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Anne Lanham, Lillian Mwanri

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Immunomodulatory Effect of Yogurt Enriched with Garlic Extract on Ulcerative Colitis Induced Rats

Mashaal Saddiqa, Huma Umbreen*, Tahir Zahoor, Nighat Bhatty, Kiran Aftab

Nutritional Sciences, Government College University, Faisalabad, Pakistan

Abstract: Immunomodulation counterbalances the inflammation in the intestines and regulates hypersensitivity in the host through gut microflora. Microflora comprises alive microorganisms living in the gastrointestinal tract that produce vitamins, short chain fatty acids (SCFA’s) and play an important role in immunomodulation. Ulcerative colitis (UC) is a chronic inflammatory disease prevalent in all parts of the world, general indications are fever and inflammation of the colon which leads to reduced absorption of nutrients and weight loss. Manifestation of UC can be reduced via immune system regulation through gut microflora by the use of prebiotics. Inulin is an established prebiotic therefore in the present study it was used with yogurt for immunomodulation. Garlic extract not only provides antioxidants, and nutrients but also contains 80% inulin which stays non-digested until it enters the colon. The present study aimed to discover the effect of inulin rich garlic extract based yogurt on the immune system of normal and DSS (Dextran Sodium Sulphate) induced colitic Sprague dawley rats and its effect on inflammatory markers. Four groups of Sprague dawley rats were used with three replicates having three animals in each replicate. Rats were given yogurt enriched with garlic extract according to their weight for three weeks and Colitis was induced with DSS earlier. Results from inflammatory markers like tumor necrosis factor (TNF-α), interleukins (IL-6), immunoglobulin (IgG), and erythrocyte sedimentation rate (ESR) showed a decreasing trend of inflammation after the use of garlic extract based yogurt, weight gain also improved after the consumption of garlic extract yogurt due to the effect of prebiotic named inulin present in the extract.

Keywords: Ulcerative colitis (UC), garlic extract, inulin, immunomodulation

Received April 11, 2022; Revised May 16, 2022; Accepted May 25, 2022

Citation: Mashaal Saddiqa, Huma Umbreen, Tahir Zahoor, Nighat Bhatty, Kiran Aftab, Immunomodulatory Effect of Yogurt Enriched with Garlic Extract on Ulcerative Colitis Induced Rats. Journal of Food and Nutrition Research, 10 (5). 377-385. January 2022. doi:10.12691/jfnr-10-5-6

1. Introduction

Ulcerative colitis (UC) is a chronic disease of the digestive tract that takes place in humans and various animal species. It is identified as mucosal inflammation of the colon with unpredictable remission and relapse occurrence. Studies on animal diseased models suggest that enteric bacteria and genetic factors can play a major part in developing ulcerative colitis; another reason is the extensive use of a westernized diet composed of fat and protein and less consumption of fruits and vegetables. The first step against colitis is the use of prebiotics and probiotics to reduce the inflammation to maintain remission and stop relapse [1,2].

According to the definition, probiotics are living microbes that provide benefits to the host other than basic nutrients. Yogurt is among the most famous fermented dairy products around the world because of its valuable potential concerning health. The presence of probiotics in yogurt is thought to provide it with health benefits like prevention of diarrhea and osteoporosis, regulation of the immune system, reduction of cholesterol, and its anti-cancerous properties [3]. Probiotics present in yogurt have been seen to prevent and modulate the immune system in Irritable Bowel Disease (IBD). IBD is characterized by Crohn’s disease and ulcerative colitis and therefore yogurt can be used to reduce the symptoms of UC and improve the quality of life [3,4].

Garlic is known to be an ancient medicinal spice due to the presence of nutritional compounds that makes it an immune booster and effective against infections [5]. Garlic is a good source of inulin (14-13 g/100 g), which is an established prebiotic and is now being used in a variety of food items to enhance their nutritional content and improve the beneficial bacteria in the intestines [6].

The gastrointestinal tract is a complex system made with the association of resident microbes, mucous layer, and immune cells. “Microbiota” is the term used for microorganisms that reside in the intestinal epithelium and can degrade dietary substances that are left undigested. This improves the efficiency of the digestive system of the host and enhances immunity, studies on laboratory animals have revealed that the immune system is influenced “outside in” by microbiota [7,8].

