DESC:1
FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
THE 5 PATENT RULES, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
10 “Curcumin compositions for osteoarthritis and joint wellness”
OMNIACTIVE HEALTH TECHNOLOGIES LIMITED
An Indian Company, registered under the Indian Companies Act, 1956 having its registered
office located at OmniActive Health Technologies Limited, T-8b, 5th Floor, Phoenix House,
15 A wing, Phoenix Mills Compound, 462, Senapati Bapat Marg, Lower Parel,
Mumbai – 400013 Maharashtra, India,
The following specification particularly describes the invention and the manner in which it is to
be performed.
20
2
Field of the Present Invention:
The present invention is related to the stable curcumin composition for prevention, improvement
and maintenance of arthritis more particularly to osteoarthritis. More specifically stable curcumin
compositions comprising curcuminoids in different polymorphic forms 5 like amorphous and
crystalline and/or in selective ratio and improved the stability of curcumin in alkaline pH
environment of the intestine for enhancement in bioavailability.
Background of the present Invention:
10 Arthritis is a more specific term that implies damage or inflammation in one or more joints. The
condition is often manifested by pain, swelling, heat, redness and limitation of movement. Knee
osteoarthritis is a type of disease that results from the degradation of articular cartilage between
the bones. As a result of this reduction in cartilage, there is more friction between the bones and
results in painful bone spurs.
15 Curcumin is derived from the rhizome of Curcuma longa and has been traditionally used in the
treatment of inflammation, skin wounds, tumors etc. Overall, curcumin is associated with several
health claims, but its therapeutic use is limited due to its low bioavailability, poor aqueous
solubility, instability at neutral and basic pH, poor absorption, rapid metabolism, and short halflife.
Curcumin is a class IV drug (low solubility and low permeability) based on the bio
20 pharmaceutics classification system (BCS). Many strategies have been developed to overcome
these limitations, particularly for oral delivery systems.
US2016089343A1 disclosed herein are formulations for the local delivery of therapeutically
effective doses of curcumin that provide sufficient serum levels of curcumin to treat diseases such
as head and neck disorders and upper aerodigestive disorders.
25 EP3275430A1 relates to an aqueous solution for intravenous infusion containing curcumin and /
or one or more curcumin derivatives, dimethylsulfoxide, one or more solubilizers, sodium selenite,
a buffer in an aqueous infusion medium, as well as a concentrate containing for their preparation
curcumin and / or one or more curcumin, dimethyl sulfoxide, one or more solubilizing agents and
sodium selenite.
3
In K .N. Pushpakumari International journal of pharmaceutical sciences and research an impact
factor (2019): 1.230 cite score (2017): 0.27), The comparative data of bio-absorption of two
turmeric formulations containing curcuminoids in a varied composition compared to regular
turmeric extract. The document is completely silent over the effect of curcumin on osteoarthritis.
In order to find a satisfactory solution to the persisting problem, there is a strong 5 felt need to
develop the stable curcumin formulation for treatment, prevention and maintenance of arthritis
more specifically to osteoarthritis. The stable curcumin compositions comprising curcuminoids in
different polymorphic forms like amorphous and crystalline and/or in selective ratio for
enhancement in bioavailability. The stable curcumin composition is comprised of curcuminoids
10 either alone or along with at least one pharmaceutically and/or nutraceutically accepted excipient
to form stable curcumin compositions having enhanced absorption and bioavailability.
Objectives of the present invention
The main objective of the present invention is to develop stable curcumin formulation for
15 prevention, improvement and maintenance of arthritis.
Another objective of the present invention is to provide the stable curcumin formulation having
enhancement in bioavailability which is available in an orally administered form.
20 Another objective of the present invention is to develop the stable curcumin formulation
comprising curcuminoids in different polymorphic form like amorphous and crystalline and/or in
selective ratio for enhancement in absorption and bioavailability.
Another objective of the present invention is to develop the stable curcumin formulation
25 comprising curcuminoids either alone and/or along with at least one pharmaceutically and/or
nutraceutically accepted excipient to form stable curcumin compositions having enhancement in
absorption and bioavailability.
4
Another objective of the present invention is to develop the stable curcumin formulation
comprising of curcuminoids either alone and/or along with at least one pH modifier and stabilizer
optionally with hydrophilic carrier, antioxidant, diluents, anticaking agent, emulsifier, fat and
surfactant to form stable curcumin compositions having enhancement in absorption and
5 bioavailability.
Further aspect of the present invention is to provide a process for preparation of the stable curcumin
composition suitable for formulating into tablets, capsules, blended powders, licaps, ointments,
pastes, lotions, liniments, mouthwashes, gargles, consumable dry syrups, liquid syrups, health
10 drinks, diet drinks, fruit juices, soft drinks and the like.
One more objective of the present invention is to provide stable curcumin compositions, use for
prevention, improvement and maintenance arthritis more particularly to osteoarthritis and its
associated conditions like articulate cartilage, joint mobility, joint flexibility, reduction in joint
15 stiffness and reduction in inflammation.
Further objective of the present invention is to provide stable curcumin compositions, use for
improvement in muscle strength and muscle performance, reduction in muscle soreness and
muscle damage and promote faster muscle recovery.
20
Summary of the present invention
According to an aspect of the present invention there is provided a stable curcumin composition
having enhanced bioavailability comprising;
i) Curcumin containing crystalline polymorphic form of curcuminoids in the range of 0
25 to 15 % and amorphous polymorphic form of curcuminoids in the range of 85 to 100
% and
ii) At least one or more pharmaceutically or nutraceutically accepted ingredient selected
from hydrophilic carrier, antioxidant, stabilizer, pH Modifier, solubilizer, fat,
anticaking agent
5
Brief description of the accompanying figures
Figure 1:X-ray diffraction (XRD) graph of the composition prepared as per example 01
Figure 2:X-ray diffraction (XRD) graph of the composition prepared as per example 02
Figure 3:X-ray diffraction (XRD) graph of the composition prepared 5 as per example 03
Figure 4:X-ray diffraction (XRD) graph of the composition prepared as per example 04
Figure 5. Effects of curcumin (Cur) on knee joint protein expression of IL-ß (A), IL-6 (B), TNF-
a (C) and NF-?ß (D) levels in monosodium iodoacetate (MIA) induced osteoarthritis (OA) rats.
