Claims:We Claim:
1. A method of preparing an anthocyanin containing nutraceutical composition, comprising
a. collecting 1000g of each fruits including Carissa carandas, Ipomoea batatas, Pithecellobium dulce, sygium cumini and Macrotyloma cumini and kept in freezer for 24 hours;
b. lyophilisation of the above fruits were done separately for 5 days at 2oC;
c. blending the dried fruits separately to obtain powder form;
d. mixing each powder in equal amount of 10g and made into suspension by mixing in distilled water
characterized in that, the composition has 295.57 mg/l of anthocyanin content in 1g/10ml of powder sample, which exhibits anti-ulcerative property against ulcerative colitis.
2. An anthocyanin containing nutraceutical composition for treating ulcerative colitis, comprising powder form of fruit extracts of Carissa carandas, Ipomoea batatas, Pithecellobium dulce, sygium cumini and Macrotyloma cumini
characterized in that, the composition has 295.57 mg/l of anthocyanin content in 1g/10ml of powder sample, which exhibits anti-ulcerative property against ulcerative colitis.
3. The composition as claimed in claim 1, wherein the anthocyanin containing nutraceutical composition inhibits the inflammatory and oxidative bio-markers in oxazolone induced ulcerative Zebra fish model.
4. The composition as claimed in claim 3, wherein the anthocyanin containing nutraceutical composition passed into body of fish through fins.
5. The composition as claimed in claim 3, wherein the anthocyanin containing nutraceutical composition is mixed with water for treating oxazolone induced ulcerative Zebra fish.
6. The composition as claimed in claim 3, wherein the anthocyanin containing nutraceutical composition of 5mg/kg reverse the levels of MPO, NO and TNF-α in oxazolone induced ulcerative Zebra fish. , Description:FIELD OF THE INVENTION
The present invention relates to an anthocyanin containing nutraceutical composition prepared from fruits extracts of Carissa carandas, Ipomoea batatas, Macrotylomacumini, Pithecellobium dulce, sygium cumini. More particularly, the invention relates to an anthocyanin containing nutraceutical composition for treating treat ulcerative colitis.

BACKGROUND OF THE INVENTION
Ulcer in colon affects the rectum and colon. Inflammation of the body part is spoken as redness, and tenderness of the body part and sigmoid as proctosigmoiditis. Ulcerative colitis or inflammatory bowel sickness could be a womb-to-tomb disease that's related to vital morbidity. Associate of individuals with colitis can have a minimum of one relapse per year [Truelove SC, Witts LJ. British medical journal.1955, 2(4947), 1041-1048]. 80% of these are delicate to moderate and regarding 20%measure severe [Travis SP, Stange EF, Lémann M, Oresland T, Bemelman WA, Chowers Y, et al. Journal of Crohn’s & colitis 2008, 2(1), 24-62]. Lethality is altered by the temporal order of interventions, as well as medical care and colectomy. Current medical approaches target curing active sickness to deal with symptoms of urgency, a frequency of defaecation and body part trauma, and additionally to enhance the quality of life, and therefore to keep up remission. The treatment chosen for active sickness is probably going to depend upon clinical severity, the extent of sickness and therefore the person's preference, and will embody the utilization of aminosalicylates, corticosteroids or biological medication. This medication may be oral or topical (into the rectum), and corticosteroids could also be administered intravenously in the population with acute severe sickness. Surgery could also be thought-about as an emergency treatment for severe colitis that doesn't answer drug treatment. Most patients receive maintenance medical care with aminosalicylates. There could also be variation within the doses of aminosalicylates and in whether or not a mixture of treatment routes is employed. Relating to immunological disorder medicinal drug or immunosuppressant, it seems that medicinal drug and immunosuppressant square measure more and more accustomed maintain remission in a population with frequently-relapsing colitis [Akshaya K, Chitra V. A Review on Pathological State and Herbal Remedies on Ulcerative Colitis, 2019.]. The exact causes of ulcerative colitis aren't legendary. Ultimately, scientists believe that there’s quite one cause which the many various factors work along to cause the unwellness. Further, the cause of one specific individual with the unwellness can be completely different from anothers. Genetics and therefore the system response might not be enough to clarify the event of inflammatory bowel disease. There is also one or additional conditions that can trigger IBD and many potential candidates which includes smoking, Nonsteroidal Anti-Inflammatory medication, Antibiotics, Contraceptive Pills, and Nutritional factors. A doctor can raise concerning symptoms and case history, and take a look at to search out whether or not any relatives have had inflammatory bowel disease, IBD or colitis. They diagnose Blood test, stool test, Colonoscopy etc.

