Antioxidant and antihyperlipidemic effects of hawthorn extract (Crataegus oxyacantha) in broiler chickens

Abstract Background One of the main problems in the poultry industry is excess body fat, and the anti‐fat effect of Cratagus extract has been confirmed in several studies. Objectives The present study was carried out to investigate the effects of hawthorn extract (Crataegus oxyacantha) on growth performance, haematological variables and hepatic gene expression in broiler chickens reared at high altitude (2100 m). Methods A total of 225‐day‐old chicks (Ross 308) were randomly distributed into three treatments. Experimental treatments were prepared by adding 0.0, 0.2 and 0.4 mL of hawthorn extract per litre of consumption of water. Results The results showed that weight gain and feed conversion ratio were significantly improved and abdominal fat decreased by consumption of two levels of Crateagus extract when compared to the control (p < 0.05). Consumption of hawthorn extract decreased circulatory levels of malondialdehyde, triacylglycerol, total cholesterol and low‐density lipoproteins cholesterol but increased ferric reducing antioxidant power and high‐density lipoproteins cholesterol (p < 0.05). Hawthorn extract caused an up‐regulation of catalase, superoxide dismutase1, glutathione peroxidase1 and peroxisome proliferator‐activated receptor alpha but reduced the expression of key lipogenic enzymes (p < 0.05). Conclusions Overall, consumption of 0.4 mL hawthorn extract per litre of drinking water, improved growth performance, suppressed lipogenesis and enhanced antioxidant response.

The excess fat in broiler chicken in the form of abdominal fat is the chief portion of body fat which constitutes approximately 2%-3% of the bird's live weight (Fouad & El-Senousey, 2014;Sanz et al., 2000).This percentage varies depending on several factors affecting fat deposition in broiler chickens, including genetics, nutrition, sex and age of the broiler chicken (Havenstein et al., 2003;Sanz et al., 2000).Physiologically, excess fat in broiler chickens is not essential for the body's vital functions, but because most fats are unsaturated and prone to oxidation, they impose a heavy burden on the body's antioxidant system (Sanz et al., 2000;Wood et al., 2008).Chickens have a higher content of polyunsaturated fatty acids in their adipose tissue than other animals, such as pigs, cattle and sheep, and therefore have a greater tendency to oxidize adipose tissue (Wang et al., 2017;Wood et al., 2008).So, lipid oxidation in poultry meat is considered to be the main cause of quality damage related to the flavour, colour, taste and nutritional composition of meat and poultry products (Sanz et al., 2000;Wang et al., 2017).Control of poultry diets with methods such as adjusting the ratio of energy to protein, reducing dietary fat content, adopt nutritional strategies, including feed restriction and the use of medicinal plant products, are related fields of research (Attia et al., 2017;Wang et al., 2017).
Hawthorn (Crataegus Sp.), also known as thornapple, maytree or hawberry, is a flowering shrub of the rose family.Common species include Crataegus monogyna, Crataegus laevigata and Crataegus oxyacantha, which is cultivated in various countries of the world.C. oxyacantha is widely distributed in different parts of Asia (Barros et al., 2011;Chang et al., 2002;Long et al., 2006).The hawthorn leaves, berries and flowers are used as herbal drugs.They contain chemicals called flavonoids, which have multiple therapeutic effects (Dahmer & Scott, 2010;Yang & Liu, 2012).C. oxyacantha is one of the most famous species of the hawthorn family that is used in traditional medicine due to its antioxidant and antihyperlipidemic effects (Ahmadipour et al., 2017, Ahmadipour, Kalantar, Kalantar, et al., 2019, Ahmadipour, Kalantar, Hosseini, et al., 2019;Long et al., 2006;Yang & Liu, 2012).
The results of the research indicated that several beneficial results of hawthorn extract were determined, including (1) lowering body and liver weight and liver to-body weight ratio; (2) improving serum parameters and liver dysfunction; (3) decreasing hepatic lipid accumulation; (4) increasing circulating adiponectin levels and up-regulating the expression of adiponectin receptors in the liver; and (5) activating AMP-activated protein kinase (AMPK) and altering AMPK-mediated SREBP-1c, peroxisome proliferator-activated receptor alpha (PPAR-α) and their downstream targets (Li et al., 2015).
Flavonoids and triterpenoids as two major constituents of hawthorn have anti-inflammatory and anti-hyperlipidemic properties.Part of the mechanism for the anti-hyperlipidemic effects of hawthorn fruit might also involve the direct protection of human LDL from oxidation (Kao et al., 2005;Kirakosyan et al., 2003;Wu et al., 2020).
There is not enough information about the antioxidant and antihyperlipidemic effects of hawthorn in broiler chickens.According to the facts that some compounds in hawthorn extract have strong antioxidant potential and some of its compounds have lowering plasma lipids effects, the objectives of the present study were to examine the effects of different consumption of levels of hawthorn flavonoid extract to prevent oxidation and hyperlipidemic of broiler chickens.To the best of our knowledge, there has been no report on the effect of hawthorn flavonoid extract in the mentioned fields.

