CA2860256A1 - Solubilization of pterostilbene and resveratrol in aqueous beverages - Google Patents
Solubilization of pterostilbene and resveratrol in aqueous beverages Download PDFInfo
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Abstract
In one embodiment, the present application discloses compositions and methods of solubilizing pterostilbene or resveratrol, or mixture thereof, in aqueous media.
Description
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
SOLUBILIZATION OF PTEROSTILBENE AND RESVERATROL
IN AQUEOUS BEVERAGES
RELATED APPLICATION:
[0001] This application claims the benefit of U.S. Provisional Application No.
61/872,971, filed September 3,2013, which is incorporated herein by reference.
SUMMARY:
ISS.000200CA
SOLUBILIZATION OF PTEROSTILBENE AND RESVERATROL
IN AQUEOUS BEVERAGES
RELATED APPLICATION:
[0001] This application claims the benefit of U.S. Provisional Application No.
61/872,971, filed September 3,2013, which is incorporated herein by reference.
SUMMARY:
[0002] Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) and resveratrol are natural dietary compounds and the primary antioxidant component of blueberries, among other fruits.
Pterostilbene has increased oral bioavailability in comparison to other stilbene compounds, which may enhance its dietary benefit and possibly contribute to advantageous clinical effect.
Multiple studies have demonstrated the antioxidant activity of resveratrol and pterostilbene in both in vitro and in vivo models illustrating both preventative and therapeutic benefits. The antioxidant activity of resveratrol and pterostilbene has been implicated in anticarcinogenesis, modulation of neurological disease, anti-inflammation, attenuation of vascular disease, and amelioration of diabetes. Solubility of pterostilbene in aqueous media is very low which is limiting of its incorporation into beverages for convenient consumption. We have discovered a methodology to solubilize resveratrol and pterostilbene, and mixtures thereof, for inclusion in beverages which provides a safe and an efficient delivery of a wide range of resveratrol and pterostilbene doses.
BACKGROUND
Pterostilbene has increased oral bioavailability in comparison to other stilbene compounds, which may enhance its dietary benefit and possibly contribute to advantageous clinical effect.
Multiple studies have demonstrated the antioxidant activity of resveratrol and pterostilbene in both in vitro and in vivo models illustrating both preventative and therapeutic benefits. The antioxidant activity of resveratrol and pterostilbene has been implicated in anticarcinogenesis, modulation of neurological disease, anti-inflammation, attenuation of vascular disease, and amelioration of diabetes. Solubility of pterostilbene in aqueous media is very low which is limiting of its incorporation into beverages for convenient consumption. We have discovered a methodology to solubilize resveratrol and pterostilbene, and mixtures thereof, for inclusion in beverages which provides a safe and an efficient delivery of a wide range of resveratrol and pterostilbene doses.
BACKGROUND
[0003] Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) is a naturally derived compound found in blueberries. The amount of daily pterostilbene consumption varies according to dietary fruit intake, and it has been estimated that pterostilbene content in blueberry varies from 99 ng to 520 ng/gram depending on the variety of berry ingested.
Substantial evidence suggests that pterostilbene may have numerous preventive and therapeutic properties in a vast range of human diseases that include neurological, cardiovascular, metabolic and hematologic disorders (D. McCormack and D. McFadden, 2013, A Review of Pterostilbene Antioxidant and Disease Modification, Oxidative Medicine and Cell Longevity, http://dx.doi.org/10.1155/2013/575482). Further benefits of pterostilbene have been reported in preclinical trials, in which pterostilbene was shown to be a potent anticancer agent in several malignancies (D. McCormack and D. McFadden, "Pterostilbene and cancer: current review,"
Journal of Surgical Research, vol. 173, no. 2, pp. 53-61, 2012). Pterostilbene is structurally PATENT APPLICATION Attorney Docket No.
