WO2020161653A2

WO2020161653A2 - Mustard extract enriched with sinigrin having increased bioactivity and bioavailability of allyl isothiocyanate

Info

Publication number: WO2020161653A2
Application number: PCT/IB2020/050940
Authority: WO
Inventors: Benny Antony
Original assignee: Benny Antony
Priority date: 2019-02-07
Filing date: 2020-02-06
Publication date: 2020-08-13
Also published as: WO2020161653A3

Abstract

The invention relates to an enteric release system for releasing glucosinolate in the colon to increase bioavailability of allyl-isothiocyanate in the body of human subject. It comprises of a) a solid core made of glucosinolate obtained from the extract prepared from prepared from mustard seed, mustard cake or mustard bran or a combination thereof and b) an enteric protective coating over said core. The glucosinolate derived from mustard extract contains 20% to 90% sinigrin which is proved to have anticancer activity. The delivery systems used here includes excipients. It is dispensed in the form of hard gel capsule, soft gel capsule, tablet, beadlets, mini tablet, granule, powder and pills and the dosage ranges from 200 mg to about 2000 mg to a human subject. The enteric coating targets the sinigrin for release in large intestine where sinigrin is converted to AITC and increases its bioactivity.

Description

MUSTARD EXTRACT ENRICHED WITH SINIGRIN HAVING INCREASED BIOACTIVITY AND BIOAVAILABILITY OF ALLYL ISOTHIOCYANATE

FIELD OF INVENTION

Invention relates to an enteric release system for releasing glucosinolate when administered to a human subject. Composition enriched with glucosinolate obtained from cruciferous vegetables. Process for enrichment of glucosinolate from cruciferous vegetables is provided, more preferably a process of extraction of mustard and enrichment with sinigrin. A method to enhance the bioavailability of allyl isothiocyanate (AITC) is provided. Invention relates to an enteric coated mustard extract composition enriched with sinigrin.The enteric coating targets the sinigrin for release in large intestine where sinigrin converted to AITC. Invention pertains to increase the bioactivity and bioavailability of AITC.

BACKGROUND OF THE INVENTION

More than million cases of cancer are reported every year. There are a lot of factors that can cause cancer, and our dietary habits have great influence over it. Fruits and vegetables rich in polyphenols reduce the risk of cancer significantly, if not completely. Herbs are known to produce a wide range of secondary metabolites such as alkaloids, terpenoids, polyacetylenes, flavonoids, quinines, phenyl propionates etc. and proved for their useful medicinal properties. Cruciferous vegetables are well known and worldwide consumed due to their health benefits and cancer prevention properties. Cruciferous vegetables such as Brussels sprouts, broccoli, cabbage, cauliflower and the seeds of mustard (especially black) contain substantial quantities of glucosinolates (chemicals naturally present in plants) including sulforaphane, sinigrin and selenium. Different glucosinolates forms a different isothiocyanate. Wasabi is the only source of 3 unique powerful glucosinolates which breakdown into 3 unique long chain methyl isothiocyanates, broccoli is a good source of glucoraphanin, the glucosinolate precursor of sulforaphane (SFN), and mustard seed is a source of sinigrin, the glucosinolate precursor of allyl isothiocyanate (AITC). Watercress is a rich source of gluconasturtiin, the precursor of phenethylisothiocyanate (PEITC), while garden cress is rich in glucotropaeolin, the precursor of benzyl isothiocyanate (BITC).

Glucosinolates are primarily found in brassicaceae and related plant families and play an important role in resistance to fungi, nematodes and other plant pathogens and herbivore. Their common structure comprises a-thioglucose group, a-sulfonated oxime moiety, and a variable side chain derived from methionine, tryptophan, or phenylalanine. The highest concentration of glucosinolates is found in reproductive organs such as seeds and siliques, followed by young leaves, while senescing rosette leaves contained the lowest concentration of glucosinolates (Ivana et al, Glucosinolates and their potential role in plant).

More than 100 glucosinolates have been identified and further more are being identified through advanced analytical methods using gas and liquid chromatography. In the plant, they coexist with an endogenous thioglucosidase enzyme called myrosinase, though glucosinolates are stored in the vacuoles known as S-cells and myrosinase in separate but adjacent cells. The glucosinolates are chemically stable and biologically inactive whilst they remain sequestered within sub-cellular compartments throughout the plant (Ian. T. Johnson, Glucosinolates in the human diet Bioavailability and implications for Health). Upon plant tissue disruption, glucosinolates are released at the damage site and hydrolyzed by the catalytic effect of myrosinase. (Ivana et al Glucosinolates and their potential role in plant)

The chemical nature of the hydrolysis products depends on the structure of the glucosinolate side chain, plant species and reaction conditions. The hydrolysis of glucosinolates forms glucose and an unstable intermediate, thiohydroximate-O-sulfate, which undergoes further transformation to yield primarily isothiocyanate, nitrile, and thiocyanate. Epithionitrile, oxazolidine-2-thione, and other less common products can be formed depending upon the nature of the glucosinolate and the conditions during its hydrolysis. As every hydrolyzed product of glucosinolate has different property, for medical purposes it will be desirable to deliver desired compound for the desired results.

Among the different glucosinolates, sinigrin is a major glucosinolate, associated with the family of glucosides present in the Brassicaceae family, such as the seeds of black mustard (Brassica nigra), brussels sprouts, and broccoli. In ancient times mustard has been used as a food and has illustrated medicinal benefits in Ayurveda. The Ayurvedic tradition established mustard as a valuable herb which has therapeutic effects. For thousands of years Indian mustard seeds and its oil have been aptly used to relieve joint pain, fever, alleviate cough and colds, lessened swelling, and in cleaning the cranial cavity. Mustard oil has also been used for the treatment of various skin diseases and wounds.

Mustard seeds are processed for oil extraction and the residue obtained is called mustard cake (spent mustard). Several attempts have been made for utilization of mustard cake. Mustard cake is used as a feed for cattle, poultry and aquatic animal. It is also used as a fertilizer and weedicide. Mustard cake is also known as a good protein source for human consumption.

Sinigrin, hydrolysis to an isothiocyanate, AITC. Sinigrin as such do not possess any health benefit or anti cancer activity that is known yet. It is found that vegetables rich in glucosinolates, when taken raw, hydrolysis occur in mouth and gastrointestinal tract. The myrosinase present in the plant tissue catalyzes the hydrolysis of glucosinolates in gastric tract. This hydrolysis is uncontrolled and the resulting product may or may not contain the desired isothiocynate. When the vegetables are cooked above 60°C, myrosinase present in it loses its enzymatic property. Other than the enzyme myrosinase, the hydrolysis of sinigrin is also triggered by intestinal microflora and other microorganisms. Study suggests that sinigrin can form AITC when it comes in contact with the intestinal microflora and later AITC is absorbed from the colon. The NMR spectroscopy results from Combourieu et al (Identification of new derivatives of sinigrin and glucotropaeolin produced by the human digestive microflora using lh NMR spectroscopy analysis of in vitro incubations); shown unambiguously that sinigrin and glucotropaeolin are transformed quantitatively by the human faecal flora into allylamine. In vitro study conducted by Krul et al (metabolism of sinigrin by human colon micro flora in a dynamic in vitro large intestine model), studied the production of allyl isothiocyanate (AITC) from sinigrin in a dynamic in vitro large intestine model after inoculation with a complex microflora of human origin. From the study Krul et al observed that peak levels of AITC were observed between 9 and 12 hour after addition of sinigrin. B. thetaiotaomicron, one of the bacteria found in human micro flora can hydrolysis sinigrin to AITC. Elfoul et al. (Formation of allyl isothiocyanate from sinigrin by BacteroidesThetaiotaomicron) showed that 2-propenyl glucosinolate (sinigrin), a common glucosinolate in Brassica vegetables, can be hydrolysed by a

Bacteroidesthetaiotaomicron strain of human origin to yield allyl isothiocyanate (AITC) in the large bowel of gnotobiotic rats inoculated with this bacterium.

