Patent Description:
The oral route of administration is typically considered the preferred and most patient-convenient means of drug delivery. With many drugs the basic goal of therapy is to achieve a steady-state blood or tissue level that is therapeutically effective and non-toxic for an extended period of time. Sustained release dosage form is an ideal strategy for the drugs with short half-lives and which require repeated dosing. These dosage forms are designed to release a drug at a predetermined rate in order to maintain a constant drug concentration for a specific period of time with minimum side effects.

Crocus sativus L. (Family: Iridaceae), commonly known as saffron or Kesar, is used in Ayurveda and other folk medicines for various purposes, such as an aphrodisiac, antispasmodic and for expectorant effects (<NPL>). Modern pharmacological studies have demonstrated that saffron extracts have anti-nociceptive, antiinflammatory (<NPL>), antitumor (<NPL>), radical scavenger (<NPL>), anticonvulsant (<NPL>), anti-ischemic (<NPL>) and anti-Alzheimer (<NPL>; <NPL>; <NPL>; <NPL>; <NPL>) effects. Recently we have shown that, in in-vivo studies Crocus sativus extract (<NUM>/kg/day, added to mice diet) improves the BBB tightness and function that was associated with reduced Aβ load and related pathological changes in 5XFAD mice. Furthermore, Crocus sativus extract upregulated synaptic proteins and reduced neuro-inflammation associated with Aβ pathology in the brains of 5XFAD mice (<NPL>).

Saffron contains more than <NUM> volatile and aroma-yielding compounds along with carotenoids (including zeaxanthin, lycopene, and various α-and β-carotenes). However, saffron's golden yellow-orange color is primarily the result of α-crocin (also called as crocin-<NUM>). It is the diester formed from the disaccharide gentiobiose and the dicarboxylic acid crocetin. When crocetin is esterified with two water-soluble gentiobioses, a water-soluble pigment (known as "crocin") is obtained. Crocins are esters formed from the conjugation of various sugars (glucose, gentiobiose, triglucose and neapolitanoside) with dicarboxylic acid of crocetin. Furthermore, geometrically crocetin exists in all-trans and <NUM>-cis forms. Thus, total of <NUM> crocins are reported, all differing via a type of sugar moiety attached and all-trans or <NUM>-cis geometry (<NPL>; <NPL>; <NPL>; <NPL>). The chemical structures of crocins are shown in <FIG>. Among various constituents of saffron, crocin and crocetin are mainly responsible for pharmacological activities. The pharmacokinetic analysis of crocin indicated that it reaches the blood circulation in the hydrolyzed form i.e. as a crocetin. The plasma half-life of crocetin is low (~ <NUM> hrs), and it gets eliminated quickly from the blood (<NPL>; <NPL>). Therefore, development of novel formulations for this botanical drug is essential, which can result in delayed or controlled release of crocin from the formulation in the gastrointestinal tract/ intestine. Furthermore, with the great number of therapeutic activities of Saffron extracts, there is a great need to have rigorously standardized extracts of C. sativus based on the presence of specific markers, and with a good quality control.

The present invention describes the preparation of standardized botanical extract of C. sativus stigma, preparation of crocin-<NUM> enriched fraction, and their novel sustained release formulations as defined by the claims, for delayed or modified release of crocin-<NUM> (an active ingredient) and inhibitory activity against NLRP3 inflammasome. The preclinical characterization of standardized botanical extract of C. sativus stigma has been published (<NPL>).

Inflammasomes are high molecular weight complexes that sense and react to injury and infection. The inflammasome is responsible for activation of inflammatory processes (<NPL>), and has been shown to induce cell pyroptosis, a process of programmed cell death distinct from apoptosis. Their activation induces caspase-<NUM> activation and release of interleukin-1β, a pro-inflammatory cytokine involved in both acute and chronic inflammatory responses. There is increasing evidence that inflammasomes, particularly the NLRP3 inflammasome, act as guardians against noninfectious material. Inappropriate activation of the NLRP3 inflammasome contributes to the progression of many non-communicable diseases such as gout (<NPL>), type-II diabetes (<NPL>), rheumatoid arthritis (<NPL>), chronic obstructive pulmonary diseases (<NPL>), kidney diseases (<NPL>), myocardial ischemia (<NPL>), cancer (<NPL>) and Alzheimer's disease (<NPL>). Therefore, inhibiting the NLRP3 inflammasome may significantly reduce damaging inflammation and is therefore regarded as a therapeutic target for these chronic inflammatory diseases (<NPL>).

