Gastroprotective and Immuno-supportive Role of Alcea kurdica against Stress Induced Lesion in Japanese Quails

: The conducted research was done in Grda rasha field (Salahaddin University) for one month to compare the impacts of Alcea kurdica powder, Rifaxmine, and Ranitidine as anti-lesion and immune-strengthening agents on stress-induced quails which are affecting their growth rate and in severe cases causing gizzard erosion and deep intestinal lesions. To do that, 75 quails (12 weeks old) were grouped into six treatments with different additives. (T0-) = Negative control (Stress-induced Without treatment), (T0+) = Positive control (No stress inducing or treatment). T1= (treated with Rifaximine 200mg/L water mixed), T2= (treated with Ranitidine 200mg/L), T3= (treated with A.kurdica extract 100mg/L). The tested groups, except for positive control, were immersed in tap cold water (17°C) as a stress-induced technique. The Macroscopic analysis showed that quails pre-treated with A.kurdica extract (T3) had significantly (p<0.05) lower relative gizzard koilin layer disruption than those of T1, T2, and T0- groups, respectively. Moreover, the Elisa results indicated higher antibody titers against ND, IBD, and IB viruses for the T3 group with significantly increased HDL and lowered LDL, VLDL, and TCHO for T3 than that of T1, T2, T0+ T0- groups, respectively. Water mixed A.kurdica extract showed positive influences on the body weight, lipid profile, immune status and minimized gizzard erosions of breeding quails, which can be considered as a medicinal plant as well as a growth-enhancing agent in the poultry industry.

Japanese quails are hardy birds that thrive very well in cages and are relatively inexpensive to maintain without stress but unfortunately in comparison with other poultries gained less agricultural interest. Despite their economic importance in animal food resources as efficient egg laying and meat providence bird, Quails can be used in studies of developmental biology as they can be a convenient animal model 1 . Quails immune system is degraded by numerous factors including mycotoxins called Aflatoxins 2 . Another mycotoxin is called Type A-trichothecenes particularly T-2 toxin causing gizzard lesions or erosion. The gizzard erosion occurs despite being a strong organ, which in severe cases causes lesions in the underlying tissues, consequently, affecting quail's growth rate, egg-laying performance, and susceptibility toward immune diseases 3 . Besides, the presence of biogenic amines and tannins in the breeder's diet or breeder age is thought to be the causative factors 4 . Other causes such as pathogenic diseases like Newcastle disease (ND), Infectious Bursal Disease (IBDV/ Gumboro), and Infectious Bronchitis viral diseases (IB) are also reported by 5 . Avian Stomach has been used as an indicator for a bird performance by many researchers 6 . The gizzard is considered as the true stomach in the avian with bigger muscularity than proventriculus, which plays a major role in the grinding food to be further digested by the small intestine 7 . Medicinal plants become more popular in recent years to improve quail's productivity and lowering mortality rates 8 .
A.kurdica, is a perennial plant mainly found on limestone in mountain slopes 9 . A. kurdica exhibited potent antioxidant activity and its aqueous extract was shown to scavenge free radicals with great antimicrobial properties against the growth of  11 . Phytochemicals are well known to be used in curing gastric disorders, weight problems and cardiac diseases, as a substitute for drugs with fewer side effects, less expense and lower toxicity 12 . Phenolic (Gallic acids) and flavonoid (Quercetin) ingredients in herbal products possess great biological effects as antiulcer, increased free radical scavenging activity (69.4% ± 1.19%) and antiinflammatory agents 13 . Pharmacologically, ranitidine and rifaximin have shown anti-lesion effectivity and healing capability against druginduced enteropathy 14,15 . The current study mainly aims to compare A. kurdica powder efficiency with rifaximin, and ranitidine as anti-ulcerative, immune-supportive, and lipid lowering agents in tap water (17°C) stress-induced quails.

Materials and Methods: Sample Collection and Preparation
A. kurdica was found in a wide geographic range near the Shaqlawa district area. The herb's identification was done in biology Dep./ College of Education/Salahaddin University-Erbil. The washing of plant parts (flowers) was done by running water and air-drying. The dried flowers (100 g) were ground using a grinder for 30 sec. Then, for the maceration of the powder, 1000 mL of boiling distilled water was used and let down for 2 hours to infuse at room temperature. The filtered extract was concentrated in the water bath and then, vacuum dried to be refrigerated at 4 °C for further analysis 16 . The extract yield was 8.6% (w/w).