AITC is known to inhibit proliferation of cancer cells by causing mitotic block associated with disruption of alpha-tubulin in a manner analogous to a number of chemotherapeutic agents. AITC also showed fungicidal activity against a variety of fungi and yeasts, mustard oil, of which 99% was AITC, was one of the strongest antifungal substances among the various natural oils examined (Yuesheng Zhang, Allyl-isothiocyanate as a cancer chemo preventive phytochemicals). AITC has antimicrobial activity against a wide spectrum of pathogens, it showed anticancer activity in both cultured cancer cells and animal models, but the sharp odour associated with AITC make it tough to commercialize. On the other hand Sinigrin has a better shelf life than AITC; Sinigrin as such is more stable than AITC in buffer solution and nutritional broths. As per one study there was a reduction in AITC around 60 to 80% in a nutritional broth with in eight hours (Stability studies of isothiocyanates and nitriles in aqueous media; Luang-In et al).

Omary et al, in their patent US6436450 B1 (Brassica vegetable composition and method for manufacture of same) had disclosed a composition includes processed dehydrated brassica vegetable containing glucosinolates and endogenous myrosinase enzyme that has not been inactivated. Endogenous myrosinase enzyme converts the glucosinolates into isothiocyanates when the composition is ingested. Omary et al does not disclose the targeted delivery of glucosinolate in large intestine and conversion of glucosinolate to isothiocyanate with the help of microflora present in the colon. Omary et al mainly focus on a composition and a process by which the endogenous myrosinase enzyme remain intact and intact enzyme coverts glucosinolates to isothiocyanate. The patent by Omary et al, is limited to adding dehydrated brassica vegetable in dietary supplement. Enrichment and purification of specific glucosinolates is not disclosed, more over the hydrolysis of glucosinolates is not controlled once administered orally. Hydrolysis can start in mouth itself and most of it be over by the time it reaches the small intestine, this will lead to the loss of potent hydrolyzed products. Targeted delivery of sinigrin in the gastric system not explored, West et al, in their patent application US20080311192 A 1 (Enteric-Coated Glucosinolates And Beta-Thioglucosidases) suggest a delivery method for glucoraphanin and myrosinase with enteric coating, which will release the two compound in small intestine. The method has a limitation when the drug reaches the large intestine. Glucoraphanin tend to convert into sulforaphane nitrile instead of sulforaphane in gut, wherein the nitrile is less potent than sulforaphane.

OBJECT OF THE INVENTION

Primary object of invention is to provide an enteric release system enriched with glucosinolate obtained from cruciferous vegetables.

Another object of invention is to provide an enteric release system enriched with sinigrin obtained from mustard.

Another object of invention is a process of extraction of spent mustard and enrichment of sinigrin to obtain a percentage of sinigrin in a range of 20% and above. Also provides methods for producing glucosinolate from cruciferous vegetables. Disclosure also provides a method of obtaining an enriched extract of mustard.

Another object of the invention is a use of extract of mustard enriched with sinigrin for anticancer activity and antimicrobial activity etc. Yet another object is a method of increasing the bioavailability of allyl isothiocyanate (AITC).

Another object of the invention is an enteric release system of mustard extract enriched with sinigrin. Yet another object of invention is to provide an enteric release system of mustard extract enriched with sinigrin and an enteric coated thioglucosidase enzyme myrosinase. Disclosure provides a method of increasing the bioactivity and bioavailability of AITC after administering enteric coated mustard extract rich in sinigrin.

SUMMARY OF THE INVENTION

Application discloses an enteric release system for releasing glucosinolate in the colon to increase bioavailability of allyl -isothiocyanate in the body. Enteric release system contains a solid core made of glucosinolate obtained from cruciferous vegetables, and an enteric protective coating over said core.

Cruciferous vegetables include mustard, mustard flowers, mustard sprout, mustard cake, mustard bran etc. Preferably, the cruciferous vegetable is mustard and more preferably from mustard cake. Preferable glucosinolate is sinigrin. Disclosure provides an enteric release system for releasing glucosinolate derived from mustard extract contains 20% to 90% sinigrin.

Disclosure also provides an enteric release system for releasing glucosinolate, enteric protective coating can be, but not limited to Methacrylic acid co-polymers, poly (methacrylic acid-co- methyl methacrylate), esters of aleurtic acid, cellulose acetate phthalate, cellulose acetate trimellitate, poly (vinyl acetate phthalate), hydroxyl propyl methyl cellulose, hydroxypropyl methylcellulose phthalate, hydroxy propyl methyl cellulose acetate succinate, acetaldehyde dimethyl cellulose acetate, chitosan, zein, fatty acids, waxes, shellac, plastics, plant fibers, or, a combination of ethyl cellulose and sodium alginate. Present invention discloses an enteric release system in which the weight gain after coating is greater than about 20 percent.

Another aspect of the disclosure is an enteric release system additionally contains an enteric coated thioglucosidase enzyme myrosinase. Disclosure provides a method of increasing the bioactivity and bioavailability of AITC after administering an enteric release system and an enteric coated thioglucosidase enzyme myrosinase. Ratio of enteric release system with enteric coated myrosinase is 1 : 100 to 100: 1.

Another aspect of the invention is a process of extraction of spent mustard and enrichment of sinigrin to obtain a percentage of sinigrin in a range of 20% and above. Disclosure also provides method of making the enteric release system. Disclosure provides a method of increasing the bioavailability of allyl isothiocyanate (AITC). Disclosure further provides an enteric release system which is found to have anticancer activity and antimicrobial activity etc.

Disclosure provides a composition contain a mixture of mustard extract with a pharmaceutically acceptable carrier. It is derived from mustard contains 25% glucosinolate. The mustard extract enriched with sinigrin is devoid of AITC. The mustard extract is derived from mustard seed, mustard cake or mustard bran or a combination thereof.

BRIEF DESCRIPTION OF DRAWING

These and other features, aspects and advantages of the present invention will become better understood when the detailed description is read with reference to the accompanying drawing. FIG.l provides method of extraction of 95% methanol extract of spent mustard.

FIG.2 provides method of preparation of ethyl acetate extract of methanol extract of spent mustard.

FIG.3 provides method of preparation of enzyme treated methanol extract of spent mustard.

FIG.4 provides method of preparation of column purified extract of methanol extract of spent mustard.

FIG.5 provides extraction and enrichment of sinigrin up to 40% from spent mustard.

FIG.6 provides extraction and purification of sinigrin up to 80% from spent mustard. DETAILED DESCRIPTION OF THE INVENTION

According to the invention there is provided an enteric release system for releasing glucosinolate in the colon to increase bioavailability of allyl-isothiocyanate in the body, when administered to a human subject. It comprises of a) a solid core made of glucosinolate obtained from cruciferous vegetables, and b) an enteric protective coating over said core.

One aspect of the invention is a composition of glucosinolate (sinigrin) content of 20% and above derived exclusively from mustard cake (spent mustard). Mustard seeds are processed for oil extraction and the residue obtained is called mustard cake (spent mustard). In said composition sinigrin ranges from about 20% to about 90%, more preferably 20% to about 80% more preferably about 20% to about 60% more preferably about 20% to about 40%. In one embodiment, composition derived from mustard contains 25% glucosinolate (sinigrin). Composition from mustard extract is devoid of AITC. Composition of mustard extract also contains a pharmaceutically acceptable carrier and excipients. The carrier and excipient used are but not limited to maltodextrin and aerosil. In the present invention glucosinolate from mustard cake is enriched by a unique extraction process.

Sinigrin itself is not active. Direct consumption of sinigrin might not lead to an efficient conversion of sinigrin by the intestinal microflora, as the final absorption happens in colon (large intestine) and to reach the large bowel intact sinigrin has to pass through the gastric juice in stomach and small intestine. So in order to protect sinigrin from degradation in stomach and small intestine, an enteric coating can provide a barrier to control the location of delivery. An enteric coated extract of mustard enriched with sinigrin reaches the colon where they can be hydrolysed by the microflora into active allyl isothiocyanate and enhancing the bioavailability of allyl isothiocynate in the body. The conversion of sinigrin to allyl isothiocyanate can be accelerated by administering enteric coated thioglucosidase enzyme myrosinase along with enteric coated extract of mustard enriched with sinigrin.

Present invention provides an enteric release system for releasing glucosinolate in the colon to increase bioavailability of allyl-isothiocyanate in the body. Enteric release system contains a solid core made of glucosinolate obtained from cruciferous vegetables, and an enteric protective coating over said core.