Normally, amyloid-beta load in the brain is low due to clearance by the BBB transporter pumps like P-gp and LRP-<NUM> but with the aging, this clearance mechanism is compromised, consequently fine balance between amyloid-beta production and its clearance from brain get disturbed which ultimately leads to accumulation of large amount of amyloid-beta in brain resulting in neurotoxicity (<NPL>; <NPL>). As the amyloid-beta load in brain is cleared either by metabolism or efflux from BBB transporter pumps like P-gp and low-density lipoprotein receptor-related protein <NUM> (LRP1) (<NPL>); therefore detoxication of amyloid-beta by increasing the P-gp and LRP1 mediated amyloid-beta efflux function could be a novel way of protecting brain from amyloid-beta toxicity.

The objective of this invention to provide novel sustained release formulations of standardized extract of Crocus sativus, as defined by the claims.

It is also an objective of this invention to provide novel sustained release formulations containing elevated concentrations of at least <NUM> specific markers obtained by means of the process, as defined by the claims.

It is another objective of this invention to provide the use and method of application of this formulation for treatment of chronic inflammatory diseases wherein NLRP3 inflammasome is involved.

The present invention seeks to provide novel and improved sustained release oral formulations of Crocus sativus as defined by the claims, for supplying optimum plasma concentrations of the biologically active compounds contained in the plant such as "crocins". There is thus provided in accordance with a preferred embodiment of the invention an orally-administrable formulation for the controlled release of "crocin-<NUM>".

In one preferred embodiment of the invention, the orally-administrable formulation for the controlled release of active ingredient 'crocin-<NUM>" comprises granulated hydroalcoholic extract or crocin-enriched fraction and at least one carrier, as defined by the claims. adjuvant or excipient thereof, and is characterized in that the total in vitro dissolution time of the formulation required for release of <NUM>% of the active ingredient available from the formulation, is between about <NUM> and about <NUM> hours, as determined by the U. basket method at a speed of <NUM> rpm, and temperature of <NUM> ± <NUM>, using <NUM> of dissolution media.

The formulation contains weight ratio of hydroalcoholic extract/fraction of Crocus sativus: polymer(s) is in the range of <NUM>:<NUM> to <NUM>:<NUM>.

In another aspect, but not claimed, the formulation is characterized in that it contains from <NUM> to <NUM>% w/w hydroalcoholic extract or crocin-<NUM> enriched fraction.

In another preferred embodiment of the invention, the formulation is in the form selected from the group consisting of a matrix tablet, which is disclosed but not claimed, or a hard gelatin two-piece capsule filled with polymeric granules or microparticles of granulated extract, as defined by the claims.

In another preferred embodiment of the invention, the formulation comprises granulated extracts mixed or coated with an excipients or polymers selected from the group consisting of hydroxypropyl methylcellulose K4M (HPMC-K4M), hydroxypropyl methylcellulose K15M (HPMC-K15M), ethyl cellulose <NUM>-<NUM> cps, hydroxy propyl methyl cellulose phthalate (HPMCP), hydroxy propyl methyl phthalate cellulose acetate succinate (HPMPCAS), cellulose acetate phthalate (CAP), eudragit S100, eudragit L100, eudragit RS <NUM>, eudragitRL <NUM>, polyethylene oxide, xanthan gum, chitosan, gelatin, sodium alginate, magnesium stearate, silicon dioxide, dicalcium phosphate, microcrystalline cellulose, lactose, starch and talc.

In another preferred embodiment of the invention, the binder is selected from group consisting of polyvinylpyrrolidone K30 and polyvinylpyrrolidone K15 and binder solution is prepared by <NUM>-<NUM>% w/v in isopropyl alcohol, methanol, ethanol or propanol.

In another preferred embodiment of the invention, said extract contains atleast <NUM>% w/w of active ingredient trans-crocetin-di-(β-D-gentiobiosyl)ester (crocin-<NUM>).

