Abstract

A reformulated turmeric extract having a 1:1 ratio of ethanol extract concentrate of oleoresin. and, ethanol extract concentrate of essential oil. The ethanol extract concentrate of oleoresin comprises curcumin, demethoxycurcumin and bisdemethoxycurcumin. The ethanol extract concentrate of essential oil comprises essential oil of turmeric having of Ar-turmerone. A method of preparing a reformulated turmeric extract having a 1:1 ratio of ethanol extract concentrate of oleoresin, and, ethanol extract concentrate of essential oil.

Information

Application ID	2356/CHE/2010
Invention Field	TRADITIONAL KNOWLEDGE CHEMICAL
Date of Application	2010-08-16
Publication Number	07/2013

Applicants

Name	Address	Country	Nationality
ANTONY  Benny	Arjuna Natural Extracts Ltd.  P.B. No.126  Bank Road  Aluva - 683 101  Kerala State  India.	India	India

Inventors

Name	Address	Country	Nationality
ANTONY  Benny	Arjuna Natural Extracts Ltd.  P.B. No.126  Bank Road  Aluva - 683 101  Kerala State  India.	India	India

Specification

FIELD OF INVENTION

The present invention relates to a pharmaceutical composition of reformulated turmeric extract comprising curcumin, demethoxycurcumin, bisdemethoxycurcumin, Ar-turmerone and other constituents of essential oil of turmeric which increase the absorption and blood level concentration of curcumin, retention of curcumin in the plasma up to 14 hours and increase the area under curve (AUC) of concentration time plots of curcumin following oral administration, and a method of preparing said pharmaceutical composition.

BACKGROUND OF THE INVENTION

Turmeric is the rhizome or underground stem of a ginger-like plant. It is an ancient spice, a native of South East Asia, used from antiquity as dye and a condiment, said to have been originated in the eastern parts of India. Interest in turmeric probably originated as a textile dye, and later as a food colorant and substitute for the costly saffron. Its use dates back nearly 4000 years, to the Vedic culture in India where it was used as a culinary spice and had some religious significance.

Susruta's Ayurvedic Compendium, dating to 250 BC, recommends an ointment containing turmeric to relieve the effects of poisoned food. Ayurveda, Unani, Siddha and Chinese medicine recommend turmeric for a large number of disorders and diseases. Traditional Indian medicine use the turmeric powder against biliary disorders, anorexia, coryza, cough, diabetic wounds, hepatic disorders, rheumatic disorders, sprains and swellings caused by injury and sinusitis.

Externally, the dried rhizome has been applied to fresh wounds and to insect stings and to help the healing process in chickenpox and smallpox. It is also applied topically for
ulcers, wounds, eczema and inflammations. In both the Ayurvedic and Siddha systems of medicine, a turmeric paste is used topically to treat ulcers and scabies. Since the Ayurvedic times, numerous therapeutic activities have been assigned to turmeric for a wide variety of diseases and conditions, including those of the skin, pulmonary and gastrointestinal systems, aches, pains, wounds, sprains and liver disorders. Modern research has confirmed and provided a scientific basis for these various health claims, unlike many other traditional medicines. Since the isolation of curcumin as the main active constituent of turmeric about two centuries ago, much of the scientific interest has shifted to this molecule rather than on turmeric. Observational studies point to the substantially reduced prevalence of Alzheimer's disease, rheumatoid arthritis and disease of the gastrointestinal tract such as colon cancer and inflammatory bowel diseases in Asian countries compared to the western world as a consequence of the daily consumption of turmeric as a curry spice.

Research on curcumin is exploding with more than 3000 reports presently available. This is because of an extremely wide array of biological activities exhibited by the molecule. Curcumin acts at multiple targets and at multiple levels. The number of transcription factors and signaling pathways modulated by curcumin is, indeed, bewildering • . For this reason, curcumin is fast emerging as a cure-all, for valid reasons. Curcumin has demonstrated benefit for most, if not all, chronic diseases afflicting mankind. It is an antioxidant several times more potent than alpha tocopherol and can effectively scavenge oxygen and nitrogen free radicals.

Curcumin is a complete anti-inflammatory modulating all the agents involved in the complex process of inflammation including cytokines, chemokines, adhesion molecules, growth factors and transcription factors such as NF-KB and AP-1 and a large number of kinases, notably the MAP kinases p38 and JNK. In heart disease, curcumin can affect all the steps believed to be involved in the pathologic process of atherosclerosis. In diabetes, curcumin can potentially reverse insulin resistance, the first clinically relevant stage of the disease. Further, curcumin can sensitize insulin by inducing the transcription factor PPAR, similar to the thiazolidinediones currently used for the purpose. Curcumin has been shown to be the only agent who can effectively address the multiple factors involved in Alzheimer's disease and rheumatoid arthritis. As an anticancer agent, curcumin is a chemo preventive, affect cell cycle progression and transformation, cause apoptosis of malignant cells by more than one mechanism, prevents angiogenesis and metastasis and is effective even against drug-resistant cancers. While common chemotherapeutic agents cause serious side effects, curcumin produces none. While the common anticancer drugs are immuno-suppressors, curcumin is an immuno-restorer3'4. Furthermore, whereas the common anticancer drugs cannot cross the bloodbrain barrier, curcumin can. Curcumin exhibits activities similar to recently discovered drugs such as TNF inhibitors, vascular endothelial cell growth factor (VEGF) blocker, human epidermal growth factor receptor (EGFR) inhibitors and the HER2 blocker, without their toxic side effects.

Turmeric may contain well over a hundred chemical compounds, most of these originating from the essential oil part of turmeric. A complete analysis of all these constituents has not so far been undertaken. However, the major and characteristic components of turmeric are the three curcuminoids and sequiterpenoids. Curcuminoids includes curcumin, demethoxycurcumin, and bisdemethoxycurcumin. Major sesqiterpenoids include Ar-Turmerone, aTurmerone, P-Turmerone, Curcumene, Zingiberene etc.

Compounds other than the three curcuminoids are constituents of the volatile oil fraction, the major components of which are the turmerones, notably Ar-turmerone5,67,8. The yield and composition are apparently influenced by the agro climatic conditions where turmeric is grown and also by the method of extraction The curcuminoids usually range about 4-5%, but up to 8% has been reported . Clinical activity of curcumin is yet to be confirmed; however, in preclinical animal models, curcumin has shown cancer chemo preventive, antineoplastic and anti¬inflammatory properties10. Especially interesting is its ability to prevent the formation of carcinogen-induced intestinal premalignant lesions and malignancies in rats ' and in the multiple neoplasia13, a genetic model of the human disease familial adenomatous polyposis.

Curcumin acts as a scavenger of oxygen species such as hydroxyl radical, super oxide anion and singlet oxygen141516, and interferes with lipid per oxidation 17' .Curcumin suppresses a number of key elements in cellular signal induction pathways pertinent to growth, differentiation and malignant transformations. Curcumin directly inhibit the cyclooxygenase-2 and also inhibits the transcription of the gene responsible for its production. Cyclooxygenases (COX) catalyze the synthesis of prostaglandins (PGs) from arachidonic acid. Curcumin is among the few agents to block both the COX and LOX (lipoxygenase) pathways of inflammation and carcinogenesis by directly modulating arachidonic acid metabolism. In a study to evaluate the effect of curcumin on the metabolism and action of arachidonic acid in mouse epidermis, it was found that topical application of curcumin inhibited arachidonic acid induced ear inflammation in mice19'20.

Curcumin (10 M) inhibited the conversion of arachidonic acid to 5- and 8-hydroxyeicosatetraenoic acid by 60% and 51%, respectively (LOX pathway) and the
metabolism to PGE2, PGF2a and PGD2 by 70%, 64% and 73%, respectively (COX pathway).

Despite this impressive array of beneficial bioactivities, the absorption and retention of curcumin in animals and humans remain low. In rodents, curcumin demonstrates poor levels in blood after oral dosing21 which may be related to its inadequate absorption and fast metabolism. Curcumin absorption and retention may also be poor in humans as seen from the results of a recent pilot study of a standardized turmeric extract in colorectal cancer patients22. Indirect evidence suggests that curcumin is metabolized in the intestinal tract. It has been suggested that the intestinal tract plays an important role in the metabolic disposition of curcumin. Intestinal mucosa as well as liver and kidney tissue from the rat can glucuronidate and sulfate curcumin, as judged by the analysis of differential amounts of curcumin present before and after treatment of tissue extracts with conjugate-hydrolyzing enzymes23. Thus, gut metabolism contributes substantially to the overall metabolic yield generated from curcumin in vivo.
Pharmacokinetic studies in animals have demonstrated upto 40-85 percent of an oral dose of curcumin passes through the gastrointestinal tract unchanged, with most of the absorbed flavonoid being metabolized in the intestinal mucosa and liver. Curcumin, when given orally or intraperitoneally to rats is mostly excreted in the feces and only a little in the urine. Only traces of curcumin are found in the blood from the heart, liver and kidney. Curcumin, when added to isolated hepatocytes is quickly metabolized and the major biliary metabolites are glucuronides of tetrahydrocurcumin and hexahydrocurcumin. Curcumin, after metabolism in the liver, is mainly excreted through bile.