The present invention provides a composition enriched with sinigrin made of extract derived from the spent of mustard, and encapsulated the composition by an enteric coating to deliver in the colon. After administering an enteric coated enriched mustard extract composition, it reaches the colon where they are hydrolysed by the indigenous microflora into active allyl isothiocyanate.

To achieve successful colonic delivery, a drug needs to be protected from absorption and /or the environment of the upper gastrointestinal tract (GIT) and then be abruptly released into the proximal colon, which is considered the optimum site for colon - targeted delivery of drugs. For targeted delivery enteric coating can be used, the enteric coating material can be, but not limited to Methacrylic acid co-polymers, poly (methacrylic acid-co-methyl methacrylate), esters of aleurtic acid, cellulose acetate phthalate, cellulose acetate trimellitate, poly (vinyl acetate phthalate), hydroxyl propyl methyl cellulose, hydroxypropyl methylcellulose phthalate, hydroxy propyl methyl cellulose acetate succinate, acetaldehyde dimethyl cellulose acetate, chitosan, zein, fatty acids, waxes, shellac, plastics, plant fibers, or, a combination of ethyl cellulose and sodium alginate.

Present invention provides an enteric release system of mustard extract composition derived from mustard spent to increase the bioavailability of Allyl isothiocyanate (AITC) by the microflora in colon. Bioavailability is increased by 2 to 20 fold, more preferably 5 to 14 fold. Invention provides combination of enteric release system of spent mustard extract composition with enteric coated myrosinase to increase the bioavailability of Allyl isothiocyanate (AITC). Enteric coated spent mustard extract composition enriched with sinigrin and combination of enteric coated spent mustard extract composition with enteric coated myrosinase is found to have anticancer activity, anti-microbial activity, anti-inflammatory activity etc. Methods of increasing the bioavailability and bioactivity of Allyl isothiocyanate (AITC) after administering enteric coated spent mustard extract composition and combination of enteric coated spent mustard extract composition with enteric coated myrosinase are provided.

In another aspect, in an enteric-coated capsule extract of mustard enriched with sinigrin (glucosinolate) and thioglucosidase enzyme myrosinase may be provided. Upon digestion, the enteric coating remains intact while passing through the stomach and small intestine and only dissolves in the large intestine to release the thioglucosidase enzyme myrosinase and glucosinolate (sinigrin) particles. The thioglucosidase enzyme converts glucosinolates into chemoprotectant allyl isothiocyanates within the large intestine (Colon). The enteric coating facilitates the glucosinolates and their conversion enzymes to reach intact in the large intestine where absorption of isothiocyanates is found to be most efficient. Preferably, glucosinolate and thioglucosidase enzyme myrosinase should be provided in a ratio of about 1 : 100 to about 100: 1, more preferably 10: 1. The thickness of coating on the composition should be sufficient to provide the desired release of both glucosinolate and thioglucosidase enzyme myrosinase in the large intestine. Preferably, the coating is greater than about 20 percent. Optionally, the uncoated materials can be mixed with fillers, vitamins, minerals, or the like, before filled into capsules. Glucosinolate is used in combination for the treatment of stomach infection, it is further noted that, glucosinolate is used in combination for the treatment of intestinal infection. Glucosinolate is used in combination for treatment or prevention of cancer, more preferably for the treat of bladder cancer and colon cancer.

The invention provides for a dosage form of the mustard extract composition for oral administration, the dosage form is selected from but not limited to capsule, tablet, mini tablet, granule, sachet, powder, paste, infusion, injection, ampoule, solution, suspension, emulsion, pills, cream etc are provided.

The disclosure provides an enteric release system of mustard extract composition in suitable dosage forms like capsule, tablet, beadlets, mini tablet, granule, sachet, powder, paste, infusion, suppository, ampoule, pills, cream etc. Further a dosage form of a mustard extract is disclosed for administering in a dosage ranging from about 200 mg to about 2000 mg to a human subject. These delivery systems may require excipients selected from the group consisting of a disintegrant, diluents, binders, fillers, a carrier, adsorbents, emulsifiers, lubricants, stabilizing agents, antiadherents, glidants, antioxidants and mixtures thereof.

Glucosinolates are extracted from one or more plant selected from the group of Cruciferous plants. One aspect of the invention is a composition with glucosinolates are blended with other plant derivatives selected from the group comprising turmeric extract, emblica extract, costus extract, ashwagandha extract, green tea extract, amaranthus, ginger extract, pepper extract, rosemary extract, garlic extract or combination thereof. It is further noted that glucosinolates blend comprises Vitamin A, Vitamin C, Vitamin D, Nitrate, Nitrite, Omega-3, essential oils, minerals or combination thereof.

Glucosinolates blend is provided with coating, the coating is for targeted delivery of glucosinolates, wherein the components of the blend may or may not be delivered together. The enteric coated glucosinolates blend is found to have higher bioactivity than just mustard extract. Method to extract glucosinolates from spent mustard cake, extracting mustard cake with methanol and the methanol part (supernatant) obtained are separated from the cake. Supernatant is concentrated in an Agitated thin film evaporator (ATFE) to form a concentrated methanol extract. The concentrated methanol extract is dried under vacuum at above 500 mm of mercury to get powder of methanol extract of mustard cake with 15% purity.

Powder of methanol extract of mustard cake with 15% purity is macerated with water and extracted with ethyl acetate. Ethyl acetate part is collected and concentrated and dried under vacuum at above 500 mm of mercury to form powder of ethyl acetate extract of methanol extract of mustard cake.

Powder of methanol extract of mustard cake with 15% purity is soaked in water, treated with protease and incubated for 6hrs at 37°C. Deactivated the enzyme by heating the slurry at 80°C for 10 minutes and enzyme treated extract is concentrated in an Agitated thin film evaporator and dried under vacuum at above 500 mm of mercury to form powder of enzyme treated extract of methanol extract of mustard cake.

Powder of methanol extract of mustard cake with 15% purity is dissolved in water and clarified to form supernatant and residue. Supernatant is loaded on a SP700 resin column. Column is initially eluted with water followed by 50% methanol. Water elute and 50% methanol elute are concentrated in an Agitated thin film evaporator (ATFE) to form concentrated water and methanol extract. Concentrated fractions are fed separately into vacuum stripper and dried under vacuum at above 500 mm of mercury to get powder of water extract and 50% methanol extract of mustard cake.

In one preferred embodiment, mustard cake is extracted with 95% methanol for 8 hours. The methanol part (supernatant) obtained are separated from the cake. Supernatant is concentrated in an Agitated thin film evaporator (ATFE) to form a concentrated methanol extract. The concentrated methanol extract is dried under vacuum at above 500 mm of mercury to get powder of 95% methanol extract of mustard cake.

Powder of methanol extract of mustard cake is fermented using yeast at 40°C for 48hrs. Then it is centrifuged to obtain a residue and centrifugate. Centrifugate is extracted with hexane in a liquid-liquid extractor. Aqueous phase is collected and concentrated to form concentrated aqueous extract. Concentrated aqueous phase is loaded in a column having amber 200 resin. Column is initially eluted with water and water fraction is collected. Then the column is eluted with methanol and the methanol fraction is collected. Water fraction is concentrated and dried under vacuum to form powder of water elute of fermented methanol cake with 40% purity.

In another preferred embodiment, powder of water elute of fermented methanol cake obtained in the above process is dissolved in water and passed through cation exchange column which is already washed with water and 5% HC1. After passing the supernatant, column is eluted with distilled water and 3% ammonium hydroxide, respectively. The water fractions and alkaline fractions are collected separately. Alkaline fraction was concentrated and dried to get powder of purified extract from spent mustard with purity of 80%.

In one embodiment mustard extract and purified mustard extract is coated with enteric coating material.

Purified spent mustard extract powder or mustard extract is passed through the Roll Compactor machine. The flakes obtained from the roll compactor are passed through an Oscillating Granulator machine to obtain granules of spent mustard extract or purified spent mustard extract. Granules of mustard extract or purified spent mustard extract is loaded into the bowl of the fluid bed extractor (FBE). Hot, filtered air up to 90 C is passed at high velocity from the bottom of the FBE bowl through the feed material (mustard extract or purified mustard extract) and feed material is fluidized.

Meantime, any enteric coating material is dissolved in suitable solvent. Coating solution is sprayed into fluidized material by using a spraying devise attached to the FBE. Through the process of fluid bed coating, fluidized particles are continuously sprayed with coating solution, depositing layers (films) of material to the surface of the particles, and yielding an even layer thickness.