In another preferred embodiment of the invention, said fraction contains atleast <NUM>% w/w of active ingredient trans-crocetin-di-(β-D-gentiobiosyl)ester (crocin-<NUM>).

The invention also comprises a process for the preparation of an orally-administrable formulation for the controlled release of a granulated extract. The steps for preparation of said formulation comprising granulated extract and at least one carrier, comprises:.

In another aspect of the present invention, said formulation comprises extract of Crocus sativus in an amount of <NUM>-<NUM>% by weight of the formulation and atleast <NUM>%of active ingredient trans-crocetin di-(β-D-gentiobiosyl)ester (crocin-<NUM>).

In another aspect of the present invention, in the comparative pharmacokinetic study conducted in SD rats, the sustained release formulation (IIIM-<NUM>-SR) displayed <NUM>-fold enhancement in the AUC of the crocetin (a bioactive metabolite, formed via enzymatic hydrolysis in the plasma/ GIT) in comparison to the plain extract.

In another aspect of the present invention, the ratio of crocin: crocetin in the plasma of rats was enhanced from <NUM>: <NUM> to <NUM>: <NUM> in SR formulation (IIIM-<NUM>-SR).

In another aspect of the present invention, the prolonged release of the bioactive constituent (a hydrolyzed metabolite crocetin) was observed up to <NUM> hrs in the rat plasma in SR formulation (IIIM-<NUM>-SR) in comparison to the plain extract.

In another aspect of the present invention, a method for enrichment of one of the active constituent is provided.

In one more embodiment of the invention, the standardization of the C. sativus extract is provided to identify and quantify amount of specified marker in the standardized extract of C. sativus by HPLC.

In another embodiment of the invention, standardized extract of C. sativus (IIIM-<NUM>-A002) displayed significant inhibition of NLRP3 inflammasome in human monocytic THP-<NUM> cells.

In one particular aspect of the present invention, an extract of C. sativus is provided, which comprises active components for NLRP3 inhibition, and related manifestations and disorders with a pharmaceutically acceptable carrier, and methods of using the same. Accordingly, the present invention is directed generally to the sustained release formulations of standardized extracts or crocin-<NUM> enriched fraction of C. sativus for treatment of chronic inflammatory diseases wherein NLRP3 inflammasome is involved.

In another aspect of the invention, chronic inflammatory diseases comprises gout, type II diabetes, rheumatoid arthritis, chronic obstructive pulmonary diseases, kidney diseases, myocardial ischemia, cancer and Alzheimer's disease.

In one more embodiment of the invention, standardized extract of C. sativus display significant increase in P-gp efflux function (as determined by Rh123) and increase in P-gp protein expression in P-gp endogenously expressing adenocarcinoma cells (LS-<NUM> cells) in-vitro.

AUC, area under the curve; aCSF, artificial cerebrospinal fluid; CAP, cellulose acetate phthalate; HBSS, Hank's buffered salt solution; HPMCP, hydroxy propyl methyl cellulose phthalate; HPMC-K15M, hydroxypropyl methyl cellulose-K15M; LS-<NUM> is a colon adenocarcinoma cell line; LRP1, low-density lipoprotein receptor-related protein <NUM>; NLRP3, NOD-like receptor (NLR) subfamily; PVP-K30, polyvinylpyrrolidone K <NUM>; P-gp, p-glycoprotein; Rh123, rhodamine <NUM>; SAM, Swiss Albino mice; STZ, streptozotocin; THP-<NUM> is a human monocytic cell line; TBST is mixture of tris-buffered saline (TBS) and Tween <NUM>; SD rat, Sprague Dawley rat.

The present invention provides novel sustained release formulations of hydroalcoholic extract and crocin-<NUM> enriched fraction of Crocus sativus stigma, as defined by the claims, for treatment of chronic inflammatory diseases involving NLRP3 inflammasome. More specifically, this invention is further directed to methods of preparation of hydroalcoholic extract or active ingredient enriched fraction from the dried stigmas of Crocus sativus using ethanol and water in ratio of <NUM>:<NUM>. The hydroalcoholic extract (IIIM-<NUM>-A002) and crocin-enriched fraction (IIIM-<NUM>-CEF) are standardized for the content of major active constituent "crocin-<NUM>". The hydroalcoholic extract and crocin-enriched fraction contain <NUM> ± <NUM> % and <NUM> ± <NUM> % of crocin-<NUM>, as determined by HPLC analysis.

