Analysis of the phytochemicals of Coriandrum sativum and Cichorium intybus aqueous extracts and their biological effects on broiler chickens

Spices and herbs can be used as feed additives and viable alternatives to antibiotics in chicken production. This study analyzed the phytochemicals, minerals, and antioxidant activity of aqueous extracts from Coriandrum sativum seeds and Cichorium intybus roots. The effects of different concentrations of C. sativum and C. intybus extracts on blood parameters, growth and carcass traits, biochemical parameters, and antioxidant activity of broiler chicks were also examined. The results showed that C. sativum aqueous extract has relatively higher contents of total flavonoids and total phenolic acids than C. intybus aqueous extract. Both extracts contain elevated mineral elements, especially iron, potassium, and sodium. Therefore, dietary supplementation of C. sativum seed and C. intybus root extracts could enhance broiler chicken growth performance, carcass characteristics, liver function, lipid profile, and antioxidant status. These extracts could be utilized as natural feed additives and growth promoters for broiler chickens.


Results and discussions
Phytochemical quantification. The chemical compounds in plants are responsible for their natural antioxidant activity, and the majority of these active compounds are natural phenols or polyphenols. Leaves, fruits, seeds, and flowers have the highest natural flavonoid and phenol contents. Increased dietary consumption of antioxidants or vegetables or fruits with antioxidant activities can improve quality of life 16 . Table 1 demonstrates that C. sativum and C. intybus extracts have different phytochemical compounds. The aqueous extract of C. sativum seeds had a significantly higher content of total flavonoids (p < 0.05, Table 1) and total phenolics (36.5 ± 1.7 mg of gallic acid/g of extract) than in the aqueous extract of C. intybus leaves (28.16 ± 1.60 mg of gallic acid/g of extract). These results agree with Nhut et al. 17 , who reported that C. sativum extract has significant phenolic contents.
Antioxidant activities of C. sativum and C. intybus aqueous extracts. The antioxidant activities of C. sativum and C. intybus aqueous extracts were examined using DPPH, ABTS, FRAP, metal chelation, and ORAC assays. Moreover, the DPPH test was performed to assess free radical scavenging, and the results were denoted by IC50, which is the proportion of antioxidants required to reduce the initial DPPH concentration by 50%. A low IC 50 indicates high antioxidant efficacy, whereas trolox was used as the reference component. As depicted in Table 2, the antioxidant activity of C. sativum was 81.9 ± 1.02 µg mL −1 in aqueous extract, 105.5 ± 1.030 µg mL −1 in aqueous extract of C. intybus. Therefore, C. sativum has an antioxidant profile that includes chelation activity, phospholipid peroxide inhibition, radical free radical scavenging activity, hydroxyl radical, and peroxided peroxidation scavenging 18 .
C. sativum and C. intybus aqueous extracts were confirmed to be rapid and efficient scavengers of ABTS radicals ( Table 2). The ABTST + scavenging activity results differed substantially (P 0.05) between C. sativum and C. intybus at 706.07 ± 16.02 and 636.27 ± 12.87 µM Trolox/mg of extract, respectively. ABTS radical cation scavenging activity is a reflection of its hydrogen-donating capacity. According to Pandey et al. 18 , high molecular weight phenolics (tannins) have a high capacity to quench free radicals (ABTST+). When combined with a nutrient, these extracts can function as potential nutraceuticals.
Antioxidants act as reductants and oxidant activators. Reducing power may be a significant indicator of possible antioxidant activity. In the present study, the antioxidant capacity of C. sativum and C. intybus extracts was determined by their ability to decrease the TPTZ-Fe (III) complex to the TPTZ-Fe (II) complex ( Table 2). The capacity to reduce ferric ions also indicated their significant FRAP activity. The aqueous extract of C. sativum had a more pronounced activity (102.91 ± 3.07 µM TE/mg of extract) than that of C. intybus (94.25 ± 7.46 µM TE/mg of extract).
The ORAC test is the only one among the analysis methods based on the hydrogen atom transfer mechanism, uses a biologically relevant radical 19 , and quantifies the inhibition time and degree of an antioxidant 20 . According to the ORAC values of the extracts (Table 2), C. sativum demonstrated the highest capacity (1398.82 ± 59.15 µM TE/mg of extract), and C. intybus had the lowest (1067.18 ± 69.10 µM TE/mg of extract).
