An Invitro Study on Predicted Glycemic Index and Bioactive Component of Forti ied-Bread Using Senna Auriculata

Bread is an affordable staple food availableworldwide. People demand for the high quality breads for consumption.Bread is considered to be a daily diet food for most of the people around the world. Foods with reduced glycemic index shows less risk of some chronic diseases like diabets and stroke. Senna auriculata a plant compound which is a potential inhibitor of diabetes. Low concentration possessed high radical scavenging activity, 90% of radicals were scavenged at lower concentrations. It further proved to have the anti-hemolytic effect. The extract inhibits the alpha amylase and alpha glycosidase activity. The bread contains large amount of digestible starch which leads to glycemic index. The foods with higher glycemic index leads to type 2 diabetes mellitus. Incorporation of Senna auriculata in bread is the effective method to produce low glycemic index foods. This study was aimed to evaluate digestability of starch and glycemic index in bread through the incorporating Senna auriculata as forti ied bread sample. At 60 mins of dialysate, the concentration of resistant starch of bread sample with Senna Auriculata exhibited to be low when compared with the control. The Senna auriculata forti ied bread sample is compared with the control which the regular bread based on their crust, colour, symmetry, texure, eating quality, and overall quality of the bread. Bread with 6% of extract was sensory evaluated. Bioactive components like phenolic, lavonoid found to be retained in forti ied bread sample. Based on the biofunctional properties developed the evaluation is analysed at the retention time of the process. The sample has higher resistant starch and lower glycemic index. This overall invitro indings gives us a promising result that due to the lower glycemic effect the Senna auriculata forti ied bread sample is situable and recommended for diabetes patients.


INTRODUCTION
Bread is an affordable staple food available world wide. People demand for the high quality breads for consumption. Using bread numerous studies have been conducted in bread making processes. Generally the breadmaking is processed using wheat lour, water, salt, sugar and yeast, mixed together in varying proportions to make a dough, which is further subjected to fermentation and baking (Sivam et al., 2010) . Bread is considered to be a daily diet food for most of the people around the world. Since bread is a common component in western diet, which delivers iber polysaccharides and antioxidants in high concentration. Normally, functional foods which have higher antioxidant and dietary ibers are considered to be the most demandable food, because of the health associated bene its, such as cancer, cardiovascular disorder, degenerative diseases and Diabetes etc. The bread contains large amount of digestible starch which leads to glycemic index. The foods with higher glycemic index leads to type 2 diabetes mellitus (Bhupathiraju et al., 2014) . Many research shows that amount and type of carbohydrate in food affects the glucose levels in the body which is the predictor of blood glucose which causes GI. To control the diabetes, the Glycemic index should be managed which is the major tool for diabetes.

Figure 1: Anti-oxidant activity of Senna auriculata extract
Foods with reduced glycemic index shows less risk of some chronic diseases like diabets and stroke (Barclay et al., 2008). There many methods and ways of breadmaking but a simple and fortiied breads process includes aqueous extract as a substitution of water. Plant extracts are used by many food industry to produce a healthy dietary products (Bucić-Kojić et al., 2011). The extraction methods differ from each other. By-products which are derived from the lowers shows high antioxidant source and cost effective too. There is an increasing demand for the plant based food products which results in substantial solid wastes (Radojkovic et al., 2012).

Figure 2: Anti -hemolytic activity of Senna auriculata extract
Senna auriculata is a evergreen shrub with yellow colour lowers which are grown in India. This plant is recognized to report its uses in Ayurvedic and Siddha system for treating various diseases. As it has the property to inhibit the carbohydrate digestive enzymes like alpha -amylase and alpha glucosidase Senna auriculata can retard the starch in food . Incorporating Senna auriculata in bread is the effective method to produce low glycemic index foods. Inspite of this inhibitory effect of Senna auriculata on the digestive enzymes, a well know health bene it is the antioxidant capacity (Aparna and Govindasamy, 2018) . The invivo oxidative stress along with associated damages can be reduced by the interactions with reactive oxygen species (ROS) (Nabavi et al., 2012). The aim of this study is to produce bread which inhibits the glycemix index, starch degradation, quality parameters, ef iciency of texture, etc.

Senna auriculata extract preparation
The lower Senna aurticulata was washed, dried and ground into a ine powder. To a inal mass of 300 grams, 100ml of water was added and stored for 3 -5 days at room temperature. The extract was then centrifuged at 4000g for 15 mins. The supernatant collected wastested for further invitro activities.