The etiology of colitis is yet unknown but it is believed to be triggered by the hyperactive immune system against the gastrointestinal tract which results in inflammation and ulceration. Macrophages and mast cells produce soluble factors, chemokines, and cytokines that are responsible for the production of inflammation [9]. It is believed that microbes in the gut are responsible for the onset of IBD (Irritable Bowel Diseases) like colitis and the accepted reason is a disturbance in microbiota irritating the immune system. The health of host depends on the diet because it can change the gut microbiota concentration which can affect inflammation indirectly through the immune system [10]. Inflammatory cytokines like IL-6 and TNF-𝛼 play the main role in the inflammation process [11].

This study aimed to obtain information about the inulin content in garlic extract and to check the effects of the use of prebiotic inulin rich extracts based yogurt on DSS (Dextran Sodium Sulphate) induced colitis in Sprague dawley rats on immunological and biochemical parameters of blood to assess the attenuation of inflammation after the use of prebiotic rich garlic extract.

2. Materials and Methods

Garlic (Allium sativum) was purchased from the local market of Faisalabad, Pakistan. For the preparation of yogurt low-fat milk was purchased from a local wall mart store. All the chemicals and testing kits were used of analytical grade and were procured from Sigma Aldrich (USA), Megazyme International (Ireland), and Randox Laboratories (UK). Male Sprague dawley rats were obtained from the National Institute of Health (NIH) Islamabad.

2.1. Preparation of Inulin Rich Extract

After peeling and washing garlic was chopped finely using a chopper. Extraction of inulin was done by hygroscopic method following the method of Toneli et al. [12] with some modifications. The ratio of chopped garlic and distilled water was 1:1, mixture was placed on a magnetic hot plate with a stirrer for two hours. Later on, the mixture was sieved and the semisolid part was removed and discarded and the liquid was concentrated. The liquid was then used as an extract; prepared extract was stored at 4°C in the refrigerator

2.1.1. Determination of Inulin in Extract Using HPLC

Inulin enriched extract from garlic was characterized to determine the inulin concentration by using the protocol of Zuleta and Sambucetti [13]. Aqueous extract of garlic and standard solution of inulin was filtered with 0.24 micro syringe filter before High Performance Liquid Chromatography (HPLC) analysis. 20 µL sample was injected, Isocratic mode was used in HPLC (LC-10A, SHIMADZU, Japan) the chromatographic separation was carried out using the column of Lichrospher (R) 100 NH₂ Purospher (R) STAR NH₂, 5 µm, Carbohydrates (mono, di, and oligosaccharides), column length was 100.00 mm, using the flow rate of 0.5 ml/min, DDH₂O was the mobile phase the detector used was RID detector and the pump was LC-10AV at room RT temperature. Column length was 100.00 mm, calibration file was ethanol, peak width was 0.200 min, the threshold was 0.050 mV, and calculation was done by ISTD2.

2.2. Preparation of Yogurt

Steps for the preparation of yogurt included pasteurization of low-fat buffalo milk, cooling till 45°C, homogenization, inoculation with a starter culture, incubation at 40°C then cooling, refrigeration, and later on the addition of Garlic inulin extract GIE [14]. GIE was added such as 1 ml yogurt containing 0.5 ml of GIE.

2.3. Experimental Animal Design

The rats were acclimatized in the animal room of the University on a basal diet for 7 days. All the protocols and procedures adopted complied with the animal care guidelines and study was approved by Ethical Review Board for Laboratory Animals of Govt. College Women University, Faisalabad. The environmental conditions as temperature (23±2°C) and relative humidity (60 ± 5%) were maintained throughout the study duration of 21 days with 12 hr light-dark period. Rats were fed an isocaloric and isonitrogenous diet and had access to tap water. The efficacy study consisted of four groups of rats with three replicates having three rats per replicate (nine in each group) as described in Table 1. Among the groups of rats, G1 was normal rats with a normal diet, G₂ was rats with colitis, G₃ included rats which received garlic extract enriched yogurt after the induction of colitis and G₄ included normal rats given garlic extract enriched yogurt. Rats of G₃ and G4 groups were given garlic extract based yogurt according to their weight by oral gavage, each rat was given 1 ml yogurt containing 0.5 ml Garlic inulin extract per 50 g of their weight daily in the morning.

