Saffron extract feed improves the antioxidant status of laying hens and the inhibitory effect on cancer cells (PC3 and MCF7) Growth

Abstract Background There have been some reports regarding supplementation of saffron petal extract on performance and egg quality in laying hens. However, the effect of saffron petal extract fed diet at different amounts on antioxidant status of laying hens and the impact of the resulting egg yolk on growth/inhibitory activity of cancer cells has not been fully studied. Objectives The effect of dried saffron petal extract on the laying performance, egg qualitative traits, antioxidant status, and its inhibitory effect on cancer cells was studied. Methods A total of 200 39‐week‐old Hy‐line W36 Leghorn laying hens were selected based on a completely randomised design in four treatments with five replications (10 hens per replication). The four treatment diets consisted of a basal diet with no supplement (control), and three diet supplement groups containing 40, 60 and 80 ppm of saffron petal extract. Results Adding 80 ppm of saffron petal extract to layer diets increased egg production (p < 0.05). Malondialdehyde,1,1‐diphenyl‐2‐picrylhydrazyl value and Superoxide dismutase significantly improved by saffron petal dietary supplementation. The yolk weight and colour, Haugh unit and shell weight and thickness were also influenced (p < 0.05) with highest values achieved in the 60 ppm saffron extract supplemented diet. Results demonstrated a significant effect of saffron extract inclusion in the diet on the growth of Michigan Cancer Foundation‐7 and Prostate Cancer Cell in a positive dose‐dependent manner (p < 0.05) and the most intense inhibitory effect on cancer cells was observed with 60–80 ppm extract. Conclusions Saffron petal extract can be used to potentially modulate the antioxidant status of laying hens and the inhibitory effect on cancer cells, best achieved with 60–80 ppm extract.


INTRODUCTION
Plants are rich in several phytochemicals or bioactive chemicals and incorporated into the animal feed for enhance productivity (Moharreri et al. (2022). Saffron has traditionally been applied as a herbal medicine. More than 150 different compounds involving carbohydrates, polypeptides, lipids, minerals and vitamins are found in saffron (Khorasany & Hosseinzadeh, 2016). Saffron contains picrocrocin, safranal, crocetin, α-crocin, lycopene, zeaxanthin and αand βcarotenes. As shown, it is rich in elements like Zn, Fe, Cu, Se, Mg, P, Ca and Mn, and several vitamins such as vitamin A and C, folic acid, riboflavin and niacin (Qadir et al., 2020). Saffron (Crocus sativus) has been considered as an additive that improves egg quality (Botsoglou et al., 2010) and oxidative stability of egg yolk (Botsoglou et al., 2005a,b). Saffron petal, as an important by-product of saffron, is annually produced in large amounts (more than 10,000 tons/year) and usually discarded as a waste product (Kafi et al., 2006). The antioxidant properties of saffron petal (Goli et al., 2012;Serrano-Díaz et al., 2012) may be related to its phenolic compounds such as crocin and kaempfrol (Zeka et al., 2015). Different pharmacological properties of saffron petal such as antibacterial, antispasmodic, immunomodulatory, antitussive, antidepressant, antinociceptive, hepatoprotective, renoprotective, antihypertensive, antidiabetic and antioxidant activity have been reviewed . Intake some of feed additives is always considered to be important in improved performance. Bentonite prevents the effects of aflatoxin present in feed that could increase uric acid absorption from the intestine (Khanedar et al. (2013), and hydroethanolic saffron petals' extract reducing the associated oxidative damages in challenged by aflatoxin (Hosseini-Vashan et al., 2018). Adding saffron petal powder to broiler diets decreased feed conversion ratio and increased feed intake, while bursa of Fabricius, body weight as well as ventricular fat were not influenced (Naghous et al., 2015). Incorporation of hydroalcoholic extract of saffron petals into quail diet enhanced the relative weight of lymphatic organs, body weight, and feed intake, while ventricular fat and feed conversion ratio decreased (Hosseini-Vashan et al., 2018). In addition, dietary inclusion of adding saffron petals decreased yolk cholesterol and increased yolk colour in laying hens (Jabbari Namroudi et al., 2021).Therefore, the present study aimed to evaluate the effects of saffron hydroalcoholic extract at various concentrations on egg qualitative traits and production function, antioxidant status, as well as the impact of the egg yolk containing saffron on growth/inhibitory activity of Michigan Cancer Foundation-7 (MCF7) and Prostate Cancer Cell (PC3).