An oral dose of 500 mg/kg given to rats resulted in a peak plasma concentration of only 1.8 ng/mL, with the major metabolites identified being curcumin sulfate and curcumin glucuronide21. A phase I clinical trial conducted on 25 patients with various precancerous lesions demonstrated oral doses of 4, 6, and 8 g curcumin daily for three months yielded serum curcumin concentrations of only 0.51 ±0.11, 0.63 ± 0.06, and 1.77 ± 1.87 uM respectively. Serum levels peaked between one and two hours post-dose and declined rapidly, showing that curcumin is not retained in the blood . Another phase I trial involving 15 patients with advanced colorectal cancer, with curcumin was given at doses of 0.45 and 3.6 g daily for four months. Only in patients taking 3.6gm per day, curcumin was detected from plasma and it was only at a single time point showing 11.1 ±0.6 nmol/L. Curcumin was not detected in the plasma of patients taking lower doses 25. In another pharmacokinetic study with oral curcuma extract in patients with colorectal cancer, oral administration of 440 to 2200 mg curcuma extract per day corresponding to 36 to 180 mg curcumin failed to get curcumin or its metabolites detected in plasma or urine even after 29 days . Although some questions remain unanswered regarding the pharmacokinetics of curcumin in humans, there is no denying the fact that considerable proportion of ingested curcumin is excreted through feces and at least about one-half of absorbed curcumin is metabolized. The quantity of curcumin that reaches tissues outside the gut is probably pharmacologically insignificant. These results have apparently dampened the spirits of researchers and halted curcumin's progress beyond Phase 1 trials.

To provide the systemic effects, curcumin must be absorbed from its oral route of administration at a suitable rate, be distributed in adequate concentration in the blood and remain in the system for a sufficient period at an effective concentration level. One measure of drug absorption is its blood serum concentration at various time intervals after administration.

Certain drugs have a correlation between blood serum concentration and the presence of drug effects. The mere administration of curcumin does not bring about its medicinal properties, rather it has to be absorbed and present in the blood and retained in detectable amounts. A recent study in rats by26 showed that, up-regulation of Glutathione S Transferase activity in the liver and decrease of MiG levels in colon mucosa after oral administration of curcumin were accompanied by measurable tissue levels of curcumin. It is conceivable that higher doses of curcumin, which furnish measurable plasma curcumin concentrations, are required to elicit a therapeutic effect in vivo.

Drug concentrations in the blood can be determined and graphed against time to obtain a drug blood level curve. In most instances, the time course of a drug's concentration in the plasma correlates well with the onset, intensity and duration of the pharmacologic effect.

Thus, the measurement of sequential plasma concentration of drugs after their administration is used to establish dosage regimens that are likely to produce the desired therapeutic levels for appropriate period of time, without the risk of drug failure or toxicity.

When a drug is administered by an oral route, it usually appears in the plasma within a short time, and its concentration rises steadily until it peaks. Once absorbed into the circulation, it is subjected simultaneously to distribution, biotransformation, and excretion. During the initial period, the rate of absorption and distribution exceeds the rate of elimination (positive slope of the curve). The peak plasma concentration is reached when absorption and elimination rates are equal (Cmax)- Thereafter, the
elimination rate exceeds the rate of absorption because fewer drugs remain available at the site of administration and plasma drug levels begin to fall (negative slope of the curve). So the rate and extent of absorption of a drug, usually from the Gl tract after administration by oral route is very important.

The important parameters to be considered for comparative analysis of blood curve levels following oral administration of a drug are Cmax, Tmax (time of peak concentration and area under the blood (serum or plasma) concentration time curve (AUC or area under curve). The AUC of a concentration time plot is considered representative of the total amount of drug absorbed into the circulation following administration of a single dose of a drug. It is a robust pharmacokinetic parameter as it is not very sensitive to small changes in concentration time data. Equivalent dose of a drug when fully absorbed produce the same AUC. If equivalent doses of drug in different formulations produce different AUC values, difference exist between the extent of absorption between the formulations.

In order to derive full benefits from the administration of curcumin in human subjects, ways and means to increase the absorption and blood level concentration of curcumin, retention of curcumin in the blood, and increase the area under curve (AUC) of concentration time plots of curcumin needs to be explored. The present invention is an effort in this direction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG: 1 provides a comparative graph of curcumin content in blood (nM/L) in albino rats following administration of DMSO alone, reformulated turmeric extract in DMSO, regular turmeric extract powder in DMSO, Raw turmeric powder in DMSO,
curcuminoid mixture alone in DMSO, essential oil of turmeric with 10- 15% Ar-turmerone in DMSO, ethanol extract concentrate of oleoresin in DMSO or ethanol extract concentrate of essential oil of turmeric in DMSO.

FiG 2 provides a comparative graph of curcumin content in blood (nM/L) following administration of reformulated turmeric extract, regular turmeric extract powder, raw turmeric powder,essential oil of turmeric having 10-15% Ar-turmerone, curcuminoid mxture alone, ethanol extract concentrate of oleoresin or ethanol extract concentrate of essential oil of turmeric.

SUMMARY

It was found that a pharmaceutical composition of reformulated turmeric extract comprising curcumin, demethoxycurcumin, bisdemethoxycurcumin, Ar- turmerone and other components of essential oil of turmeric increase the absorption and blood level concentration of curcumin, retention of curcumin in the plasma up to 14 hours, and increase the area under curve (AUC) of concentration time plots of curcumin.

Accordingly, a pharmaceutical composition of reformulated turmeric extract is disclosed.

One embodiment provides a reformulated turmeric extract having a 1:1 ratio of ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil. The ethanol extract concentrate of oleoresin includes curcumin, demethoxycurcumin and bisdemethoxycurcumin. The ethanol extract concentrate of essential oil includes essential oil of turmeric having of Ar-turmerone.

An embodiment of the present invention provides a pharmaceutical composition of
reformulated turmeric extract wherein, the curcumin content ranges, by weight, from
about 290 mg to about 375 mg; demethoxy curcumin content ranges, by weight, from
about 84 mg to about 125 mg; bisdemethoxy curcumin content ranges, by weight, from
about 8 mg to about 40 mg; Ar-turmerone content ranges, by weight, from about 16
mg to about 33 mg; and other sesqiterpenoids of essential oil of turmeric content
ranges, by weight, from about 13 mg to about 30 mg.

An embodiment of the present invention discloses a pharmaceutical composition of
reformulated turmeric extract wherein, the peak blood curcumin level, Cmax of the
reformulated turmeric composition is 3-14 fold higher compared to regular turmeric
extract capsules containing 95% curcuminoid.

An embodiment of the present invention discloses a pharmaceutical composition of
reformulated turmeric extract wherein, the absorption of curcumin from the disclosed
composition is 60- 98% compared to regular turmeric extract capsules.

An embodiment of the present invention discloses a pharmaceutical composition of
reformulated turmeric extract wherein, curcumin is retained in the blood after oral
administration of a reformulated turmeric extract up to 14 hours post drug compared to
regular turmeric extract capsules.

An embodiment of the present invention discloses a pharmaceutical composition of
reformulated turmeric extract wherein, the AUC of curcumin from reformulated
turmeric extract is 3-14 times greater than from composition of regular turmeric extract
capsules.

In one embodiment, the ethanol extract concentrate of oleoresin is prepared by the
process including powdering dried rhizomes of turmeric to form turmeric powder. The
turmeric powder is extracted with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain a first residue and n-hexane extract. The first residue and the n-hexane extract are separated. The first residue is dried to obtain a second dried residue. The second dried residue is extracted with ethanol to obtain an ethanol extract and a third residue. The ethanol extract is concentrated to obtain the ethanol extract concentrate of oleoresin. In one embodiment, the ethanol extract concentrate of essential oil is prepared by the process including powdering dried rhizomes of turmeric to form turmeric powder. The turmeric powder is extracted with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain a residue and n-hexane extract. The residue and the n-hexane extract are separated. The n-hexane extract is concentrated to form a concentrated hexane extract. The concentrated hexane extract is mixed with ethanol in the presence of silica to obtain an ethanol extract. The ethanol extract is concentrated to obtain the ethanol extract concentrate of essential oil. One embodiment discloses a method of preparing a reformulated turmeric extract comprising a 1:1 ratio of ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil. The method discloses mixing the ethanol extract concentrate of oleoresin and the ethanol extract concentrate of essential oil in a 1:1 ratio to obtain a blend. Ethanol is added to the blend in 2:1 ratio and kept for 4 days at 27°C to obtain a residue and a filtrate. The resulting residue and the filtrate are separated. The residue is dried and powdered to obtain the reformulated turmeric extract. An embodiment of the present invention discloses a method of preparing a reformulated turmeric extract wherein, the peak blood curcumin levels Cmax of the reformulated composition was 3-14 fold higher compared to regular turmeric extract capsules.

An embodiment of the present invention discloses a method of preparing a
reformulated turmeric extract wherein, the absorption of curcumin from the disclosed
composition is 60- 98% compared to regular turmeric extract capsules.

An embodiment of the present invention discloses a method of preparing a
reformulated turmeric extract wherein, curcumin is retained in the blood after oral
administration of a reformulated turmeric extract up to 14 hours post drug compared to
regular turmeric extract capsules.

An embodiment of the present invention discloses a method of preparing a
reformulated turmeric extract wherein, the AUC of curcumin from reformulated
turmeric extract is 3-14 times greater than from composition of regular turmeric extract
capsules.

An embodiment of the present invention discloses a method of preparing a capsule containing the above composition for oral administration to mammals, especially humans.

DETAILED DESCRIPTION

An embodiment of the present invention relates to a pharmaceutical composition of reformulated turmeric extract to increase the absorption and blood level concentration of curcumin, retention of curcumin in the plasma up to 14 hours and increase the area under curve (AUC) of concentration time plots of curcumin comprising of curcumin, demethoxycurcumin, bisdemethoxycurcumin, Ar turmerone and other components of essential oil of turmeric.