Although iron is required for proper physiology, its excessive amount can induce cellular damage. If the reduced metals go through the Fenton reaction, they may generate reactive hydroxyl radicals that contribute to oxidative stress 21 . The capacity to chelate/deactivate transition metals, catalyze hydroperoxide breakdown, Elemental analysis. The presence of inorganic components is essential for the survival of bioactive chemical entities. In addition to the four-building elements, carbon, hydrogen, nitrogen, and oxygen (forming the main organic molecules), living organisms require many inorganic components to function properly. Numerous elements are fundamentally necessary for normal physiological function 23 . Twenty-two inorganic elements with an essential role in biological activities were detected in C. sativum and C. intybus extracts (Table 3). Inductively coupled plasma-optical emission spectrometry was used to analyze these elements, which is one of the most effective techniques for performing multi-element analysis quickly and with high sensitivity.
The most abundant element in the samples was calcium, followed by iron, potassium, and sodium. This element is critical for various physiological processes and, along with phosphorus, serves as a structural bone component. Calcium supplementation contributes to the prevention of bone fractures and calcium-deficient diseases 24 . Inorganic elements, calcium, was the most abundant inorganic element in C. sativum and C. intybus extracts (686,97 ± 101.3 and 616,34 ± 112.1 mg/kg, respectively).
Iron, potassium, and sodium were identified as possible nutritional components (Table 3). Iron is a critical element that contributes to hemoglobin's oxygen-carrying ability and is present in various essential enzymes, including the cytochrome p450 enzyme. The amount of iron in C. sativum and C. intybus extracts was 15,630 ± 181.1 and 1317 ± 71.3 mg/kg, respectively (Table 3).
Potassium was found in high concentrations in C. sativum and C. intybus extracts (11,200 ± 200.5 and 12,500 ± 111.1 mg/kg, respectively) and contributed to fluid balance, nerve impulses, and muscular contraction. A high-potassium diet lowers blood pressure, prevents kidney stones, water retention, osteoporosis, and protects against stroke.
Sodium is one of the beneficial elements that the body needs in trace amounts to perform regular biological functions. This substance aids in the transmission of nerve impulses, the maintenance of proper water and mineral balance, and the contraction and relaxation of muscles. The human body requires 500 mg of sodium each day. In this study, 360.54 ± 12.7 and 500.76 ± 13.1 mg/kg of sodium were detected in C. sativum and C. intybus extracts, respectively. Excess sodium intake is linked to increased risks of hypertension, stroke, and heart diseases.
Phosphorus, chromium, manganese, and strontium were among the other important trace elements detected in C. sativum and C. intybus extracts (Table 3). Phosphorus is a significant energy source for the body and is a mineral that is necessary for the formation of bones, teeth, DNA, RNA, and cell membrane. Phosphorylation is included in various carbohydrates and proteins found in the body. In this study, phosphorus was detected in significant amounts in C. sativum and C. intybus extracts (702.53 ± 11.29 and 675.32 ± 14.1 mg/kg, respectively). Se, known for its ability to protect against oxidative damage 25 , was found in high concentrations (0.32 ± 0.01 mg/kg) in C. intybus but was not detected in C. sativum.
Copper is the third most prevalent trace element (after zinc and iron) in the human body and is a critical component of oxygen-transporting blood cells 26 , bones, the heart, the brain, connective tissues, and other body organs 27 . Copper concentrations were high in all tested plant materials, particularly in C. intybus plant sample (6.81 ± 0.89 mg/kg) ( Table 3).
Zinc concentration was high in C. intybus (46.7 ± 0.89 mg/kg) but low in C. sativum (36.21 ± 2.1 mg/kg). Zinc is a trace element or micronutrient required to foe the growth and development of microbes, animals, and www.nature.com/scientificreports/ plants. This element is essential for protein synthesis and DNA and collaborates with copper as a cofactor in several crucial enzyme systems 28 .