ISS.000200CA
similar to resveratrol, a compound found in red wine that has comparable antioxidant, anti-inflammatory, and anticarcinogenic properties; however, pterostilbene exhibits increased absorption due to the presence of two methoxy groups which cause it to exhibit increased lipophilicity and oral bioavailability. In animal studies, pterostilbene was shown to have 80%
bioavailability compared to 20% for resveratrol making it potentially advantageous as a therapeutic agent. The multiple benefits of pterostilbene in the treatment and prevention of human disease have been attributed to its antioxidant, anti-inflammatory, and anticarcinogenic properties leading to improved function of normal cells and inhibition of malignant cell. The evidence reviewed by McCormack and McFadden shows that pterostilbene reduces oxidative stress (OS) and production of reactive oxygen species (ROS), such as hydrogen peroxide (H202) and superoxide anion (02¨), which are implicated in the initiation and pathogenesis of several disease processes. In addition, various cell lines treated with pterostilbene have shown increased expression of the antioxidants catalase, total glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD).
Substantial evidence suggests that pterostilbene may have numerous preventive and therapeutic properties in a vast range of human diseases that include neurological, cardiovascular, metabolic and hematologic disorders (D. McCormack and D. McFadden, 2013, A Review of Pterostilbene Antioxidant and Disease Modification, Oxidative Medicine and Cell Longevity, http://dx.doi.org/10.1155/2013/575482). Further benefits of pterostilbene have been reported in preclinical trials, in which pterostilbene was shown to be a potent anticancer agent in several malignancies (D. McCormack and D. McFadden, "Pterostilbene and cancer: current review,"
Journal of Surgical Research, vol. 173, no. 2, pp. 53-61, 2012). Pterostilbene is structurally PATENT APPLICATION Attorney Docket No.
ISS.000200CA
similar to resveratrol, a compound found in red wine that has comparable antioxidant, anti-inflammatory, and anticarcinogenic properties; however, pterostilbene exhibits increased absorption due to the presence of two methoxy groups which cause it to exhibit increased lipophilicity and oral bioavailability. In animal studies, pterostilbene was shown to have 80%
bioavailability compared to 20% for resveratrol making it potentially advantageous as a therapeutic agent. The multiple benefits of pterostilbene in the treatment and prevention of human disease have been attributed to its antioxidant, anti-inflammatory, and anticarcinogenic properties leading to improved function of normal cells and inhibition of malignant cell. The evidence reviewed by McCormack and McFadden shows that pterostilbene reduces oxidative stress (OS) and production of reactive oxygen species (ROS), such as hydrogen peroxide (H202) and superoxide anion (02¨), which are implicated in the initiation and pathogenesis of several disease processes. In addition, various cell lines treated with pterostilbene have shown increased expression of the antioxidants catalase, total glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR) and superoxide dismutase (SOD).
[0004] Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a stilbenoid, a type of natural phenol and a phytoalexin produced naturally by several plants.
[0005] That the many observed benefits of pterostilbene ingestion be realized for people, it is necessary that convenient dosage forms be developed. Various beverages are popular with consumers for consumption; sports drinks, vitamin waters, fruit juices, energy drinks, etc.
However, the near insolubility of pterostilbene in aqueous solutions precludes the ready incorporation of resveratrol or pterostilbene into these popular beverages.
SUMMARY OF THE INVENTION:
However, the near insolubility of pterostilbene in aqueous solutions precludes the ready incorporation of resveratrol or pterostilbene into these popular beverages.
SUMMARY OF THE INVENTION:
[0006] In one embodiment, the application discloses a stable and homogeneous aqueous composition comprising: a) a compound selected from the group consisting of resveratrol and pterostilbene, or a mixture thereof,; and b) an emulsifying agent in an amount sufficient to solubilize resveratrol or pterostilbene, or a mixture thereof, to form the stable and homogeneous composition. In one aspect, the stable and homogeneous aqueous composition further comprises caffeine. In one aspect, the composition is water soluble. In another aspect of the composition, the resveratrol and caffeine is a co-crystal complex or the pterostilbene and caffeine is a co-crystal complex. In another aspect, the composition further comprises an emulsifying agent selected from the group consisting of TPGS, TPGS-300, TPGS-500, TPGS-600, TPGS-750, PATENT APPLICATION Attorney Docket No.