Gas chromatography/mass spectrometry (GC/MS) procedure
The bioactive molecules in hawthorn extracts were determined using a gas chromatograph attached to a mass spectrometer and displayed in Table 1.The HP-5MS 5% phenylmethyl siloxane capillary column  Information System), oil components were identified (Adams, 2007).

Determination of total phenol content
The total phenolic content of the samples was determined using the Folin-Ciocalteu method described by Singleton et al. (1999).Briefly, 50 µL of Crataegus-Drop (concentration 1 mg/mL in methanol) was mixed with 3.95 mL of distilled water and 250 µL of Folin-Ciocalteu, respectively.Results are presented in Table 2 as mg gallic acid equivalents/g lyophilized extract (mg GAE/g E).

Measurement of total flavonoid content
Total flavonoid content (TCF) was determined according to the method described by Cottica (2015).Briefly, 0.25 mL of 5% aluminium chloride (ethanol) was mixed with 0.5 mL of Crataegus-Drop (1 mg/mL concentration in methanol).Total flavonoids are expressed in Table 2 as mg quercetin equivalent/g lyophilized extract (mg QE/g 128 E).

Antioxidant evaluation (DPPH free radical scavenging assay)
The antioxidant capacity of Crataegus-Drop was measured according to the method of Mensor et al. (2001) using 1,1-diphenyl-2-picrylhydrazyl (DPPH).A volume of 200 µL of Crataegus-Drop (concentration 1 mg/mL in methanol) was added to 80 µL of 0.3 mM methanolic DPPH solution.DPPH was expressed as the inhibition rate (%) of the lyophilized extract in Table 2.

Birds and experimental facility
The experiment was conducted in the experimental facility of Shahrekord University, Shahrekord, Iran.A total of 225-day-old broilers (Ross 308) were randomized across 15-floor pens.Each pen measured 2 m 2 (15 birds per pen) and was equipped with a bell drinker and a feed trough.All chicks were kept, maintained and treated according to the accepted standards for the human treatment of animals.
One-day-old chicks were then allocated to pens (15 birds per pen with a weighted 47.8 g on average) so that all pens had equal initial body weights (717 ± 10 g).For all experimental groups, a basal diet of the corn-soybean meal was formulated for starting (1-3 weeks of age) and growing (3-6 weeks of age) periods according to National Research Council (NRC) (1994) recommendations (Table 3).
Experimental treatments were prepared by adding 0.0, 0.