The bars point out the standard deviation. Blots were repeated at least three times (n = 3) and a
10 representative blot is shown. Asterisks above the lines indicate statistical differences among the
groups (ANOVA and Turkey's post-hoc test; **P<0.01; ***P<0.001; **** P<0.0001). OA,
osteoarthritis; Cur, curcumin; MIA, monosodium iodoacetate; IL-1ß, interleukin-1ß; IL-6,
interleukin-6; TNF- a, tumor necrosis factor a; NF-?ß, nuclear factor kappa ß.
Figure 6. Effects of curcumin (Cur) on knee joint protein expression of collagen type 2 (A), MMP-
15 3 (B), COX-2 (C) and LOX-5 (D) levels in monosodium iodoacetate (MIA) induced osteoarthritis
(OA) rats. The bars point out the standard deviation. Blots were repeated at least three times (n = 3)
and a representative blot is shown. Asterisks above the lines indicate statistical differences among
the groups (ANOVA and Turkey's post-hoc test; **P<0.01; ***P<0.001; **** P<0.0001). OA,
osteoarthritis; Cur, curcumin; MIA, monosodium iodoacetate; MMP-3, matrix metalloproteinase-
20 3; COX-2, cyclooxygenase-2; LOX-5, 5-lipoxygenase.
Figure 7. Effects of curcumin (Cur) on the knee joint in monosodium iodoacetate (MIA) induced
osteoarthritis (OA) rats. Representative radiographic images (A) obtained at the end of the
experiment are shown. Mean values of Kellgren- Lawrence scores are demonstrated with ±
25 standard deviations (B). Asterisks indicate statical differences of Kellgren-Lawrence scores among
the groups represented with bar (Kruskal-Wallis followed by Mann-Whitney U; *P<0.05 **P<0.01;
compared as OA group).
6
Figure 8. Effects of curcumin (Cur) on histopathology of the knee joint in monosodium
iodoacetate (MIA) induced osteoarthritis (OA) rats. Representative histopathologic images of
hematoxylin-eosin (A) and toluidin blue (B) staining obtained at the end of the experiment are
shown. Mean values of Mankin scores are demonstrated with ± standard deviations. Asterisks
above the line indicate statical differences of Mankin scores among the groups 5 represented (C)
with bar (Kruskal-Wallis followed by Mann-Whitney U; *P<0.05; compared as OA group).
Figure 9. Effects of curcumin (Cur) on knee swelling (A), left knee joint diameter (B) right knee
joint diameter (C) and the ratio of right to left diameter values (D) in monosodium iodoacetate
(MIA) induced osteoarthritis (OA) rats. Asterisks above the lines indicate statistical differences
10 among the groups (ANOVA and Turkey's post-hoc test; **P<0.01; ***P<0.001; **** P<0.0001).
Figure 10. Effects of curcumin (Cur) on paw area (B) and stride length (D) in monosodium
iodoacetate (MIA) induced osteoarthritis (OA) rats. Representative measures of paw area (A) and
stride length (C) are shown. Asterisks above the lines indicate statistical differences among the
groups (ANOVA and Turkey's post-hoc test; *P<0.05; **P<0.01; ***P<0.001; **** P<0.0001).
15 Figure11: Plasma total curcuminoids concentration of Curcumin composition vs Reference
Detailed description of the present invention
According to embodiment of the present invention, the stable curcumin formulation comprises
20 curcuminoids in different polymorphic form like amorphous and crystalline in selective ratio for
enhancement in bioavailability. According to further embodiment of the present invention the
stable curcumin formulation comprising curcuminoids either alone and/or along with at least one
excipient to form stable curcumin composition having enhancement in absorption and
bioavailability for prevention, improvement and maintenance of arthritis more particularly to
25 osteoarthritis.
Within the context of this invention the terminology the “curcumin composition” is commonly
used in the specification to refer composition comprising curcuminoids in different polymorphic
form like amorphous and crystalline in selective ratio either alone and/or along with at least one
7
excipient to form stable curcumin composition having enhancement in absorption and
bioavailability.
Curcumin (1, 7 bis (4-hydroxy-3-methoxy phenyl)-l, 6 heptadiene-3, 5- dione), is a principal
curcuminoid of the popular Indian spice Curcuma longa commonly known as turmeric, 5 a perennial
herb of Zingiberaceae (ginger) family. It is to be appreciated that the term “curcumin” can be
interpreted to be within the scope of the term curcuminoids, which can in general include
components of curcumin such as curcumin, demethoxy curcumin and bisdemethoxy curcumin.
Commercial products which may be referred to as “curcumin” may have these components, along
10 with other components belonging to the class curcuminoids. The curcumin extract contains total
curcuminoids in the range of 40-99%.
According to further embodiment of the present invention the curcumin composition comprising
curcuminoids present in the range of 10-90 % w/w of the composition.
15
According to further embodiment of the present invention the stable curcumin formulation
comprising curcuminoids either alone and/or along with at least one excipient to form stable
curcumin composition having enhancement in absorption and bioavailability. More preferably,
stable curcumin composition is formulated using excipients selected from the group of, but not
20 limited to a hydrophilic carrier, an antioxidant, solvent , emulsifier, surfactant, solubilizer,
stabilizer, pH modifier, binder, anticaking agent, fat and/or the combination thereof.
In one more embodiment, the solid hydrophilic carrier employed in preparation of curcumin
compositions is selected from the group such as, but not limited to, cellulose derivatives,
25 polyacrylates, polyethylene glycols, povidones, starch and starch derivatives, gums, sugars, pectin,
plant polysaccharides, protein such as casein, whey protein, soy protein, pea protein, sugar alcohol
,glucose, polyvinyl pyrrolidone, and the like.
According to preferred embodiment the hydrophilic carrier is Hydroxypropyl methyl cellulose.
According to further embodiment, the hydrophilic carrier is present in the range of 1-90 % w/w of
30 the composition.
8
In one more embodiment, the antioxidant in preparation of curcumin compositions is selected from
the group such as, but not limited to natural tocopherols, mixed tocopherol, ascorbyl palmitate,
rosemary extract, epigallocatechin gallate, catechins, and ascorbic acid.
According to further embodiment, the antioxidant is present in the range of 0.1-5 10 % w/w of the
composition.