As disclosed herewith the term "reformulated turmeric extract" is a mixture of curcumin, demethoxycurcumin, bisdemethoxy curcumin, Ar-turmerone and other components of essential oil of turmeric. The term "essential oil of turmeric" is also referred to as "volatile oil" or "volatile oil of turmeric." Apart from Ar-turmerone, other constituents of essential oil of turmeric includes sesquiterpenoids The reformulated turmeric extract of the preferred embodiment of the present invention is made from turmeric by a process comprising of: Dried rhizomes of turmeric is powdered in a pulverisor to obtain turmeric powder. Next n-hexane in the amount of four times the quantity of turmeric powder is added to the turmeric powder for hexane extraction. The extraction is performed using an extractor with reflux condenser. The bottom of the extractor is fitted with a polypropylene (100 microns) filter cloth. The mixture is refluxed at the boiling temperature (50-70°C) of hexane for one hour to obtain a residue (Yield 92.5% and moisture content 6%) and a hexane extract (yield 6%). After first extraction, the residue is further extracted two more times with four times the quantity of n-hexane. The residue and hexane extracts are separated by draining out the hexane extract from the extractor bottom through the polypropylene filter cloth using a centrifugal pump. The residue is dried in a rotary vacuum drier. The dried residue is extracted with 3 times the quantity of ethanol. The ethanol extraction of the residue is performed at 60-70 °C temperature for 1 hour using an extractor with reflux condenser. The extractor is fitted with polypropylene (100 microns) filter cloth at the bottom of extractor to separate the ethanol extract and the residue. The ethanol extract is concentrated at 60-70°C under vacuum (550mm Hg) to form ethanol extract concentrate containing Oleoresin (yield 20%). The oleoresin includes curcuminoids.

The hexane extract is concentrated at 50-70°C under vacuum (500mm Hg) to form concentrated hexane extract. Then 2 times the quantity of silica (200-400mesh size) is mixed with the concentrated hexane extract in a sigma mixer for one hour and is refluxed with 2 times the quantity of 90% ethanol for 1 hour by using extractor with reflux condenser and fitted with polypropylene (l00microns) filter cloth at the bottom of extractor. Silica is removed by draining out the ethanol extract from the extractor bottom through the polypropylene filter cloth using a centrifugal pump. The ethanol extract is concentrated at 60-70°C under vacuum (550mm Hg) to form ethanol extract concentrate (yield 10%). The ethanol extract concentrate includes the essential oil of turmeric. Next, a 1:1 ratio of ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil is blended in a double cone blender to obtain blend. Then, ethanol is added to the blend at a ratio of 2:1 of the ethanol to the blend to obtain a mixture. The mixture is kept for 4 days in a stainless steel (SS-316) holding vessel with conical bottom with jacket for circulating cooling water at 27°C. After 4 days, the mixture is transferred to a plate & frame filter press by using a stainless steel screw pump. This transfer separates the mixture into a residue (yield 4%) and a filtrate. The filtrate has the resin and fat. The residue is dried by using rotary vacuum drier (700mm Hg) to obtain a dried residue. The dried residue is powdered in a pulverisor to form a fine powder of reformulated turmeric extract containing of curcumin, demethoxycurcumin, bisdemethoxycurcumin, Ar turmerone and other components of essential oil of turmeric.

One embodiment provides a reformulated turmeric extract having a 1:1 ratio of ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil. The ethanol extract concentrate of oleoresin includes curcumin, demethoxycurcumin and
bisdemethoxycurcumin. The ethanol extract concentrate of essential oil includes essential oil of turmeric having of Ar-turmerone.

An embodiment of the present invention provides a composition of reformulated turmeric extract which when given orally, significantly increase the absorption and blood level concentration of curcumin, retention of curcumin in the plasma up to 14 hours and increase the area under curve (AUC) of concentration time plots of curcumin. Another embodiment of the present invention provides a method of preparing the reformulated turmeric extract which when given orally, significantly increase the absorption and blood level concentration of curcumin, retention of curcumin in the plasma up to 14 hours and increase the area under curve (AUC) of concentration time plots of curcumin.

Another embodiment of the present invention, the composition of a reformulated turmeric extract wherein, the curcumin content ranges, by weight, from about 290 mg to about 375mg, demethoxy curcumin content ranges, by weight, from about 60 mg to about 125 mg bisdemethoxy curcumin content ranges, by weight, from about 8 mg to about 40 mg, Ar- turmerone content ranges, by weight, from about 6 mg to about 36 mg and other sesqiterpenoids of essential oil of turmeric content ranges, by weight, from about 10 mg to about 30 mg

In some embodiments of the present invention, the composition of reformulated turmeric extracts the curcumin ranges, by weight, from about 325 mg to about 360 mg. In some embodiments of the composition of turmeric extract the demethoxycurcumin ranges, by weight, from about 84 mg to about 93 mg. In some embodiments of the composition of turmeric extract the bisdemethoxy curcumin ranges, by weight, from about 9 mg to about 40 mg. In some embodiments of the composition of turmeric extract the Ar turmerone ranges, by weight, from about 16 mg to about 33 mg and other sesqiterpenoids of essential oil of turmeric content ranges, by weight, from about 13 mg to about 25 mg.

In some embodiments of the reformulated turmeric extract, curcumin ranges from about 58 % to about 75 %. In some embodiments of the reformulated turmeric extract, demethoxycurcumin ranges from about 12 % to about 25 %. In some embodiments of the reformulated turmeric extract, bisdemethoxycurcumin ranges from about 1.6 % to about 8 %. In some embodiments, of the reformulated turmeric extract Ar-turmerone ranges from about 1.2 % to about 7.2 %. In some embodiments of the reformulated turmeric extract, curcumin ranges from about 290 mg to about 375 mg. In some embodiments of the reformulated turmeric extract, demethoxycurcumin ranges from about 60 mg to about 125 mg. In some embodiments of the reformulated turmeric extract, bisdemethoxycurcumin ranges from about 8 mg to about 40 mg. In some embodiments of the reformulated turmeric extract of claim 1, Ar-turmerone ranges from about 6 mg to about 36 mg.

The following are examples of preferred combinations of curcuminoids, Ar- turmerone and other sesqiterpenoids of essential oil of turmeric.

1. Curcumin 59.3%, demethoxycurcumin 23.6%, bisdemethoxycurcumin 7.9%,Ar-turmerone 3.6% and other sesqiterpenoids of essential oil of turmeric 5.46%.

2. Curcumin 60.4%, demethoxycurcumin 22.6%, bisdemethoxycurcumin 7.7%,Ar-lurmerone 4.2% and othersesqiterpenoids of essential oil of turmeric 4.9 %.

3. Curcumin 61.9%, demethoxycurcumin 21.9%, bisdemethoxycurcumin 6.8%,Ar-turmerone 4.7 % and other sesqiterpenoids of essential oil of turmeric 4.6%.

4. Curcumin 62.8%, demethoxycurcumin 20.4%, bisdemethoxycurcumin 6.2%,Ar-turmerone 5.7 % and other sesqiterpenoids of essential oil of turmeric 4.9%.

5. Curcumin 63.6%, demethoxycurcumin 20.8%, bisdemethoxycurcumin 6.4%,Ar-turmerone 4.2% and other sesqiterpenoids of essential oil of turmeric 5%.

6. Curcumin 64.5%, demethoxycurcumin 19.6%, bisdemethoxycurcumin 6.7%,Ar-
turmerone 3.9% and other sesqiterpenoids of essential oil of turmeric 5.14%.

7. Curcumin 67.1%, demethoxycurcumin 24.4%, bisdemethoxycurcumin 1.9%,Ar-
turmerone 3.2% and other sesqiterpenoids of essential oil of turmeric 3.3%.

8. Curcumin 69.6%, demethoxycurcumin 17%, bisdemethoxycurcumin 4.7%,Ar- turmerone 6.1% and other sesqiterpenoids of essential oil of turmeric 2.6%.

9. Curcumin 72.6%, demethoxycurcumin 15.1%, bisdemethoxycurcumin 3.2%,Ar-turmerone 3.4% and other sesqiterpenoids of essential oil of turmeric 5.7%.

10. Curcumin 74.4%, demethoxycurcumin 12.6%, bisdemethoxycurcumin 4.1%,Ar-turmerone 3.1% and other sesqiterpenoids of essential oil ofturmeric 5.9%.

The present invention is the optimal composition among different composition, which
showed maximum absorption of curcumin, increase the blood level concentration of
curcumin, retention of curcumin in the plasma up to 14 hours and increase the area
under curve (AUC).

In some embodiments of the present invention, absorption of curcumin from the reformulated turmeric extract composition ranges from 60% to 98%.

In one embodiment of the present invention, the increases in the blood level concentration of curcumin from the reformulated turmeric extract ranges from 3 times to 14 times.

In some embodiments, oral administration of the reformulated turmeric extract resulted in an increase in absorption of curcumin ranging from about 2 fold to about 40 fold as compared to oral administration of raw turmeric, essential oil of turmeric, regular turmeric extract, ethanol extract concentrate of oleoresin or ethanol extract concentrate of essential oil.

In some embodiments, oral administration of the reformulated turmeric extract resulted
in about a 10-fold increase in blood level of curcumin compared to oral administration
of regular turmeric extract. The regular turmeric extract includes curcumin,
demethoxycurcumin and bisdemethoxycurcumin.

In some embodiments, oral administration of the reformulated turmeric extract resulted
in retention of curcumin in blood for 10 hours.

In one embodiment of the present invention, the retention of curcumin from the
reformulated turmeric extract in blood ranges from 5 hrs to 14hrs.

In one embodiment of the present invention, the increase in the AUC of curcumin from
concentration time plots from the reformulated turmeric extract ranges from 3 times to
14 times.