In addition to these basic elements, bromine, lead, cadmium, and barium were found in the extracts of C. sativum and C. intybus. These inorganic elements are hazardous, though their concentrations were within the acceptable range.
Cadmium and lead have permissible limits of 0.3 and 10 mg/kg, respectively 29 . Table 3 lists the additional elements detected in trace amounts.
Based on these results, C. sativum and C. intybus can be recommended as raw or processed supplements for food products.
Growth performance. Table 4 lists the average body weight gain, feed consumption, feed conversion ratio, and mortality rate of broiler chicks fed with diets supplemented with 1000 mg of C. sativum, 500 mg of C. intybus, or a mixture of 500 mg of C. sativum plus 250 mg of C. intybus extracts/kg of diet during starter, finisher, and entire experimental periods. The inclusion of C. sativum and C. intybus extracts in broiler chick diets significantly improved body increase weight during starter, finisher, and entire periods. This finding could be attributed to the presence of antioxidants and phenolic substances in C. sativum and C. intybus extracts that enhanced growth performance. Jang 30 adding coriander oil to broiler diets contributed significantly to increasing weight. Comparable findings were revealed by Faramarzzadeh et al. 31 , who added 4.5% of chicory powder to the broiler diet.
Consumption of feed during starter and finisher periods was not significantly affected by the supplementation of C. sativum and C. intybus extracts in the broiler chick diet. Meanwhile, the addition of 500 mg of C. intybus extract/kg of diet significantly increased feed consumption during the entire experimental period (0-6 weeks). This increase could be attributed to the phytogenic feed additives that improved the flavor and palatability of feeds 32,33 . The improvement in feed consumption induced by the powder of coriander seeds can be due to essential oils as well as their primary ingredient, which is linalool, in coriander seeds. Çabuk et al. 34 illustrated that linalool has an appetizing impact on diets and promotes the digestion process in animals. In addition, previous studies have reported the positive impacts of essential oils on feed intake 35 .
Dietary supplementation of C. sativum and C. intybus extract significantly improved the ratio of feed conversion during all three periods. This improvement could be attributed to the appetite-enhancing, digestivestimulating, and antimicrobial effects of the two extracts 36 . According to the present findings, the fed diet of hens supplied with chicory powder is characterized by a substantially diminished feed conversion ratio than the unsupplemented group 34 . Similarly, Cabuk et al. 34 noted a considerable increase in the conversion ratio of feed in the diet of broilers supplied with a blend of herbal plants. Additionally, Liu et al. 37 demonstrated that supplementing a baseline diet with chicory powder induced a considerable improvement in the consumption of feed during the first 13 days of the feeding interval, which is consistent with the current study results. Improved broiler development performance on a diet supplied with the chicory powder might be attributable to the increase in number, length, as well as the surface area of intestinal villi that are paralleled with an enhanced absorptive and digestive ability of jejunum 38 .
Furthermore, adding chicory powder to diets will benefit younger broiler chicks more than older ones owing to the stimulation of villi development 37 . The findings of Al-Jaff 39 are consistent with the present study results. The addition of coriander seed to broiler chickens' diet induced an improvement in FCRs, due to the active component (linalool) in coriander, causing greater efficiency in feed utilization, resulting in enhanced growth 40 . Morover, Przybilla and Weiss 41 illustrated that the herbal blend's route of action feed conversion occurs via enhancing the digestive processes. According to Rajeshwari and Andallu 42 , coriander is an efficient appetizer www.nature.com/scientificreports/ and contributes to the production of enzymes as well as digestive juices in the stomach, stimulating peristaltic motion and digestion, and consequently enhancing FCR. Dietary supplementation of these extracts significantly decreased the mortality rate of broiler chicks during the experimental period. This finding could be attributed to the positive results of the two extracts for tannins, alkaloids, terpenoids, flavonoids, and saponins and the negative for glycosides. Adisa et al. 36 indicated that tannins possess antiviral and antibacterial activities. Taraz et al. 43 concluded that coriander seeds inhibit pathogenic microorganisms in the digestive system, decreasing birds' mortality or disease infection.