Measurements
Body weight gain and feed intake were recorded in 1-20, 21-40 and 1-40 days of age periods.The feed conversion ratio (FCR) was also calculated for the same periods.At 40 days, 10 birds were selected from each group for blood collection and processing.The selected birds had body weights within ∼5% of the average pen body weight.Blood samples (∼3 mL) were collected from the brachial vein and centrifuged at 2500 × g for 10 min to separate sera.Serum samples were tested for TC, TG, HDL, LDL and malondialdehyde (MDA).
TC, TG, HDL and LDL were measured using commercial laboratory kits following the manufacturer's manuals (Pars Azmoon).MDA, as a biomarker of lipid peroxidation, was assayed according to Nair and Turner (1984).Ferric reducing antioxidant power (FRAP) assay was measured according to the procedure described by Sudha et al. (2012) with some modifications.The FRAP reagent contained 2.5 mL of a 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, 2.5 mL of 10 mM FeCl 3 •6H 2 O and 25 mL of 300 mM acetate buffer (pH 3.6).

Quantitative real-time PCR analysis
The liver of 10 birds per treatment was harvested, homogenized and treated by a digestion buffer (RNX-Plus reagent; Sinaclon Bioscience).

Target
centrifuged at 16,000 × g for 15 min (under a cool temperature, 4 • C).
The supernatant was taken to precipitate RNA using the equal volume of isopropanol.The samples were then rinsed with 75% ethanol, centrifuged and the resulting pellet dissolved in the appropriate amount of diethyl dicarbonate-treated water.The extracted RNA was treated with a DNAase kit (Sinaclon Bioscience) to remove residual DNA.The quantity of RNA was checked by O.D. (A260/280) using a biophotometer (Eppendorf).RNA with an absorbance ratio between 1.8 and 2.2 was used for the synthesis of cDNA.
Total RNA was converted to cDNA by means of the PrimeScript RT Reagent Kit (Takara Bio Inc.).The levels of superoxide dismutase 1 (SOD1), catalase (CAT), Glutathione peroxidase1 (GPX1), PPARα and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) transcripts were determined by real-time RT-PCR using SYBR Premix Ex Taq II kit (TliRnase H Plus; Takara Bio Inc.).YWHAZ was used as an endogenous reference gene to normalize the input load of cDNA across samples (Hassanpour et al., 2018).

Statistical analysis
The ANOVA procedure of SAS Institute Inc (2007) software was adopted to analyse the data in a completely randomized design.
Duncan's test compared significant differences among the means, and the level of probability less than 0.05 was considered statistically different.Linear and quadratic effects of Crateagus extract levels were studied using orthogonal polynomial contrasts to compare with the control group.

RESULTS
The effects of consumption of treatments on the growth performance of broiler chickens are presented in hawthorn extract per litre of consumption of water when compared to the control (p < 0.05).The proportions of liver and abdominal fat to live body weight were linearly reduced by the consumption of hawthorn extract, and there was significant difference between the control and hawthorn extract at 0.2 and 0.4 mL (p < 0.05).that antioxidant genes (SOD1, GPX1 and CAT) had up-regulation under the effects of using 0.4 mL of hawthorn extract.
Table 9 represents the effects of hawthorn extract on the expression of lipogenic genes in the liver of broiler chickens.The results showed that genes involved in lipid metabolism were significantly down-regulated by the consumption of hawthorn extract.Orthogonal contrasts of the expression fatty acid synthase, acetyl CoA carboxylase, lipoprotein lipase and malic enzyme between the control vs. Crateagus extract groups were significant.This evidence is that Crateagus extract consumption significantly prevents expression of genes in a linear manner.