In one embodiment the reformulated turmeric extract comprising of curcumin,
demethoxycurcumin, bisdemethoxycurcumin, Ar-turmerone and other components of essential oil of turmeric is in a dosage forms selected from the group consisting of a capsule, tablet, granule, sachet, powder, paste, ointment, infusion, injection, ampoule, solution, suspension, emulsion, pill, sustained release formulation and combinations thereof. In some embodiments of the present invention, the composition of
reformulated turmeric extract can also be added with other ingredients like cellulose,
lecithin, vitamin premixes, DMSO, vegetable oil and other extracts of natural or herbal in origin.

In some embodiments of the present invention, a composition of reformulated turmeric extract is capsulated within hard / soft gelatin / vegetable shell capsules. Reformulated turmeric extract pharmaceutical composition is encapsulated into hard gelatin capsules. The disclosure provides a method of preparing a hard gelatin capsule with the composition. Dried rhizomes of turmeric are powdered in a pulverisor to obtain turmeric powder. Next n-hexane in the amount of four times the quantity of turmeric powder was added to the turmeric powder for hexane extraction. The extraction is performed using an extractor with reflux condenser. The bottom of the extractor is fitted with a polypropylene (100 microns) filter cloth. The mixture was refluxed at the boiling temperature (50-70°C) of hexane for one hour to obtain a residue and a hexane extract. After first extraction, the residue is further extracted two more times with four times the quantity of n-hexane. The residue and hexane extracts are separated by draining out the hexane extract from the extractor bottom through the polypropylene filter cloth using a centrifugal pump. The residue is dried in a rotary vacuum drier. The dried residue is extracted with 3 times the quantity of ethanol. The ethanol extraction of the residue is performed at 60-70 °C temperature for 1 hour using an extractor with reflux condenser.

The extractor is fitted with polypropylene (100 microns) filter cloth at the bottom of extractor to separate the ethanol extract and the residue. The ethanol extract is concentrated at 60-70°C under vacuum (550mm Hg) to form ethanol extract concentrate containing Oleoresin The oleo resin includes curcuminoids.

The hexane extract is concentrated at 50-70°C under vacuum (500mm Hg) to form concentrated hexane extract. Then 2 times the quantity of silica (200-400mesh size) is mixed with the concentrated hexane extract in a sigma mixer for one hour and is refluxed with 2 times the quantity of 90% ethanol for 1 hour by using extractor with reflux condenser and fitted with polypropylene (l00microns) filter cloth at the bottom of extractor. Silica is removed by draining out the ethanol extract from the extractor bottom through the polypropylene filter cloth using a centrifugal pump. The ethanol extract is concentrated at 60-70°C under vacuum (550mm Hg) to form ethanol extract concentrate.

The ethanol extract concentrate includes the essential oil of turmeric. Next, a 1:1 ratio of ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil are blended in a double cone blender to obtain blend. Then, ethanol is added to the blend at a ratio of 2:1 of the ethanol to the blend to obtain a mixture. The mixture is kept for 4 days in a stainless steel (SS-316) holding vessel with conical bottom with jacket for circulating cooling water at 27°C. After 4 days, the mixture is transferred to a plate & frame filter press by using a stainless steel screw pump. This transfer separates the mixture into a residue and a filtrate. The filtrate has the resin and fat. The residue is dried by using rotary vacuum drier (700mm Hg) to obtain a dried residue. The dried residue is powdered in a pulverisor to form a fine powder of reformulated turmeric extract. The powder is charged into a hopper of a semi¬automatic capsule filling machine in an air-conditioned and de-humidified room. A '0' size hard gelatin capsule shell is loaded into the tray of the capsule filling machine. The powder of the reformulated turmeric extract is filled into the capsule shell and is compressed into hard gelatin capsule shell of '0' size.

The filled capsules are sorted by a sorting machine and polished with the help of a polishing machine to obtain the reformulated turmeric extract in capsule form.

In one embodiment, the ethanol extract concentrate of oleoresin is prepared by the process including powdering dried rhizomes of turmeric to form turmeric powder. The turmeric powder is extracted with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain a first residue and n-hexane extract. The first residue and the n-hexane extract are separated. The first residue is dried to obtain a second dried residue. The second dried residue is extracted with ethanol to obtain an ethanol extract and a third residue. The ethanol extract is concentrated to obtain the ethanol extract concentrate of oleoresin. In one embodiment, the ethanol extract concentrate of essential oil is prepared by the process including powdering dried rhizomes of turmeric to form turmeric powder. The turmeric powder is extracted with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain a residue and n-hexane extract. The residue and the n-hexane extract are separated. The n-hexane extract is concentrated to form a concentrated hexane extract. The hexane extract is mixed with ethanol in the presence of silica to obtain an ethanol extract. The ethanol extract is concentrated to obtain the ethanol extract concentrate of essential oil. One embodiment discloses a method of preparing a reformulated turmeric extract comprising a 1:1 ratio of ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil. The method discloses mixing the ethanol extract concentrate of oleoresin and the ethanol extract concentrate of essential oil in a 1:1 ratio to obtain a blend. Ethanol is added to the blend in 2:1 ratio and kept for 4 days at 27°C to obtain a residue and a filtrate. The residue and the filtrate are separated. The residue is dried and powdered the residue to obtain the reformulated turmeric extract.

One embodiment provides a method of increasing absorption of curcumin in blood by
administering the reformulated turmeric extract.

One embodiment provides a method of increasing blood level concentration of
curcumin by administering the reformulated turmeric extract.

One embodiment provides a method of increasing the retention time of curcumin in blood by administering the reformulated turmeric extract.

One embodiment provides a method of increasing the area under the curve of curcumin in blood by administering the reformulated turmeric extract.

The disclosed pharmaceutical composition of reformulated turmeric extract can be used for attaining a therapeutically significant level of curcumin in the human body for the treatment of various disease conditions. it will be readily understood by the skilled artisan that numerous alterations may be made to the examples and instructions given herein.

These and other objects and features of present invention will be made apparent from the following examples. The following examples as described are not intended to be construed as limiting the scope of the present invention.

EXAMPLES

Example 1

Method of preparation of reformulated turmeric extract
The turmeric rhizomes (1000Kg) were dried to obtain 250Kg and powdered by using pulverisor (stainless steel SS-304). The powdered turmeric of 10-20 mesh 250Kg was
extracted with four times with 1000L of n-hexane by using extractor with reflux condenser and fitted with polypropylene (l00microns) filter cloth of bottom of extractor. The extraction was carried out by refluxing at boiling temperatures of hexane of about 50° C to 70°C for one hour to obtain residue 231 Kg and hexane extract 15Kg. The residue and hexane extract were separated by draining out the hexane extract from the extractor bottom through the polypropylene filter cloth using a centrifugal pump. After first extraction, the residue was further extracted two more times with four times the quantity of n-hexane. The residue was dried by rotary vacuum drier to obtain 231 Kg dried material. The dried residue was extracted with 3 times the quantity of ethanol. The ethanol extraction of the residue was performed at 60-70 °C temperature for 1 hour using an extractor with reflux condenser. The extractor was fitted with polypropylene (100 microns) filter cloth at the bottom of extractor to separate the ethanol extract and the residue. The ethanol extract was concentrated at 60-70°C under vacuum (550mm Mg) to form ethanol extract concentrate containing Oleoresin (46 Kg). The oleo resin includes curcuminoids. The hexane extract was concentrated at 50-70°C under vacuum (500mm Hg) to form concentrated hexane extract 15Kg. Silica 30Kg (200-400mesh size) was mixed with the concentrated hexane extract (15Kg) in a Sigma Mixer-100kg (GMP) (manufacturer: Pharma fab industries, Mumbai, volume capacity: 300LTS, Working capacity: l00Kg) for one hour and the mixture was extracted with 30L 90% ethanol for 1 hour by using extractor with reflux condenser and fitted with polypropylene (l00microns) filter cloth of bottom of extractor. The silica was separated from the ethanol extract by draining out the ethanol extract from the extractor bottom through polypropylene filter cloth using a centrifugal pump. The ethanol extract was concentrated at 60-70°C under vacuum (550mm Hg) to form ethanol extract concentrate containing essential oil of turmeric (1.5 Kg).

The ethanol extract concentrate containing oleoresin and ethanol extract concentrate containing essential oil of turmeric was blended in 1:1 ratio (1.5 Kg Ethanol extract concentrate of essential oil and 1.5 Kg ethanol extract concentrate of Oleoresin) by using Double Cone Blender (stainless steel SS-316, capacity 2000 litre, manufacturer: Zebra Pharma, Mumbai) for 1 hour. Ethanol (2:1) was added to the blend and kept at 27 for four days. After four days, mixture was transferred to Plate & Frame Filter Press (polypropylene body, dimension: 2ftx2ftx8ft, manufacturer: HYDRO PRESS, Coimbatore) by using a stainless steel screw pump to separate residue (0.12Kg) and filtrate (resin and fat). The separated residue was dried by using rotary vacuum drier (700mm Hg) and the dried extract was powdered in a pulverisor to form fine powder of reformulated turmeric extract containing of curcumin, demethoxycurcumin, bisdemethoxy curcumin, Ar-turmerone and other components of essential oil of turmeric. 500mg of the reformulated turmeric extract powder had a curcumin content of 318 mg, demethoxycurcumin content of 104 mg, bisdemethoxycurcumin content of 32mg, Ar-turmerone content in the composition was 21 mg and other sesqiterpenoids of the essential oil in the composition was 25mg.