Evaluation of invitro -antioxidant activity
Based on the protocol (Brand-Williams et al., 1995) the antioxidant activity of the aqueous extract was evaluated. 0.1mM DPPH solution of was prepared using ethanol and 100 micro-liter of DPPH was added to the Senna auriculata aqueous extract solutions of different concentrations. The mixture was shaken vigorously and allowed to stand at room temperature for 30 mins. The absorbance was measured at 517 nm using a UV-Vis spectrophotometer. DPPH alone serves as control. Gallic acid was used for comparison. The triplicates value obtained were calculated.

HRBC membrane stabilization assay
Based on the protocol (Varghese et al., 2017). 2 ml of blood was drawn and added to EDTA to prevent coagulation. To 100 µL of blood was added to the different concentrations of the Senna auriculata aqueous extract. The mixture was incubated at 37 • C for 30 mins. Blood with distilled water serves as control. The sample mixture was then centrifuged at 300 rpm for 10 mins. The supernatant was removed

HRLC-MS investigations
HRLCMS of Senna Auriculata were analysed using Agilent 6200 series liquid chromatography system at SAIF IIT Mumbai. The solvent system comparised of 95% water (Solvent A): 5% aceonitrile (Solvent B) with applied gradient, low rate of 0.2 ml/min with column temperature of 25 • C. Hypersil gold 3 micron of size 100 x 2.1 MM was used. Injection volume was set to 5µL with run time of 30 mins where the sam-ple ionization was achieved through ESI interface in both positive and negative mode (Kadam et al., 2018;Agme-Ghodke et al., 2016).

a -amylase inhibitory assay
According to the previous method (Zhang et al., 2010;Suthindhiran et al., 2009). The extract was mixed with di-methyl sulfoxide solution and different concentrations of the samples were used. The alpha amylase was dissolved in phosphate buffer and the pH was adjusted to 6.8. After pre-incubation 1% of starch solution was added to all the tubes. The reaction mixture was incubated for 15 minutes. Finally, after the incubation time the reaction was stopped using 1 ml of di-nitro salicylic acid reagent and the tubes were kept for boiling in water bath for 10 minutes. The contents were cooled and 10 ml of distilled water was added to all the tubes. Absorbance was measured at 540 nm using microplate reader. Acarbose serves as positive control.

Percentage of inhibition =
a-glucosidase inhibitory assay α-glucosidase inhibitory assay was determined using protocol (Li et al., 2005;Suthindhiran et al., 2009). 112µL of potassium phosphate buffer of pH 6.8, 20 µL of enzyme solution (1 unit / ml), 8µL of the aqueous extract of SA were mixed and incubated in 96 well plate at 37 • C for 15 minutes. Further 20 µL of p-NPG was added to each well and incubated for 15 minutes at 37 • C. The reaction was terminated using 80µL of Na 2 CO 3 solution. Absorbance was determined at 405 nm. Instead of sample, 8µL of DMSO was added to control and blank. Acarbose is used as positive control.

Preparation of Senna auriculata forti ied -bread sample
The bread was prepared using ingredients which is shown in Tables 1, 2 and 3 . The ingredients were mixed thoroughly until smooth, which was followed by the rest period of 15 minutes. The dough was then moulded into into cylindrical shape into a container of 0.2 mm thick. Then the mould was baked at 120 • C for 45 minutes. After baking, the bread loaves were cooled and crust and crumb were separated (Ezhilarasi et al., 2013). The Senna auriclata was added to the wheat lour as levels 2%, 4% and 6% to form Senna auriculata forti ied bread sample for investigationl studies. The amount of Senna auriculata in bread before digestion is determined using crumb samples of bread. The crumbs were lyophilised, blended into powder form and further seived using mesh for extraction. 0.5 grams of ine bread powder was     (7) Symmetry (7) Colour (7) Texture (7) Eating quality (7) Overall acceptability (7) Normal white bread (control) 6 6 5 6 5 5 Senna auriculata forti ied bread (2%) 6 6 5 5 4.5 4 (4%) 6 5 6 5 4 4.5 (6%) 6 6 6 5 4.5 5.8 The paranthesis values indicates the maximum rating in the score card  weighed and mixed with 8 ml of methanol homogenously using vortex in 15 ml tube. Followed which the tubes were incubated at 60 • Cfor 5 mins in a water bath. The tubes were then kept in orbital shaker at 300 rpm for 10 mins. The liquid extract and solid fraction were obtained using centrifugation at 3000 rpm for 5 mins using Thermo Fisher Scienti ic, cooling centrifuge. The liquid extract is used for GCMS analysis.