DISCUSSION
The effects of hawthorn on antioxidant and antihyperlipidemic conditions of broiler chickens demonstrate that treatment with hawthorn in a dose of 0.4 mL/L of consumption of water results in a significant reduction in TC and LDL and a significant increase in HDL.Hawthorn extract has the ability to demonstrate a lipid-lowering effect in animal models of hyperlipidemia (Kausar et al., 2011;Kwok et al., 2013;Yoo et al., 2016).Hawthorn treated with n-ethanol and ethyl acetate alleviated hyperlipidemia by regulating disorders of lipid, energy and amino acid metabolism and reducing oxidative stress, resulting in a decrease in plasma levels of TC, triglycerides and low-density lipoprotein cholesterol (LDL-C) in hyperlipidemic rats (Kausar et al., 2011;Zhang et al., 2022).Similarly, hawthorn extract was able to improve hyperlipidemia by improving the lipid profile, reducing oxidative stress and lowering total and LDL cholesterol levels in the serum of ovariectomized rats (Yoo et al., 2016).Moreover, the alcoholic extract of Zhongtian hawthorn promotes cholesterol elimination (Hu et al., 2016).Several previous preclinical studies reported that the hawthorn extract reduces concentrations of TC as well as LDL, and TG in different laboratory animals as well as humans with hyperlipidemia (Tassell et al., 2010;Venskutonis, 2018;Wu et al., 2020).The results of Table 7 in this experiment also prove that hawthorn extract had a serum lipidlowering effect on broiler chickens.Moreover, hawthorn extract also causes a significant decrease in lipid deposits in rabbit liver and aorta (Verma et al., 2007).It has been found that reductions in TC in hamsters fed a high-fat diet following treatment with hawthorn were accompanied by an increased expression of the liver enzyme cholesterol hydroxylase (the rate-limiting enzyme in the synthesis of bile acid from cholesterol) and lower levels of intestinal acyl-CoA: cholesterol acyltransferase (an enzyme involved in cholesterol uptake and absorption by the intestine), resulting in decreased blood lipid concentrations (Zhang et al., 2002).Furthermore, the treatment of the human hepatocyte cell line with hawthorn extract induced a 5.6-fold up-regulation of LDL-receptor transcription in addition to downregulated ApoB (ApoB is a structural component of serum LDL particles) synthesis in a concentration-dependent manner (Xu et al., 2009).These in turn modulate both lipogenesis and lipolysis, resulting in reduced blood lipid concentrations.The changes in lipid parameters observed in our study are in accordance with the previous preclinical studies that demonstrate the antihyperlipidemic effect of hawthorn extract in animal models of hyperlipidemia (Hu et al., 2016;Verma et al., 2007;Xu et al., 2009;Zhang et al., 2022).
Phenolic compounds, flavonoids and triterpenoids are important components of natural antioxidant substances (Venskutonis 2018).
Hawthorn polyphenol extract can regulate the apoptosis of HaCaT cells by reducing reactive oxygen species production, DNA damage and p53 activation, effectively reducing UVB-induced photodynamic and non-photodynamic damage and providing protection to the skin (Liu et al., 2019;Zhang et al., 2022).Hawthorn total flavonoid extract and (−)-epicatechin inhibited acute cellular oxidative stress triggered by the free radical initiator Aminobenzyl phsphonic acid (ABPA).Both and muscle tissue, where it plays different roles (Wang et al., 2017;Wood et al., 2008).Hawthorn flavonoids increase LPL expression through a PPAR-dependent mechanism directed towards the identification of the components (Fan et al., 2006).Along with our study, Akila and Devaraj (2008) showed that hawthorn extract significantly restored the activity of antioxidant enzymes such as superoxide dismutase, CAT, GPX and glutathione, thereby restoring the antioxidant status of the organism to almost normal levels.
Alcoholic extract of C. oxyacantha (AEC) pretreatment maintained mitochondrial antioxidant status and prevented mitochondrial lipid per-oxidative damage and decrease in Krebs cycle enzymes induced by isoproterenol in rat heart (Wang et al., 2011(Wang et al., , 2013)).AEC can act on myocardial tissue to reduce iNOS expression and downregulate COX-2 to exert anti-inflammatory effects (Wang et al., 2013).Hawthorn flavonoids exert antihyperlipidemic effects by acting on the PPARγ pathway to increase LDL expression in blood vessels (Fan et al., 2006).
According to a report by Venskutonis (2018), methanol and ethanol extracts of Crataegus azarolus exerted substantial antioxidant, antiinflammatory and antiproliferative capacities, which were evaluated by measuring the secreted amounts of the proinflammatory mediator prostaglandin E2, and by assaying the mRNA levels of the proinflammatory cytokines (IL-α, IL-β and Il-6), chemokines (CCL3 and CCL4) and inflammation-sensitive COX2 and iNOS enzymes.
In agreement with our data, Yoo et al. (2016) also confirmed that hawthorn extract could improve body performance, lipid profile, potentiate enzymatic antioxidants and modulate oxidative stress in rat.
In the study of Pirany et al. (2020) differential expression of genes implicated in liver lipid metabolism in broiler chickens differing in weight was investigated.Lipid parameters and expression of ACACA, APOA1, CPT1A, FASN, FOXO1, LIPG, PPARα and SIRT1 genes involved in lipid metabolism were investigated in two groups of high (HW) and low (LW) weight broilers from the same strain.In this regard, according to the results, blood cholesterol and liver triglyceride levels were significantly increased in HW chickens compared to LW broilers, whereas other parameters, that is blood triglyceride, blood HDL/LDL, liver cholesterol and total liver fat showed no significant changes in either group.Moreover, the relative expression of ACACA, APOA1 and CPT1A genes was significantly lower in the liver tissues of HW broilers than in the LW group.The mRNA levels of these three genes showed a significant negative correlation with abdominal fat deposition and the live weight of broilers.However, relative expression of FASN, FOXO1, LIPG, PPARα and SIRT1 hepatic genes did not differ among broilers.
So it was concluded that of eight hepatic genes implicated in lipid metabolism, only the expression of three (ACACA, APOA1 and CPT1A) was significant for fat and leanness within the same strain of chicken.
As reducing body fat is a major goal in the broiler industry, these data can provide fresh insight into the molecular processes underlying the regulation of fat deposition in broilers.
It is concluded that hawthorn extract may improve the weight gain and FCR and decrease the abdominal fat in broiler chickens.Consumption of this extract can also attenuate oxidative stress through decreasing the lipid peroxidation and potentiate the total antioxidant capacity especially via up-regulation of CAT, SOD1 GPX1 enzymes.On the other hand, hawthorn through improving lipid profile and reducing the expression of key lipogenic enzymes is capable of suppressing lipogenesis and imply its antihyperlipidemic effect.However, a few studies have been published about the effects of hawthorn on antioxidant and antihyperlipidemic conditions as well as antioxidant and lipogenic gene expression in the liver of broiler chickens.So, further systematic in vivo and in vitro research is warranted to explore and verify the potential effect of hawthorn extract to provide precise guidance for clinical use and new drug discovery.