Example: 2

Method of preparation of capsules containing reformulated turmeric extract
A 500 mg capsule containing reformulated turmeric extract was prepared by encapsulating the reformulated turmeric extract powder from Example 1 into hard gelatin capsules. The encapsulation was done in a 21 °C air-conditioned and de- humidified room. 0.1 kg of powdered reformulated turmeric extract was charged into the hopper of a semi- automatic capsule filling machine. '0' size hard gelatin capsule shell was loaded to the tray and the powder was filled into the shell. The filled weight of capsules was checked simultaneously and these capsules were sorted by a sorting machine and polished with the help of a polishing machine to obtain 200 capsules of 500 mg of reformulated turmeric extract powder per capsule.

Example 3

Method of preparation of Regular turmeric extract
The rhizomes of turmeric (300Kg) were dried. The dried turmeric rhizomes were powdered to form powdered turmeric. The powdered turmeric was treated with ethyl acetate (900 L) to form a solution. The extraction was carried out at 78°C temperature for 1 hr. After initial extraction, the extraction process was repeated 4 more times and the resultant solution was filtered and the solvent was stripped from the filtered solution to form an extract. This extract was cooled to about 4° C to obtain crystals of curcuminoid (12Kg) and a liquid. The crystals of curcuminoid were isolated from the liquid by filtration. The crystals were powdered to form powdered curcuminoid mixture having 95% curcuminoids. The powdered curcuminoid mixture was referred to as regular turmeric extract.

Example 4

Method of preparation of capsules containing regular turmeric extract
A 500 mg capsule containing regular turmeric extract was prepared by encapsulating the powdered curcuminoid mixture having 95% curcuminoids from Example 3 into hard gelatin capsules. The encapsulation was done in a 21 °C air-conditioned and dehumidified room. 3kg of powdered curcuminoid mixture having 95% curcuminoids was charged into the hopper of a semi- automatic capsule filling machine. '0' size hard gelatin capsule shell was loaded to the tray and the powder was filled into the shell. The filled weight of capsules was checked simultaneously and these capsules were sorted by a sorting machine and polished with the help of a polishing machine to obtain 6000 capsules of 500 mg each.

Example 5

Method of preparation of raw turmeric powder
The raw turmeric rhizomes (10Kg) were collected and cleaned. The rhizomes were dried and pulverized to get turmeric powder (2.5Kg). The turmeric powder was sieved through 20 meshes to obtain the raw turmeric powder. The raw turmeric powder had 5% curcuminoids

Example 6
Method of preparation of capsules containing raw turmeric powder
A 500 mg capsule with raw turmeric powder (curcuminoids 5%) was prepared by encapsulating the raw turmeric powder from Example 5 into hard gelatin capsules. The encapsulation was done in an air-conditioned (21°C) and de- humidified room. 2.5 kg of raw turmeric powder was charged into the hopper of a semi- automatic capsule tilling machine. '0' size hard gelatin capsule shell was loaded to the tray and the raw turmeric powder was filled into the shell. The filled weight of capsules were checked simultaneously and these capsules were sorted by a sorting machine and polished with the help of a polishing machine to obtain 5000 capsules of 500 mg of raw turmeric powder per capsule.

Example 7
Method of preparation of Essential oil of turmeric with 10-15%Ar-turmerone
The rhizomes of turmeric (50Kg) were dried. The dried turmeric rhizomes were powdered to form powdered turmeric. The powdered turmeric was treated with ethyl acetate (150 L) to form a solution. The extraction was carried out at 78°C temperature for 1 hr. After initial extraction, the extraction process was repeated 4 more times on the powdered turmeric and the resultant solution was filtered. The solvent was stripped from the filtered solution to form an extract. This extract was cooled to about 4° C to obtain crystals of curcuminoid mixture (2Kg) and a liquid. The crystals of curcuminoid mixture were isolated from the liquid by filtration. The filtered liquid had essential oil of turmeric and a resin. The filtered liquid was then steam distilled to obtain 2.5 Kg of essential oil of turmeric. The essential oil of turmeric had 10-15 %Ar- turmerone.

Example 8
Method of preparation of capsules containing essential oil of turmeric with 10-15
% Ar-turmerone.

A 500 mg capsule with essential oil of turmeric containing 10-15% Ar- turmerone was prepared by encapsulating the essential oil of turmeric from Example 7 in soft gelatin capsules. The encapsulation was done in an air-conditioned (24°C) and relative humidity 40-60%. 2.5 kg essential oil of turmeric having 10-15 % Ar-turmerone was charged into the hopper of a semi- automatic capsule filling machine. '0' size soft gelatin capsule shell was loaded to the tray and the blended extract powder was filled into the shell. The filled weight of capsules was checked simultaneously and these capsules were sorted by a sorting machine and polished with the help of a polishing machine to obtain 5000 capsules of 500 mg essential oil of turmeric per capsule.

Example 9

Method of preparation of curcuminoid mixture alone
The rhizomes of turmeric (300Kg) were dried. The dried turmeric rhizomes were powdered to form powdered turmeric. The powdered turmeric was treated with ethyl acetate (900 L) to form a solution. The extraction was carried out at 78°C temperature for 1 hr. After initial extraction, the extraction process was repeated 4 more times and the resultant solution was filtered and the solvent was stripped from the filtered solution to form an extract. This extract was cooled to about 4° C to obtain crystals of curcuminoid mixture (12Kg) and a liquid. The crystals of curcuminoid mixture were isolated from the liquid by filtration. The crystals obtained were recrystallised in ethanol. The recrystallized crystals of curcuminoid mixture were separated from ethanol by filtration .The recrystallied crystals were powdered to form powdered curcuminoid mixture. The powdered curcuminoid mixture was found to contain about 97% curcuminoid mixture.

Example 10

Method of preparation of capsules containing curcuminoid mixture alone with
97% curcuminoid
A 500 mg capsule containing curcuminoid mixture alone was prepared by encapsulating the powdered curcuminoid mixture having 97% curcuminoids from Example 9 into hard gelatin capsules. The encapsulation was done in a 21 °C air-conditioned and de-humidified room.3 kg of powdered curcuminoid mixture having 97% curcuminoids was charged into the hopper of a semi- automatic capsule filling machine. '0' size hard gelatin capsule shell was loaded to the tray and the powder was filled into the shell. The filled weight of capsules was checked simultaneously and these capsules were sorted by a sorting machine and polished with the help of a polishing machine to obtain 6000 capsules of 500 mg each.

Example 11

Method of preparation of ethanol extract concentrate of Oleoresin
The turmeric rhizomes (500Kg) were dried to obtain 125Kg and powdered by using pulverisor (stainless steel SS-304). The powdered turmeric of 10-20 mesh 125Kg was extracted with four times with 500L of n-hexane. The extraction was carried out by refluxing at boiling temperatures of hexane of about 50° C to 70°C for one hour to obtain residue 115 Kg and hexane extract 7.5Kg. The residue and hexane extract were separated. After first extraction, the residue was further extracted two more times with four times the quantity of n-hexane. The residue was dried by rotary vacuum drier to obtain 115 Kg dried material. The dried residue was extracted with 3 times the quantity of ethanol. The ethanol extraction of the residue was performed at 60-70° C temperature for 1 hour using an extractor with reflux condenser. The ethanol extract and the residue were separated. The ethanol extract was concentrated at 60-70°C under vacuum

(550mm Hg) to form ethanol extract concentrate and powdered to form ethanol extract containing Oleoresin (23 Kg).

Example 12

Method of preparation of capsules containing ethanol extract concentrate of
Oleoresin having 94% curcuminoids

A 500 mg capsule containing ethanol extract concentrate of Oleoresin was prepared by encapsulating the powdered ethanol extract concentrate of Oleoresin having 94% curcuminoids from Example 11 into hard gelatin capsules. The encapsulation was done in a 21 °C air-conditioned and de- humidified room. 3 kg of powdered ethanol extract concentrate of Oleoresin having 94% curcuminoids was charged into the hopper of a semi- automatic capsule filling machine. '0' size hard gelatin capsule shell was loaded to the tray and the powder was filled into the shell. The filled weight of capsules was checked simultaneously and these capsules were sorted by a sorting machine and polished with the help of a polishing machine to obtain 6000 capsules of 500 mg each.

Example 13

Method of preparation of ethanol extract concentrate of Essential oil of turmeric
The turmeric rhizomes (lOOOKg) were dried to obtain 250Kg and powdered by using pulverisor (stainless steel SS-304). The powdered turmeric of 10-20 mesh 250Kg was extracted with four times with 1000L of n-hexane. The extraction was carried out by refluxing at boiling temperatures of hexane of about 50° C to 70°C for one hour to obtain residue 231 Kg and hexane extract 15Kg. The residue and hexane extract were
separated. After first extraction, the residue was further extracted two more times with four times the quantity of n-hexane.

The hexane extract was concentrated at 50-70°C under vacuum (500mm Hg) to form concentrated hexane extract 15Kg. Silica 30Kg (200-400mesh size) was mixed with the concentrated hexane extract (15Kg) in a Sigma Mixer-lOOkg (GMP) for one hour and the mixture was extracted with 30L 90% ethanol for 1 hour. The silica was separated from the ethanol extract by draining out the ethanol extract from the extractor bottom through polypropylene filter cloth using a centrifugal pump. The ethanol extract was concentrated at 60-70°C under vacuum (550mm Hg) to form ethanol extract concentrate containing essential oil (1.5 Kg).