In one more embodiment, the solvent in preparation of stable curcumin composition is selected
from the group such as, but not limited to, isopropyl alcohol, acetone, methanol, alcohol, ethyl
10 acetate, ethanol, methylene dichloride, water and mixtures thereof. The temperature maintained
for obtaining a homogenous mass may range from ambient to 80°C; preferably 25°C to 60°C.
According to further embodiment, the solvent is present in the range of 40-98 % w/w of the
composition.
15
In one more embodiment, the emulsifier in preparation of stable curcumin composition is selected
from the group such as, but not limited to polysorbate, sugar alcohols, glycerol & derivatives
thereof.
20 According to further embodiment, the emulsifier is present in the range of 0.1-10 % w/w of the
composition.
In one more embodiment, the surfactant in preparation of stable curcumin composition is selected
from the group such as, but not limited to sugar alcohols, sugar esters (such as sucrose stearate and
25 others), esters of sugar alcohol (such as sorbitan and others)
According to further embodiment, the surfactant is present in the range of 0.1-10 % w/w of the
composition.
In one more embodiment, the solubilizer in preparation of curcumin compositions is selected from
30 the group such as, but not limited to propylene glycol alginate, sugar alcohols, sugar esters,
9
phospholipid, Lecithin, d-limonene, Vitamin E TPGS (d-a-Tocopheryl polyethylene glycol 1000
succinate), sodium lauryl sulphate and beta cyclodextrin.
According to further embodiment, the solubilizer is present in the range of 0.1-40 % w/w of the
5 composition.
In one more embodiment, the stabilizer in preparation of curcumin compositions is selected from
the group such as, but not limited to glyceryl monostearate, sugar alcohols, triglycerides,
antioxidants, monoglycerides and phospholipids.
10
According to further embodiment, the stabilizer is present in the range of 0.1-10 % w/w of the
composition.
In one more embodiment, the pH modifier in preparation of curcumin compositions is selected
15 from the group such as, but not limited to citric acid, trisodium citrate, lactic acid, L-arginine,
calcium carbonate and magnesium carbonate.
According to further embodiment, the pH modifier is present in the range of 0.1-10 % w/w of the
composition.
20
In one more embodiment, the binder in preparation of curcumin compositions is selected from the
group such as, but not limited to hydroxy propyl cellulose, pregelatinized and starch.
According to further embodiment, the binder is present in the range of 0.1-10 % w/w of the
25 composition.
In one more embodiment, the anticaking agent in preparation of curcumin compositions is selected
from the group such as, but not limited to colloidal silicon dioxide, magnesium stearate, stearic
acid, and mannitol.
30
10
According to further embodiment, the anticaking agent is present in the range of 0.1-10 % w/w of
the composition.
In one more embodiment, the fat employed in preparation of curcumin compositions is selected
from the group such as, but not limited to, medium chain triglycerides, long chain 5 triglycerides,
vegetable oils, esters of fatty acids, hydrocarbons such as terpenes, monoglycerides
According to further embodiment, the fat is present in the range of 0.1-40 % w/w of the
composition.
10 According to further embodiment of the present invention stable curcumin composition described
herein exhibit enhanced bioavailability and the compositions can be available in orally
administrable solid, semisolid, liquid forms, selected from, but not limited to dosages such as,
powders, granules, pellets, beadlets, caplets, tablets, capsules, soft gel capsules, solution,
emulsions, suspensions, dispersions and the like.
15
According to one more embodiment of the present invention the curcumin composition is in the
form of granule.
According to one more embodiment of the present invention the curcumin composition is in the
20 form of powder.
In some embodiment, a process for the preparation of a stable curcumin composition having
enhancement in stability and bioavailability use for osteoarthritis comprises:
(i) Dissolving one or more excipients in solvent or mixture thereof with continue stirring for 10-
25 15 min at 50-80 °C;
(ii) Dissolving curcumin in solvent or mixture thereof with continue stirring for 5-10 min at 50-
80°C followed by cool down to temperature to 30-40°C; and
(iii) adding, other excipients such as hydrophilic carrier, an antioxidant, diluents, stabilizer, pH
modifier, anticaking agent, fat, emulsifier and surfactant, in a solvent to form a homogenous mass;
30 (iv) removing the solvent by evaporation to form a dry mass; and
(v) pulverizing the dry mass to form a fine powder.
11
The removal of solvent in step (ii) can be performed in vacuum distillation or evaporation
technique, or by spray drying technique. The resultant dry mass is pulverized by using, for
example, mortar and pestle, mixer-grinder, multi-mill, ball mill, jet mill and the like.
5
According to further embodiment of the present invention provides the stable curcumin
composition is use for prevention, improvement and maintenance of arthritis more specifically
osteoarthritis.
10 According to further embodiment of the present invention provides the stable curcumin
composition is use for prevention, improvement and maintenance of osteoarthritis.
According to further embodiment of the present invention provides the stable curcumin
composition is used for maintenance of Joint wellness.
15
According to further embodiment of the present invention provides the stable curcumin which
exhibits increased joint mobility, comfortable movements for ease of daily activities, improve
walking performance, increased joint flexibility, reduction in joint stiffness, reduced joint
discomfort/pain, reduced inflammation and reduced cartilage breakdown.
20
According to further embodiment of the present invention the stable curcumin composition uses
for improvement in muscle strength and muscle performance, reduction in muscle soreness and
muscle damage and promote faster muscle recovery.
25 Example 01:
Sr. No. Ingredient % w/w
1 Curcumin Extract 24
2 Hydroxypropyl methyl cellulose 67.1
3 Lecithin 2
4 Medium Chain Triglyceride (MCT Oil) 1
5 Glyceryl Monostearate 0.5
12
6 Citric acid anhydrous 0.5
7 Mixed Tocopherol 0.5
8 Silicon Dioxide 0.5
9 d-limonene 2
10 Sodium Lauryl Sulphate 1.9
11 IPA 190
12 MDC 760
Total(solid content) 100
(Total Curcuminoids. in %) = Qty. of extract (in %)* strength of extract (% Curcuminoids)/100=
24*95/100= 22.8%
The process for preparation of composition has defined in the stepwise manner as follows:
i) Dissolve Citric Acid anhydrous, Glyceryl monostearate, Lecithin 5 in mixture of
Isopropyl alcohol & Methylene Dichloride under heating at 55-60 °C. Add curcumin
extract containing from curcumin, demethoxy curcumin and bisdemethoxy curcumin
under heating at 55-60 °C and stir for 25-40 min until dissolve completely. The above
solution was cooled down to 30-40°C.