Table 1. Bio evaluation trial

2.4. Induction of UC (Ulcerative Colitis)

Colitis was induced by 4% Dextran Sodium Sulphate (DSS) in drinking water for 5 days following the protocol of Videla et al. [15]. After five days of consumption of DSS water rats were fasted overnight and isoflurane anesthesia was used prior to get blood samples from aorta to confirm colitis.

2.5. Physical Parameters

To check the effect of treatment on body weight and feed intake, the weight was recorded on weekly basis to access the effect of treatment on weight. Feed intake was measured on daily basis and the food spilled was excluded (daily) during the study period to calculate feed intake during treatment.

2.6. Collection of Samples

After the study period of 21 days rats were decapitated in the morning at 9:00 am in 12 hour fasting condition under isoflurane as anesthesia. Jugular vein in neck area of rats was cut using sharp scalpel and blood was collected in sterilized tubes to get blood for erythrocyte sedimentation rate and serum. For plasma blood was collected in separate tubes containing heparin and centrifuged, after the separation of plasma it was stored in separate aliquots at 20°C for analysis [16].

2.7. Analysis of Blood

Blood was analyzed for Complete Blood Count (CBC) and differential White Blood Cell count (WBC) with hematology analyzer (Mythic 18), samples were run in triplicates to ensure precision [17].

2.8. Pro-Inflammatory Markers

Plasma collected in sterilized tubes was used and pro-Inflammatory mediators like IL-6 and TNF-α and IgG were analyzed with ELISA Kits from MERK whereas blood was used to determine ESR of rat groups.

2.8.1. Interleukin-6 (IL-6)

The sensitivity of the essay for IL-6 was 1.0 pg/ml and detective range of ELISA was 10-480 pg/ml, plasma sample of 10 µl was diluted with 40 µl sample diluent later on added with 100 µl HRP-conjugate reagent and incubated for 60 minutes at 37°C. Washing solution was used to wash the wells and then choromogen was added and incubated for 15 minutes at 37°C in dark after the addition of stop solution microtiter plate reader was used to observe the Optical Density at 450 nm in spectrophotometer. Tests were conducted in triplicates [18].

2.8.2. Tumor Necrosis Factor- α (TNF- α)

After the addition of, standard solution in standard well plasma sample of Sprague dawley rats was taken 10 µl and diluted with 40 µl of sample diluent. In each well (horseradish peroxidase) HRP- conjugate reagent was added covered and later on incubated for 60 minutes at 37°C. Chromogen solution was added and then stop solution changed the color of the samples which was noted at 450 nm Optical Density using microtiter plate reader the range of TNF-α was 1-80 pg/ml in this experiment. Triplicate samples were tested to get the best results [18].

2.8.3. Immunoglobulin G (Ig-G)

Samples of plasma of rats were taken in triplicates are diluted and then incubated for one hour at 37°C. HRP-conjugated anti-rat IgG was added after one hour washing was done samples were mixed with 50 µl of stop solution and optical ratio density was noted at 450 nm all the steps were taken according to manufacturer’s instructions [18].

2.8.4. Erythrocyte Sedimentation Rate (ESR)

Blood sample of 120 μl was taken and plunged in 30 μl of sodium citrate after mixing sample was moved in a capillary tube (1.0 mm×100 mm). Capillary tube was held at the angle of 45°C and results were seen after 15 minutes [18].

2.9. Safety Parameters

Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were analyzed for liver safety. Plasma sample (50 µl) was used and mixed with 400 µl reagent and rested for 30 minutes at 37°C using kinetic method. Routine chemistry analyzer (Microlab 300) was used for liver function tests by using SGPT and SGOT kinetic method [19].

2.10. Composition of Rat Basal Diet and Its Nutrient Contents

Rat groups were fed basal diet on the basis of g/kg which was composed of Sucrose (100 g), Corn starch (230 g), Soya bean meal (420 g), Maltodextrin (100 g), Soyabean oil (50 g), Maize bran (50 g), Choline bitartarate (2 g), AIN93G Min Mix (35 g) and AIN93G Vit Mix (10 g) per 1000 g moreover this diet has total calories of 3822 Kcal. The nutritional value of this diet on dry matter basis is Moisture 88.2%, Crude protein 18.9%, Crude fat 5.9%, Crude fiber 7.2%, Ash 8.7% and NFE (Nitrogen free extract, 47.5%).

