey et al. [12] reported that dietary incorporation of 0.1%, 0.15%, and 0.2% spirulina increased the egg yolk color score (RYCF) from 6.3 (0.1%) and 6.7 (0.15%) to 7.6 (0.2%). Zahroojian et al. [26] also found that supplementation of 1.5%, 2%, and 2.5% spirulina increased the egg yolk color score from 10.55 (1.5%) and 11.43 (2%) to 11.66 (2.5%) compared to the control. Dietary inclusion of 0.1%, 0.2%, and 0.3% spirulina increased the egg yolk color score from 6.11 (0.1%) to 6.89 (0.2%) and 7.33 (0.3%) [36]. Anderson et al. [27] also evaluated the effect of dietary addition of 0.25%, 0.5%, 1.2%, and 4% spirulina, which increased quail egg yolk color measured at the 2nd and 23rd day of treatment. Park et al. [41] reported that incorporation of marine microalgae (*schizochytrium*) at 0.5% and 1% in laying hens' diet increased the egg yolk color score after 6 weeks of treatment, with a mean value of 9 and 8.8 compared to 8.7 corresponding to the control group.

#### *4.4. Egg Yolk Cholesterol Concentration*

Dietary supplementation of spirulina did not affect the egg yolks' total cholesterol concentration. The absence of the effect of spirulina on egg yolk total cholesterol was in agreement with the results reported by Zahroojian et al. [26], who reported that dietary addition of 1.5%, 2%, and 2.5% spirulina did not affect the egg yolk concentration of cholesterol, with mean values of 10 (1.5%), 10.59 (2%), and 11.81 mg/g (2.5%). By contrast, Dogan et al. [35] reported a reduction in egg yolk cholesterol concentration per gram of yolk from 19.65 to 18.93 when laying hens' diet were supplemented with 1% and 2% spirulina. Mariey et al. [12] also reported that egg yolk concentration of cholesterol decreased from 13.50 to 10.20 mg/g with dietary addition of 0.2% spirulina. Total egg cholesterol also decreased from 12.9 to 9.9 mg/g when spirulina was supplemented at a level of 0.3% [28].

Selim et al. [36] reported that dietary incorporation of 0.1%, 0.2%, and 0.3% spirulina reduced egg yolk cholesterol concentration from 13.6 mg/g (control) to 13.1 (0.1%), 12.4 (0.2%), and 11.7 mg/g (0.3%). Park et al. [41] also found that incorporation of *schizochytrium*, a marine microalgae, in laying hens' diets at a level of 0.5% and 1% reduced serum cholesterol from 133.8 to 118.5 mg/dl. These authors attributed this reduction to the high contents of polyunsaturated fatty acids in spirulina. In fact, omega-3 fatty acids stimulate the activity of LCAT (lecithin cholesterol acyltransferase) [42], an enzyme

responsible for the serum cholesterol esterification [43], so that most newly formed cholesterol esters are initially incorporated in HDL (High-Density Lipoprotein) [44].

Chen et al. [45] also reported that the docosahexanoic acid (DHA) of microalgae may inhibit the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase by reducing cholesterol synthesis so that the serum cholesterol concentration decreases.

It can be concluded that incorporation of spirulina in 44-week-old Lohman White laying hens' diets was without effects on laying hens' performances and increased egg weight, shell thickness, albumen weight and height, and Haugh unit. The use of *Spirulina platensis* as a laying hens' feed additive increased egg yolk color, as measured by a chromameter, and did not affect the total cholesterol concentration. Further investigations are needed to evaluate the impact of spirulina on egg yolk

**Author Contributions:** B.O., M. K., and A.S., have conceived the work. B.O., M.A., A.T. and M.K. have carried out the experimental study and analyzed the data. B.O., M.A., A.T., M.K., M.L., A.D., N.C. and A.S. wrote the manuscript. All authors have made a substantial contribution to revise the work, and approved it for publication.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*