10 ii) Further add hydroxypropyl methylcellulose and sodium lauryl sulphate under stirring
condition.
iii) Add Medium Chain Triglyceride oil, d-limonene and Tocopherol in step (ii) solution
under stirring. The solution was stirred for 35-45 min to obtain homogeneous solution.
iv) The homogeneous solution was used as feed liquid for spray dryer. The powder
15 obtained from spray dryer was further dried in vacuum dryer at temperature 50-90oC
which is then sifted and blended after adding colloidal silicon dioxide. The product
obtained was yellowish orange free flowing homogeneous powder.
v) Figure 01 shows the X-ray powder diffraction (XRD) of the curcumin composition
having 1.2% of crystalline polymorph and 98.8% of the amorphous polymorph of the
20 curcuminoid.
13
Example 02:
Sr. No Ingredient % w/w
1 Curcumin Extract 24.0
2 Mixed Tocopherol 0.5
3 Hydroxypropyl Methylcellose 67.1
4 Citric Acid anhydrous 1.5
5 Lecithin 4.9
6 Medium Chain Triglycerides(MCT Oil) 1.0
7 Glyceryl monostearate 0.5
8 Colloidal Silicon Dioxide 0.5
9 Isopropyl alcohol 190
10 Methylene Dichloride 760
Total (Solid content) 100
(Total Curcuminoids. in %) = Qty. of extract (in %)* strength of extract (% Curcuminoids)/100=
24*95/100= 22.8%
The process for preparation of composition has defined in the stepwise 5 manner as follows:
i) Dissolve Citric Acid anhydrous, Glyceryl monostearate, Lecithin in mixture of
Isopropyl alcohol & Methylene Dichloride under heating at 55-60 °C. Add curcumin
extract containing from curcumin, demethoxy curcumin and bisdemethoxy
curcuminunder heating at 55-60 °C and stir for 25-40 min until dissolve completely.
10 The above solution was cooled down to 30-40°C.
ii) Further add hydroxypropyl methylcellulose under stirring condition.
iii) Add Medium Chain Triglyceride oil and Tocopherol in step (ii) solution under stirring.
The solution was stirred for 35-45 min to obtain homogeneous solution.
iv) The homogeneous solution was used as feed liquid for spray dryer. The powder
15 obtained from spray dryer was further dried in vacuum dryer at temperature 50-90oC
which is then sifted and blended after adding colloidal silicon dioxide. The product
obtained was yellowish orange free flowing homogeneous powder.
14
v) Figure 02 shows the X-ray powder diffraction (XRD) of the curcumin composition
having 1.2% of crystalline polymorph and 98.8% of the amorphous polymorph of the
curcuminoid.
5 Example 03:
Sr. No. Ingredient % w/w
1 Curcumin Ext 24
2 Hydroxypropyl methyl cellulose 69
3 Lecithin 2
4
Medium Chain Triglyceride(MCT
Oil) 1
5 Glyceryl Monostearate 0.5
6 Citric acid anhydrous 0.5
7 d-limonene 2
8 Mixed Tocopherol 0.5
9 Silicon Dioxide 0.5
10 Ethanol 240
11 Ethyl acetate 960
(Total Curcuminoids. in %) = Qty. of extract (in %)* strength of extract (% Curcuminoids)/100=
24*95/100= 22.8%
i) Dissolve Citric Acid anhydrous, Glyceryl monostearate, Lecithin in mixture of ethanol
10 and ethyl acetate under heating at 55-60 °C. Add curcumin extract under heating at 55-
60 °C and stir for 25-40 min until dissolve completely. The above solution was cooled
down to 30-40°C.
ii) Further add hydroxypropyl methylcellulose under stirring condition.
iii) Add Medium Chain Triglyceride oil, d-limonene and Tocopherol in step (ii) solution
15 under stirring. The solution was stirred for 35-45 min to obtain homogeneous solution.
15
iv) The homogeneous solution was used as feed liquid for spray dryer. The powder
obtained from spray dryer was further dried in vacuum dryer at temperature 50-90oC
which is then sifted and blended after adding colloidal silicon dioxide. The product
obtained was yellowish orange free flowing homogeneous powder.
v) Figure 03 shows the X-ray powder diffraction (XRD) of the curcumin 5 composition
having 2% of crystalline polymorph and 98 % of the amorphous polymorph of the
curcuminoid.
Example 04:
Sr. No. Ingredient % w/w
1 Curcumin Ext 71.15
2 Hydroxypropyl methyl cellulose 21.25
3 Lecithin 4.6
4 Medium Chain Triglyceride(MCT Oil) 1
5 Glyceryl Monostearate 0.5
6 Citric acid anhydrous 0.5
7 Mixed Tocopherol 0.5
8 Silicon Dioxide 0.5
9 Ethanol 300
10 Ethyl acetate 900
11 Water 200
10 (Total Curcuminoids. in %) = Qty. of extract (in %)* strength of extract(% Curcuminoids)/100=
71.15*90/100= 64.04%
i) Dissolve Citric Acid anhydrous, Glyceryl monostearate, Lecithin in mixture of ethanol
and ethyl acetate under heating at 55-60 °C. Add curcumin extract under heating at 55-
15 60 °C and stir for 25-40 min until dissolve completely. The above solution was cooled
down to 30-40°C.
ii) Further add hydroxypropyl methylcellulose under stirring condition.
iii) Add Medium Chain Triglyceride oil and Tocopherol in step (ii) solution under stirring.
The solution was stirred for 35-45 min to obtain homogeneous solution.
16
iv) The homogeneous solution was used as feed liquid for spray dryer. The powder
obtained from spray dryer was further dried in vacuum dryer at temperature 50-90oC
which is then sifted and blended after adding colloidal silicon dioxide. The product
obtained was yellowish orange free flowing homogeneous powder.
v) Figure 04 shows the X-ray powder diffraction (XRD) of the curcumin 5 composition
having 4% of crystalline polymorph and 96% of the amorphous polymorph of the
curcuminoid.