3. Results

3.1. Inulin in Garlic Extract

Table 2 indicates the amount of inulin in garlic extract according to the results obtained by HPLC was 92.4±0.1%. Inulin standard was used to equate the results of inulin, retention time (min) and area (mV.s) depict that higher concentration of inulin is present in garlic extract (Table 2).

Table 2. Inulin content in garlic extract

3.2. Pro-Inflammatory Markers

3.2.1. Tumor Necrosis Factor- α and Interleukins-6 (TNF-α, IL-6)

Inflammatory markers i.e. TNF-α, IL-6 were calculated and their mean values are presented in Figure 1 and Figure 2. The highest level of IL-6 (319.05±2.894 pg/l) was seen in diseased group of rats (G₂) due to the presence of inflammation as indicated by Figure 2. IL-6 was observed to be reduced in G₃ and G₄ (colitis rats which received yogurt containing garlic extract and normal rats consuming garlic extract based yogurt) as compared to negative control group (G₁) (Figure 2). Tumor Necrosis Factor-α was seen to be the highest in diseased group of rats G₂ (5.38±0.105 g/dl) as presented in Figure 1. However blood level of TNF- α significantly reduced in G3 and G4 group of rats that consumed the garlic extract based yogurt and their values are 1.47±0.059, 2.90±0.192 g/dl respectively (Figure 1).

Figure 1. Mean Tumor Necrosis Factor- α (TNF- α) (g/dL±SEM) in rat groups

Figure 2. Mean Interleukin-6 (IL-6) (pg/L±SEM) in rat groups

3.2.2. Immunoglobulin-G and Erythrocyte Sedimentation Rate (IgG, ESR)

The concentrations of Immunoglobulin-G (IgG) in different rat groups are presented in Figure 3 as their mean values along with standard error of mean. Negative control group of rats G₁ exhibited the lowest value of IgG (5.34±0.358 g/L), whereas diseased group of rats G₂ has maximum value of IgG (5.38±0.105 g/L), however amount of IgG has been seen to be reduced in G₃ (colitic rats on garlic extract yogurt) and G₄ (normal rats on garlic extract yogurt) as has been shown in Figure 3 reflecting the process of immunomodulation.

Mean values of Erythrocyte Sedimentation Rate (ESR) values are presented in Figure 4 and indicate significant difference (p≤0.05) among positive and negative control groups of rats. It can be seen in Figure 4 that highest value of 7.16±0.333 mm/1st hr was noted in positive control group of rats (G₂) and the use of garlic extract yogurt in G₂ and G₃ (colitis rats on garlic extract yogurt and normal rats on garlic extract yogurt) gradually decreased ESR amount with reduction in inflammation. Figure 4 also indicates that the lowest amount of ESR, 5.23±0.352 mm/1st hr was seen in normal rats on placebo (G₁) due to absence of disease.

Figure 3. Mean Immunoglobulin G (IgG) (g/L±SEM) in rat groups

Figure 4. Mean Erythrocyte Sedimentation Rate (ESR) (mm/1st hr±SEM) in rat groups

3.3. Blood Parameters

The value of plasma heamoglobin g/dL has been presented in Table 3 as mean values. Results indicate that there is significant difference in heamoglobin concentration between the diseased group of rats (G₂) and the rest of the groups which include the positive and negative control groups (G₂ and G₁) and the group which includes normal rats consuming garlic extract yogurt (G₃). The lowest value was observed in G₂ (colitic rats) which was 10.30±0.473 g/dL (Table 3).

Red blood cells also called erythrocytes (RBC) are key part of blood the mean values are presented in Table 3. It can be seen that the lowest value 5.550±0.166 (×1012/L) is of the group of colitis rats on basal diet (G₂) whereas G₁, G₃ and G₄ (normal rat group, colitis rats consuming garlic extract yogurt and normal rats consuming garlic extract yogurt) represent RBC values that are non-significantly different from one another. The mean values of platelets are presented in Table 3. Significant difference (P≤0.05) has been noted in different groups of rats. The highest amount of platelets was seen in diseased group on basal diet i.e. G₂ whereas, both groups consuming garlic extract G₃ and G₄ presented second the highest amounts of platelets which were not significantly different from one another while lowest amount was seen in negative control group G₁.