OBJECT OF THE INVENTION
An object of the invention relates to an anthocyanin containing nutraceutical composition for treating ulcerative colitis.
Another object of the present invention relates to the method of preparing an anthocyanin containing nutraceutical composition from fruit extracts of Carissa carandas, Ipomoea batatas, Macrotyloma cumini, Pithecellobium dulce, sygium cumini.
Yet another object of the invention relates to a method for detecting the total anthocyanin content and presence of phytochemical in fruit extracts and evaluate the ulcerative properties of selected plants in zebra fish model.

SUMMARY OF THE INVENTION
The present invention relates to an anthocyanin containing nutraceutical composition for treating ulcerative colitis. The composition is prepared from the fruits extracts of Carissa carandas, Ipomoea batatas, Macrotylomacumini, Pithecellobium dulce, sygium cumini .
In one embodiment, the present invention relates to the method of preparing an anthocyanin containing nutraceutical composition from fruit extracts of Carissa carandas, Ipomoea batatas, Macrotyloma cumini, Pithecellobium dulce, sygium cumini. The selected fruits and tubers were rinsed well and the pulp was removed from seeds sliced into small pieces and spreaded in a tray covered with cling wraps and kept in deep freezer and dried below 2°C for 24 hours and lyophilized for 5 days to get powder. Obtained fine powder of all five plants is mixed with equal quantities and stored in freezer.
In other embodiment, the present invention relates to the detection of total anthocyanin content in the obtained fine powder from the selected plant extract, namely Carissa carandas, Ipomoea batatas, Macrotyloma cumini, Pithecellobium dulce, sygium cumini. The total anthocyanin content in 1g/10ml of powder sample is determined using pH differential method and it is found to be 295.57 mg/l and they are screened to have saponins, alkaloids, proteins, carbohydrate, tannins, flavanoids, glycosides, sterols.
In another embodiment, the present invention illustrates the anti-inflamatory, anti oxidative, anti-ulcerative properties of an anthocyanin containing nutraceutical composition in a oxazolone induced ulcerative zebra fish model. The zebra fish model is treated with anthocyanin containing nutraceutical composition mixed with water. Treatment with anthocyanin containing nutraceutical composition significantly reversed the levels of MPO, NO and TNF-α. It is observed that 5mg/kg was more potent, therefore the high dose intake of nutraceuticals has beneficial effect on UC.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a chart showing body weight changes in Zebrafish
Figure 2 shows histopathology of Zebrafish in ulcerative colitis
Figure 3 depicts a chart showing free radical scavenging nitric oxide assay
Figure 4 depicts a chart showing tumor necrosis factor α
Figure 5 depicts a chart showing Myeloperoxidase assay