Example 05
10
Sr. No Ingredient % w/w
1 Curcumin Extract 86.5
2 Mixed Tocopherol 0.5
3 Hydroxypropyl Methylcellose 7
4 Citric Acid anhydrous 0.5
5 Lecithin 4
6 Medium Chain Triglycerides(MCT Oil) 0.5
7 Glyceryl monostearate 0.5
8 Colloidal Silicon Dioxide 0.5
9 Isopropyl alcohol 380
10 Methylene Dichloride 1520
Total (Solid content) 100
(Total Curcuminoids. in %) = Qty. of extract (in %)* strength of extract(% Curcuminoids)/100
= 86.5*95/100 = 82.18%
i) Dissolve Citric Acid anhydrous, Glyceryl monostearate, Lecithin in mixture of ethanol
15 and ethyl acetate under heating at 55-60 °C. Add curcumin extract under heating at 55-
60 °C and stir for 25-40 min until dissolve completely. The above solution was cooled
down to 30-40°C.
ii) Further add hydroxypropyl methylcellulose under stirring condition.
17
iii) Add Medium Chain Triglyceride oil, d-limonene and Tocopherol in step (ii) solution
under stirring. The solution was stirred for 35-45 min to obtain homogeneous solution.
iv) The homogeneous solution was used as feed liquid for spray dryer. The powder
obtained from spray dryer was further dried in vacuum dryer at temperature 50-90oC
which is then sifted and blended after adding colloidal silicon dioxide. 5 The product
obtained was yellowish orange free flowing homogeneous powder.
At the outset of the description that follows, it is to be understood that the ensuing description only
illustrates a particular form of this invention. However, such a particular form is only an exemplary
10 embodiment and is not intended to be taken restrictively to imply any limitation on the scope of
the present invention.
Clinical experimentation study 1:
Objective: The present study has been assessed for the efficacy of curcumin composition (test
product) in a monosodium iodoacetate (MIA) induced osteoarthritis.
15 Test product: The curcumin composition comprising of curcuminoids manufactured by
OmniActive Health Technologies Limited, India.
Dose 1: 100 mg/kg of formulation (20 mg/kg of total curcuminoids) (Example 1: 1.2% of
crystalline polymorph and 98.8% of the amorphous polymorph of the curcuminoid)
20 Dose 2: 200 mg/kg of formulation (40 mg/kg of total curcuminoids) (Example 1: 1.2% of
crystalline polymorph and 98.8% of the amorphous polymorph of the curcuminoid)
Experimental Design: Female wistar rats (8 weeks) randomly allocated into groups as defined
below (n=7 in each):
I. Normal Control group
25 II. Osteoarthritis group
III. Osteoarthritis, curcumin composition dose1 (100 mg/kg of formulation) group and
IV. Osteoarthritis, curcumin composition dose2 (200 mg/kg of formulation) group.
18
In order to induce osteoarthritis in rat model, the right knee of the rats was shaved and disinfected
with 70% alcohol following anaesthetization using with xylazine (10 mg/kg) and ketamine
hydrochloride (50 mg/kg). 3 mg of MIA was dissolved in 50 µL saline and injected into right knee
joints through the infrapatellar ligament using a 0.3 ml insulin syringe fitted with a 29-G needle.
Control group was given an injection of 50 µL saline. Two weeks after injection 5 with MIA, the
formulation 1 and formulation 2 were dissolved in 1 mL saline given orally for 4 weeks. All rats
were observed every other alternate day to assess knee joint swelling.
Four weeks after the rats were sacrificed, and blood and the specimens of the knee joint was
10 collected for the follow-up experiment. The blood samples were centrifuged at 3,000 rpm for 10
min, and the harvested sera was kept at -20 °C until the day of analysis.
Sample preparation:
Curcumin compositions according to the present invention are as follows:
15 The test sample for 100 mg/kg & 200 mg/kg has prepared as per the example 1 with the process
parameters provided.
Biochemical Analysis
Serum of MDA (malondialdehyde) was analysed by HPLC. Antioxidant enzymes (SOD, CAT,
GSHPx), IL-1ß, IL-6, IL-10, and TNF-a were measured using the relevant commercial kits
20 according to the enzyme-linked immunosorbent assay (ELISA) method.
Western Blot Analysis
Articular cartilage samples were analysed for the expression of TNF-a, IL-6, IL-1ß, nuclear factor
kappa B, cyclooxygenase-2 (COX-2), collagen type2, CRP, 5-LOX, MMP3, Cartilage oligomeric
25 matrix protein (COMP) using the Western blot technique.
Histological Analysis
Histological changes were assessed to confirm the effects of product on cartilage degeneration in
the knee joints of MIA-induced osteoarthritis rats. Following the rat sacrifice, each knee joint was
30 fixed in 10% formalin for 24 h at 4°C, and decalcified with 5% hydrochloric acid for 4 days at
19
4°C. Following decalcification specimens were dehydrated in graded acetone and embedded in
paraffin. Sections (thickness, 2–3 µm) were stained with 0.2% hematoxylin and 1% eosin for 5
min and 3 min, respectively. The histological preparations were analysed and photographed with
a microscope using a digital image capture camera.
5
Example 1
The objective of the study was to demonstrate the effect of curcumin composition as per this
invention on serum inflammation markers.
10 Method: Animals were sacrificed, and serum samples collected and assessed for serum biomarkers
measured using the relevant commercial kits according to the enzyme-linked immunosorbent assay
(ELISA) method as shown in the table 1 and illustratively provided in fig 5.
Table 01:
Marker
Groups
--P--*
Control OA OA+Cur1 OA+Cur2
TNF-a, pg/mL 21.29±3.14d 72.05±8.61a 55.48±7.93b 43.71±6.90c 0.0001
IL-1ß, pg/mL 18.61±1.67d 54.79±4.64a 41.03±2.96b 31.96±2.62c 0.0001
IL-6, pg/mL 10.58±1.02d 63.93±5.38a 48.40±3.91b 36.93±2.90c 0.0001
IL-10, pg/mL 98.12±6.33a 34.06±2.61d 47.58±4.12c 57.09±6.39b 0.0001
COMP, ng/mL 7.77±0.88d 27.68±2.99a 20.11±2.08b 14.44±1.98c 0.0001
CRP, mg/L 1.25±0.33d 11.27±1.55a 5.81±0.70b 4.21±0.84c 0.0001
15 Abbreviations:
OA: Osteoarthritis group
Cur1: Curcumin composition dose 1 (100 mg/kg of formulation)
Cur2: Curcumin composition dose 2 (200 mg/kg of formulation)
TNF-a: tumor necrosis factor a; IL-1ß: interleukin-1ß; IL-6: interleukin-6; IL-10: interleukin-10;
20 COMP: cartilage oligometrix matrix protein; CRP: c-reactive protein.
20
Statistical comparisons are indicated with different superscript (a-d) in the same row (P < 0.05;
*ANOVA and Turkey's post-hoc test). Mean values of items are demonstrated with ± standard
deviations.