Total leukocyte count (TLC) in rat groups has been presented in Table 3 as mean values along with SEM. G₂ which is the group of colitic rats on basal diet, showed the highest amount of TLC (9.90±0.120×109 /L) whereas the lowest amount (6.96±0.045×109 /L) was seen in G₃, which is the group of colitic rats consuming garlic extract yogurt. Hematocrit or HCT has been shown in Table 3 as their mean values for rat groups. The lowest value was observed in diseased rat group G₂ with value of 45.88±1.576% whereas the highest percentage was noted in negative control group (52.56±0.817%). Amount of Mean Cell Volume (MCV) of rat groups is displayed in Table 3 as mean values. The highest value of 64.75±0.694 fl is observed in negative control group G1 whereas MCV values or G₂, G₃ and G₄ are non-significantly different from each other. MCH is Mean Cell Heamoglobin and the mean values are presented in Table 3. Amount of MCH in rat groups is significantly different from one another highest amount (18.86±0.166%) was notes in G₃ which is colitis rats consuming garlic extract based yogurt, G₂ contained the second highest amount of MCH as displayed in Table 3. MCHC is the abbreviation of Mean cell heamoglobin concentrate and the values of MCHC in different rat groups have been presented in Table 3. The highest value of MCHC was noted in G1 normal rats on placebo which was 25.36±0.144% whereas G₂, G₃ and G₄ displayed non-significantly different values of MCHC.

Mean lymphocyte percentage in rat groups is presented in Table 3. Lymphocytes were noted the highest in G₂ (Colitis rats), its mean value was 93.10±0.895% where as other rat groups G₁, G₃ and G₄ displayed non significantly different value among one another but their lymphocyte count was significantly different (P≤0.05) from G₂. Amount of monocytes in rat groups has been presented in Table 3 in percentage mean value. The highest value has been noted in diseased group of rats G₂ which is 6.18±0.535% which decreases in other groups of rats. Moreover, the amount of monocytes in G₂ is significantly different from other groups of rats (G₁, G₃ and G₄) and monocyte percentage in G₃, G₁ and G₄ is non-significantly different from one another. Serum eosinophils percentage of different rats groups is presented in Table 3 as mean values (P≤0.05). Eosinophil percentage in colitic rat group (G₂) is significantly different from all other rat groups (G₁, G₃, G₄) as the highest value of eosinophils has been noted in G₂ of 1.50±0.233%.

Table 3. Blood Parameters (Mean±SEM) in rat groups

3.4. Physical Parameters

Weight gain of rat groups in weeks is presented in Table 4. Significant difference (P≤0.05) was observed between the weight gain of rat groups and among the weeks. Maximum weight gain (121.10±1.456 g) was seen in third week of study whereas weight gain in first and second week was noticeably less than the third week. Weight gain among the rat groups displays that lowest weight gain (108.73±1.079 g) was seen in colitic rat group (G₂) whereas weight gain gradually increased (117.83±1.996 g) in colitic rats fed with garlic extract yogurt (G₃).

Feed intake of rat groups in three weeks was noted and displayed in Table 5. Significant difference (P≤0.05) was observed between the treatments and weeks. The maximum feed intake (17.811±0.136 g) was seen in week three whereas first week displayed the lowest amount of feed intake which was 17.371±0.185g. When considering the feed intake among treatments diseased group of rats consumed the lowest feed which was 16.947±0.039 g whereas negative control group G₁ and normal rats consuming garlic yogurt (G₄) displayed the highest intake of food (18.122±0.025, 18.192±0.051 g respectively).

Table 4. Weight gain (Mean±SEM) of rat groups

Table 5. Feed intake (Mean±SEM) of rat groups

3.5. Liver Safety Parameters

Serum aspartate aminotransferase (AST) of different rat groups including positive and negative control groups is presented in Table 6. A decreasing trend has been noticed between diseased rats (G₂) and rats treated with garlic extract. Significant difference (P≤0.05) has been noticed in colitic rats and rats that were induced with colitis but they were given garlic extract based yogurt (G₃). The highest value of AST was notes in diseased rat group (G₂) which was 131.00±0.683 U/L whereas the normal rats that were on placebo (G₁) and normal rat group consuming garlic extract yogurt (G₄) showed lowest values of AST.