DETAILED DESCRIPTION OF THE INVENTION
The following description is presented to enable any person skilled in the art to make and use the embodiments, and is provided in the context of a particular application and its requirements. Various modifications to the disclosed embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the scope of the present invention. Thus, the present invention is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.
The present invention relates to an anthocyanin containing nutraceutical composition for treating ulcerative colitis. The composition is prepared from the fruits extracts of Carissa carandas, Ipomoea batatas, Macrotylomacumini, Pithecellobium dulce, sygium cumini .
The present invention relates to the method of preparing an anthocyanin containing nutraceutical composition from fruit extracts of Carissa carandas, Ipomoea batatas, Macrotyloma cumini, Pithecellobium dulce, sygium cumini. The selected fruits and tubers were rinsed well and the pulp was removed from seeds sliced into small pieces and spreaded in a tray covered with cling wraps and kept in deep freezer and dried below 2°C for 24 hours and lyophilized for 5 days to get powder. Obtained fine powder of all five plants is mixed with equal quantities and stored in freezer.
The present invention relates to the detection of total anthocyanin content in the obtained fine powder from the selected plant extract, namely Carissa carandas, Ipomoea batatas, Macrotyloma cumini, Pithecellobium dulce, sygium cumini. The total anthocyanin content in 1g/10ml of powder sample is determined using pH differential method and it is found to be 295.57 mg/l and they are screened to have saponins, alkaloids, proteins, carbohydrate, tannins, flavanoids, glycosides, sterols.
The present invention illustrates the anti-inflamatory, anti oxidative, anti-ulcerative properties of an anthocyanin containing nutraceutical composition in an oxazolone induced ulcerative zebra fish model. The zebra fish model is treated with anthocyanin containing nutraceutical composition mixed with water. Treatment with anthocyanin containing nutraceutical composition significantly reversed the levels of MPO, NO and TNF-α. It is observed that 5mg/kg was more potent, therefore the high dose intake of nutraceuticals has beneficial effect on UC.

Experimental Details
Preparation of nutritional supplements from selected fruits and tubers (Carissa carandas, Ipomoea batatas, Macrotylo macumini, Pithecellobiumdulce, sygium cumini). The plants were obtained from Thathalur village, Thirukazhukundram Taluk, Chengalpattu District, Tamil Nadu, India. The selected plants were rinsed well and the pulp was removed from seeds sliced into small pieces and spreaded in a tray covered with cling wraps and kept in deep freezer for 24 hours and lyophilized for 5 days to get powder. Obtained Fine powder of all 5 plants is mixed with equal quantities and stored in freezer for further investigation. Table 1 show plants and their yield in grams.
Table 1 Medicinal plants and their yield in grams
Medicinal plants Amount used Yield
Carissa carandas 1000gms 188gms
Ipomoea batatas 1000gms 220gms
Pithecellobium dulce 1000gms 223.5gms
Syzygium cumin 1000gms 180gms
Macrotyloma cumini 1000gms 500gms

Preliminary phytochemical analysis:
Phytochemical constituents is tested and found to be positive for alkaloids, Terpenoids, Reducing Sugars, Saponins, Tannins, Flavanoids, Anthocyanins, and Steroids shown table 2.
Table 2 Phytochemical Analysis
S.No Phytochemicals Results
1 Alkaloids +
2 Carbohydrate +
3 Flavanoids +
4 Glycosides +
5 Protein +
6 Sterols +
7 Saponins +
8 Tannins +
9 Triterpenoids -

Total anthocyanin content:
Overall anthocyanin content was estimated by the pH-differential spectrophotometric technique Take two 10ml standard flask and add One ml of sample in each and made up to volume by adding Kcl (0.025M) at ph 1 in and sodium acetic acid (0.4M) at ph 4.5 and kept to equilibrate for 15 min before identification by spectrophotometer. The absorbance was estimated at 520 nm and 700 nm.
The distinction in the absorbance at varying pH level and wavelengths was determined as:
𝐴 = (𝐴520𝑛𝑚 − 𝐴700𝑛𝑚)𝑝𝐻1.0 − (𝐴520𝑛𝑚 − 𝐴700𝑛𝑚)𝑝𝐻4.5
𝑇𝑜𝑡𝑎𝑙 𝑎𝑛𝑡ℎ𝑜𝑐𝑦𝑎𝑛𝑖𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (𝑚𝑔/𝑙) = (𝐴 × 𝑀𝑊 × 𝐷𝐹 × 1000)/Ɛx1
Where, MW = molecular weight DF = dilution factor MW = 449.2g mol-1 Ɛ = 26,900 Lcm-1mol-1 of cyanidin-3-glucoside were utilized. Recognizable proof of anthocyanins.