Conclusion:
From the table 1 and fig 5, it was observed that serum inflammatory markers such 5 as TNFa, IL1ß,
IL-6, COMP, CRP which are responsible for induction of inflammation and associated pathology
were increased during OA and treatment with curcumin composition 1 and curcumin composition
2 reduced these levels in a statistically significant manner. Levels of cytokine IL-10 reduced
during OA were restored by treatment.
10
Example 2
The objective of the study was to demonstrate effect of curcumin composition on serum
antioxidant markers.
15 Method: Animals were sacrificed, and serum samples collected and assessed for serum
antioxidant markers. Serum of MDA was analyzed by HPLC. Antioxidant enzymes (SOD, CAT,
GSHPx) were measured using the relevant commercial kits according to the enzyme-linked
immunosorbent assay (ELISA) method and data shown in the table below.
20 Table 02:
Marker
Groups
--P--*
Control OA OA+Cur1 OA+Cur2
MDA, nmol/mL 0.63±0.07d 1.97±0.08a 1.65±0.09b 1.19±0.18c 0.0001
SOD, U/mL 50.45±4.07a 21.86±3.43d 31.35±2.35c 38.03±3.59b 0.0001
CAT, U/mL 142.68±6.58a 60.64±7.48d 71.76±5.74c 89.77±6.05b 0.0001
GSH-Px, U/mL 117.94±5.68a 59.82±3.32c 63.78±6.69c 85.84±4.98b 0.0001
MDA: malondialdehyde; SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; CAT:
catalase. Statistical comparisons are indicated with different superscript (a-d) in the same row (P
21
< 0.05; *ANOVA and Turkey's post-hoc test). Mean values of markers are demonstrated with ±
standard deviations
Conclusion:
MDA which is a marker of oxidative stress that plays a key role in induction of pathology of OA
was significantly increased during OA and were significantly reduced during treatment. 5 Similarly,
treatment significantly improved the levels of antioxidant enzymes, SOD, CAT and GSH-Px as
shown in the table 2.
Example 3
The objective of the study was to demonstrate effect of curcumin composition on inflammation
10 markers in synovial joint tissue.
Method: Animals were sacrificed, and synovial tissue samples collected and assessed for
inflammatory markers by western blot as shown in figure 05.
Conclusion:
15 It was observed that protein levels of inflammatory markers as shown below; IL-ß (A), IL-6 (B),
TNF-a (C) and NF-?ß (D) were increased in OA rats as measured by western blot followed by
densitometric analysis of the relative intensity according to the control group after ß-actin
normalization to ensure equal protein loading. Blots were repeated at least three times (n = 3) and
a representative blot (E) is shown.
20 It was observed that treatment with curcumin composition at two doses (100 & 200 mg/kg) reduced
the levels of inflammatory markers in the joint tissue in a statistically significant manner.
Example 4
The objective of the study was to demonstrate effect of curcumin composition on cartilage
degradation and inflammation markers in synovial joint tissue.
25
Method: Animals were sacrificed, and synovial tissue samples collected and assessed for markers
of cartilage degradation and inflammation by western blot as shown in figure 06.
22
Conclusion:
It was observed that protein levels of markers of collagen degradation such as Collagen Type 2
(A), MMP-3 (B) and inflammatory markers COX-2 (C) and LOX-5 (D) shown below were
increased in OA rats as measured by western blot followed by densitometric analysis of the relative
intensity according to the control group after ß-actin normalization to ensure equal 5 protein loading.
Blots were repeated at least three times (n = 3) and a representative blot (E) is shown.
It was observed that treatment with curcumin composition at two doses reduced the levels of
markers of cartilage degradation and inflammation in the joint tissue in a statistically significant
manner
10 Example 5
The objective of the study was to demonstrate effect of curcumin composition on joint architecture
of synovial joint as assessed by X-ray image analysis
Method: Animals were evaluated for structural integrity of synovial joint by radiography analysis
15 as shown in figure 07 and table 04.
Table 4. Kellgren- Lawrence scoring system (Kellgren and Lawrence, 1957).
Stage Radiologic Findings
0 None
1
Doubtful: Suspicious narrowing of the joint space and possible
osteophyte formation.
2
Minimal: Definite osteophyte and possible narrowing of the joint
space.
3
Moderate: Numerous moderate osteophytes, definite narrowing of the
joint space, some sclerosis and possible deformity of the bone ends.
4
Severe: Large osteophytes, marked narrowing of the joint space,
sclerosis and deformity of the bone ends
23
Conclusion:
It was observed that induction of OA is associated with loss of joint structural integrity as observed
by radiographic images (A) obtained at the end of the experiment. Significant improvement in the
joint architecture was observed after treatment with curcumin composition 1 & curcumin
composition 2. The above results were further validated by measuring mean values 5 of Kellgren-
Lawrence scores (B) with ± standard deviations.
Example 6
The objective of the study was to demonstrate effect of curcumin composition on morphological
10 analysis of synovial joint tissue.
Method: Animals were sacrificed, and synovial tissue samples collected, fixed in 10% formalin
for 24 h at 4°C, decalcified with 5% hydrochloric acid for 4 days at 4°C, dehydrated in graded
acetone and embedded in paraffin. Sections of 2–3 µm thickness were stained with 0.2%
15 hematoxylin and 1% eosin for 5 min and 3 min, respectively and histological preparations were
analyzed and photographed with a microscope using a digital image capture camera as shown in
figure 8.
Conclusion:
20 It was observed that there was a significant loss of joint structure and inflammatory infiltration of
joint in OA condition as observed after histopathologic images of hematoxylin-eosin (A) and
toluidine blue (B) staining obtained at the end of the experiment. This was further validated by
mean values of Mankin scores (C).
25 Example 7
The objective of the study was to demonstrate effect of curcumin composition on morphological
analysis of synovial joint tissue.
Method: Knee was visually assessed for morphological changes of joint pathology such as knee
30 swelling, knee joint diameter, and ratio of right to left diameter values as shown in figure 09.