GC-MS investigation
The samples obtained from the centrifugation was analysed using JEOL GCMATE II GC, with maximum resolution of 6000, and maximum caliberated mass 1500 daltons. It is used to analyse complex organic and biochemical mixtures. The compounds from the spectra were collected as they exit the column, which are then identi ied and quanti ied based on their mass -to -charge ratio (m/z) (Neelamegam and Ezhilan, 2012).   2014 was used to perform invitro digestion study. In this protocol the amount of alpha amylase was modiied in the absence of the salivary amylase. The fresh bread samples were sieved using 2 mm sieve from which 5 grams was mixed with the reaction mixture which contains 4 ml of stimulated salivary luid, 25 µL of 0.3 M CaCl 2 and 975 µL of water. The mixture was subjected to vortex for 20 sec with further incubation of 2 mins using magnetic stirrer. The mixture was further used for gastric and intestinal phase. This phase is noted as oral phase.

Invitro digestion of bread
Gastric phase: The samples of oral phase was further mixed with pepsin 8 ml using stimualted gastric luid whose inal concentration is 2000 U/ml, 5µL of 0.3 M CaCl 2 solution, 1.3 ml of water, 0.65 ml of 1 M HCl (pH 3). The mixture was incubated with stirring at 37 • C for 2 hrs.
Intestinal phase: The samples from gastric digestion was mixed using reaction mixture which contains stimulated intestinal luids (100 U/ml for the activity of trypsin), 16 ml of pancreatin and pH was adjusted to 7. The samples were kept in shaker for 2 hrs at 37 • C.
All the sample tubes of the intestinal phase were prepared for 7 digestion time points such 0, 20, 40, 60, 80, 100, 120 mins. All the samples was the centrifuged at 4 • C for 10 mins. 4 ml of ethanol was added to the 1 ml supernatant to terminate the reaction. Then they are used for glucose analysis.

Reducing sugar released
The resultant mixture obtained from the intestinal phase was transferred to the dialysis tube and dialysed for 5 hrs at 37 • C using PBS buffer. 0.5 ml of dialysate was withdrawn at every 10 mins. The reduced sugar was identi ied using the method described by Miller, 1959. 0.5 ml of DNS reagent is added to dialysed samples and boiled for 10 mins. The absorbance was determined using Spectrophotometer at 540 nm. Reduced sugar was expressed and calculated in terms of GE, mg/ml.

Sensory evaluation of bread
A score card was used for sensory evaluation for the desirability and undesirability of bread. They are evaluated based on colour, texture, taste, along with quality characeristics like colour of crust, crumb, mouthfeel and overall quality. This was given to panelist consisting for both the genders of age 25 -50 years. The score for each parameter was assigned by panelist which is evaluated against the maximum score using 7 point hedonic rating scale: excellent as 7, very good as 6, good as 5, satisfactory as 4, fair as 3, poor as 2, very poor as 1 (Rathi et al., 2004;Makinde and Akinoso, 2014).

Estimation of total sugars
The total sugars was estimated based on the protocol (Albalasmeh et al., 2013). 10 µL of sample was combined with 300 µL of 5% phenol. The tube is incubated for 5 mins, further the incubation 1.8 ml of concentrated sulphuric acid was added. The tubes were cooled and absorbance read at 490 nm. Total sugar content was expressed as mg glucose/g of extract.

Estimation of total phenolic content
Modi ied method of Henriquez et al., 2010 was used. The reaction mixture containing 100 µL of sample, 1 ml of folin -ciocalteau reagent , 2 ml of 10% sodium carbonate solution was incubated for 60 mins at room temperature. Absorbance was measured at 765 nm using UV-Vis spectrophotometer. The values obtained are compared with standard gallic acid. The total phenolic content is expressed as mg gallic acid equivalent/ g of extract.

Estimation of lavonoids content
Based on the protocol (Lallianrawna et al., 2013). 75 µL of 5% of NaNO 2 solution was added to 0.9 ml of sample. 150 µL of 10% AlCl 3 .6H 2 O was added to the mixture after 5 mins, and incubated at room temperature for 5 mins. Followed by 0.5 ml of 1 M NaOH and total volume was made to 2.5 ml using water. The solution was mixed and at 510 nm the absorbance was measured using UV-Vis spectrophotometer. In blank the extracts were replaced by distilled water. The total lavonoids content is expressed as catechin equivalent/g of extract.