Total anthocyanin content(mg/l)=(A×MW×DF×1000)/Ɛx1
= 0.199×449.2×100×1000/26900×1
=295.57mg/l

Experimental in Zebra fish Model
Zebra fish was obtained from Pentagrit, chennai and housed in tanks at 28˚C (14 hours light 10-hours dark cycle) . the experimentation was permitted by the institutional animal ethical committee. The IAEC ethical clearance certificate no. is. IAEC/198/2018 the animals were acclimatized for period of 10 days and were differentiated into 4 groups having 10 animals in every group.
Grouping of animals:
Zebra Fishes were divided into four groups with 10 in each group
Table 3: Grouping of Animals
S.no Animal Group Chemical
1
Zebra Fish
(Daniorerio) Group- I Oxazolone induction
(5µl) Intrarectal + Saline (5µl) oral
2 Group- II Oxazolone induction
(5µl) Intrarectal + Prednisolone (Std) will be dissolved in tank
3 Group- III Oxazolone induction
(5µl) Intrarectal + Low Dose (1mg/ml) oral
4 Group- IV Oxazolone induction
(5µl) Intrarectal + High Dose (5mg/ml) oral

Induction of enterocolitis in zebra fish
Adult zebra fish was instilled intrarectally with 0.2%oxazolone in 50 percent C2H5OH in a whole volume of 0.6µl/100mg body weight. Previously zebra fish abdomen was slightly pressed in order to determine the anal opening. oxazolone was injected with a Hamilton glass syringe mounted with silicone tubing to ensure minimal mechanical damage.
Example 1
Forty fishes were divided into four groups with 10 fishes in each group in separate tank. With Group I (control) Group II (1mg/g) Group III (5mg/g) Group IV (standard) Table 4. Nutraceuticals and prednisolone are pre-treated for 14 days and followed by induction of colitis by 37 intrarectal injection of 0.2% oxazolone in 50%ethanol. After 14 days fishes are sacrificed colons are isolated for further evaluation. Figure 1 shows body weight changes in zebra fish.
Table 4 Body weight changes in zebra fish
Groups Week 1 Week 2 Week 3 Week 4
I 0.296 ±0.09 0.287 ±0.07 0.232 ±0.07 0.189 ±0.09
II 0.274 ±0.07 0.276 ±0.08 0.274 ±0.1 0.276 ±0.08
III 0.283 ±0.07 0.276 ±0.08 0.278 ±0.08 0.280 ±0.06
IV 0.287 ±0.1 0.298 ±0.06 0.312 ±0.12 0.314 ±0.14