Experimental Treatments
Adult quails (12 weeks old) were distributed into 5 treatments, each treatment with 3 replicates: 15 chicks per treatment. T -0 = Negative control (Immersed in tap cold water (17°C) with no pretreatment), T0+= Positive control (Without pretreatment nor immersion). T1= (Immersed in tap cold water (17°C) with Rifaximine 200mg/L water mixed pretreatment), T2= (Immersed in tap cold water (17°C) with Ranitidine 200mg/L water mixed pretreatment), T3= (Immersed in cold tap water (17°C) with A.kurdica extract 100mg/L water mixed pretreatment) 17  -On the 15th day and 30th day of the experiment Body weight were measured, and Serum separated from collected blood to estimate blood glucose with a blood glucose analyzer (Accu-Chek Compact, Roche Diagnostics, Mannheim, Germany) 19 and then, the serum was taken to the immunity laboratory to evaluate the determined antibodies titers against Newcastle (ND), Gamboro (IBD) and Infectious bronchitis viral diseases (IB) by using ELISA 5 . To find the quail's lipid profile, we purchased kits known as DiaSys (Diagnostic System, Germany) 20 . At the end of the experiment, 5 quails were randomly chosen from each group, kept at room temperature and starved for 24hours, then Slaughtered, and necropsied after two hours following stress inducing by immersion in tap cold water (17°C) for 5 seconds 21 . Intestinal tracts and the gizzards were dissected and washed with distilled water for observation. Macroscopic screening for intestinal lesion and gizzard erosion was made following the same method done by Džaja et al. 21

Results and Discussion:
Treating quails with mixed water additives has caused some changes in their Body weight and blood sugar as in (Table 1). On the 15th-day, the bodyweight of all treatments showed insignificant differences, but after the 30th day of treatments, significant reductions in body weight were seen in T0-, T0+, T2, and T1 groups, respectively, but the T3 group showed no changes in the body weight. A previous study showed that adding plant additives did not significantly cause differences in the bodyweight of quails 23 . Based on the results, adding A. kurdica powder did not interfere with quail's body weight, unlike anti-ulcer drugs such as Ranitidine and Rifaximine which reduced the quail's body weight and eventually decreased their growth rate and immune system capacity. Our data is in accordance with 24 , who proved undesired effects of these drugs as H2 receptor blocking agents causing reduction in stomach acid secretions and appetite loss leading to decrease in the body weight. Our data are also following the conclusion that declared adding organic feed containing mackerel, cassava leaves and turmeric powder to quails' diet have no significant effect on their bodyweight 25 . Following the 15 th and 30 th days of treating groups with additives, blood sugar was nonsignificantly lower in A.kurdica (T3) group than that of (T1), (T1), (T0+), and (T0-), respectively. The anti-diabetic activity of Alcea genus was previously shown in alloxan-induced diabetic rates 26 . The hypoglycemic effect of A.kurdica can be linked with their antioxidant activity and phenolic contents that have been reported by Dar et al. 27 ( Table 1).  Table 2. represents the effect of adding A.kurdica extract, Rifaximine and Ranitidine mixed water on quails' lipid profile, in which the T3 group showed significantly lower (P≤0.05) TCHO and VLDL values than that of T2, T1, T0+, T0groups, respectively. The LDL and TG values were lower significantly in T3 than that of T1, T2, T0+, T0-, respectively. The HDL values were significantly higher in the T3 group than that of the T2, T1, T0+, T0-groups respectively. The lipid profile of the T1 and T2 groups were not significantly changed by water mixed additives. On the whole, Our results showed the effectiveness of A.kurdica as a hypocholesterolemic agent that may be a good choice to treat obese or hyperlipidemia patients which were not reported before. Herbal medicine gained popularity in recent years, motivating researchers to investigate different plant parts in searching for new efficient phytochemicals 28 . The positive impact of A.kurdica extract on the quails' lipid profile can be explained by its natural contents with different phytochemicals like Phenolic (Gallic acids) and flavonoid (Quercetin), illustrated to possess different bioactivities including free radical scavenging as well as an immunomodulatory role on peripheral blood mononuclear cells (PBMCs), in addition, flavonoids known to induce innate and adaptive B-and T-cell responses, including TH1, TH2 and regulatory T cells 27,10 . The flavonoid's capability to improve birds' lipid profile is due to the presence of Functional hydroxyl groups that mediate their antioxidant effects by scavenging free radicals and/or by chelating metal ions 29 . In addition, previous research also concluded that adding plant extracts to quails' diet showed significant improvement in the lipid profile, oxidative stress status, body weight, and egg-yolk cholesterol of quails under stress conditions 30,31 .
A.kurdica as an antiulcer agent to cure irritation and mucosa membrane lesions were purchased for many years 11 . Figure 1. shows that gizzard ulcer score distribution in which (T0+ group) had very mild lesions, then increased progressively in other groups with significantly lower lesion scores for A. Kurdica (T3) group. Figure 6. shows that quails drinking A.kurdica mixed water (T3) had lower disruption of gizzard koilin layer in comparison with other treated groups shown in Figs. 2,3,4, and 5. The levels of damage are considered as discoloration and gross lesions in the surface of the intestinal tract and severe disruption of the eroded gizzard koilin layer (arrow), penetrating submucosa tissues. Our results regarding stressed-induced gizzard erosion are approved by GRABAREVIĆ et al. 17 . The present scores show significant protection of gizzard and intestine lining by A.kurdica mixed water, which was disrupted with multiple lesion areas in other tested groups (T1, T2, and T0-).