Example: 14

Method of preparation of capsules containing ethanol extract concentrate of essential oil of turmeric

A 500 mg capsule containing ethanol extract concentrate of essential oil of turmeric was prepared by encapsulating the powdered ethanol extract concentrate of essential oil of turmeric from Example 13 into soft gelatin capsules. The encapsulation was done in an air-conditioned (24°C) and relative humidity 40-60%. 1 kg ethanol extract concentrate of essential oil of turmeric was charged into the hopper of a semi automatic capsule filling machine. '0' size soft gelatin capsule shell was loaded to the tray and the blended extract powder was filled into the shell. The filled weight of capsules was checked simultaneously and these capsules were sorted by a sorting machine and polished with the help of a polishing machine to obtain 2000 capsules of 500 mg essential oil of turmeric per capsule.

Example 15

Animal study to assess the efficacy of the disclosed composition of reformulated turmeric extract in blood, the concentration of curcumin in blood was compared to following administration of regular turmeric extract, raw turmeric powder, curcuminoid mixture alone, essential oil of turmeric having 10-15% Ar-turmerone, ethanol extract concentrate of oleoresin, ethanol extract concentrate of essential oil of turmeric and . reformulated turmeric extract.

Albino rats weighing 150 - 200 gm of both sexes were used for the study. The rats were kept individually in polypropylene cages and maintained in well ventilated room under normal and uniform conditions like 12 hours light and dark cycle and at 26 ± 2°C. Water and feed were given ad-libitum.The animals were divided into 8 groups and 3 animals were used for each group. The animals were divided as shown in table 1.

Table 1: Segregation of rats for experimental study.

Method

Group 1 animals consisted of animals which were given vehicle, DMSO. Regular turmeric extract having 95% curcuminoids of which curcumin73 %( 365mg), Demethoxy curcumin 16.2 %( 81 mg) and Bisdemethoxy curcumin 5.8 %( 29mg) (compound 1) was solubilized in DMSO and fed to group 2 animals as shown in table 1. This was the curcumin control. To group 3 animals, reformulated turmeric extract having Curcumin 63.6%(318mg), Demethoxycurcumin 20.8%(104mg), Bisdemethoxy Curcumin 6.4%(32mg), Ar-turmerone 4.2% (21mg) and other sesqiterpenoids of essential oil of turmeric 5% (25mg) (compound 2) was solubilized in DMSO and fed. Raw turmeric powder with 5% curcuminoids of which Curcumin 2.5% (12.5 mg), Demethoxycurcumin 1.6% (8mg)and bisdemethoxycurcuminO.9% (4.5mg) (compound 3) was mixed with DMSO and fed to group 4 animals. To Group 5 animals, curcuminoid mixture having 97% curcuminoid mixture of which curcumin 75% (375mg), Demethoxy curcumin 17.2% (86mg) and Bisdemethoxycurcumin 4.8%
(24mg) (compound 4) solubilized in DMSO was given. To Group 6 animals, essential oil of turmeric with 10- 15% Ar-turmerone (compound 5) in DMSO was given. To group 7 animals, ethanol extract concentrate of oleoresin having 94%curcuminoid mixture of which curcumin 72.3% (361.5mg), Demethoxy curcumin 17.2% ( 86mg) and Bisdemethoxy curcumin4.5% (22.5mg) (compound 6) solubilized in DMSO was given.

To group 8 animals, ethanol extract concentrate of essential oil of turmeric (compound 7) solubilized in DMSO was fed. The animals were able to access drinking water freely. The study drugs were given by oral route. Two hours post drug the blood was collected for analysis of curcumin levels in the blood by HPLC method. The blood was extracted exhaustively with ethyl acetate to recover curcumin. The ethyl acetate extract was analyzed by HPLC on a RP-C18 column (25x4.5mm) using methanol as solvent and UV detection at 420nm. The eluent flow rate was lml/min.

The results are summarized in Fig: 1 which shows the results of blood levels of
curcumin in albino rats.

The results as shown in Fig. 1 indicates that the level of curcumin in the blood was higher in albino rats fed with reformulated turmeric extract compared to rats fed with regular turmeric, curcuminoid mixture alone and ethanol extract concentrate of oleoresin. The blood level concentration of curcumin was very low for animals fed with raw turmeric powder. The curcumin content in the blood in animals fed with essential oil of turmeric with 10 -15% Ar-turmerone and ethanol extract concentrate of essential oil of turmeric were not detectable.

Example 16

Animal study to assess the efficacy of the disclosed composition of reformulated turmeric extract in absorption of curcumin compared to regular turmeric extract, raw turmeric powder, curcuminoid mixture alone, essential oil of turmeric with 10-15% Ar- turmerone, ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil of turmeric in rats

Albino rats weighing 150 - 200 gm of both sexes were used for the study. The rats were kept individually in polypropylene cages and maintained in well ventilated room under normal and uniform conditions like 12 hours light and dark cycle and at 26 + 2°C. Water and feed were given ad-libitum. The animals were divided into 8 groups and 2 animals were used for each group. The animals were divided as shown in table 2.

Table 2: Segregation of rats for experimental study.

Method

Group 1 animals consisted of animals which were given vehicle, DMSO. Regular
turmeric extract with 95% curcuminoids of which curcumin 73%(365 mg),Demethoxy
curcumin 16.2%(81mg) and Bisdemethoxy curcumin 5.8%(29 mg)(compound 1) was
solubilized in DMSO and fed to group 2 animals as shown in table 2. This was the
curcumin control. To group 3 animals, reformulated turmeric extract with having
Curcumin 63.6%(318mg),demethoxycurcumin 20.8%( 104mg), Bisdemethoxy
Curcumin 6.4%(32mg),Ar-turmerone 4.2%(21mg) and other sesqiterpenoids of essential oil of turmeric 5%(25mg) (compound 2) was solubilized in DMSO and fed. Raw turmeric powder with 5% curcuminoids of which Curcumin 2.5%(12.5mg), Demethoxycurcuminl.6%(8mg) and Bisdemethoxycurcumin 0.9% (4.5mg)(compound 3) was mixed with DMSO and fed to group 4 animals. To Group 5 animals, curcuminoid mixture having 97% curcuminoid mixture of which curcumin 75% (375mg), Demethoxycurcumin 17.2% (86mg) and Bisdemethoxycurcumin 4.8%
(24mg) (compound 4) solubilized in DMSO was given. To Group 6 animals, essential oil of turmeric with 10- 15% Ar-turmerone as in Example 7 (compound 5) in DMSO was given. To group 7 animals, ethanol extract concentrate of oleoresin having 94% curcuminoid mixture of which curcumin 72.3% (361.5mg), Demethoxy curcumin 17.2%(86mg) and Bisdemethoxycurcumin 4.5%(22.5.mg) (compound 6) solubilized in DMSO was given. To group 8 animals, ethanol extract concentrate of essential oil of turmeric (compound 7) solubilized in DMSO was fed. The animals were able to access drinking water freely. The study drugs were given by oral route. After 24 hrs the fecal matter from the eight groups were collected, dried and weighed accordingly. The amount of curcumin in fecal matter was determined by HPLC method on a RP-C18 column (25x4.5mm) using methanol as solvent and UV detection at 420nm. The eluent How rate was lml/min. The results are summarized in Table 3 which shows the results of curcuminoid absorption in albino rats.

Table: 3 Results of absorption of curcumin by albino rats

The results as shown in Table 3 indicate that the dietary absorption of curcumin from the regular turmeric extract powder(having 95% curcuminoid mixture) by albino rats was only 7.2mg, which corresponds to about 16.58% of the curcuminoid mixture, absorption of curcumin from ethanol extract concentrate of oleoresin (having 94% curcuminoid mixture) was only 6.2mg, which corresponds to about 15.2% of the curcuminoid mixture and curcuminoid mixture alone(having 97% curcuminoid mixture) was only 7.3mg, which corresponds to about 18.6%> of the curcuminoid mixture. Whereas the absorption of curcumin from the reformulated turmeric extract was increased to about 35.1mg, which corresponds to about 96.6% of the curcuminoid .The raw turmeric powder alone or essential oil of turmeric with 10-15% Ar-turmerone alone or ethanol extract concentrate of essential oil of turmeric or the addition of oil of ginger to curcuminoid mix did not significantly increase the absorption of curcumin. Thus the absorption of curcumin given orally can be significantly increased by the reformulated turmeric extract obtained as per Example 1

Example 17

Human study to assess the efficacy of the disclosed composition of reformulated turmeric extract compared to regular turmeric extract, raw turmeric powder, essential oil of turmeric with 10-15% Ar-turmerone, curcuminoid alone, ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil of turmeric. Twenty one healthy male human volunteers weighing 55-60 kg were enrolled in the study. They were randomly divided into seven groups of three subjects each. The volunteers abstained from the consumption of aspirin or other NSAIDS and all foods containing turmeric two days prior to the study date. Written informed consent was obtained from each participant before enrolment.

Group A: Subjects given four 500mg capsules of regular turmeric extract powder containing 95% curcuminoid mixture prepared as per Example 4 (Compound 1). Group B: Subjects given four 500mg capsules of the disclosed composition of reformulated turmeric extract prepared as per Example 2 (Compound 2). Group C: Subjects given four 500mg capsules of raw turmeric powder containing 5% curcuminoids prepared as per Example 6 (Compound 3).

Group D: Subjects given four 500mg capsules of essential oil of turmeric with 10-15% Ar-turmerone prepared as per Example 8 (Compound 4).