In another embodiment combination of enteric coated extract of purified mustard extract and enteric coated purified myrosinase in 10: 1 ratio is provided.

A study is conducted to test if intestinal microbiota can efficiently convert sinigrin to AITC. Rats were inoculated with microbiota derived from human gastrointestinal system. The animals were fed with different sinigrin compositions and the presence of AITC was tested. Quantity of AITC at different stages of gastrointestinal tract of the animal was recorded. AITC was detected in the colon more than other parts of the gastrointestinal system; this might be because of the high microbiota concentration in the intestine. It was also observed that when sinigrin was delivered directly into large intestine AITC detection in colon increased at least by two fold. Bioavailability of AITC was tested on rabbits, the study animals were administered different forms of sinigrin composition and after few hours their blood was tested for AITC. Bioavailability of AITC for over 10 hours was observed only in groups where sinigrin was delivered directly to the large intestine. The increase in bioavailability is 2 to 20 fold after administering enteric release system.

In a similar study the animals were administered with a mixture of sinigrin and myrosinase. The combination of enteric coated sinigrin and myrosinase which delivered in large intestine showed presence of AITC in blood over 12 hours. When the same combination of sinigrin and myrosinase was delivered in small intestine AITC was detected only till 8 hours.

The anticancer activity of sinigrin was evaluated in an orthotropic rat bladder cancer model. After the study it was observed bladder tumours formed in nearly all rats. Untreated animals the tumour was over two third of the bladder. Clinically significant inhibitions in tumour growth were observed when animals were administered with high purity sinigrin specifically when it was delivered directly to the large intestine of animals. Sinigrin in combination with myrosinase also showed promising results.

Another animal study using diethylnitrosamine induced cancer was also conducted. During the course of the study body weight of each study animal was recorded every four 4 weeks. It was observed that within 24 weeks animals starts losing weight except those animals which were not induced with diethylnitrosamine and the animals which were fed with enteric coated sinigrin, which delivered the sinigrin to the colon. After some week the animals were sacrificed and ratio of liver weight and body weight were calculated. Animals treated with enteric coated purified sinigrin or combination of enteric coated purified sinigrin with enteric coated myrosinase reported a very good decrease in the liver weight/body weight ratio. Liver of each animal is tested for surface tumours. Enteric coated purified spent mustard extract which delivers the sinigrin to the colon was able to inhibit the growth of cancer cells. AST and ALT enzyme were also tested, injection of carcinogen produced severe liver toxicity as evidenced by AST and ALT levels of untreated control group animals. Treatment with all the extracts was effective in reducing these liver enzymes levels and the most significantly effective extract was combination of enteric coated spent mustard extract that delivers the extract directly to the colon. Antimicrobial activity of sinigrin is also tested. Sinigrin compositions were tested against different microbial strain. The inhibition of different bacteria was directly proportional to the percentage of sinigrin present in the extract. The maximum inhibition was noted with purified mustard methanol extract containing 80% sinigrin against all the seven bacteria tested. The extract containing 40% sinigrin was slightly less effective than extract containing 80% sinigrin. The Minimum Inhibitory concentration (MIC) against different bacterial strains was directly proportional to the percentage of sinigrin present in the extract. The low MIC value was noted with mustard methanol extract containing 80% sinigrin against all the seven bacteria tested. The extract containing 40% sinigrin was slightly less effective than extract containing 80% sinigrin. Details of some of the method of preparation of the enteric coated composition enriched with glucosinolate obtained from cruciferous vegetables especially with reference to one prepared by a process of extraction of mustard and enrichment with sinigrin are given here by way of examples.

Example 1

Method of extraction of 95% methanol extract of spent mustard

100 Kg mustard cake (spent mustard) were filled in the Soxhlet extractor and extracted with 300 L of 95% methanol. The extraction was carried out for 8 hrs at a temperature of about 80°C. After the completion of extraction, the supernatant was filtered and concentrated in an Agitated thin film evaporator (ATFE) at a temperature of 60°C to form concentrated methanol extract. Concentrated methanol extract was dried under vacuum at above 500 mm of mercury to get 17.32 Kg powder of methanol extract of mustard cake with purity 15%. The process was explained in FIG 1.

Example 2

Method of preparation of ethyl acetate extract of methanol extract of spent mustard

A methanol extract of mustard cake with a purity of 15% obtained by the method shown in Example 1 was taken. The powder of methanol extract of mustard cake was macerated with water and transferred into a liquid-liquid extractor and extracted with ethyl acetate. Ethyl acetate phase and aqueous phase were separated. After extraction ethyl acetate phase was collected. Ethyl acetate phase was concentrated in an Agitated thin film evaporator to form concentrated ethyl acetate extract. Ethyl acetate concentrate was fed into vacuum stripper and dried under vacuum at above 500 mm of mercury to obtain 2.5 kg of powder of ethyl acetate extract of methanol extract of mustard extract with a purity of 19%. The process was explained in FIG 2.

Example 3

Method of preparation of enzyme treated methanol extract of spent mustard

A methanol extract of mustard cake with a purity of 15% obtained by the method shown in Example 1 was taken. 17 kg of methanol extract of mustard cake was soaked in water. Treated with 1% enzyme protease and incubated for 6 hrs at 37°C. The enzyme was deactivated by heating the slurry at 80°C for 10 minutes. Protease treated extract was concentrated in an Agitated thin film evaporator (ATFE) to form concentrated extract. Concentrate fraction was fed into vacuum stripper and dried under vacuum at above 500 mm of mercury to get powder of enzyme treated extract of methanol extract of mustard cake with a purity of 15%. The process was explained in FIG 3.

Example 4

Method of preparation of column purified extract of methanol extract of spent mustard

A methanol extract of mustard cake with a purity of 15% obtained by the method shown in Example 1 was taken. 17 kg of methanol extract of mustard cake was dissolved in water and clarified to form supernatant and residue. Supernatant was loaded on a SP700 resin column. After passing the supernatant through the column, column was initially eluted with water followed by 50% methanol. Water elute and 50% methanol elute was collected. Water fraction and 50% methanol fraction was concentrated in an Agitated thin film evaporator (ATFE) to form concentrated water and methanol extract. Concentrated fractions were fed separately into vacuum stripper and dried under vacuum at above 500 mm of mercury to get 2.5 kg powder of water extract with a purity of 19% and 2 kg powder of 50% of mustard methanol extract with a purity of 20%. The process was explained in FIG 4.

Example 5

Extraction and enrichment of sinigrin from spent mustard A methanol extract of mustard cake with a purity of 15% obtained by the method shown in Example 1 was taken. 17 Kg methanol extract of mustard cake was fermented by using 1% yeast at 40°C for 48 hrs. Fermented methanol extract solution was centrifuged at 20000 rpm for 20 min to obtain a residue and centrifugate. Centrifugate was transferred into a liquid-liquid extractor and extracted with hexane. After extraction hexane phase and aqueous phase were separated. 7.65 kg of the aqueous phase with a purity of 30% was collected through side valve. Aqueous phase was concentrated in an Agitated thin film evaporator to form concentrated aqueous phase. Concentrated aqueous phase was loaded in a column having amber 200 resin. Column was initially eluted with water and water fraction was collected. Then the column was eluted with methanol and collected the methanol fraction. Water fraction was concentrated and dried under vacuum to form 5.75 kg powder of water elute of fermented methanol cake with a purity of 40%. The process was represented in FIG 5.

Example 6

Extraction and purification of sinigrin from spent mustard

Powder of water elute of fermented methanol cake from Example 5 was dissolved in 25F water and centrifuged 5000 RPM for 10 minutes to form supernatant and residue. Supernatant was passed through cation exchange column. Before passing through column, column (Amberlite IR120H) was washed with distilled water and aqueous solution of 5% HC1, respectively, for 2 hour. After acidifying the resin, distilled water was passed through the column until pH of elute became neutral. After passing the supernatant, column was eluted with distilled water and 3% ammonium hydroxide, respectively. The water fractions and alkaline fractions were collected separately. The pH of water fraction was adjusted to neutral by using weak alkali and pH of alkaline fraction was adjusted to neutral by using weak acid. Alkaline fraction was concentrated and dried to get 0.5 kg powder of purified extract from spent mustard with purity of 80%. The process was represented in FIG 6.