24
Conclusion:
Both right and left knee joint swelling was observed when OA was induced in rats and this was
significantly reduced by treatment with curcumin composition 1 and curcumin composition 2 (A).
This was further validated by measuring left knee joint diameter (B) right knee joint diameter (C)
and the ratio of right to left diameter values and a significant treatment effect 5 was observed with
curcumin composition 1 & curcumin composition 2.
Example 8
The objective of the study was to demonstrate effect of curcumin composition on paw area and
stride length.
10
Method: The hind paws of rats were brushed with ink and the animals were allowed to run on a 60
cm-long, 7 cm-wide track covered with white paper. A dark chamber was placed at the end of the
track to entice rats. Upon completion of the test, the paper was scanned at 300 dpi. The
measurement around the paw was defined as paw area (cm²), the distance between the first and
15 fifth toes as paw width (cm), the distance of the same hind paw between two steps as stride length
(cm), the horizontal distance between the left and right paw as the base (cm), the distance between
the third toe and the heel as paw length (cm) and the paw angle as the angle through the hind legs
(°). The measures of footsteps were quantified by ImageJ software as shown in figure 10.
20 Conclusion:
Paw area and stride length were significantly reduced due to osteoarthritis. There was a significant
improvement in both paw area and stride length with curcumin composition 1 and curcumin
composition 2.
25 Clinical experimentation study 2:
Objective: The purpose of this study was to evaluate the plasma pharmacokinetic profile of the
curcumin composition in adult Male Sprague Dawley.
Dose selection
25
The dose of 1000 mg/kg body weight (200 mg/kg of total curcuminoids) was selected for oral
route of administration.
Test product: Curcumin composition as per example 01
Reference: Curcumin extract (Curcumin extract without any formulation and polymorphic form)
Curcumin extract (Regular Curcumin) extract without any formulation and polymorphic 5 in general
prepared by dring turmeric rhizome extracted with solvent and evaporated to form oleoresin
turmeric which further oleoresin crystallized using suitable solvent. The obtained crystals are
dried and powdered which contain 95% of curcuminoids
Time point (hr.): 0.00, 0.25, 0.50, 1.00, 2.00, 3.00, 4.00, 6.00, 8.00 and 24.00
10 Table 05:
Reference
Curcumin
composition 20%
Fold increase of
Curcumin composition
20% over Reference
Parameters Mean ± SD Mean ± SD
Cmax (ng/mL) 28.169 ± 28.604 1522.184 ± 257.839 54.04X
Tmax (h) 0.656 ± 0.719 1.938 ± 0.863 2.95X
AUClast (hr*ng/mL) 95.349 ± 55.134 6170.474 ± 1071.967 64.71X
t1/2 (h) 2.19 ± 0.81 5.103 ± 0.725 2.33X
Cmax : Peak plasma concentration
Tmax : Time to reach the peak plasma concentration
AUC0-last: Area under the concentration-time curve from time zero to last quantifiable
15 concentration
t1/2 : Half-life
26
Conclusion:
Curcumin composition showed 64.71 folds higher AUC compared to reference curcumin extract.
Based on the results and conclusions provided on osteoarthritis model, we here by concluded that
the same curcumin composition will also have a beneficial effect on muscle health. The cell line
study model with further evidenced by the study protocol has been illustrated 5 below to study the
efficacy on muscle health.
Further study to demonstrate the effect of curcumin composition on muscle health as follows.
10 In-Vitro Study:
Objective: Demonstration of ergogenic potential curcumin composition (test product) for
improving physiological or metabolic responses that may help in muscle development (prevent
loss of muscle protein as in age-related sarcopenia), enhance exercise performance, ameliorate
muscle injury and muscle inflammation (DOMS)
15 Cell model: C2C12 murine myoblasts
Experimental approaches: As per the studies were conducted on basic assays and generate more
information after we identify selected ingredients for development C2C12 myoblasts was cultured
at 37°C in humidified 5% CO2 were plated in 24 well-plate and incubated for 24h before adding
treatment
20 1. Cytotoxicity assay
2. Mitochondrial density assay: Effect of ingredients on mitochondrial biogenesis is analyzed
by measuring cellular respirations accompanied by increase in mitochondrial mass is
calculated.
3. Antioxidant Capacity: After 24h of treatment, the total antioxidant capacity was
25 determined in C2C12 homogenates
4. Lactate dehydrogenase and Creatine Kinase activity: LDH and CK activities are used as
indicator of the occurrence of muscle cell damage and is assessed by determining the
release of the cytosolic enzyme lactate dehydrogenase (LDH) and creatine kinase (CK)
activity
27
5. IGF-1 Elisa assay: IGF-I exerts acute anabolic actions on protein and carbohydrate
metabolism by increasing the cellular uptake of amino acids in cells and plays a major role
in the regulation of skeletal muscle growth.
5 In-Vivo Study:
Objective: To evaluate the effect of curcumin composition (test product) formulation on
endurance, grip strength and muscle metabolism in rats.
Animals and Experimental Design:
21 Wistar Albino or Sprague Dawley male rats (20% extra animals), age: 8 weeks, weight: 180 ±
10 20 g were housed in a controlled environment with a 12:12-h light-dark cycle at 22°C and were
provided with rat chow and water ad libitum.
Rats were randomly divided into 3 treatment groups, each containing 7 animals.
1. Control (Normal and exercised)
2. Curcumin composition 1 (100 mg/kg of formulation)
15 3. Curcumin composition 2 (200 mg/kg of formulation)
At screening period, the grip strength was performed on day 3. On day -2, Treadmill Test will be
performed.
Grip Strength Test:
The grip strength of the rats was evaluated using a force measurement system. Combined forelimb
20 and hind limb grip strength and forelimb grip strength were measured at the end of the oral
administration period. The system has an electronic digital force gauge that determines the peak
force. Each rat was held by the tail until it released the pull bar. Five consecutive tests were
performed on each rat to obtain the peak value.