Serum alanine aminotransferase (ALT) values are presented in Table 6 and are significantly different (P≤0.05) among the rat groups. The maximum value was noted in group of colitic rats (G₂) which was 50.71±0.335 U/L which reduced in G₃ (colitic rats given garlic extract yogurt) and further decreased in G₄ (normal rats given garlic extract).

Amount of Serum alkaline phosphatase (ALP) in rat groups is presented in Table 6. Diseased group (G₂) displays the highest amount of 35.75±0.300 U/L which significantly reduced in colitic rats given garlic extract yogurt (G₃) and remains 35.23±0.091U/L.

Table 6. Serum aspartate aminotransferase (AST), Serum alanine aminotransferase (ALT) and Amount of Serum alkaline phosphatase (ALP) (Mean±SEM) in rat groups

4. Discussion

The use of garlic has its important health benefits recognized all around the world and this study aims to validate that there is a strong connection in the consumption of garlic extract and immune system modulation [20]. DSS induced colitic rats were tested for inflammatory markers like IL-6, TNF-α and ESR. These are the communal markers of systemic inflammation. Cells that are affected by TNF-α include the cells of intestinal epithelium and the main action of TNF-α is activation of nuclear factor (NF)-κB and reduction of signaling pathway which results in cell death and deterioration of barrier function of intestine these both points are hallmarks of ulcerative colitis (UC) [21]. Previous studies also report the similar results of increased level of TNF-α in colitic rats [21,22]. Erythrocyte sedimentation rate (ESR) is also known to be a marker of infection and inflammation and studies suggest the increased levels of ESR in DSS induced colitis which is parallel to our results [23]. Interleukin 6 increases in ulcerative colitis and decreases when inflammation is reduced these results of present study are in line with Kanauchi et al. [24], who also studied markers of inflammation after modifying the diet by inulin as prebiotic in DSS induced colitic rats. Immunoglobulin-G concentration in rat groups as presented in graph explains that IgG results show G₂ >G₃>G₄>G₅ whereas G₂ is colitic group of rats which shows highest concentration of IgG and the amount reduced in colitic group consuming yogurt enriched with garlic extract, similar results were seen in the study conducted by Faseleh et al. [25]. They found the immunomodulatory effect by prebiotic supplement in rat model and concluded that in immune function, gene expression is controlled and therefore immunoglobulin concentration is enhanced. The increased IgG in colitis were also observed in studies of Wedd et al. [26] whereas Shokryazdan et al. observed the amplified IgG levels through prebiotic supplementation by enhancing immune system [27].

Heamoglobin carries oxygen in red blood cells (RBC) from heart to the organs and vice versa, less heamoglobin and RBC show anemia which is indicative of bleeding in colon [28]. Results of this study reveal that low heamoglobin and RBC level were seen in colitic group of rats (G₂) which improved after the use of garlic extract yogurt and similar results were observed by several other studies [29,30,31].

Elevated amount of platelets was seen in diseased group of rats (G₂) which is in response to the cytokines presence due to inflammation however, as the ingestion of garlic extract with yogurt is reducing the level of cytokines and therefore, platelets level is reducing in G₃ (Colitic group consuming yogurt enriched with garlic extract). Parallel results were observed by Samson et al. [32]. Hematocrit (HCT) is also an indicator of anemia in colitis and its decreased value in colitic group (G₂) of this study indicates the intimation of anemia which was also noted in study of Larrosa et al. [28]. Whereas, level of HCT increased even in colitic rats after the use of inulin rich garlic extract yogurt (G₃) similar results were reported by Samolinska and Grela who presented improved HCT percentage in animals treated with inulin as prebiotic [31]. Total leukocyte count in rat groups was noted to be significantly different with the highest value of total leukocytes in colitis group (G₂) due to the manifestation of colitis and is in accordance with the findings of Mawdsley et al. and Hori et al. [33,34].

Mean corpuscular heamoglobin (MCH) level indicates microcytic anemia (reduction in cell size to carry enough oxygen), in our study the lowest MCH was observed in G₂ (colitic group of rats) and its amount improved in G₃ (colitic groups consuming yogurt enriched with garlic extract) signifying the use of prebiotic rich extract similar to the findings of Augusti [35]. Mean corpuscular volume (MCV) indicates the small size of red blood cells, in our study the negative control group displays the highest MCV percentage and other groups have MCV percentage which is non-significantly different from one another indicating that the anemia is not due to the less intake of iron the results are in accordance with Samson et al. [32].