Study design
In the current study, 200 39-week-old Hy-line W36 Leghorn laying hens based on a completely randomised design (CRD) were allocated into four treatments with five repetitions (10 hens per replication).This

Diets
The diets were adjusted based on the requirements recommended for the strain Hyline by using UFFDA software, the components of which are presented in Table 1. The present experiment was performed for 12 weeks since the beginning of laying hen production until the end of its peak.
Further, There were four experimental treatments that consisted of a basal diet + 0 (control), 40, 60 or 80 ppm of saffron petal extract. The extract powder was stored in dark bottles at 4 • C until use. In this regard, the content of kaempferol and crocin, anthocyanin, and total phenolic compounds was respectively measured according to Zeka et al. (2015) and Goli et al. (2012).

Total anthocyanin content
Total anthocyanin content (TAC) was evaluated by pH differential method (Giusti & Wrolstad, 2003). Hence, 0.2 g of each sample was dissolved in 10 ml of distilled water in a volume flask far from the light.
Absorbance was measured at 510 nm and 700 nm.

Total phenolic content (TPC)
Folin-Ciocalteau method was used to measure the total phenolic content (TPC) of the extracts (Singleton et al., 1999). TPC was presented as mg gallic acid (GA) equivalents per g. For 100 μl test sample solution made from 100 mg POPx in 10 ml of methanol, 6 ml of double-distilled water and 500 μl of Folin-Ciocalteau reagent were added.

Determination of total flavonol content (TFC)
The TFC content was determined according to the colorimetric assay of Spigno et al. (2007). Briefly, 0.5 ml of extracts was added into a 2.5 ml of water. At zero time, 150 μl of 5% NaNO2 was added to the mixture.
After 6 min, 0.3 ml of 10% AlCl3 was added into the flask. At 5 min, 1 ml of 1 M NaOH was added to the mixture. The reaction flask was immediately diluted to volume with the addition of 550 μl of distilled water and thoroughly mixed.

Egg production performance
Diet was formulated according to guidelines and was ad libitum provided. Daily feed intake (g), egg production (% /hen/ day production) and feed conversion ratio were calculated during the experiment. In each treatment, daily feed intake was determined by subtracting the feed level remaining at the next day from the total feed for the treatment. A digital scale with the precision of 0.01 was applied to measure egg weight. The weights of feed and eggs were recorded using an electronic weighing scale with an accuracy of 10 kg × 0.5 g (Model DT580, Atrontec Electronic Tech Co., Ltd., Jiangsu, China).

Qualitative traits of egg
In each month, four eggs were selected from each repetition and weighted to assess some qualitative traits. Then, egg shape, yolk index and colour (Vuilleumier, 1969), shell thickness (mean shell thickness in the wide and narrow end of egg, as well as its equator with the precision of ± 0.01 mm), shell weight (±0.01 g), Haugh unit and egg weight were measured (Holder & Bradford, 1979).
Shape index was obtained by using the following equation.
where SI represents shape index, EW refers to egg weight and EL refers to its length.

Antioxidant activities
To evaluate the antioxidant status, the activity of serum superoxide dismutase (SOD) enzyme was evaluated using Randsel SOD diagnostic kit (Randox, Crumlin, Uk) in Mad laboratory. Malondialdehyde was determined as the final product of lipid peroxidation in chicken hepatocytes using thiobarbituric acid reactant (TBARS). The measurement of free radical scavenging activity was performed according to previous research (Xie, 2014 (Rezaeian & Pourianfar, 2018). Based on the methods, MCF7 and PC3 cells were cultured in RPMI and DMEM media, respectively.
Both media (as a solution containing sodium bicarbonate) were mixed with heat-inactivated FBS (10%), filtered through a 0.22-μm filter, and utilised for cell culture. The cells were cultured in the sterile flasks having filter cap with the growth area of 75 cm 2 by considering the experiences acquired by cell passage. Then, they were frozen and reproduced as detailed in the previous study (Rezaeian & Pourianfar, 2018). Briefly, the freezing process was employed for the cells at exponential growth phase in a fresh freezing medium consisting of FBS and DMSO (Merck, Darmstadt, Germany) in a 9:1 ratio. Finally, the effect of egg yolk at different concentrations on the inhibition of cancer cell growth and proliferation was evaluated.