The improved growth performance of broilers fed with both extracts could be attributed to coriander. Chicory extracts have some components with antimicrobial properties, such as flavonoids and phenolics (Table 1). Deans and Waterman 44 concluded that the addition of plant extracts to poultry diets could enhance the growth performance by stimulating endogenous enzymes and regulating intestinal microflora balance. Table 5 depicts the carcass traits of broiler chicks fed with diets supplemented with 1000 mg of C. sativum, 500 mg of C. intybus, or 500 mg of C. sativum + 250 mg of C. intybus extracts/kg of diet. These results indicated that dietary supplementation with C. sativum and C. intybus extracts significantly increased liver and carcass weight and decreased abdominal fat percentage. In contrast, the other carcass organs were not significantly affected. These findings are in agreement with prior studies 45 . The increased dressing percentage may be linked to the stimulatory effects of coriander on pancreatic secretions, which in turn boost digestive enzyme secretion and produce additional nutrients, such as amino acids that are digested and absorbed from the digestive system 46 . Generally, the accumulation of body fats can be the net result of the balance between fat catabolism, fat synthesis (lipogenesis), as well as dietary absorbed fat via β-oxidation (lipolysis).

Carcass characteristics.
Consequently, when the quantity of ingested fat remains constant, reduced deposition of body fats can be due to diminished synthesis of fatty acids, elevated fat catabolism, or both mechanisms. Hence, carcass characteristics are enhanced. Antioxidants and phenolic substances in herbous extracts enhanced the broiler chicken's carcass reaching 1.2% 47 .
Panda et al. 48 and Faramarzzadeh et al. 31 demonstrated marked improvement in the proportion of dressing by supplementation of chicory powder (3.0%) to broiler diet. The current findings also agree with Yusrizal and Chen 49 , who illustrated that the fed diets of broiler chickens supplied with chicory fructans (1%) were heavier than controls. In addition, they detected a positive correlation between the addition of chicory supplementation as well as carcass characteristics in poultry species 50 . www.nature.com/scientificreports/ Hematological parameters. Table 6 presents the effects of supplementing the broiler chick diet with 1000 mg of C. sativum, 500 mg of C. intybus, or 500 mg of C. sativum + 250 mg of C. intybus extracts/kg of diet on hematological plasma parameters. These results revealed that this supplementation significantly increased RBCs, hemoglobin concentration, and hematocrit percentage. In contrast, WBC: heterophil and heterophil: LYM ratios were significantly reduced at six weeks of age, while LYM and MON percentages were not significantly affected. Khubeiz and Shirif 51 reported that adding coriander seed powder to broiler chick diets significantly reduced heterophil percentage and heterophil, whereas the LYM ratio significantly increased the LYM percentage. The increase in the H/L ratio indicated that the birds were under acute stress. In the present work, the addition of C. sativum and C. intybus extracts to the broiler chick diet improved the hematological parameters at 6 weeks of age.
Liver and kidney function. The effects of supplementing the broiler chick diet with 1000 mg of C. sativum, 500 mg of C. intybus, or 500 mg of C. sativum + 250 mg of C. intybus extract/kg of diet on total protein, albumin, globulin, A/G ratio, urea uric acid, AST, and ALT are depicted in Table 7. These results indicate that this supplementation did not significantly affect total plasma protein, albumin globulin, and A/G ratio. Plasma AST and ALT were significantly affected by the dietary addition of the two extracts (Table 7). Similar results were obtained by Al-Jaff 39 , who reported that supplementing broiler diets with coriander seeds significantly decreased AST and ALT. This reduction in plasma AST and ALT indicated that plant extracts do not negatively affect liver function and have no adverse influence on the growth and health of broiler chicks. Table 7 shows that plasma urea and uric acid concentrations in broiler chicks were significantly decreased when fed with a diet supplemented with C. sativum and C. intybus extracts. Therefore, adding C. sativum and C. intybus extracts to the broiler chick diet did not adversely affect kidney function, and the extracts do not have any toxic substances.
These findings support the hepatoprotective impact of C. intybus and C. sativum extracts, suggesting enhanced kidney and liver functions, which is consistent with Khubeiz and Shirif et al. 51 and Hosseinzadeh et al. 52 . Aromatic plant supplements in the chicken are characterized by stimulatory impacts on the digesting process by increasing digestive enzymes production and enhancing the use of digestive products through an improved liver function 52 .