Particularly, this invention provides the method for preparation of novel sustained release formulations wherein the extract is wet-granulated using excipients, biodegradable polymers and/ or non-biodegradable polymers alone or in combination, as defined by the claims, and the said granules are either filled into a capsule or compressed into a tablet. The said formulation comprising a granulated extract of Crocus sativus with polymers results in sustained release of the extract in the gastrointestinal tract.

Moreover, this invention provides formulations wherein the total in-vitro dissolution time of said formulations required for release of <NUM>-<NUM>% of the active ingredient "crocin-<NUM>" is between <NUM> to <NUM> hours, as determined by the U. dissolution apparatus by basket method at a speed of <NUM> rpm, and temperature of <NUM> ± <NUM>, using <NUM> of dissolution media. The sustained release of the bioactive constituent was observed in the rat pharmacokinetic study; which validated the in-vitro dissolution results. After oral administration of crocin-enriched fraction (IIIM-<NUM>-CEF) as well as IIIM-<NUM>-SR formulation in rats, it was observed that crocin, the major constituent of Crocus sativus extract, gets metabolized to "crocetin", which is the bioactive metabolite. The crocin-enriched fraction (IIIM-<NUM>-CEF) and its SR formulation, when administered orally at equivalent dose (a dose equivalent to <NUM>/kg of crocin), the significantly higher AUC for "crocetin" was observed in case of SR formulation in comparison to the plain extract. This result indicated that SR formulation controls the release of extract, leading to the release of bioactive constituent for prolonged time. As an overall effect of this, the higher amount of crocetin is available in the blood circulation, which ultimately results in improved therapeutic effect.

The significant inhibition of NLRP3 inflammasome in human monocytic THP-<NUM> cells by IIIM-<NUM>-A002 is depicted in <FIG>. The nigericin induced release of IL-1beta was significantly suppressed by hydroalcoholic extract (IIIM-<NUM>-A002) at the test concentration of <NUM>µg/ml. The effect was further enhanced in a concentration dependent manner from <NUM> to <NUM>µg/ml.

The spectrums of conditions for which the inventive extract can be used for inflammatory conditions where NLRP3 inflammasome is involved, includes, but not limited to gout, type II diabetes, rheumatoid arthritis, chronic obstructive pulmonary diseases, kidney diseases, myocardial ischemia, cancer, Alzheimer's disease.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Following examples are given by way of illustration and should not construe to limit the scope of invention.

EXAMPLE <NUM>. Preparation of hydroalcoholic extract (IIIM-<NUM>-A002) and crocin-enriched fraction (IIIM-<NUM>-CEF). The authentic plant material of Crocus sativus (stigma) was purchased from local market of Srinagar (Jammu and Kashmir State, India). The plant material was taxonomically characterized, and a voucher specimen was deposited in the Janaki Ammal Herbarium at the CSIR-IIIM, Jammu. Driedmaterial was extracted with water: ethanol (<NUM>:<NUM>) mixture and then freeze dried.

The active ingredient [trans-crocetin di-(β-D-gentiobiosyl) ester] (crocin-<NUM>) enriched extract was prepared using following procedure:.

The hydroalcoholic extract is primarily a mixture of crocins, which are crocetin glycosides. The HPLC/ LCMS analysis indicated that amongst the total <NUM> crocetin-esters (crocins) reported (chemical structures are shown in <FIG>), trans-<NUM>-GG-crocin is the major crocetin-ester. The HPLC chromatogram (<FIG>) showed presence of primarily six crocins including trans-<NUM>-ng-crocin (tR = <NUM>), trans-<NUM>-GG-crocin (tR = <NUM>)trans-<NUM>-Gg-crocin (tR = <NUM>), trans-<NUM>-gg-crocin (tR = <NUM>), cis-<NUM>-GG-crocin (tR = <NUM>) and cis-<NUM>-Gg-crocin (tR = <NUM>). The trans-<NUM>-GG-crocin being the major crocin, it was isolated and characterized. The % content of trans-<NUM>-GG-crocin in hydroalcoholic extract (IIIM-<NUM>-A002) and crocin-<NUM> enriched fraction (IIIM-<NUM>-CEF) was determined using HPLC analysis.