Total starch (TS)
Total starch content is determined by the method using protocol (Goñi et al., 1997). 50 mg of ground sample was mixed in 6 ml of 2 M KOH and kept in shaker at room temperature for 30 mins. 3 ml of 0.4 M sodium acetate buffer (pH 4.7) and 60 µL amyloglucosidase (sigma-aldrich) was added. The mixture was incubated at 60 • C for 45 mins in water bath. Using glucose oxidase -peroxidase kit the glucose was measured.

Glycemic index (GI)
A modi ied protocol was used (Goñi et al., 1997). 100 mg of ground sample was incubated with 10 ml of HCl-KCl buffer (pH1.5), 200 µL pepsin solution at 40 • C for 1 hour with continuous shaking. pH is raised by adding 200 µL of pancreatic alpha amylase solution, further incubated for 45 mins at room temperature. The reaction of enzyme was stopped by adding 70 µL of Na 2 CO 3 solution. 25 ml of tris maleate buffer was added to dilute the samples. 5 ml of pancreatic alpha amylase solution and incubated at room temperature with constant shaking. Aliquots of 1 ml were taken from samples at different time intervals and kept for boiling water with constant shaking for 5 mins. This inactivates the enzyme reaction. Samples were refrigerated. The alliquots were then treated using 3 ml of 0.4 M sodium acetate buffer and 60 µL of amyloglucosidase and further incubated at 60 • C, 45 min with shaking. The volume was adjusted to 10 ml using distilled water after incubation period and centrifuged. 0.1 ml of aliquot is transferred into glass tube for glucose measurement using glucose oxidase -peroxidase kit. Absorbance was measured at 510 nm against reagent using UV-Vis spectrophotometer. The values were plotted and concentration over time curve was analysed using sigmaplot 10.0. The samples GI were estimated by the equation of pGI = 39.71 + 0.549 HI. Hydrolysis index (HI) is calculated as percentage of total content of glucose released is compared to the standard glucose that is released (0-180 mins) (Barine and Yorte, 2016).

Moisture content
Moisture content of the bread was analysed using the procedure as described by AOAC, 2002.

Crude protein
Protein was determined using micro-Kjeldahl protocol method in AOAC, 2002. The digested protein in the sample was determined spectrophotometrically and calculated as % crude protein =

Crude fat
This content analysis was carried out using standard method as described in AOAC, 2002.

Crude iber and ash content
The crude fat and ash content in bread is analysed using the standard method of AOAC, 2002.

Carbohydrate
Using AOAC, 2002 protocol difference method of analysis the bread samples estimation was determined. the crude iber, protein and fat were subtracted from organic matter; the remaining is calculated as carbohydrate content.

Statistical analysis
The experiments were conducted in triplicates. Using SPSS 20.0 the one way analysis of variance (ANOVA) with Ducan test, which showed p < 0.05 to evaluate the sigi icance of the sample.

Evaluation of invitro -antioxidant activity
The DPPH radical scavenging activity results are shown in Figure 1 and compared with antioxidant Gallic acid. Low concentration possessed high radical scavenging activity, 90% of radicals were scavenged at lower concentrations. From the analysis, we can conclude that the scavenging effects of Senna auriculata extract on DPPH radicals were excellent.

Evaluation of anti-hemolytic activity
Hemolysis shows that Senna Auriculata aqueous extract is non toxic in its nature and can be used for further studies. Different concentrations of the extract were analysed for its toxicity studies against human RBC cells. The results showed that control cell which is treated with Triton X 100 which resulted in 100% lysis. The assay results revealed that the Senna auriculata extract doesnot possess any toxicity, which is shown in Figure 2.

HRLC-MS
There are around 100 compounds were identi ied in the extract of Senna auriculata by HRLC-MS. Based on the retention time, molecular formula, database formula, most prevailing compounds are identi ied and the relative compounds found to be with many medicinal properties and the positive and negative ionisations are also depicted below in Figure 3.

a -amylase inhibitory assay
The anti diabetic activity of Senna auriculata showed the highest inhibition rate of 77.67% was seen at 100 µg/ml concentration and the lowest inhibition rate of 23.67% was seen at 10 µg/ml concentration as shown in Figure 4. Acarbose was taken as positive control. The result showed that the extract is dose dependent and revealed the increase in inhibitory activity against amylase enzyme.
a -glucosidase inhibitory assay Alpha glucosidase plays a major role in modulating PP hyperglycemia. The aqueous extract of Senna auriculata was studied for the alpha -glucosidase inhibitory activities which was detected by using pNGP as substrate. The present data revealed that 10, 25, 50 µg/ml showed < 50 % but 100 and 500 µg /ml showed > 50 % of its inhibitory activity as shown in Figure 5.