28
Treadmill Test:
An animal treadmill was used to measure the running endurance of the rats. The rats were
subjected to overnight fasting and sacrificed by cervical dislocation and blood and gastrocnemius
muscle were collected. Serum samples was obtained by taking blood samples to gel biochemical
tubes after centrifugation (5000 rpm at 4°C for 10 minutes). Samples of the 5 liver and from the
gastrocnemius muscle (taken from approximately the same location each time) was quickly
removed, placed on ice, and kept at -80°C until analyses. For biochemical assays, tissues samples
were homogenized within 10 min in 10 volumes of cold Tris 10 mM (pH 7.4). Muscle
homogenates was centrifuged at 4000 ×g at 4 °C for 10 min to yield the low-speed supernatant
10 fraction that was used for the lipid peroxidation analyses.
Biochemical Analysis:
Serum glucose, lipid profile, aspartate aminotransferase (AST), alanine aminotransferase (ALT),
urea, creatinine levels will be analyzed with a portable automated chemistry analyzer (Samsung
LABGEO PT10V, Samsung Electronics Co., Suwon, Korea). Rat Lactate Assay Kit (Cayman
15 Chemical Co., Ann Arbor, MI, USA) will be used to measure the serum lactate concentrations
through enzyme-linked immunosorbent assays (ELISA, Elx-800, Bio-Tek Instruments Inc,
Vermont, USA). ELISA (MyBioSource, San Diego, CA, USA) will also be used in measuring
serum myoglobin concentration.
The malondialdehyde (MDA) level in muscle tissue was measured by high-performance liquid
20 chromatography (Shimadzu, Tokyo, Japan) using a Shimadzu UV-vis SPD-10 AVP detector and
C18 ODS-3, 5 µm, 4.6 mm ×250 mm column. The activities of superoxide dismutase (SOD),
catalase (CAT), and glutathione peroxidase (GSH-Px) was determined using commercially
available kits (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s
procedure.
25 It has to evident to those skilled in the art that the invention is not limited to the details of the
illustrative examples and that the present invention may be embodied in other specific forms
without departing from the essential attributes thereof. It is therefore desired that the present
embodiments and examples be considered in all respects as illustrative and not restrictive,
reference being made to the appended claims, rather than to the foregoing description, and all
29
changes which come within the meaning and range of equivalency of the claims are therefore
intended to be embraced therein.

,CLAIMS:We claim:
1) A stable curcumin composition having enhanced bioavailability comprising;
i) Curcumin containing crystalline polymorphic form of curcuminoids is in the range of
0 to 15 % and amorphous polymorphic form of curcuminoids is in the range of 85 to
5 100 % and
ii) At least one or more pharmaceutically or nutraceutically accepted ingredient selected
from hydrophilic carrier, antioxidant, stabilizer, pH Modifier, solubilizer, fat,
anticaking agent.
10 2) The stable curcumin composition as claimed in claim 1, wherein the curcuminoids are selected
from one or more of curcumin, demethoxycurcumin, bisdemethoxycurcumin or mixture
thereof.
3) The stable curcumin composition as claimed in claim 1, wherein the curcuminoids is present
15 in the range of 10-90 % w/w of the composition.
4) The stable curcumin composition as claimed in claim 1, wherein the composition has
increased bioavailability of 60 -70 folds against reference.
5) The stable curcumin composition as claimed in claim 1, wherein the composition comprises
20 hydrophilic carrier are selected from Hydroxypropyl Methyl cellulose, cellulose derivatives,
starch and starch derivatives in the range of 1-90 % w/w of the composition.
6) The stable curcumin composition as claimed in claim 1, wherein the composition comprises
antioxidant are selected from tocopherols, mixed tocopherol, ascorbyl palmitate, catechins,
25 and ascorbic acid present in the range of 0.1-10% w/w of the composition.
7) The stable curcumin composition as claimed in claim 1, wherein the composition comprises
stabilizer are selected from glyceryl mono stearate, sugar alcohols, or triglycerides present in
the range of 0.1-10 % w/w of the composition.
30
31
8) The stable curcumin composition as claimed in claim 1, wherein the composition comprises
pH Modifier are selected from citric acid, trisodium citrate, lactic acid, magnesium carbonate
in the range of 0.1-10% w/w of the composition.
9) The stable curcumin composition as claimed in claim 1, wherein the composition 5 comprises
solubilizer selected from lecithin, d-limonene, propylene glycol alginate, sodium lauryl
sulphate in the range of 0.1-40 %w/w of the composition.
10) The stable curcumin composition as claimed in claim 1, wherein the composition comprises
10 fat are selected from medium chain triglycerides, terpene, vegetable oil in the range of 0.1-40
% w/w of the composition.
11) The stable curcumin composition as claimed in claim 1, wherein the composition comprises
anticaking agent are selected from Colloidal Silicon Dioxide, magnesium stearate, steric acid,
15 mannitol in the range of 0.1-10 % w/w of the composition.
12) The stable curcumin composition as claimed in claim 1, wherein the composition is in the
form of tablets, capsules, blended powders, licaps, ointments, pastes, lotions, liniments,
mouthwashes, gargles, consumable dry syrups, liquid syrups, health drinks, diet drinks, fruit
20 juices, soft drinks and the like.
13) The stable curcumin composition as claimed in claim 1, wherein the composition is in the
form of granule and/ or powder or mixture thereof.
25 14) The stable curcumin composition as claimed in claim 1, wherein the composition is used for
prevention, improvement and maintenance of osteoarthritis.
15) The stable curcumin composition as claimed in claim 1, wherein the composition improves
joint mobility, joint flexibility, reduction in joint stiffness, improve walking performance,
30 reduction in cartilage breakdown and reduction in inflammation.
32
16) The stable curcumin composition as claimed in claim 1, wherein the composition decrease
tumor necrosis factor-alpha (TNF-a), interleukin1ß (IL-1ß), interleukin-6 (IL-6), Collagen
Type 2, matrix metalloproteinase-3 (MMP3), cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-
LOX), cartilage oligometrix matrix protein (COMP); c-reactive protein(CRP),
malondialdehyde 5 (MDA).
17) The stable curcumin composition as claimed in claim 1, wherein the composition increases
interleukin-10 (IL-10), superoxide dismutase (SOD), glutathione peroxidase(GSH-Px),
catalase (CAT).
10
18) The stable curcumin composition as claimed in claim 1, wherein the composition is use for
improvement in muscle strength and muscle performance, reduction in muscle soreness and
muscle damage and promote faster muscle recovery.
15 19) The stable curcumin composition as claimed in claim 1, wherein the subject is human and/or
animal.
20) The stable curcumin composition as claimed in claim 1, wherein dose is 10-1000 mg/kg.