HPLC analysis was performed on the Shimadzu HPLC system connected to a PDA detector, and C8 (Intersil, <NUM> x <NUM>, <NUM>µ) column. Mobile phase consisted of acetonitrile (A) and <NUM>% formic acid in water (B). The gradient system comprised of <NUM>% B (<NUM>), <NUM>% B (<NUM>), <NUM>% B (<NUM>), <NUM>% B (<NUM>), <NUM>% B (<NUM>), <NUM>% B (<NUM>) at a flow rate of <NUM>/min.

The hydroalcoholic extract was found to contain <NUM> ± <NUM> (average of six different experiments) of trans-<NUM>-GG-crocin. Thus, the % content of trans-<NUM>-GG-crocin in Crocus sativus (stigma) dried material was found to be <NUM>%. The trans-<NUM>-GG-crocin enriched fraction (IIIM-<NUM>-CEF) was found to contain <NUM> ± <NUM> % of trans-<NUM>-GG-crocin. The chemical structures of all crocins are provided in <FIG>.

EXAMPLE <NUM>. Preparation of novel sustained release formulations. The hydroalcoholic extract (IIIM-<NUM>-A002) or crocin-<NUM> enriched fraction (IIIM-<NUM>-CEF) and excipient(s) were weighed accurately and mixed thoroughly using mortor and pestle. This mixture was kneeded using <NUM>% PVP-K30 solution in isopropanol (as a binder) to form a dough. This dough was then passed through sieve #<NUM>. The obtained granules were dried in vacuum dessicator at room temperature. The formulations were assayed for crocin-<NUM> content using HPLC method as mentioned in example <NUM>. Based on the results of assay, formulation equivalent to <NUM> of crocin-<NUM> was filled into the hard gelatin capsules of size '<NUM>'. These capsules were analyzed for in-vitro dissolution profile.

Some of the composition of sustained release formulations are provided in Table <NUM>.

EXAMPLE <NUM>. Dissolution profile of sustained release formulations. The dissolution profile of plain hydroalcoholic extractor crocin-<NUM> enriched fraction and their sustained release formulations was studied using USP dissolution apparatus as per the protocol given in USP <NUM> (<NPL>). Lab-India Dissolution Tester (Model: DS <NUM>; apparatus <NUM> - Basket Apparatus) was used for this study. Various parameters are: RPM = <NUM>; Temp. = <NUM> ± <NUM>; Volume of dissolution medium = <NUM>; Dissolution medium = (A) Hydrochloric acid buffer (pH <NUM>) for first <NUM>, (B) Phosphate buffer pH <NUM>; Sampling time points (h) = <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

The percent release of trans-crocetin di-(β-D-gentiobiosyl) ester ("crocin-<NUM>") from developed formulations was determined by HPLC analysis (<FIG>). Results showed that crocin-<NUM> gets <NUM>% released in <NUM> in case of capsules filled with plain hydroalcoholic extract (CSHA-<NUM>) as well as in crocin-<NUM> enriched fraction (CSE-<NUM>). However, capsules filled with novel sustained release formulations CSHA-<NUM>, CSHA-<NUM>, CSHA-<NUM>, CSE-<NUM>, CSE-<NUM>, CSE-<NUM> and CSE-<NUM> resulted in a delayed release of crocin-<NUM> with <NUM>-<NUM>% release up to <NUM> hrs.

The dissolution profiles of formulations of hydroalcoholic extract of Crocus sativus are shown in <FIG>. From these formulations, the best formulation CSHA-<NUM> was studied for dissolution profile in triplicate, and the release profile is shown in <FIG>. Thus, the CSHA-<NUM> was considered as the optimum batch with sustained release profile over a period of <NUM>. Similarly, amongst various combination of polymer to IIIM-<NUM>-CEF attempted, the best four formulations CSE-<NUM>, CSE-<NUM>, CSE-<NUM> and CSE-<NUM> resulted in a delayed release of crocin-<NUM> with <NUM>-<NUM>% release up to <NUM> hrs (<FIG>). The % release of crocin-<NUM> after <NUM> and <NUM> hrs. is tabulated in Table <NUM>.