Statistical analysis
This experiment was conducted in the form of simple CRD and CRD with several observations (qualitative traits of egg) in each replication by using the following models, respectively.
where μ demonstrates mean trait, T i indicates the effect of treatment and ɛ ijk and e ijk illustrate the effect of sampling and experimental error, respectively. [Correction added on 23 September 2022, after first online publication: Equation 3 was corrected.] The data were analysed by using GLM in SAS statistical software. Duncan test was utilised to compare the mean of various treatments when the mean difference was significant at p < 0.05.
Additionally, the statistical analysis of cell culture was carried out in SPSS 22 software. All of the cell tests were performed with at least three completely independent replications on separate days according to the instruction for cell cultures (Lazic et al., 2018). In each independent repetition (each 96-well plate), three wells were assigned to each treatment (as sub-sample). The main factor as a treatment (independent variable) included four diets, which was studied in the form of the eggs from the chickens fed with the diets. Furthermore, yolk was removed and tested as a treatment on the cell. The trait under study (dependent variable) was the level of the decrease in cell growth compared to the negative control (untreated).Two different cell lines were examined, to which each diet level was provided in five consecutive concentrations. The statistical analyses were implemented to assess the effect of diet level on cell growth reduction.  Jadouali et al. (2018).The reasons for the differences between our reported values and that of other researchers are probably attributed to the genetic variation, ecological conditions and/or the differences in the extraction method. Table 3 outlines the effects of saffron petal hydroalcoholic extract on egg production performance in layers. As shown, a significant increase was observed in egg production percentage (p < 0.05) in the treatment 4. Adding 80 ppm of saffron petal extract to layer diets increased egg production due its bioactive compounds, minerals and various vitamins that affects feed intake and with a positive effect on nutrient absorption efficiency improves body weight and feed conversion ratio. In addition, saffron petals had high levels of camphor, flavonoids, quercetin and minerals, which decreased the activity of free radicals and break down fats, leading to an increase in the availability of nutrients, along with the growth of poultry (Hosseini & Mollafilabi, 2017;Omidi et al., 2014). However, the treatments had no effect on feed intake, conversion ratio, and egg weight and mass (p > 0.05). Linear and quadratic effects were also not significant.

RESULTS AND DISCUSSION
The effects of saffron petal hydroalcoholic extract on the qualitative traits of the egg from layers are summarised in SEM, standard error of the mean.
Linear effect for colour yolk and Quadratic effects for yolk weight, Haugh unit, and shell weight was significant. Saffron is rich in carotenoids (Bolhassani et al., 2014), like crocin and crocetin that are considered to be the main and crocin and crocetin as the two main natural carotenoids include were considered to be the main contributor for the colour enhancement (Bolhassani et al., 2014;Daniel, 2006;Mohajeri et al., 2010). Thus, this improvement in yolk colour reflects the transfer of crocins, lycopenes and carotenes, as the colouring constituents of saffron, from chicken diet to egg yolk (Tarantilis et al., 1995).