Mean cell heamoglobin concentration (MCHC) percentage was slightly high in colitis group treated with yogurt enriched with garlic extract indicating the potential of garlic in reducing inflammation as presented by Suleria et al. [36].

The results of lymphocyte count shows that diseased group of rats (G₂) have the highest amount of lymphocytes which indicate the presence of inflammation because lymphocytes increase in response to the inflammatory conditions whereas the colitic group treated with garlic extract (G₃) have less amount of lymphocytes as compared to G₂ which indicates the consumption of prebiotic rich garlic extract yogurt has reduced inflammation and therefore lymphocyte count; these results correlate with previous studies [5,37]. Eosinophil count of rat groups as presented in Table 3 suggest the increased level of eosinophil in colitic group of rats (G₂) when compared to other rat groups agreeing with previous studies where eosinophil pass in the gut and release granular mediators that play important role in pathological process of UC (ulcerative colitis) where they remain activated to repair the diseased condition [38]. Neutrophils and monocytes were both noted as high in concentration in colitic rat group (G₂) and decrease in colitic group of rats consuming yogurt enriched with garlic extract (G₃) and in normal rat groups (G₁, G₄). Results of study conducted by Zhu et al. also depict the same results with reduced count of monocytes and neutrophils in colitis group and another study by Mirabeau and Samson which shows that consumption of garlic extract by rats improves their count of neutrophils in blood [5,39].

The results of weight gain among the rat groups in three weeks display an increasing trend as 1st and 2nd week < 3rd week weight which is due to the induction of colitis in first week. Whereas among the treatments colitic group G₂ has the lowest weight among other treatments which is due to the use of DSS for induction of colitis and the decrease of absorption of nutrients in colon due to the presence of inflammation which ameliorated after the use of garlic extract based yogurt [36].

According to the present study results of feed intake throughout the weeks showed an increasing trend from 1st <2nd <3rd week food intake and significant difference was observed, which explained that reduction of feed intake is due to the suppression of hunger by prostaglandin in colitic rats and the association of anorexia with GIT (Gastrointestinal tract) inflammation. Another reason which has been seen is that garlic burns calories and is famous for enhancing metabolism also the extract of garlic is rich in inulin which helps in increased satiety [37,38].

Serum aspartate aminotransferase (AST) in rat groups displayed a decreasing trend with the highest value in (diseased group) G₂ >G₃>G₁≥G₄. Increased value of AST is anticipated after change in serum concentration of enzyme due to the breakdown of liver cells, similar results were seen by Rajendiren et al. in DSS induced colitic rats [39]. Another study by Mansoub suggests that serum AST level can be reduced by supplementation of yogurt with prebiotics which is in accordance with our results of G₃ (colitic rats consuming yogurt enriched with garlic extract) [40].

Serum alanine aminotransferase (ALT) amount in rat groups show the highest amount of ALT in diseased group (G₂) which indicates presence of disease. Moreover a decreasing trend was observed in amount of ALT in G₃ (colitic rats consuming garlic yogurt) indicating that supplementation with garlic extract as prebiotic improves liver health, similar results were seen in some other studies [32,41].

Serum alkaline phosphatase (ALP) values of rat group show the highest value in diseased group of rats (G₂) and the value improves in G₃ (colitic rats consuming garlic enriched yogurt). Increased amount of ALP directs liver disease or bile duct blockage, present study shows the hepatoprotective effect of garlic as prebiotic similar findings were seen in the study of Usmani et al. [42]. In another study raw garlic was used for antioxidant effect and decrease in ALP amount in serum [43].

5. Conclusion

On the basis of present study it can be concluded that gastrointestinal track is the largest organ in the body that responds to the antigens and modify the immune system. Prebiotics like inulin help in modulation of immune system and management of inflammation. Garlic was used as low cost source of inulin and we incorporated it with yogurt to increase its nutritional properties. Inulin rich garlic extract yogurt proved to be a marvelous remedy to improve the inflammatory markers of colitis rats. This study recommends use of inulin rich yogurt to modify the intestinal microbiota for immunomodulation in intestinal diseases like ulcerative colitis (UC) for human trial.

Acknowledgements

Not applicable.

Conflict of Interest

The Authors declare no conflict of interest.

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