ISS.000200CA
TPGS-1000, TPGS-M, TPGS-300-M, TPGS-500-M, TPGS-600-M. TPGS-750-M and TPGS-1000-M, or a mixture thereof In another aspect of the above the composition is an emulsion, a concentrated emulsion or a clear solution in water. As disclosed herein, the composition is stable, that is, does not result in decomposition and/or precipitation for a period of at least 6 months or 12 months at RT, or at least 12 months at about 12-15 C. The stability of the solution is also noted where the solution is clear, or not cloudy or hazy. Clarity may be determined by turbidity units that may be measured as Nephelometric Turbidity Units (NTU) as known in the art.
ISS.000200CA
TPGS-1000, TPGS-M, TPGS-300-M, TPGS-500-M, TPGS-600-M. TPGS-750-M and TPGS-1000-M, or a mixture thereof In another aspect of the above the composition is an emulsion, a concentrated emulsion or a clear solution in water. As disclosed herein, the composition is stable, that is, does not result in decomposition and/or precipitation for a period of at least 6 months or 12 months at RT, or at least 12 months at about 12-15 C. The stability of the solution is also noted where the solution is clear, or not cloudy or hazy. Clarity may be determined by turbidity units that may be measured as Nephelometric Turbidity Units (NTU) as known in the art.
[0007] In one embodiment, the present application discloses a method of solubilizing resveratrol or pterostilbene, or a mixture thereof, in an aqueous media comprising: a) dissolving the resveratrol or pterostilbene, or a mixture thereof, and an emulsifying agent in a suitable solvent to form a homogeneous solution; b) evaporating the solvent to form a mixture of resveratrol or pterostilbene, or a mixture thereof, and the emulsifying agent;
c) dissolving the resveratrol or pterostilbene mixture, or mixture thereof, in the aqueous medium by stirring the mixture with a warm medium to form a stable emulsion of resveratrol or pterostilbene, or a mixture thereof, and emulsifying agent.
c) dissolving the resveratrol or pterostilbene mixture, or mixture thereof, in the aqueous medium by stirring the mixture with a warm medium to form a stable emulsion of resveratrol or pterostilbene, or a mixture thereof, and emulsifying agent.
[0008] In one aspect of the above embodiment, the emulsifying agents are GRAS
(Generally Recognized as Safe). In another aspect, the emulsifying agent has an HLB of greater than 9. In another aspect of each of the above, the emulsifying agent is TPGS
(tocopherol polyethylene glycol succinate). In another aspect, the ratio of emulsifying agent to resveratrol or pterostilbene is in the range of 3:1 to 10:1. In another aspect of the above, the ratio of emulsifying agent to resveratrol or pterostilbene is from 4:1 to 6:1. In another aspect, the ratio of emulsifying agent to resveratrol or pterostilbene is from 1:1 to 2:1. In another aspect, the resultant emulsion comprises a particle size of less than 250 nanometers. In yet another aspect, the resultant emulsion comprises a particle size of less than 100 nanometers.
In another aspect, the resultant emulsion comprises a particle size of between 25 and 100 nanometers. In yet another aspect, the resultant emulsion comprises a particle size of less than 50 nanometers. In another aspect, the resultant emulsion comprises a particle size of between 15 and 50 nanometers.
(Generally Recognized as Safe). In another aspect, the emulsifying agent has an HLB of greater than 9. In another aspect of each of the above, the emulsifying agent is TPGS
(tocopherol polyethylene glycol succinate). In another aspect, the ratio of emulsifying agent to resveratrol or pterostilbene is in the range of 3:1 to 10:1. In another aspect of the above, the ratio of emulsifying agent to resveratrol or pterostilbene is from 4:1 to 6:1. In another aspect, the ratio of emulsifying agent to resveratrol or pterostilbene is from 1:1 to 2:1. In another aspect, the resultant emulsion comprises a particle size of less than 250 nanometers. In yet another aspect, the resultant emulsion comprises a particle size of less than 100 nanometers.
In another aspect, the resultant emulsion comprises a particle size of between 25 and 100 nanometers. In yet another aspect, the resultant emulsion comprises a particle size of less than 50 nanometers. In another aspect, the resultant emulsion comprises a particle size of between 15 and 50 nanometers.
[0009] In another embodiment, there is provided a method for solubilizing resveratrol or pterostilbene from a co-crystal complex of resveratrol or pterostilbene and caffeine in aqueous media comprising: a) dissolving the resveratrol or pterostilbene complex and an emulsifying PATENT APPLICATION Attorney Docket No.