F I G U R E 1
Microscopic image related to the growth reduction effects of the yolk received the treatment 4 at the concentration of 5000 μg/ml on MCF7 cancer cells (right) compared to the negative control (left), which was taken 72 h after treatment and a few min before MTT when the cells were washed with PBS buffer other treatments led to the high inhibition at 5000 μg/ml of egg yolk.
However, there was a significant difference between the concentrations of 5000 μg/ml compared to the egg yolk obtained using T4  Following the second treatment T2, the concentrations of 1250 and 5000 μg/ml resulted in maximising the percentage inhibition. Although the difference was insignificant (p > 0.05). The highest and lowest inhibition percentages in the last treatment T4 was 5000 μg/ml so that an increase in concentration led to an increase in the percentage inhibition. In this treatment, a significant inhibition was observed at 5000 μg/ml, which is consistent with those obtained for MCF7
Based on the results the inhibition percentages on PC3 cancer cells in different concentrations were not significant in T1 and T2 diets. In T3 diet, the inhibition percentage of 625 μg/ml was significant compared to the negative control, which was about 20%. Although no significant difference was reported between 625, 1250 and 2500 μg/ml in terms of inhibition percentage (p ≥ 0.05), 20% inhibition at 625 μg/ml was considered as important since it has a very low concentration.
The results may indicate that PC3 cells are very sensitive to even low TA B L E 6 Mean inhibition (percentage relative to the negative control) of the various concentrations of the egg yolk from different diets against PC3 cancer cell (n = 3, p < 0.05) The means with various letters in each row are significantly different (p < 0.05). Recently, some researchers have highlighted many therapeutic roles for saffron and its components such as anticancer, antitumour and antineuropathic properties (Amin & Hosseinzadeh, 2012;Hosseinzadeh, 2014;Hosseinzadeh et al., 2005). Furthermore, saffron plays an essential role in cell proliferation inhibition, apoptosis induction, antioxidant and free radical inhibitory effects, gene conservation, lipid peroxidation prevention, and anti-inflammatory processes. The results of the above-mentioned mechanisms indicated the potential therapeutic properties of saffron for liver, gastric, colorectal, pancreatic and ileum cancers (Khorasany & Hosseinzadeh, 2016). Given that crocetin is known as an anticancer compound, it exhibits antioxidant, antiproliferative and apoptosis activities against cancer cells . Reducing the level of malondialdehyde and promoting the level of glutathione and antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase are the main functions of crocin against oxidative stress (Sun et al., 2014). Furthermore, it, as another active constituent of saffron, represents anticancer effects similar to crocetin . and its components do not affect normal cells (Milajerdi, et al., 2015).

F I G U R E 2
However, conducting further research is necessary for testing these effects on several normal human cell lines.

ANTIOXIDANT STATUS
The antioxidant effects of saffron petal extract in laying hens are reported in was significantly decreased in treatments containing saffron at 60 and 80 ppm compared to the control and saffron at 40 ppm (p < 0.05).
Also, the percentage of DPPH in experimental treatments, especially diets containing saffron, increase significantly compared to the basic diet (p < 0.05). Also, the amount of SOD in the control diet compared to other diets and the control showed a significant decrease (p < 0.05). SOD, the primary antioxidant enzyme that protects cells from oxidative stress, increases the antioxidant ability to eliminate overactive reactive oxygen species (ROS) and reduce lipid peroxidation in poultry (Zhu et al., 2017). In addition, it increased serum antioxidant status by increasing T-AOC content, T-SOD and Mn-SOD activities and decreasing MDA content (Zhang et al., 2020). Eggs can be enriched with antioxidants through manipulating poultry feed (Surai et al., 2006).

CONCLUSION
In general, the addition of saffron petal hydroalcoholic extract into diet leads to an improvement in egg production percentage (at 80 ppm in layers diet) and egg quality (yolk colour and Haugh unit). Further, a significant effect of the diet on the growth/inhibition of MCF7 and PC3 cancer cells was observed in a positive concentration-dependent manner (p < 0.05). Some treatment levels indicated a dose dependent effect on the cancer cell inhibition, with higher inhibition observed at lower concentrations of egg yolk and some reduced effects at higher concentrations. Like the results of the previous studies, some egg yolk components actually could support the growth of cancer cells when used at higher concentration levels. Therefore, it is recommended to consume the diet of saffron petal at 80 ppm in layers.
Although this level of inhibition is below 50% and egg yolk cannot naturally be considered as an anticancer drug, egg yolk is a human food (not a drug). Thus, its food safety requires that the bioactive effects should not have too much bioactivity. Finally, malondialdehyde, DPPH and SOD significantly improved by treatments including saffron petal hydroalcoholic extract . On the basis of these observations, we conclude that saffron petal hydroalcoholic extract can be used as a new feed additive to potentially modulate the antioxidant status of laying hens and improve their production performance and egg quality and the inhibitory effect on cancer cells, best achieved with 60-80 ppm extract.