The summarized overview of best formulations with their T<NUM>%, T<NUM>% and T<NUM>% values are shown in Table <NUM>. The release half-life (T<NUM>%) for crocin-<NUM> in plain extract and fraction is < <NUM> hr, whereas it is <NUM> and <NUM> hrs. in novel sustained release formulations CSHA-<NUM> and CSE-<NUM>. The T<NUM>% (time taken to release <NUM>% of drug from the formulation) for crocin-<NUM> in plain extract and fraction is < <NUM> hr, whereas it is <NUM> and <NUM> hrs. in novel sustained release formulations CSHA-<NUM> and CSE-<NUM>. This is indicative of the delayed release of crocin-<NUM> from novel formulations.

EXAMPLE <NUM>. Pharmacokinetic study of IIIM-<NUM>-A002 extract and its SR formulation in SD rats. For comparative oral pharmacokinetic study of plain extract (IIIM-<NUM>-A002) and SR formulation (filled in 9el capsules), the dose equivalent to <NUM>/kg of crocin was administered in SD rats in both these groups. Blood samples were collected (n=<NUM>/time point) at <NUM> (IV only), <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, post-dose. At each time point about <NUM>µL of blood was collected by jugular vein into a labeled microfuge tube containing <NUM> K2EDTA solution (<NUM>µL per mL of blood) and equivalent volume of heparinized saline was replaced following sample collection. The blood samples were processed to obtain the plasma samples within <NUM> of scheduled sampling time. All plasma samples were stored below -<NUM> until bioanalysis. The plasma samples were analyzed for crocin and crocetin content using a fit-for purpose LC-MS/MS method with a lower limit of quantification (LLOQ) of <NUM> ng/mL. The pharmacokinetic parameters of crocin and crocetin were calculated using the non-compartmental analysis tool of validated Phoenix® WinNonlin® software (version <NUM>).

The PK parameters of crocin-<NUM> and crocetin after administration of crocin-<NUM> enriched fraction (IIIM-<NUM>-CEF) and its sustained release formulation (IIIM-<NUM>-SR) at a dose equivalent to <NUM>/kg of crocin-<NUM> in SD rats are shown in Table <NUM>. The time-plasma concentration curve of extract and formulation is depicted in <FIG>. The PK results indicate that when the crocin-<NUM> enriched fraction is administered in rats, the majority of crocin-<NUM> gets metabolized to crocetin. The AUCinf of crocin-<NUM> and crocetin after administration of crocin-<NUM> enriched fraction (IIIM-<NUM>-CEF) were found to be <NUM> and <NUM>µg. h/mL, respectively (the ratio of crocin-<NUM> to crocetin in plasma is <NUM>: <NUM>). When the equivalent dose of the extract IIIM-<NUM>-CEF was administered in the form of SR formulation, the crocetin concentration in plasma was increased by <NUM> fold. The AUCinf of crocetin was changed from <NUM> to <NUM>µg. Similarly, the Cmax was also increased by <NUM> fold. Interestingly, the ratio of crocin-<NUM> to crocetin in plasma was also increased, from <NUM>: <NUM> to <NUM>: <NUM>, indicating that more amount of crocetin (which is a bioactive constituent) is available in the blood circulation to display therapeutic effect at the site of action.

EXAMPLE <NUM>. P-gp induction activity of IIIM-<NUM>-A002 in LS-<NUM> cells. IIIM-<NUM>-A002 was screened for its ability to induce P-gp using rhodamine123 (Rh123) cell exclusion method. In this method, P-gp function was evaluated in terms of rhodamine <NUM> (Rh123) accumulations and efflux. Briefly, the protocol used is as follows: Colorectal LS-<NUM> cells [obtained from ECACC (European Collection of Cell Cultures) catalogue number: <NUM>; passage number <NUM>] were seeded at a density of <NUM>×<NUM><NUM> per well of <NUM> well plate and were allowed to grow for next <NUM>. Cells were further incubated with the test samples, and were diluted to the final desired series of concentrations and rifampicin (standard) to a final concentration of <NUM> in complete media for <NUM>. The final concentration of DMSO was kept at <NUM>%. Test sample and standard rifampicin were removed and cells were incubated with HBSS solution for <NUM> minutes before further incubation with HBSS solution (containing <NUM> of Rh123 as a P-gp substrate) for <NUM> minutes. At the end of Rh123 treatment cells were washed four times with cold PBS followed by cell lysis for <NUM> by using <NUM>µl of lysis buffer (<NUM>% Triton X-<NUM> and <NUM> N NaOH). A total of <NUM>µl of lysate was used for reading fluorescence of Rh123 at <NUM>/ <NUM>. Samples were normalized by dividing fluorescence of each sample with total protein present in the lysate.