(
30.00 m 0.25 mm, 0.25 m film thickness) with an Agilent 7890A gas chromatograph (Agilent Technologies) was used to evaluate Crataegus-Drop.The temperature was initially connected to the stove at 60 • C for 4 min, then increased by 4 • C per minute until it reached 260 • C. The detector and injector had respective temperatures of 300 and 290 • C. The carrier gas streamed at a rate of 2 mL/min and was helium.In the split mode, samples (1 µL) were manually injected.Peak area percentages were used to produce the numerical data.A mass-specific detector (Agilent Technologies) was coupled to the gas chromatograph.The ionization voltage, 70 eV, and ion source temperature, 200 • C, were among the EI-MS working parameters.A similar arrangement of n-alkanes (C5-C24) was used to analyse maintenance records for all components, while maintaining the same environmental conditions as the samples.By comparing mass spectral fragmentation 2 and 0.4 mL of HFE (HE 00152, Crataegus-Drop 6260) per litre of consumption of water (measured pH = 7.05 and total dissolved solids = 2000 ppm).The HFE contained 0.25 and 0.50 mg/L of total flavonoid compounds.In this way, birds in 0.1 and 0.2-mL HFE groups received 0.05-0.10mg of total flavonoid compounds daily.C. oxyacantha (common hawthorn) is an endemic member of the Rosaceae family that grows in Europe, Africa and Asia, where it is commonly found as a shrub or small tree 5-10 m tall(Chang et al., 2002).Hawthorn as a traditional medicinal plant is a plant locally called Sorkh-e-valik or Zalzalak that is found in the western and central regions of Iran(Salehi et al., 2009).Oral Crataegus-Drop is a well-known herbal drug therapy that is available at the pharmacy under an approved code and supervision of the Iranian Food and Drug Administration (HE 00152, Crataegus-Drop 6260).The flavonoid extract of this product contains biologically active flavonoid compounds (polyphenols) such as anthocyanidins and proanthocyanidins (also known as bioflavones or procyanidins).Each millilitre of oral Crataegus-Drop 6260 contained 2.5 mg of total flavonoid compounds in the form of hyperoside (21.4% polyphenols and 19.7% procyanidins), produced by Iran Darouk Pharmacy Co, under the production code of 3067-88-02.The determination of total phenolic compounds in Crataegus-Drop 6260 was carried out through the colorimetric method in accordance with the standard extraction procedure of the manufacturer(Ahmadipour, 2018).
total flavonoid extract and (−)-epicatechin at all concentrations tested were taken up by cells and both significantly inhibited the oxidation of DCFH(Zhang et al., 2022).The report ofWang et al. (2011) indicated that flavonoid fraction showed inhibitory effects on TG and glucose absorption and accelerating effects on gastrointestinal transit in vivo and suppressed the accumulation of TG and free fatty acid.It also suppressed the gene expressions of C/EBP, PPAR, SREBP 1c, aP2 and adiponectin in vitro.As we know, LPL plays an important role in lipoprotein metabolism and is expressed in various tissues, especially adipose