Example 7

Method of extraction of sinigrin from mustard seed

100 Kg mustard seed were filled in the Soxhlet extractor and extracted with 300 F of 95% methanol. The extraction was carried out for 8 hrs at a temperature of about 80° C. After the completion of extraction, the supernatant was filtered and concentrated in an Agitated thin film evaporator (ATFE) at a temperature of 60°C to form concentrated methanol extract. Concentrated methanol extract was dried under vacuum at above 500 mm of mercury to get 1 kg of powder of methanol extract of mustard cake with a purity of 4%.

Example 8

Method of making enteric coated purified mustard extract enriched with sinigrin.

lKg of purified spent mustard extract powder with 40% sinigrin was passed through the Roll Compactor machine. The flakes obtained from the roll compactor were passed through an Oscillating Granulator machine fitted with 16 mesh screen to obtain granules of spent mustard extract.

Granules of spent mustard extract were loaded into the bowl of the fluid bed extractor (pam glatt pharma technologies). The bowl has a fine Stainless steel mesh at the bottom. The air used for drying/fluidizing was successively filtered through HEPA (High-efficiency particulate arrestance) filters (EU 13 grade, 0.3 micron rating, 99.99% efficiency).

Hot, filtered air up to 90°C was passed at high velocity from the bottom of the FBE bowl through the feed material (spent mustard extract with 40% sinigrin) and feed material was fluidised. Meantime, lOOg coating material (Poly-methacrylicacid-co-methyl methacrylate (Eudragit) was dissolved in 900 ml water. Coating solution was sprayed into fluidised material by using a spraying devise attached to the FBE (spray speed 0.5 L in 1 Hr, pump rpm range 10-12). Through the process of fluid bed coating, fluidized particles are continuously sprayed with coating solution, depositing layers (films) of material to the surface of the particles, and yielding an even layer, 20% weight gain with 6 mg/cm thickness.

Same enteric coating process was repeated for making enteric coated mustard extract with 10% weight gain of coating material to release in small intestine.

Above coating process was repeated with other mustard extracts with different percentage of sinigrin for making enteric coated mustard extract.

Example 9

Method of extraction of myrosinase from white mustard 300g of white mustard seeds were washed with distilled water and homogenized with 2L of distilled water. The non soluble material was removed by centrifugation at 17700g for 20 minutes. Supernatant formed was filtered and dialyzed against distilled water. During dialysis non active proteins precipitated and removed by centrifugation. Supernatant light yellow transparent crude extract was then dialysed against 20mM Tris-HCl (pH 7.4) containing 0.5 M NaCl. Centrifuged the dialysed extract and supernatant obtained was loaded on a Con A- Sepharose column equilibrated with buffer used for last dialysis. Column was initially washed with buffer used in the dialysis until the absorbance reached zero at 280 nm. Then column was eluted with different eluent solutions, viz. 0.25 M glucose, mannose, methyl a-D-glucoside and methyl a-D-mannoside, all in the buffer used for dialysis. Each fraction was collected. Maximum myrosinase activity was found in elution with methyl a-D-mannoside (90% recovery).

Enteric coated purified myrosinase was prepared by a process as described in example 8.

Example 10

Method of making combination of enteric coated extract of purified mustard extract and enteric coated purified myrosinase in 10:1 ratio.

Enteric coated purified mustard extract enriched with sinigrin prepared as per example 8 and enteric coated myrosinase from white mustard prepared as per example 9 are blended in 10: 1 ratio to form combination of enteric coated extract of purified mustard extract and enteric coated purified myrosinase.

The enteric coated composition enriched with glucosinolate obtained from cruciferous vegetables especially prepared by a process of extraction of mustard and enrichment with sinigrin was evaluated for its capability to increase the bioactivity and bioavailability of AITC.

Example 11

Forty six germ-free adult male rats, originating from isolated environment fed with sterilized nutrients, were orally inoculated with a 24-h culture of microbiota. The microbiota was derived from human gastrointestinal system, and was cultured in a controlled environment. Animals were given free access to a sterilized palliated diet, and to tap water, sterilized by steaming (120°C, 40 min). After 2 weeks adaptation to the diet and the flora, two rats were sacrificed and the levels of bacterial population in the stomach, small intestine, and colon were determined by anaerobically growing serial 10-fold dilutions of the contents.

The other rats were randomly allocated to eleven groups of four animals each and dosed by stomach tube under light ether anaesthesia. Each dose was flushed into the stomach with 0.5 ml distilled water. Solutions used for the gavages were prepared and filter-sterilized immediately prior to administration.

After dosing, rats were sacrificed, at 0 h for the control group and at 6, 12, and 18 hours for the treated groups. The stomach, small intestine, and colon were collected separately and their contents was transferred into a 12ml amber glass vial cooled in a water-ice bath, along with an appropriate volume of chilled phosphate buffer 100 mM, pH 7.0, to allow for a 4ml headspace volume. The vessels were tightly closed and stored at -20°C.

AITC being volatile compounds was analyzed by gas chromatography (GC), following headspace solid-phase micro-extraction.

Table 1 : Grouping of rats and treatment.

Table 2: AITC present in stomach, small intestine, and colon in different time points after administering different mustard extracts

AITC was not detected in the control animals. The maximum level of AITC was found in colon of animals treated with combination of enteric coated purified spent mustard extract (sinigrin 80%) with enteric coated purified myrosinase. In this group AITC (195 nmol/g) was maximum in colon at 12 hour time point and AITC was not detected in stomach and small intestine. The level of AITC was found in the animals treated with combination of enteric coated purified spent mustard extract (sinigrin 40%) with enteric coated purified myrosinase was 147 nmol/g in colon at 12 hour time point. The level of AITC was also high in the animals treated with enteric coated purified spent mustard extract (sinigrin 40%) and enteric coated purified mustard methanol extract (sinigrin 80%) and the values found to be 120 and 159 nmol/g in colon at 12 hour time point. There was no detection of AITC in stomach and small intestine of animals treated with enteric coated extract groups. Animals treated with spent mustard extract or mustard seed extract without coating showed small detection of AITC in stomach till 6 hrs and in small intestine till 12 hrs.

Example 12

Rabbits weighing 2 - 2.5 Kg were used for the study. Animals were divided into 7 groups and 6 animals were used for each group. The animals were divided as shown in Table 1. The dosage administered was 100 mg/Kg body weight of the rabbit.

Table 1 : Grouping of rabbits and treatment.

The study c rugs were given by oral route. Two hours post drug, blood was collected from the ear vein of each rabbit followed by 4, 6, 8, 10 and 12hrs. The blood samples were centrifuged at 3000 rpm for 15 minutes and plasma was separated. AITC was analyzed in plasma by gas chromatography and reported as ng/ml of plasma.

The AITC was not detected in control rats at any time point (Group 1). When purified mustard extract was fed to rabbits (Group 2), low level of AITC was detected up to 4 hours. In case of enteric coated purified extract to deliver in small intestine (Group 3), AITC was detected from 4 to 6 hour and maximum concentration was detected as 90 ng/ml at 6 hr. In group 4, the purified extract was coated to deliver the actives in large intestine and AITC was detected from 6 to 12 hour and 8 hour being maximum. Combination of purified mustard methanol extract with purified myrosinase produced detectable level of AITC at up to 4 hour time point (Group 5) and detection was better than Group 2. When extract as well as myrosinase was coated to deliver in small intestine, AITC was detected from 4 to 10 hours and maximum level (160 ng/ml) was detected at 6 hour (Group 6). The maximum concentration of AITC was detected as 360 ng/ml at 8 hour when enteric coated purified mustard methanol extract was fed along with enteric coated purified myrosinase (Group 7).

Example 13

Bioavailability study

Rabbits weighing 2 - 2.5 Kg were used for the study. Animals were divided into eleven groups and 4 animals were used for each group. The animals were divided as shown in Table 1. The dosage administered was 100 mg/kg body weight of the rabbit. The samples were administered to rabbits after 12 hours of fasting and food was withdrawn till the last blood sample draw. Water was given ad libitum. Blood samples were collected at 2, 4, 6, 8, 10 and 12 hour post dose and plasma was separated by centrifugation. The plasma samples were analyzed for the AITC content.

Table 1 : Segregation of rabbits for experimental study.