IIIM-<NUM>-A002 treatment in LS-<NUM> colon cancer cells at various concentrations ranging from <NUM>µg/ml to <NUM>µg/ml led to significant increase in the efflux of substrate rhodamine <NUM> dye as determined by decrease (by <NUM>-<NUM>%) in intracellular % Rh123 levels (<FIG>). Rifampicin was used as a positive control in this study. Rifampicin at <NUM>µg/mL showed decrease in intracellular accumulation of Rh123 levels (by <NUM>%) in LS180 cells, in comparison with control (<NUM>%).

EXAMPLE <NUM>. Western blot analysis of IIIM-<NUM>-A002 in LS-<NUM> cells. The protein lysates were prepared and total protein content in lysate were measured employing Bio-Rad protein assay kit using bovine serum albumin as standard. Proteins aliquots (<NUM>µg) were resolved on SDS-PAGE and then electro transferred to PVDF membrane overnight at <NUM> at 30V. Nonspecific binding was blocked by incubation with <NUM> % non-fat milk in Tris-buffered saline containing <NUM>% Tween-<NUM> (TBST) for <NUM> at room temperature. The blots were probed with P-gp antibody for <NUM> and washed three times with TBST. Blot was then incubated with horseradish peroxidase conjugated anti-mouse secondary antibody for <NUM>, washed again three times with TBST and signals detected using ECL plus chemiluminescence's kit on BioRadChemiDoc XRS system.

Western-blot results clearly indicate that IIIM-<NUM>-A002 induces P-gp expression by <NUM>-<NUM> fold in LS-<NUM> colon cancer cells at concentrations ranging from <NUM>-<NUM>µg/ml, respectively as shown in <FIG>, which might be the possible explanation for the increased efflux of Rh123 dye. Rifampicin was used as a positive control in this study. Rifampicin at <NUM>µg/mL showed <NUM>-fold increase in the P-gp expression.

EXAMPLE <NUM>. Inhibition of NLRP3 inflammasome. THP-<NUM> cells were differentiated with phorbol-<NUM>-myristate-<NUM>-acetate (PMA) (<NUM> ng/ml) for <NUM>. The media was changed after <NUM> followed by rest of two days in complete RPMI containing <NUM>% heat inactivated FCS. Cells were primed with LPS (<NUM>µg/ml) for <NUM> followed by pre-treatment with different concentration of hydroalcohlic extract of Crocus sativus for <NUM>. Then, cells were stimulated with nigericin, <NUM> for <NUM>. Supernatant was collected and stored at -<NUM> for ELISA of IL-1β. Secretion of IL-1β with nigericin was measured by BD OptEIA for IL-1beta (human) and was considered as readout for NLRP3 activation.

IIIM-<NUM>-A002 showed strong inhibition of NLRP3 inflammasome at the low concentration of <NUM>µg/ml. Results are shown in <FIG>. IIIM-<NUM>-A002 at <NUM>µg/mL showed <NUM>% inhibition of IL-1β release in comparison to LPS+ nigericin (<NUM>%). MCC-<NUM> (<NPL>) was used as a positive control. MCC-<NUM> at <NUM> ng/mL showed <NUM>% inhibition of IL-1β release in comparison to LPS+ nigericin (<NUM>%).