TA B L E 1
Composition of hawthorn extract (Crataegus oxyacantha).
Total phenolic, flavonoid content and antioxidant activity for hawthorn extract.Ingredients and chemial composition of experimental diets (1-42 days).

Table 5
Effects of Crateagus extract (CE) on broiler's growth performance.Effect of Crateagus extract on carcass variables in broiler chickens measured at 42 days of age.Each mean represents values from 10 replicates.Superscripts in the same row with different letters are significantly different (p < 0.05).Abbreviations: FRAP, ferric reducing antioxidant power; HDL, high-density lipoproteins cholesterol; LDL, low-density lipoproteins cholesterol.
. Broilers' weight gain and FCR were significantly improved by adding 0.2 and 0.4 mL of hawthorn extract per litre of consumption of water when compared to the control (p < 0.05).Data for carcass characteristics are presented in Table 6.Carcass, breast and thigh yield were significantly increased by adding 0.4 mL of TA B L E 5 Note: Superscripts in the same row with different letters are significantly different (p < 0.05).Each mean represents values from 10 replicates. 1 Orthogonal contrast.TA B L E 7 Effect of Crataegus extract (CE) on serum and blood variables in broiler chickens measured at 42 days of age. 1 Orthogonal contrast.

Table 7
Effect of Crataegus extract (CE) on antioxidant gene expression in the liver of broiler chickens measured at 42 days of age.Effect of Crataegus extract (CE) on lipogenic gene expression in the liver of broiler chickens measured at 42 days of age.
depicts serum and blood variables in broilers receiving 0.2 and 0.4 mL of hawthorn extract per litre of consumption of water.Regression analysis indicated a significant linear response for serum variables when Crateagus extract consumption.Two-steps treatments level while increasing HDL compared to the group that received 0.4 mL HFE and the control group (p < 0.05).Orthogonal contrast between the control vs. Crateagus extract groups was significant for serum variables.Table8depicts the effects of hawthorn extract on the expression of antioxidant genes in the liver of broiler chickens.The results describeTA B L E 8Note: Superscripts in the same row with different letters are significantly different (p < 0.05).Abbreviations: CPT1A, Carnitine palmitoyltransferase 1A; FAS, fatty acid synthase; LPL, Each mean represents values from 10 replicates; PPARα, peroxisome proliferator activated receptor alpha. 1 Orthogonal contrast.