Figure 1. Macroscopic Distribution of Score Lesion in the Experiment treatments
The negative control group of quails (T0-) showed surface lesions on the intestinal tract with disruption and erosion in the gizzard koilin layer, penetrating submucosa tissues as shown in Fig. 2. The positive control (T0+) with no stress inducer had few intestinal damages with a smooth inner surface of gizzard mucosa as shown in Fig. 3. Quails pretreated with Rifaximine (T1) and Ranitidine (T2) had a moderate disruption in the gizzard koilin layer with intestinal lesions as shown in Fig. 4 and Fig. 5.    There is no doubt that feeding birds immersed in tap water (17°C) as stress inducer transfers birds into a state with the depression of an optimal immune response: disrupting intestinal mucosa and the koilin layers of gizzard 21 . Lower score lesions in quails under A.kurdica mixed water in Fig.1, maybe due to flavonoids and phenolic content of this plant as shown to have antimicrobial, antioxidant activates by scavenging free radicals and immunomodulatory efficacy 27 .
Our data results are supported by a previous research article which mentioned that the flowers of Alcea kurdica are served as antiulcer and antiinflammatory agents 12 . Researchers previously mentioned that phytochemicals such as flavonoids, tannins, triterpenoids, saponins, and coumarins possessed significant antiulcer activities 32 . In contrast, some anti-ulcer drugs like Ranitidine may possess several side effects as shown in our study causing a decrease in the quail's body weight in comparison to other tested groups. Making birds more susceptible to various avian diseases affects their growth rate and egg-laying performance. These antiulcer drug side effects have been linked with decreasing gastric acid secretion and appetite loss 7 .
The data in Fig. 7 summarizes the Geometric Mean Titers (GMT) for the treated quails against ND, IBD and IB viruses. The GMTs obtained reached the protection level and varied significantly among treated groups and ranged from 845 to 2056. Quails drunk A.kurdica mixed water had significantly higher antibody titers against ND, IBD, and IB viruses than that of Ranitidine and Rifaximine respectively. A previous study explained that the development of a bird's specific immunity is reduced by the water that quails drink due to an imbalance between acid and base status 33 .
The highest immune titers against ND, IBD, and IB were recorded in the T3 group followed by T2 and T1 respectively. The lowest Immune titers against ND, IBD, and IB were recorded for T0group. The ELISA results for the immune titers of T1 and T2 against the mentioned diseases did not significantly differ. The data analysis demonstrated that immune titers of T3 quail groups against ND, IBD, and IB were significantly higher more than 2 times than that of the T0+ group under a diet with no additives. Only one group (T3 against ND) had protective antibody titers (GMT > 2000) and the rest showed immune titers (GMT<2000) As shown in Fig. 7. Monitoring serologic antibodies against diseases in the poultry industry is considered an efficient way to evaluate the response of the immunity system toward additives and stressinduced immune diseases 33 . Possessing high immune titers by quails drinking A.kurdica mixed water may be due to its phytochemical contents like flavonoids and phenolic compounds. These bioactive compounds possess great antimicrobial and antioxidant properties by their scavenging and redox properties, through neutralizing and quenching free radical and pathogenic microbes attacking animals' digestive and immune systems 27 . In addition to its terpenoid contents, which are known for their capability in disrupting membranes of microbes with efficient antioxidant and immunomodulatory properties by induction of immune cells 34 .

Conclusion:
A.kurdica is a natural efficient plant with valuable phytochemicals. Their flavonoid and phenolic contents possess various biological impacts, acting as anti-cholesterolemia, anti-ulcer, and immune-supportive agents by scavenging free radicals and immune cell activation. Treating quails with the A.kurdica extract improves their immune system under stress conditions against various avian diseases, which are considered as risk factors for intestinal lesions and gizzard erosion, causing a decrease in the growth rate and egg-laying performance. Moreover, A.kurdica extract can be considered as a substitute for anti-ulcer drugs with fewer side effects as shown to not interfere with the quails' body weight. Further investigations are required to study the long-term physiological impact of A.kurdica as an antiulcer and immune booster agent that may be of bird breeder's interest to maximize productivity and decrease mortality rates of birds.