The study drugs were given by oral route. Two hours post drug, blood was collected from the ear vein of each rabbit followed by 4, 6, 8, 10 and 12 hrs.

AITC was not detected in control group of animals at any time point till 12 hr. Maximum concentration of AITC was found as 780 nmol/ml at 8 hr in animals treated with combination of spent mustard extract enriched with 80% sinigrin with enteric coating and enteric coated purified myrosinase. When animals fed with enteric coated spent mustard extract enriched with 80% sinigrin, maximum concentration of AITC was 420 nmol/ml at 8 hr. In group 6 animals treated with combination of enteric coated spent mustard extract enriched with 40% sinigrin and enteric coated purified myrosinase also showed an AITC content of 330 nmol/ml. In group 5 animals treated with enteric coated spent mustard extract purified (sinigrin 40%), maximum concentration of AITC was found as 160 nmol/ml at 8 hr. Animals fed with enriched extract of spent mustard with 40% and 80% sinigrin showed minimum AITC concentration at 2 and 4^th hour. The animals treated with enteric coated spent mustard extract (sinigrin 4%) and combination of enteric coated spent mustard extract (sinigrin 4%) with enteric coated purified myrosinase showed a maximum AITC concentration 95 nmol/ml and 114 nmol/ml at 8^th hr.

Example 14

Bioactivity study

Orthotopic rat bladder cancer model

The anticancer activity of sinigrin was evaluated in an orthotopic rat bladder cancer model. Twelve female F344 rats (8-10 weeks of age) were inoculated orthotopically via a urethra catheter with AY-27 cells (lxlO⁶ cells in 0.5 ml serum-free medium per rat). One day after the inoculation, the rats were randomly assigned to receive by gavage vehicle control or sinigrin that was freshly prepared in an equal volume of water, once daily for 3 weeks. The sinigrin solution was given to the animals within 30 min of preparation. The animals were monitored and weighed daily and were euthanized 24 h after the last dose, and the bladders were quickly removed and weighed. Approximately half of each bladder was fixed in formalin for histological analysis and the other half was frozen in liquid nitrogen for western blot analysis.

Western blot analysis

Cells were grown in 10 cm plates for 24 h (1.5xl0⁶ cells per plate in 10 ml medium), treated with sinigrin (dissolved in culture medium) for 24 h and then harvested for analysis. Cells after harvest were washed with ice-cold PBS and lysed in radio immunoprecipitation assay buffer supplemented with a protease inhibitor cocktail. Bladder tumor samples were thoroughly washed in ice-cold PBS, frozen with liquid nitrogen, reduced to powder with a biopulverizer and finally homogenized in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail mentioned above in glass homogenizers. Cell lysates and tissue homogenates were cleared of debris by low-speed centrifugation and measured for protein contents using the bicinchoninic acid kit (Pierce, Rockford, IL). The samples were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, which were probed by specific antibodies and visualized using SuperSignal West Pico Chemiluminescence Detection System (Thermo Scientific, Rockford, IL).

Histological analysis

Rat bladders fixed in formalin were paraffin embedded, cut to 4 pm thickness and stained with standard hematoxylin and eosin. The slides were examined for bladder and tumor histology using a light microscope. Tumor muscle invasion was assessed at high magnification.

Results

Table 1: Tumor weight (calculated by subtracting the average normal bladder weight from tumor-bearing bladder weight)

Table 2: Percentage of bladders where the tumor invaded the muscle tissue

Animals in all the groups behaved normally and there was no significant difference in body weight gain over the experimental period among the groups. Bladder tumours formed in nearly all rats. The tumours in the vehicle control group weighed 336 mg (Table 1), which is five times the normal bladder weight (67 mg), showing the explosive cancer growth rate. Moreover, tumours invaded the musculature in 70% of the tumour bearing bladders (Table 2). Treatment with mustard methanol extract purified (sinigrin 80%) at doses of 100 mg/kg body weight reduced tumour weight 230 mg only and 45% of the tumour-bearing bladders of rats showed muscle invasion. Treatment with enteric coated purified mustard methanol extract (sinigrin 80%) at doses of 100 mg/kg body weight reduced tumour weight 105 mg and 13% of the tumour bearing bladders of rats showed muscle invasion. Treatment with combination of enteric coated purified mustard methanol extract (sinigrin 80%) and enteric coated purified myrosinase at doses of 100 mg/kg body weight reduced tumour weight 76 mg which is almost near to normal bladder weight (67 mg). More interestingly, only 4% of the tumour-bearing bladders of rats showed muscle invasion, whereas the muscle invasion rate in the control group was 70%. Treatment with mustard methanol extract purified prepared as per Example 5 (sinigrin 40%) at doses of 100 mg/kg body weight reduced tumour weight 265 mg only and 50% of the tumour-bearing bladders of rats showed muscle invasion. Treatment with enteric coated purified mustard methanol extract (sinigrin 40%) at doses of 100 mg/kg body weight reduced tumour weight 155 mg and 29% of the tumour-bearing bladders of rats showed muscle invasion. Treatment with combination of enteric coated purified mustard methanol extract (sinigrin 40%) and enteric coated purified myrosinase at doses of 100 mg/kg body weight reduced tumour weight 120 mg. 19% of the tumour-bearing bladders of rats showed muscle invasion, whereas the muscle invasion rate in the control group was 70%. Treatment with mustard methanol extract (sinigrinl5%) at doses of 100 mg/kg body weight reduced tumour weight 279 mg only and 55% of the tumour-bearing bladders of rats showed muscle invasion. Mustard seed methanol extract (sinigrin 4%) was least effective among the extracts group since tumour weight was 294 mg and about 62% of the tumour-bearing bladders of rats showed muscle invasion. Whereas enteric coated mustard seed methanol extract (sinigrin 4%) and combination of enteric coated mustard seed methanol extract (sinigrin 4%) with enteric coated myrosinase at doses of 100 mg/kg body weight reduced tumour weight 210 mg and 180 mg respectively. Thus, the anticancer efficacy of combination of enteric coated mustard methanol extract purified (sinigrin 80%) with enteric coated myrosinase appears to be more robust, particularly in blocking muscle invasion.

Example 15

Carcinogen-induced hepatocarcinogenesis in rats

Male SD rats (100-120 g body weight) were housed in the animal room with a 12 hour light- dark cycle and constant temperature of 25±2°C. Food and water were given ad libitum. Sixty rats were randomly divided into 12 groups comprising of one normal group (5 rats), one untreated control group (5 rats) and 10 treatment groups containing 5 rats in each.

Rats of untreated control group (Group 2) and all the treatment groups (Group 3 to Group 12) were induced with carcinogens by a single i.p. injection of 200 mg/kg of diethylnitrosamine (DEN). All the rats were treated for 24 weeks as below:

Group 1: Normal control.

Group 2: Untreated control.

Group 3: Mustard methanol extract as per Example 1 (sinigrinl5%) 100 mg/kg p.o.

Group 4: Mustard seed methanol extract as per Example 7 (sinigrin 4%) 100 mg/kg p.o.

Group 5: Mustard methanol extract purified prepared as per Example 5 (sinigrin 40%) 100 mg/kg p.o.

Group 6: Enteric coated purified mustard methanol extract prepared as per Example 8 (sinigrin 40%) 100 mg/kg p.o.

Group 7: Combination of enteric coated purified mustard methanol extract (sinigrin 40%) with enteric coated purified myrosinase prepared as per Example 10 (100 mg/kg p.o). Group 8: Purified mustard methanol extract prepared as per Example 6 (sinigrin 80%) 100 mg/kg p.o.

Group 9: Enteric coated purified mustard methanol extract prepared as per Example 8 (sinigrin 80%) 100 mg/kg p.o.

Group 10: Combination of enteric coated purified mustard methanol extract (sinigrin 80%) with enteric coated purified myrosinase prepared as per Example 10 (100 mg/kg p.o).

Group 11: Enteric coated mustard seed methanol extract prepared as per Example 8 (sinigrin 4%) 100 mg/kg p.o.

Group 12: Combination of enteric coated mustard seed methanol extract (sinigrin 4%) with enteric coated purified myrosinase prepared as per Example 10 (100 mg/kg p.o).