Lipid profile and glucose. Table 8 shows the average levels of plasma TC, TG, LDL, HDL, and Glucose of the broiler chicks supplemented with 1000 mg of C. sativum, 500 mg of C. intybus, or 500 mg of C.  56 , who demonstrated that chicory-based fructan addition to the broiler diet induced a decline in serum total cholesterol, as well as LDL and triglycerides in broilers. This finding is consistent with Elson 57 , who illustrated that these isoprenoids inhibit the synthesis of cholesterol by suppressing 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase production, the rate-controlling enzyme of the synthetic cholesterol pathway. At the same time, adding chicory extract resulted in alleviated cholesterol absorption reaching 41% (P < 0.05) in the ileum as well as 30% (P < 0.05) in the jejunum compared to controls 58 . Glucose concentration was significantly decreased upon the supplementation of C. sativum and C. intybus extracts in the diet of broiler chicks at six weeks of age ( Table 8). The present paper's results are consistent with those of Khoobani et al. 56 , who revealed that the addition of chicory to the broiler diet resulted in diminished serum glucose. Hosseinzadeh et al. 52 reported that coriander extracts a significant decrease in serum glucose in broilers in the control group. The decrease in glucose concentration might be attributed to the action of coriander in stimulating insulin secretion and enhancing glucose uptake and metabolism by the muscle 42 . Table 9 depicts the effects of dietary supplementation with different levels of extracts (1000 mg of C. sativum, 500 mg of C. intybus, or 500 mg of C. sativum + 250 mg of C. intybus ) on SOD, MDA, TAC, plasma GSH, and CAT levels in broiler chicks. SOD was significantly decreased, and TAC was significantly increased. MDA, plasma GSH, and CAT were not significantly influenced by extract supplementation. A high plasma antioxidant capacity was obtained upon supplementation of 500 mg of C. sativum + 250 mg of C. intybus extracts/ kg of diet.

Antioxidant activity.
Modifications in antioxidant enzyme parameters, as well as a decline in some nonenzymatic antioxidants, may induce oxidative stress. In addition, Natural biological processes constantly generate reactive oxygen species, neutralized by the antioxidant enzymes in the human body, such as SOD and GSH-PX, besides certain protective substances 59 The antioxidant state of poultry may be determined by testing four main antioxidant enzymes: GSH-PX, T-AOC, MDA, and T-SOD. Total superoxide dismutase and GSH-PX are natural scavengers to free radicals by the conversion of oxygen radicals to hydrogen peroxide in order to alleviate the damaging reactive oxygen species from the cellular environment 60 . Malondialdehyde has been frequently used as an indicator to assess lipid peroxidation in biological processes; nevertheless, it is observed that it is correlated with free radical damage along with increased varying diseases 61 . In general, antioxidant defense systems, like the nonenzymatic and enzymatic systems, can be indicated by T-AOC, with elevated T-AOC in broiler chicks supplied with C. intybus and C. sativum extracts, reflecting enhanced antioxidant defense. Ahmed 62 reported that supplementing the broiler chick diet with plant products with high antioxidant compounds contents could improve the reactive oxygen scavenging activity and conserve fat sources.

Conclusion
This study demonstrated that C. intybus and C. sativum extract enhanced broiler chicks' performance indices, hematobiochemical profiles, as well as carcass characteristics. In addition, they improve animal health and well-being through their contribution to the antioxidant defense mechanism against free radical generation. Consequently, it is evident that these two herbs (Cichorium intybus and Coriandrum sativum) can improve performance without inducing any adverse effects on studied features.

Materials and methods
Extracts preparation. C. sativum seeds and C. intybus roots were purchased from a local market, dried, separately ground to a fine powder, and added 500 mL of distilled water to prepare C. sativum and C. intybus extracts. The mixture was heated for 30 min in a water bath at 65 °C after 24 h of maceration at room temperature. Following filtration, the extracts were concentrated using a rotary evaporator and stored at 4 °C for later use. The collection of herbs complies with relevant institutional, national, and international guidelines. www.nature.com/scientificreports/ Estimation of total flavonoid content and total phenolic content. Total phenolic content was measured using Folin-Ciocalteu colorimetric technique 63 and expressed using gallic acid equivalents (mg gallic acid/g of extract). Colorimetry with aluminum chloride determined total flavonoid content 64 and expressed using quercetin equivalents (mg of quercetin/g of extract).