EXAMPLE <NUM>. In-vivo efficacy of IIIM-<NUM>-A002 on intra-cerebroventricular streptozotocin induced dementia in SD rats using Morris Water Maze test. Animals were randomized into <NUM> groups based on body weight, each group with n=<NUM>-<NUM>. Artificial cerebrospinal fluid (a CSF - <NUM> NaCl, <NUM> mMKCl, <NUM> MgCl2, <NUM> CaCl2, <NUM> dextrose) was injected to group <NUM> or STZ (<NUM>/kg) to group <NUM>-<NUM> animals through intra-cerebroventricular (ICV) route on day <NUM> and <NUM>. On 15th -18th day, vehicle was administered to group <NUM> - <NUM>, test compound IIIM-<NUM>-<NUM> at a dose of <NUM> and <NUM>/kg, p. to group <NUM> and <NUM> animals respectively. Rats were subjected to MWM test <NUM> after dosing, <NUM> trials were conducted each day for <NUM> days i.e. from 15th to 18th day. If animal failed to locate the hidden platform in stipulated time, they were gently pushed to the hidden platform and kept on platform for <NUM> seconds. Time (seconds) to locate the hidden platform was recorded as escape latency (latency time). Escape latency were recorded for retention session i.e. from day <NUM>-<NUM>. The difference (% change) in escape latency was compared with aCSF.

There was significant increase in latency time to locate the hidden platform in STZ treated rats as compared with aCSF treated rats suggesting memory impairment. A significant increase in latency time was observed in ICV- STZ treated rats at day <NUM> & <NUM> indicating memory deficit. Further, treatment with test compound (IIIM-<NUM>-<NUM>) decreased the STZ induced memory impairment in rats as demonstrated by reduction in latency time at dose of <NUM>/kg p. for <NUM> days. Results are shown in <FIG>.

EXAMPLE <NUM>. In-vivo efficacy of IIIM-<NUM>-A002 on scopolamine induced dementia in Swiss albino mice (SAM) using passive avoidance test. Animals were randomized to their body weight (n=<NUM>-<NUM>). All animals were then habituated to experimental apparatus (Passive avoidance instrument of make - UGO Basile Biological Research Apparatus Italy) prior to the experiment. For habituation, animals were placed gently in the light compartment of the apparatus. After <NUM> seconds guillotine door was opened and animal allowed entering the dark compartment without giving the shock. Animals that took more than <NUM> seconds to enter the dark room were eliminated from the experiments. For acquisition trial the animals were placed in light compartment and allowed to enter the dark compartment through guillotine door. As soon as the animal entered to the dark compartment, door was closed and animal was delivered a foot shock (<NUM> mA current) immediately for the duration of <NUM> seconds. Animals were then removed from apparatus after <NUM> seconds and placed temporarily in its home cage. Repeated the same procedure (keeping the gap between acquisition trials to <NUM> minutes) till the animal remained in the light compartment for consecutive <NUM> seconds. Retention was recorded at day <NUM> without shock in dark chamber. Increase in transfer latency time from light to dark chamber indicated learning in animals. Rivastigmine was used as positive control in this study. The data were acquired as transfer latency (in sec) i.e. time taken by each mice to move from light compartment to dark compartment (Table <NUM>). Percentage (%) change in transfer latency as compared with control on retention trial was calculated. The scopolamine group was compared with control while test/standard groups were compared with scopolamine treated animals. For statistical analysis, one way ANOVA followed by Tukey test was used.

This study suggests that scopolamine administration results in memory impairment in mice as demonstrated by significant reduction in transfer latency as compared with control in retention trial. Administration of test compound IIIM-<NUM>-<NUM> at <NUM> mpk for <NUM> day significantly recovered the memory as shown by increase in transfer latency as compared with scopolamine treated mice. Positive control rivastigmine at a dose of <NUM>/kg resulted in a significant reduction in latency time (<FIG>).

Claim 1:
A sustained release formulation comprising granules of Crocus sativus hydroalcoholic extract or fraction; biodegradable polymers and/ or non-biodegradable polymers alone or in combination; optionally an excipient, selected from carrier, adjuvant and binder; wherein the hydroalcoholic extract/fraction comprises a mixture of at least <NUM> crocins including trans-<NUM>-ng-crocin, trans-<NUM>-GG-crocin, trans-<NUM>-Gg-crocin, trans-<NUM>-gg-crocin, cis-<NUM>-GG-crocin and cis-<NUM>-Gg-crocin and wherein weight ratio of the hydroalcoholic extract/fraction of Crocus sativus: polymer(s) is in the range of <NUM>:<NUM> to <NUM>:<NUM>.