During the experiment, the body weight of each rat was monitored daily. At the end of the experiment, all rats were killed by carbon dioxide asphyxiation and the blood was collected. Serum was separated by centrifugation and analyzed for AST (SGOT) and ALT (SGPT) activities. The rat liver was perfused with ice old PBS and quickly removed, washed and weighed. The rat livers were histologically analyzed. The liver slices were fixed with 10% formaldehyde solution for 24 hours and stored in 75% ethanol for histological analysis.

Table 1 : Body weight of rats

Increase in body weight over time got arrested due to the injection of carcinogen (diethylnitrosamine). Untreated control rats suffered maximum and body weight was increased to 140 gm from 108 gm in 24 weeks. The animals treated with combination of enteric coated purified mustard methanol extract (sinigrin 80%) with enteric coated myrosinase reported a very good increase in body weight among the extract treated groups. In this group the body weight was increased from 109 to 265 gm in 24 weeks. The animals treated with enteric coated purified mustard methanol extract (sinigrin 80%) shows increase in body weight from 109 to 250 gm in 24 weeks. In animals treated with enteric coated purified spent mustard extract (sinigrin 40%) shows increase in body weight from 107 to 222 gm in 24 weeks.The animals treated with combination of enteric coated purified spent mustard extract (sinigrin 40%) with enteric coated myrosinase shows increase in body weight from 109 to 235 gm in 24 weeks. Animals treated with purified spent mustard extract (sinigrin 80%) reported increase in body weight from 109 to 182 gm. Animals treated with purified spent mustard extract (sinigrin 40%) reported increase in body weight from 110 to 164 gm. Animals treated with spent mustard extract (sinigrin 15%) was also reported better body weight (108 to 155 gm) than untreated control. Animals treated with mustard seed extract (sinigrin 4%) reported body weight increase from 109 to 145 only which was the least among extract treated groups.

Table 2: Effects of sinigrin treatment on liver weight/body weight ratio in different treatment groups

The liver/body weight ratio was maximum (6.7%) in untreated control rats. The animals treated with combination of enteric coated purified spent mustard extract (sinigrin 80%) with enteric coated myrosinase reported a very good decrease in the liver weight/body weight ratio and reported as 3%. Animals treated with combination of enteric coated purified spent mustard extract (sinigrin 40%) with enteric coated myrosinase showed a decrease in the ratio of 3.7%. Animals treated with enteric coated purified spent mustard extract with sinigrin 40% and 80% showed a decrease in liver weight/body weight ratio 4.1% and 3.4% respectively. Animals treated with spent mustard extract (sinigrin 15%) were reported a ratio of liver weight/body weight and it was 5.7%. Animals treated with mustard seed methanol extract (sinigrin 4%) reported the ratio 6.3% showing that it was least active among extract treated groups.

Table 3: Effects of sinigrin treatment on the number of surface tumors in the rat.

The surface tumors were maximum in untreated control group and found as 4. The animals treated with combination of enteric coated purified spent mustard extract (sinigrin 80%) with enteric coated myrosinase reported only 0.2 surface tumors showing that it is very effective as anticancer. The animals treated with combination of enteric coated purified spent mustard extract (sinigrin 40%) with enteric coated myrosinase reported only 1.2 surface tumors. Animals treated with enteric coated purified spent mustard extract with sinigrin 40% and 80% was reported 1.6 and 0.7 surface tumors. Whereas animals treated with purified spent mustard extract with 40% and 80% sinigrin without enteric coating showed high number of surface tumors (2.9 and 2.6 respectively) compared to same extract with enteric coating. Number of surface tumors in animals treated with spent mustard extract (sinigrin 15%) and animals treated with mustard seed extract (sinigrin 4%) were found as 3.3 and 3.6 respectively. Number of surface tumors in animals treated with enteric coated mustard seed methanol extract (sinigrin 4%) and animals treated with combination of enteric coated mustard seed methanol extract (sinigrin 4%) with enteric coated myrosinase were found as 2.3 and 1.9 respectively.

Table 4: Effects of sinigrin treatment on AST and ALT enzyme activities

Injection of carcinogen produced severe liver toxicity as evidenced by AST (262.6 U/L) and ALT (140.2 U/L) levels of untreated control group animals. Treatment with all the extracts was effective in reducing these liver enzymes levels and the most effective extract was combination of enteric coated spent mustard extract purified (sinigrin 80%) with enteric coated purified myrosinase since the rats of this group were having almost normal AST (65.2 U/L) and ALT(27.2 U/L) levels compared to normal control. Animals treated with combination of enteric coated spent mustard extract purified (sinigrin 40%) with enteric coated purified myrosinase shows almost normal AST (98.4 U/L) and ALT (46.6 U/L) levels compared to normal control. Enteric coated spent mustard extract with 40% and 80% sinigrin also showed better AST and ALT levels compared to purified spent mustard extract without coating.

Example 16

Antimicrobial Activity Seven bacterial strains Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Clostridium difficile, Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhi were used in the study. All cultures were maintained at -80 °C in brain heart infusion (BHI) broth with 30% glycerol until experimental use. Before in vitro assay, the isolates were cultured for 18 h at 30 °C in BHI broth. Cultures were then diluted in fresh BHI broth and adjusted to a final concentration of 105 CFU/mL.

Test Samples

Sample 1: spent mustard extract as per Example 1 (sinigrin 15%), 500 pg/disc

Sample 2: mustard seed extract as per Example 7(sinigrin 4%), 500 pg/disc

Sample 3: spent mustard extract purified prepared as per Example 5 (sinigrin 40%), 500 pg/disc

Sample 4: spent mustard extract purified prepared as per Example 6 (sinigrin 80%), 500 pg/disc Sample 5: Vancomycin (standard), 30 pg/disc

The samples were prepared in 10% dimethyl sulfoxide (DMSO) and diluted to required concentration before use.

Antimicrobial Activity by Disc diffusion method

The test samples were screened against seven bacterial strains by in vitro disc diffusion method. Isolated colonies from pure strains were transferred from the BHI solid medium grown overnight and inoculated into 4.0 mL of 0.9% NaCl solution. From these stocks, suspensions were prepared by adjusting the turbidity to 0.5 McFarland standard units. A loop of bacteria from suspension was spread with a sterile cotton swab into Petri dishes (90 mm of diameter) containing 20 mL of Mueller-Hinton Agar (Oxoid, Basingstoke-Hampshire, UK) and a sterile filter paper discs (6 mm in diameter) (Oxoid, Basingstoke-Hampshire, UK) impregnated with 15 pL of sample were then placed on the agar plate. Then the plates were incubated overnight at 37°C. A negative control (15 pL of solvent, DMSO) and positive control (commercial antibiotic of vancomycin in discs (30 pg) were used. At the end of the incubation, the diameter of inhibition zones were measured on the plates with a ruler and recorded in mm. All tests were performed in triplicate and the antibacterial activity was expressed as the mean of inhibition zone diameters.

Table lA.Inhibitory diameter zone in mm for extracts against different bacterial strains.

Table lB.Inhibitory diameter zone in mm for extracts against different bacterial strains

The inhibition of different bacteria was directly proportional to the percentage of sinigrin present in the extract. The maximum inhibition was noted with purified mustard methanol extract containing 80% sinigrin against all the seven bacteria tested. The extract containing 40% sinigrin was slightly less effective than extract containing 80% sinigrin. The extract containing sinigrin 15% and 4% were also effective but less than the high purity extracts. The standard vancomycin was also effective against all the microorganisms.

Antimicrobial Activity Minimum Inhibitory Concentration (MIC)

For the MIC assay a modified resazurinmicrotitre-plate assay was used. Briefly, 100 pL of each test sample (500 pg/well) and standard antibiotic (Vancomycin, 30 pg/well) in 10% DMSO solution was pipetted into the first row of the 96 well microplates. To all other wells, 100 pL of nutrient broth was added. Two fold serial dilutions were performed using a multichannel pipette such that each well had 100 pL of the test material in serially descending concentrations. Twenty microliters of bacterial suspension was added to each well to achieve a concentration of 5 x 10⁵cfu/mL. Finally, 20 pL of resazurin indicator solution (270 mg of pure resazurin in 40 mL of sterile bi-distilled water) were added to each well. At the end, the microplate was covered with cling film to avoid dehydration of bacteria. Each microplate had a set of controls: a column with VA as positive control; a column with DMSO solution as a negative control; a column with all components except bacterial suspension (contamination control) and column with bacteria suspension (bacteria growth control). The plates were prepared in triplicate and incubated at 37 °C for 24 h. The color change was assessed visually. The bacterial growth was indicated by color changes from purple to pink (or colorless) and the lowest concentration at which color change occurred was considered MIC value. Each assay was performed in three replicates and the mean value was recorded.