In vitro antioxidant activity of extracts. Antioxidant capacity was evaluated using the assays of scavenging free radicals (ABTS and DPPH), ferric-reducing antioxidant power (FRAP), oxygen radical antioxidant capacity (ORAC), and metal chelation, which was performed following the methods of Brand-Williams et al. 65 for DPPH, Benzie et al. 66 for FRAP, Arnao et al. 67 for ABTS, Chew et al. 68 for metal chelation, and Liang et al. 69 for ORAC.

Elemental analysis by ICP-MS.
Inductively coupled argon plasma (ICAP 6500 Duo, Thermo Scientific, England) was used to identify the components in the extracts and digests. The stock solution for instrument standardization was a 1000 mg/L multi-element certified standard solution from Merck, Germany. Leggett and Westermann's technique 70 was followed for sample digestion.
Animal welfare. The committee of the Poultry Production Department at Faculty of Agriculture, Minia University, approved the current research procedures. These procedures recommend animal rights and welfare by assuring minimal stress to animals. All methods were carried out in accordance with relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines.
Growth performance trial. A total of 240-day-old unsexed broiler chicks (Ross 308) were randomly divided into 60 birds per treatment group. Each group was randomly allocated into five replicates (12 chicks each), kept in a wire cage (100 × 50 × 50 cm), and provided with a feeder and drinker. For the first 3 days, the brooding temperature was set at 34 °C and then decreased gradually to 24 °C at the end of the experimental period. The chicks were reared under a continuous program with 24 h of light during the 1st week and 23 h of light and 1 h of darkness for the remaining experimental period. The first group was fed on a basal diet (control), and the remaining groups were fed on a basal diet supplemented with 1000 mg of C. sativum (2nd group), 500 mg of C. intybus (3rd group), and 500 mg of C. sativum + 250 mg C. intybus extracts/kg of diet (4th group). All broiler chicks were fed with starter (0-3 weeks) and finisher (4-6 weeks) diets formulated according to the National Research Council (NRC, 1994). Table 10 presents starter and finisher diets' ingredients and chemical composition. Feed and water were supplied ad libitum during the experimental period. The birds were vaccinated against Newcastle disease at 1 and 18 days of age and Gumboro disease at 14 and 24 days of age. Bodyweight gain feed consumption and feed conversion ratio were measured during starter (0-3 weeks), finisher (4-6 weeks), and whole experimental periods (0-6 weeks).
Carcass traits and blood samplings. Ten birds were selected from each experimental group at six weeks of age and hand slaughtered. Carcass, liver, gizzard heart, and abdominal fat were removed and individually weighed, and values were expressed as percentages of preslaughter live weight. Blood was collected at slaughter time in heparinized tubes to determine the following hematological parameters: red blood cells (RBCs), hemoglobin concentration (Hb), hematocrit (HC), white blood cells (WBCs), lymphocytes (LYMs), monocytes www.nature.com/scientificreports/ (MONs), and heterophil. A total of 100 leukocytes for each bird were counted to calculate heterophil: MONand heterophil: LYM ratios. Blood samples were centrifuged at 3000 rpm for 15 min. The plasma was maintained at − 20 °C until the estimation of the following biochemical parameters using commercial colorimetric kits (Biodiagnostic Company, Giza, Egypt): total protein, albumin, globulin, alanine transaminase (ALT), aspartate transaminase (AST), urea, uric acid, total cholesterol (TC), and triglyceride (TG). Plasma total antioxidant capacity (TAC), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were determined using commercial kits and a spectrophotometer (Shimadzu, Japan).
Statistical analysis. Data were assessed by one-way ANOVA in the SAS Institute. Differences between means at p < 0.05 were compared using Duncan's multiple range test.

Data availability
The datasets utilized and analyzed during this investigation are available upon reasonable request from the corresponding author.