Group E: Subjects given four 500mg capsules of curcuminoid mixture alone with 97% curcuminoids prepared as per Example 10(Compoi»nd 5)

Group F: Subjects given four 500mg capsules of ethanol extract concentrate of oleoresin having 94% curcuminoids prepared as per Example 12 (Compound 6) Group G : Subjects given four 500mg capsules of ethanol extract concentrate of essential oil of turmeric prepared as per Example 14 (compound 7) After overnight fasting, subjects consumed four study drug capsules having a dosage of 500mg each to obtain a single dose of 2000 mg. Blood was drawn from each volunteer just prior to dosing and at 0.5, 1, 2, 3, 4, 5, 6, 8 and 10 hours post drug. After wash out period of one week the subjects crossed-over to the other drug as given below in table
A

Table 4:

The blood samples of patients at different time points were taken during the cross over study and the curcumin content in blood was analyzed in a HPLC. The blood was extracted exhaustively with ethyl acetate to recover curcumin. The ethyl acetate extract
was analyzed by HPLC on a RP-C18 column (25x4.5mm) using methanol as solvent and UV detection at 420nm. The eluent flow rate was lml/min. The level of curcumin in blood was plotted in y axis and the time period in hours is plotted in x axis and given in Fig: 2.

The curcumin content in the blood of volunteers given the reformulated turmeric extract, regular turmeric extract powder, raw turmeric powder, essential oil of turmeric with 10-15% Ar-turmerone, curcuminoid mixture alone, ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil of turmeric at given time point was taken and the average values of all such time points were taken and plotted on a
graph as shown in Fig: 2. The results showed that the level of curcumin in the blood was increased in subjects taking the reformulated turmeric extract compared to the regular turmeric extract powder , raw turmeric powder, essential oil of turmeric with 10-15%Ar-turmerone, curcuminoid mixture alone, ethanol extract concentrate of oleoresin and ethanol extract concentrate of essential oil of turmeric orally as capsules. The max level of curcumin in the blood after oral consumption of reformulated turmeric extract was 610 nmol/L, from regular turmeric extract powder was 63nmol/L and from raw turmeric powder was only 3.1nmol/L. The maximum level of curcumin in the blood after oral consumption of curcuminoid mixture alone was 73nmol/L and from ethanol extract concentrate of oleoresin was 59nmol/L. Curcurmin was not detected following administration of essential oil of turmeric having 10-15% Ar-turmerone and ethanol extract concentrate of essential oil of turmeric. The Cmax of curcumin from reformulated turmeric extract was 10 times greater than the Cmax of regular turmeric extract powder.

In subjects taking regular turmeric extract powder capsules, the level of curcumin in the blood was detected in small amounts and peaked at lhr following which the level decreased and touched baseline by 3 hours. In subjects taking curcuminoid mixture alone and ethanol extract concentrate of oleoresin capsules, the maximum level of curcumin in the blood was found in second hour. In subjects taking raw turmeric powder, small detection of curcumin was observed at half and third hour. In subjects taking essential oil of turmeric with 10 -15% Ar-turmerone capsules and ethanol extract concentrate of essential oil of turmeric, the level of curcumin in the blood was not detectable. But in subjects given oral reformulated turmeric extract capsules, the level of curcumin in blood increased sharply within 30 minutes of administration and reached peak at 4.5 hours with a Cmax of 610 nmol/L. Following administration of reformulated turmeric extract, the level of curcumin in blood was retained for 10 hours post drug with a gradual decline in the curcumin concentration.

The area under the curve from the average graph plotted for subjects taking reformulated turmeric extract was 14 times more than the AUC of concentration time curve of curcumin in subjects given oral dose of regular turmeric extract powder capsules.

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I claim:

1. A reformulated turmeric extract comprising:
a 1:1 ratio of ethanol extract concentrate of oleoresin, and, ethanol extract
concentrate of essential oil,
wherein the ethanol extract concentrate of oleoresin comprises curcumin,
demethoxycurcumin and bisdemethoxycurcumin, and,
wherein the ethanol extract concentrate of essential oil comprises essential oil of
turmeric having of Ar-turmerone.

2. The reformulated turmeric extract of claim 1, wherein curcumin ranges from
about 58 % to about 75 %.

3. The reformulated turmeric extract of claim 1, wherein demethoxycurcumin ranges from about 12 % to about 25 %.

4. The reformulated turmeric extract of claim 1, wherein bisdemethoxycurcumin ranges from about 1.6 % to about 8 %.

5. The reformulated turmeric extract of claim 1, wherein Ar-turmerone ranges from about 1.2 % to about 7.2 %.

6. The reformulated turmeric extract of claim 1, wherein curcumin ranges from
about 290 mg to about 375 mg.

7. The reformulated turmeric extract of claim 1, wherein demthoxycurcumin ranges from about 60 mg to about 125 mg.

8. The reformulated turmeric extract of claim 1, wherein bisdemethoxycurcumin ranges from about 8 mg to about 40 mg.

9. The reformulated turmeric extract of claim 1, wherein ar-turmerone ranges from about 6 mg to about 36 mg.

10. The reformulated turmeric extract of claim 1, wherein the ethanol extract concentrate of oleoresin is prepared by the process comprising:
powdering dried rhizomes of turmeric to form turmeric powder,
extracting the turmeric powder with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain a first residue and a n-hexane extract,
separating the first residue and the n-hexane extract,
drying the first residue to obtain a second dried residue,
extracting the second dried residue with ethanol to obtain an ethanol extract and a third residue, and,
concentrating the ethanol extract to obtain the ethanol extract concentrate of oleoresin.

11. The reformulated turmeric extract of claim 1, wherein the ethanol extract
concentrate of essential oil is prepared by the process comprising:
powdering dried rhizomes of turmeric to form turmeric powder,
extracting the turmeric powder with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain a residue and a n-hexane extract,
separating the residue and the n-hexane extract,
concentrating the n-hexane extract to form a concentrated hexane extract,
mixing the concentrated hexane extract with ethanol in the presence of silica to obtain an ethanol extract, and,
concentrating the ethanol extract to obtain the ethanol extract concentrate of essential oil.

12. A hard gelatin capsule comprising the reformulated turmeric extract of claim 1.

13. The reformulated turmeric extract of claim 1, wherein oral administration of the reformulated turmeric extract results in increase in absorption of curcumin ranging from about 2 fold to about 40 fold as compared to oral administration of raw turmeric, essential oil of turmeric, regular turmeric extract, ethanol extract concentrate of oleoresin or ethanol extract concentrate of essential oil.

14. The reformulated turmeric extract of claim 1, wherein oral administration of the reformulated turmeric extract results in about a 10-fold increase in blood level of curcumin compared to oral administration of regular turmeric extract,
wherein the regular turmeric extract comprises curcumin, demethoxycurcumin and bisdemethoxycurcumin.

15. The reformulated turmeric extract of claim 1, wherein oral administration of the reformulated turmeric extract results in retention of curcumin in blood for 10
hours.

16. The reformulated turmeric extract of claim 1, wherein oral administration of the reformulated turmeric extract results in about a 14-fold increase in area under the curve (AUC)nanograms of curcumin per gram of blood as compared to oral administration of regular turmeric extract, wherein the regular turmeric extract comprises curcumin, demethoxycurcumin and bisdemethoxycurcumin.

1 7. A method of preparing reformulated turmeric extract comprising:
mixing an ethanol extract concentrate of oleoresin and an ethanol extract concentrate of essential oil in a 1:1 ratio to obtain a blend,
adding ethanol to the blend in 2:1 ratio at 27°C to obtain a residue and a filtrate,
and,
drying and powdering the residue to obtain the reformulated turmeric extract,
whereinthe ethanol extract concentrate of oleoresin comprises curcumin,
demethoxycurcumin and bisdemethoxycurcumin, and,
wherein, the ethanol extract concentrate of essential oil comprises essential oil
of turmeric having Ar-turmerone.

18. The method of claim 17, wherein the ethanol extract concentrate of oleoresin is
prepared by the method comprising:
powdering dried rhizomes of turmeric to form turmeric powder,
extracting the turmeric powder with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain first residue and a n-hexane extract,
drying the first residue to obtain a second dried residue, extracting the second dried residue with ethanol to obtain a first ethanol extract and a third residue, and,
concentrating the first ethanol extract to obtain the ethanol extract concentrate of oleoresin.

19. The method of claim 17, wherein the ethanol extract concentrate of essential oil
is prepared by the process comprising:
powdering dried rhizomes of turmeric to form turmeric powder, extracting the turmeric powder with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain a fourth residue and a n-hexane extract,
concentrating the n-hexane extract to form a concentrated hexane extract, mixing the concentrated hexane extract with ethanol in the presence of silica to obtain a second ethanol extract,
concentrating the second ethanol extract to obtain an ethanol extract concentrate of essential oil.

20. A method of preparing a hard gelatin capsule comprising reformulated turmeric
extract, the method comprising:
mixing an ethanol extract concentrate of oleoresin and an ethanol extract
concentrate of essential oil in a 1:1 ratio to obtain a blend,
adding ethanol to blend in 2:1 ratio at 27°C to obtain a residue and a filtrate,
drying and powdering the residue to obtain the reformulated turmericextract,
and,
encapsulating the reformulated turmeric extract into a hard gelatin capsule,
whereinthe ethanol extract concentrate of oleoresin comprises curcumin,
demethoxycurcumin and bisdemethoxycurcumin,
wherein the ethanol extract concentrate of essential oil, comprises essential oil
of turmeric having Ar-turmerone.

21. The method of preparing a hard gelatin capsule comprising reformulated turmeric extract of claim 20, wherein the ethanol extract concentrate of oleoresin is prepared by the process comprising:
powdering dried rhizomes of turmeric to form turmeric powder,
extracting the turmeric powder with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain a first residue and a n-hexane extract,
drying the first residue to obtain a second dried residue,
extracting the second dried residue with ethanol to obtain a first ethanol extract and a third residue, and,
concentrating the first ethanol extract to obtain the ethanol extract concentrate of oleoresin.