Table 2A.Minimum Inhibitory concentration (MIC) (pg/mL) of test samples against different bacterial strains

Table 2B.Minimum Inhibitory concentration (MIC) (pg/mL) of test samples against different bacterial strains

The MIC against different bacterial strains was directly proportional to the percentage of sinigrin present in the extract. The low MIC value was noted with mustard methanol extract containing 80% sinigrin against all the seven bacteria tested. The extract containing 40% sinigrin was slightly less effective than extract containing 80% sinigrin. The extract containing sinigrin 15 and 4% were also effective but less than the high purity extracts (MIC was high). The standard vancomycin was also effective against all the microorganisms.

Claims

m:

1. An enteric release system for releasing glucosinolate in the colon to increase bioavailability of allyl-isothiocyanate in the body, when administered to a human subject, comprises of:

a) a solid core made of glucosinolate obtained from cruciferous vegetables, and b) an enteric protective coating over said core.

2. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein the cruciferous vegetable is mustard, from which glucosinolate is derived.

3. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein glucosinolate is derived from a mustard extract prepared from mustard seed, mustard cake or mustard bran or a combination thereof.

4. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein the glucosinolate derived from mustard extract contains 20% to 90% sinigrin.

5. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein the glucosinolate derived from mustard extract contains 20% to 80% sinigrin.

6. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein the glucosinolate derived from mustard extract contains 20% to 60% sinigrin.

7. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein the glucosinolate derived from mustard extract contains 20% to 40% sinigrin.

8. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein the protective coating composition is prepared from coating materials selected from ;

Methacrylic acid co-polymers,

poly (methacrylic acid-co-methyl methacrylate),

esters of aleurtic acid,

cellulose acetate phthalate,

cellulose acetate trimellitate,

poly (vinyl acetate phthalate),

hydroxyl propyl methyl cellulose,

hydroxypropyl methylcellulose phthalate,

hydroxy propyl methyl cellulose acetate succinate, acetaldehyde dimethyl cellulose acetate,

chitosan,

ethyl cellulose,

sodium alginate, zein, fatty acids, waxes, shellac, plastics, plant fibers, and a combination there of.

9. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein the weight gain after coating is greater than 20 percent.

10. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein the protective coating composition consisting of a combination of ethyl cellulose and sodium alginate.

11. The enteric release system for releasing glucosinolate as claimed in claim 1 , wherein the delivery systems includes excipients selected from the group consisting of a disintegrates, diluents, binders, fillers, a carrier, adsorbents, emulsifiers, lubricants, stabilizing agents, antiadherents, glidants, antioxidants and mixtures thereof.

12. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein the dosage form is selected from hard gel capsule, soft gel capsule, tablet, beadlets, mini tablet, granule, powder and pills and dosage is in the range of 200 mg to about 2000 mg to a human subject.

13. The dosage form as claimed in claim 12, wherein beadlet form is a sugar bead coated with a glucosinolate composition.

14. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein bioavailability is increased by 2 to 20 fold.

15. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein bioavailability is increased by 5 to 14 fold.

16. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein solid core contain thioglucosidase enzyme myrosinase in addition to glucosinolate.

17. The enteric release system for releasing glucosinolate as claimed in claim 16, wherein thioglucosidase enzyme myrosinase in the enteric release system is provided with enteric coating.

18. The enteric release system for releasing glucosinolate as claimed in claim 16, wherein bioavailability is further increased by combining enteric release system with enteric coated myrosinase in a ratio 1 : 100 to 100: 1.

19. The enteric release system for releasing glucosinolate as claimed in claim 16, wherein the enteric release system is combined with enteric coated myrosinase in the ratio of 10: 1.

20. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein glucosinolates are blended with other plant derivatives selected from the group comprising turmeric extract, emblica extract, costus extract, ashwagandha extract, green tea extract, amaranthus, ginger extract, pepper extract, rosemary extract, garlic extract or combination thereof.

21. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein, glucosinolates are blended with Vitamin A, Vitamin C, Vitamin D, Nitrate, Nitrite, Omega-3, essential oils, minerals or combination thereof.

22. The enteric release system for releasing glucosinolate as claimed in claim 1, wherein, enteric release system has anticancer activity, anti -microbial activity and anti inflammatory activity.

23. A method of making an enteric release system for releasing glucosinolate comprising the steps of :

a) preparing the flakes of sinigrin rich plant extract powder obtained from the

cruciferous vegetables in powder form by passing through a roll compactor;

b) granulating the resultant flakes obtained from the roll compactor by passing through an Oscillating Granulator machine to obtain granules of glucosinolate; c) loading resultant granules into the bowl of the fluid bed extractor (FBE);

d) passing filtered air heated up to 90°C at high velocity from the bottom of the bowl of the fluid bed extractor through the glucosinolate granules; and

e) continuously spraying the enteric coating material solution prepared by dissolving the selected enteric coating material in a suitable solvent into fluidized material by using a spraying devise attached to the FBE, so as to deposit layers (films) of material to the surface of the particles and yielding an even layer thickness.

24. The method of making an enteric release system for releasing glucosinolate as claimed in claim 23, wherein the cruciferous vegetables used for obtaining sinigrin rich extract is mustard.

25. The method of making an enteric release system for releasing glucosinolate as claimed in claim 23, wherein glucosinolate is derived from a mustard extract prepared from mustard seed, mustard cake or mustard bran or a combination thereof.

26. The method of making an enteric release system for releasing glucosinolate as claimed in claim 23, wherein the protective coating composition is prepared from:

Methacrylic acid co-polymers,

poly (methacrylic acid-co-methyl methacrylate),

esters of aleurtic acid,

cellulose acetate phthalate,

cellulose acetate trimellitate,

poly (vinyl acetate phthalate),

hydroxyl propyl methyl cellulose,

hydroxypropyl methylcellulose phthalate,

hydroxy propyl methyl cellulose acetate succinate,

acetaldehyde dimethyl cellulose acetate,

chitosan,

ethyl cellulose,

27. The method of making an enteric release system for releasing glucosinolate as claimed in claim 23, wherein sinigrin rich plant extract from mustard is obtained by a process comprising the steps of:

a) adding water to methanol extract of mustard and fermenting the solution;

b) centrifuging the fermented solution to obtain residue and supernatant;

c) washing the supernatant with Hexane in a liquid-liquid extractor to obtain an aqueous phase; and

d) purifying the aqueous phase by loading into a column and eluting with water to obtain the enriched sinigrin extract.

28. The method of making as claimed in claim 27, wherein the methanol extract is from spent mustard cake.

29. The method of making an enteric release system for releasing glucosinolate as claimed in claim 23, wherein the sinigrin rich spent mustard extract with 20% to 80% purity of sinigrin is prepared by a process comprising the steps of:

a) adding water to methanol extract of mustard and fermenting the solution;

b) centrifuging the fermented solution to obtain residue and supernatant,

c) washing the supernatant with Hexane in a liquid-liquid extractor to obtain a aqueous phase, and

d) purifying the aqueous phase by loading into a column and eluting with water to obtain the water fraction

e) concentrating and drying the resultant water fraction to form water elute of

fermented methanol extract,

f) dissolving the water elute of fermented methanol extract in water and centrifuging to form supernatant,

g) passing through cation exchange column,

h) eluting with water and 3% ammonium hydroxide, and

i) concentrating 3% ammonium hydroxide fraction and drying to get powder of purified extract from spent mustard.

30. The process of claim 29, wherein the solution is fermented with yeast for two days.

31. A pharmaceutical composition comprises a mixture of mustard extract with a

pharmaceutically acceptable carrier and excipient, wherein said composition has 25% glucosinolate.

32. The composition of claim 31, wherein the mustard extract is devoid of AITC.

33. The composition of claim 31, wherein the mustard extract is derived from mustard seed, mustard cake or mustard bran or a combination thereof.

34. The composition of claim 31 , wherein the pharmaceutically acceptable carrier is

maltodextrin and excipient is aerosil.

35. The composition of claim 31, wherein spent mustard extract contains 20% to 90% sinigrin.