Quanti ication GCMS
The GC/MS analysis both organic and biochemical mixtures. The name and retention time was shown in Figure 6, exhibits the phytochemical constituents of Senna Aurticulata through GC/MS analysis.

Invitro digestability study
Invitro digestion of bread Figure 7 shows a dose dependent inhibition of Senna auriculata during the release of reduced sugar dur-ing intestinal digestion of bread. Gastric chyme transit which happens through whole small intestine takes about 4 -5 hrs (DeSesso and Jacobson, 2001). At 60 mins of dialysate, the concentration of resistant starch of bread sample with Senna auriculata exhibited to be low when compared with the control. At the end of 250 mins of dialysis, when compared to control the concentration of resistant starch for Senna Auriculata forti ied bread sample 2%, 4% and 6% was reduced.

Sensory evaluation
Sensory evaluation of bread samples was given in Table 4. The results were analysed using 7 point hedonic scale. The Senna auriculata forti ied bread sample is compared with the control which the regular bread based on their crust, colour, symmetry, texure, eating quality, and overall quality of the bread. The global smell was lower in breads prepared using Senna aurticulata when compared to normal breads. There is a decrease in the lavour of Senna auriculata forti ied bread when compared with the control due to the taste of the lower, which could be more signi icant to the sensory attributes and overall accetability of bread. The forumlated forti ied bread which is preared using above said concentraion was found to have positive effect on consumers overall acceptability, even more among diabetes patients.

Bioactive components
Based on the biofunctional properties developed the evaluation is analysed at the retention time of the process. Since they are recommended for the diabetic patients the total sugar and reducing sugar are analysed carefully. The increase in total and reducing sugars depends on the content of Senna auriculata which is added on bread. (Table 5) Normal white bread shows a high range of sugar content (6.26 mg/100g), when compared with Senna auriculata forti ied bread which exhibits low range of sugar content. Reducing sugar also exhibited the same pattern of decrease. Thus the addition of Senna auriculata to bread reduces the sugar content.
Total phenolic and lavonoids in bread sample of Senna auriculata showed a prominent levels of both phenolic content and lavonoids. Many people showed interest in plants for the potential health bene its. Based on those research bioactive components like phenolic, lavonoids etc are more effective and acts as inhibitors of diabetes (alpha glucosidase) and more related health problems (Henríquez et al., 2010).

Total starch and glycemic index
Based on the analysis made through sensory attributes 6% Senna auriculata was used for the starch digestability studies (invitro). The levels of total starch, resistant starch, digestible starch of the formed bread products are exhibited in Table 6 Further they even con irm that these biofunctional components act effectively and inhibits the enzyme in post prandial hypoglycemia (Shen et al., 2012). Therapeutic value of low GI in diet of diabetic both type 1 and type 2 are ef icient. The low GI as well as high resistant starch reduces the insulin resistance by lower the blood glucose levels in diabetic patients as well as improves their lipid metablosim and to prevent cardiovascular diseases.

Quality evaluation of bread
The moisture content of bread shows the life of bread for consumption. The moisture content of bread made with Senna auriculata is found to be more when compared to normal bread. To keep the quality of bread moisture content is more important. These moisture content has adverse effect on the storage. The protein content of the wheat lour is 12.12±0.01 and the Senna auriculata fortiied bread seems to have 10.40±0.68 (Table 7). This results shows that the mixed lour has reduce pro-tein content when compared to the normal bread sample. Even the mixture reduced the fat (1%) content in the lour. There is a decrease in ash content and increase in iber content when analysed. High ash and crude contents make digestion dif icult in human body. There is increase in carbohydrate content due to the extract which makes the bread soft in its texture.

CONCLUSIONS
This study indicates that incorporation of Senna auriculata into bread reduces the starch digestion and also lowers the glycemic index. It also proves that Senna auriculata has a potential to bakery products with lots of health supplements. The bioactive components of bread like phenolic, lavonoids are found to be higher in the forti ied bread sample. Involvement of carbohydrate hydrolysing enzyme which is involved in post prandial hypoglycemia a important cause of type 2 diabetes. This overall invitro indings gives us a promising result that due to the lower glycemic effect the Senna auriculata forti ied bread sample is situable and recommended for diabetes patients.