22. The method of preparing a hard gelatin capsule comprising reformulated
turmeric extract of claim 20, wherein the ethanol extract concentrateof essential
oil is prepared by the process comprising:
powdering dried rhizomes of turmeric to form turmeric powder,
extracting the turmeric powder with n-hexane by adding n-hexane to the turmeric powder in a ratio of 4:1 of n-hexane to turmeric powder to obtain a fourth residue and a n-hexane extract,
concentrating the n-hexane extract to form a concentrated hexane extract,
mixing the concentrated hexane extract with ethanol in the presence of silica to obtain a second ethanol extract,
concentrating the second ethanol extract to obtain an ethanol extract concentrate of essential oil.

23. A method of increasing absorption of curcumin in blood comprising administering the reformulated turmeric extract of claim 1.

24. A method of increasing blood level concentrationof curcumin comprising administering the reformulated turmeric extract of claim 1.

25. A method of increasing the retention time of curcumin in blood comprising administering the reformulated turmeric extract of claim 1.

26. A method of increasing the area under the curve of curcumin in blood comprising administering the reformulated turmeric extract of claim 1.

Documents

Name	Date
2356-CHE-2010 FORM-5 11-08-2011.pdf	2011-08-11
2356-CHE-2010 DRAWINGS 11-08-2011.pdf	2011-08-11
2356-CHE-2010 FORM-3 11-08-2011.pdf	2011-08-11
2356-CHE-2010 FORM-2 11-08-2011.pdf	2011-08-11
2356-CHE-2010 CORRESPONDENCE OTHERS 11-08-2011.pdf	2011-08-11
2356-CHE-2010 ABSTRACT 11-08-2011.pdf	2011-08-11
Form-1.docx	2011-09-04
2356-CHE-2010 CLAIMS 11-08-2011.pdf	2011-08-11
Drawings.pdf	2011-09-04
2356-CHE-2010 FORM-18 12-12-2011.pdf	2011-12-12
2356-CHE-2010 CORRESPONDENCE OTHERS 12-12-2011.pdf	2011-12-12
2356-CHE-2010-Power of Attorney-080216.pdf	2016-03-05
2356-CHE-2010-Form 2(Title Page)-080216.pdf	2016-03-05
2356-CHE-2010-Form 13-080216.pdf	2016-03-05
2356-CHE-2010-Form 1-080216.pdf	2016-03-05
2356-CHE-2010 DESCRIPTION (COMPLETE) 11-08-2011.pdf	2011-08-11
2356-CHE-2010-Amended Pages Of Specification-080216.pdf	2016-03-05
2356-CHE-2010-Abstract-080216.pdf	2016-03-05
2356-CHE-2010-Examination Report Reply Recieved-080216.pdf	2016-03-05
2356-CHE-2010-Claims-080216.pdf	2016-03-05
Correspondence by Applicant_National Bio-Diversity_20-06-2017.pdf	2017-06-20
2356-CHE-2010_EXAMREPORT.pdf	2016-07-02
2356-CHE-2010-HearingNoticeLetter.pdf	2018-01-25
2356-CHE-2010-ExtendedHearingNoticeLetter_26Mar2018.pdf	2018-02-27
2356-CHE-2010-ExtendedHearingNoticeLetter_24Apr2018.pdf	2018-03-26
2356-CHE-2010-REQUEST FOR ADJOURNMENT OF HEARING UNDER RULE 129A [23-02-2018(online)].pdf	2018-02-23
2356-CHE-2010-REQUEST FOR ADJOURNMENT OF HEARING UNDER RULE 129A [23-03-2018(online)].pdf	2018-03-23
2356-CHE-2010-Response to office action (Mandatory) [28-06-2018(online)].pdf	2018-06-28
Marked up Claims_Granted 321323_25-09-2019.pdf	2019-09-25
2356-CHE-2010-Annexure (Optional) [28-06-2018(online)].pdf	2018-06-28
Drawings_Granted 321323_25-09-2019.pdf	2019-09-25
Claims_Granted 321323_25-09-2019.pdf	2019-09-25
2356-CHE-2010-Written submissions and relevant documents (MANDATORY) [03-05-2018(online)].pdf	2018-05-03
Abstract_Granted 321323_25-09-2019.pdf	2019-09-25
2356-CHE-2010-PETITION u-r 6(6) [23-02-2021(online)].pdf	2021-02-23
2356-CHE-2010-Covering Letter [23-02-2021(online)].pdf	2021-02-23
2356-CHE-2010-RELEVANT DOCUMENTS [27-09-2021(online)].pdf	2021-09-27
2356-CHE-2010-IntimationOfGrant25-09-2019.pdf	2019-09-25
2356-CHE-2010-PETITION u-r 6(6) [27-09-2021(online)].pdf	2021-09-27
2356-CHE-2010-Covering Letter [27-09-2021(online)].pdf	2021-09-27
2356-CHE-2010-FORM-28 [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-FORM-16 [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-PatentCertificate25-09-2019.pdf	2019-09-25
Description_Granted 321323_25-09-2019.pdf	2019-09-25
2356-CHE-2010-FORM FOR SMALL ENTITY [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-NBA Approval Submission(Mandatory) [24-09-2019(online)].pdf	2019-09-24
2356-CHE-2010-POWER OF AUTHORITY [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-EVIDENCE FOR REGISTRATION UNDER SSI [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-ASSIGNMENT WITH VERIFIED COPY [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010 DRAWINGS 11-08-2011.pdf	2011-08-11
2356-CHE-2010 FORM-5 11-08-2011.pdf	2011-08-11
2356-CHE-2010 FORM-3 11-08-2011.pdf	2011-08-11
2356-CHE-2010 CORRESPONDENCE OTHERS 11-08-2011.pdf	2011-08-11
2356-CHE-2010 DESCRIPTION (COMPLETE) 11-08-2011.pdf	2011-08-11
Drawings.pdf	2011-09-04
2356-CHE-2010 ABSTRACT 11-08-2011.pdf	2011-08-11
2356-CHE-2010 CORRESPONDENCE OTHERS 12-12-2011.pdf	2011-12-12
2356-CHE-2010 FORM-18 12-12-2011.pdf	2011-12-12
2356-CHE-2010-Power of Attorney-080216.pdf	2016-03-05
Form-1.docx	2011-09-04
2356-CHE-2010-Form 13-080216.pdf	2016-03-05
2356-CHE-2010-Examination Report Reply Recieved-080216.pdf	2016-03-05
2356-CHE-2010-Amended Pages Of Specification-080216.pdf	2016-03-05
Correspondence by Applicant_National Bio-Diversity_20-06-2017.pdf	2017-06-20
2356-CHE-2010_EXAMREPORT.pdf	2016-07-02
2356-CHE-2010-Claims-080216.pdf	2016-03-05
2356-CHE-2010-Abstract-080216.pdf	2016-03-05
2356-CHE-2010-ExtendedHearingNoticeLetter_26Mar2018.pdf	2018-02-27
2356-CHE-2010-HearingNoticeLetter.pdf	2018-01-25
2356-CHE-2010-REQUEST FOR ADJOURNMENT OF HEARING UNDER RULE 129A [23-02-2018(online)].pdf	2018-02-23
2356-CHE-2010-Response to office action (Mandatory) [28-06-2018(online)].pdf	2018-06-28
2356-CHE-2010-ExtendedHearingNoticeLetter_24Apr2018.pdf	2018-03-26
2356-CHE-2010-REQUEST FOR ADJOURNMENT OF HEARING UNDER RULE 129A [23-03-2018(online)].pdf	2018-03-23
Claims_Granted 321323_25-09-2019.pdf	2019-09-25
Marked up Claims_Granted 321323_25-09-2019.pdf	2019-09-25
2356-CHE-2010-Annexure (Optional) [28-06-2018(online)].pdf	2018-06-28
Drawings_Granted 321323_25-09-2019.pdf	2019-09-25
2356-CHE-2010-PatentCertificate25-09-2019.pdf	2019-09-25
2356-CHE-2010-IntimationOfGrant25-09-2019.pdf	2019-09-25
2356-CHE-2010-NBA Approval Submission(Mandatory) [24-09-2019(online)].pdf	2019-09-24
2356-CHE-2010-Covering Letter [23-02-2021(online)].pdf	2021-02-23
2356-CHE-2010-PETITION u-r 6(6) [23-02-2021(online)].pdf	2021-02-23
Abstract_Granted 321323_25-09-2019.pdf	2019-09-25
2356-CHE-2010-PETITION u-r 6(6) [27-09-2021(online)].pdf	2021-09-27
2356-CHE-2010-Covering Letter [27-09-2021(online)].pdf	2021-09-27
2356-CHE-2010-RELEVANT DOCUMENTS [27-09-2021(online)].pdf	2021-09-27
2356-CHE-2010-Written submissions and relevant documents (MANDATORY) [03-05-2018(online)].pdf	2018-05-03
2356-CHE-2010-FORM-16 [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-FORM-28 [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-FORM FOR SMALL ENTITY [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-RELEVANT DOCUMENTS [15-09-2022(online)].pdf	2022-09-15
2356-CHE-2010-POWER OF AUTHORITY [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-EVIDENCE FOR REGISTRATION UNDER SSI [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-ASSIGNMENT WITH VERIFIED COPY [03-11-2021(online)].pdf	2021-11-03
2356-CHE-2010-Form 1-080216.pdf	2016-03-05
2356-CHE-2010 FORM-2 11-08-2011.pdf	2011-08-11
2356-CHE-2010 CLAIMS 11-08-2011.pdf	2011-08-11
Description_Granted 321323_25-09-2019.pdf	2019-09-25
2356-CHE-2010-Form 2(Title Page)-080216.pdf	2016-03-05

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