Hisopathology:
Figure 2 shows histopathology of Zebra Fish in ulcerative colitis. Group-I shows severe loss of mucosa with disruption of intestinal villi, loss of globlet cells and leucocyte infiltration. Group-2 shows mild loss of mucosa with disruptions of intestinal villi, mild loss of globlet cells and leucocyte infiltration. Group-3 shows no histopathological changes are observed and Group-4 shows minimal loss of mucosa.
Effects of anthocyanin containing nutraceutical composition in zebra fish
Ulcerative colitis (UC) is a chronic inflammatory bowel disease that causes inflammation and scores called ulcers in the lining of the rectum and colon. Major cause is multifactorial. There is a significant proof for T-cells in the lamina propria add to commencement in the lamina propria and progression of disorder. T-Lymphocytes in particular CD4 + T-cells play an important role in all immune regulatory process in the gastrointestinal tract. Production of proinflammatory cytokines such as IFN- γ,TNF-α IL-5, IL-6,IL12,IL-13, IL-9 is due to the activation of NF-KB pathways because of disruption of mucus film covering the epithelium. In the present study zebra fish is used as experimental model science they are easier to cultivate and maintain when compared with other models. Zebrafish has high similarities with human beings in gastrointestinal system with a gallbladder, liver & intestinal tract with similar absorptive and secretory functions. Here UC was chemically induced by oxazolone mixed in 50% ethanol and was found to induce marked colitis associated with weight loss. Histopathological analysis of colon obtained from zebra fish with disease control revealed superficial colonic inflammation, epithelial, cell erosion, ulceration, infiltration of lymphocytes a rise in production of TNF-α by NK-Tcell. Protection against UC is produced by Nutraceuticals prepared from selected 5 medicinal plants of nutritional value plants rich in anthocyanin levels. Anthocyanin are water soluble pigments that might appear as red, purple or blue pigments according to their pH levels. Since Nutraceuticals are thermostable and might decompose at higher temperature, they are prepared by collecting fresh fruits and freeze drying them to powdered form. Total anthocyanin content was determined by using pH differential method and it was found to be 295.57 mg/l and they are screened to have saponins, alkaloids, proteins, carbohydrate, tannins, flavanoids, glycosides, sterols
Antiulcerogenic effect is seen in the zebrafish by oral administration of Nutraceuticals in two doses (1mg/g, 5mg/g) which was compared with standard drug prednisolone and the above investigation resulted that 5mg/kg was found to be more potent in healing. Inflammatory cytokines play a major role in modulating musical immune system where the neutrophils and macrophages responsible for disrupting epithelialintegrity and causing colon injury. Migration of granulocytes & macrophages are responsible for disrupting epithelial integrity and causing colon injury. The mechanism of UC is characterized by migration of granulocytes and other leukocytes to the inflamed mucosa leading to increased levels of TNF-α and other interleukins. In control group there is a rise in TNF-α (4984 ± 1.29) which is evidence for epithelial cell necrosis edema and neutrophil infiltration which is supported by histopathological results followed by reduction in levels in other treatment group. Oxidative stress also plays a major role in UC initiation & progression. Imbalance in oxidant and antioxidant levels are seen in colitis. Most of the studies pointed vital role of Free radical in the pathogenesis of mucosal injuries. Myeloperoxidase (MPO) serves as a important tool in assessing UC status. Myeloperoxidase is a lysosomal protein that is released into the phagosome of neutrophils during the degranulation process and combines with hydrogen peroxide and halide to form hypochlorous acid or with tyrosine to form tyrosyl radicals. These products are highly cytotoxic and can be released from the cell to destroy foreign microorganisms. These agents might also damage normal tissue and contribute to inflammation. MPO levels in diseased group is elevated (28.72 ± 0.94) and in both treatment group the level is reduced Present study displayed that nutraceutical protected oxazolone induced ulcerative colitis by inhibiting inflammatory and oxidative bio-markers. Finally results also produced assuring outcomes for future clinical usage as a natural and harmless effective supplement for UC.
UC is a chronic medical condition that may require patient to take medication throughout their lives to prevent relapse and reduce the risk of colorectal cancer by improving quality of life. In recent times focus on plant research has increased all over the world and a large body of evidence has collected to show enormous prospective of medicinal plants used in various traditional systems. Today we are witnessing a great deal of public interest in the use of herbal remedies. Now a days herbal medicine are the major components in Ayurvedic, homeopathic Naturopathy and other medicinal systems. Synthetic drugs may give instant relief from symptoms but have lot of undesirable effects. From present invention one can conclude that intrarectal injection of oxazolone in zebra fish induced severe mucosal damage and inflammation with rise in MPO, NO and TNF-α. Treatment with nutraceuticals significantly treated oxazolone induced inflammation of the zebra fish colon and reversed the levels of MPO, NO and TNF-α (Table 5, 6 & 7 and figure 3,4 & 5). It is observed that 5mg/kg was more potent, therefore we can presume that the high dose intake of nutraceuticals has some beneficial effect on UC.
Table 5 Free Radical scavenging Nitric oxide assay
S. NO Concentration (µg/ml) % Free radical scavenging activity of nutraceuticals %Free radicals scavenging activity of ascorbic acid
1 100 15.53 42.41
2 200 26.4 50.2
3 400 54.2 84.19
4 800 61.6 94.68

Table 6 Tumor Necrosis Factor α
GROUPS TNF alpha(pg/ml)
Group I 4984 ± 1.29
Group II 983.72 ±1.72
Group III 1452 ± 1.29
Group IV 2651 ± 1.44

Table 7 Myeloperoxidase assay
GROUPS MPO Units/mg
Group I 28.72 ± 0.94
Group II 17.80 ± 1.09
Group III 9.0 ± 0.69
Group IV 10.75 ± 0.5

It may be appreciated by those skilled in the art that the drawings, examples and detailed description herein are to be regarded in an illustrative rather than a restrictive manner.