ISS.000200CA
agent in a suitable solvent to form a homogeneous solution; b) evaporating the solvent to form a mixture of the resveratrol or pterostilbene complex and the emulsifying agent;
and c) dissolving of the resveratrol or pterostilbene mixture in the aqueous medium by stirring the mixture with a warm medium to form a stable emulsion of resveratrol or pterostilbene and emulsifying agent.
Also provided are the above methods for solubilizing a mixture of both pterostilbene and resveratrol.
ISS.000200CA
agent in a suitable solvent to form a homogeneous solution; b) evaporating the solvent to form a mixture of the resveratrol or pterostilbene complex and the emulsifying agent;
and c) dissolving of the resveratrol or pterostilbene mixture in the aqueous medium by stirring the mixture with a warm medium to form a stable emulsion of resveratrol or pterostilbene and emulsifying agent.
Also provided are the above methods for solubilizing a mixture of both pterostilbene and resveratrol.
[0010] In one aspect of the above, the warm medium is provided by heating the mixture above RT for a sufficient amount of time to form the stable emulsion. In another aspect, the warm medium is obtained by heating the mixture to about 30 C to about 80 C, 30 C to 70 C, 30 C to 60 C, 30 C to 50 C, or 30 C to 40 C. In another aspect, the warm medium is obtained by heating the mixture to 40 C to 70 C, or 40 C to 60 C. In one aspect of the above embodiments, the emulsifying agents are GRAS (Generally Recognized as Safe).
In another aspect, the emulsifying agents have an HLB of greater than 9. In another aspect, the emulsifying agent is TPGS (tocopherol polyethylene glycol succinate), TPGS-300 (D-alpha-tocopheryl polyethylene glycol 300 succinate), TPGS-500, TPGS-600, TPGS-750 and TPGS-1000, or a mixture thereof. In another aspect, the emulsifying agent is TPGS-M
(tocopherol polyethylene glycol methyl ether succinate), TPGS-300-M (D-alpha-tocopheryl polyethylene glycol methyl ether 300 succinate), TPGS-500-M, TPGS-600-M, TPGS-750-M and TPGS-1000-M, or a mixture thereof. In another aspect, the emulsifying agent is selected from the group consisting of TPGS, TPGS-300, TPGS-500, TPGS-600, TPGS-750, TPGS-1000, TPGS-M, TPGS-300-M, TPGS-500-M, TPGS-600-M, TPGS-750-M and TPGS-1000-M, or a mixture thereof. In another aspect of the above, the ratio of emulsifying agent to resveratrol or pterostilbene is in the range of 3:1 to 10:1; 1:1 to 3:1; 4:1 to 5:1; or 5:1 to 8:1. In another aspect, the resultant emulsion has a particle size of less than 250 nanometers (nm), less than 100 nm; between 25 and 100 nm, below 50 nm or between 15 and 50 nm. In one aspect of each of the above embodiments and aspects, the composition, formulation or emulsion comprises a mixture of resveratrol and pterostilbene.
In one variation, the mixture of resveratrol to pterostilbene is in a ratio of about 95:5, 90:10, 85:15, 80:20, 75: 25, 70:30, 65:35, 60: 40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90 or 5:95.
In another aspect, the emulsifying agents have an HLB of greater than 9. In another aspect, the emulsifying agent is TPGS (tocopherol polyethylene glycol succinate), TPGS-300 (D-alpha-tocopheryl polyethylene glycol 300 succinate), TPGS-500, TPGS-600, TPGS-750 and TPGS-1000, or a mixture thereof. In another aspect, the emulsifying agent is TPGS-M
(tocopherol polyethylene glycol methyl ether succinate), TPGS-300-M (D-alpha-tocopheryl polyethylene glycol methyl ether 300 succinate), TPGS-500-M, TPGS-600-M, TPGS-750-M and TPGS-1000-M, or a mixture thereof. In another aspect, the emulsifying agent is selected from the group consisting of TPGS, TPGS-300, TPGS-500, TPGS-600, TPGS-750, TPGS-1000, TPGS-M, TPGS-300-M, TPGS-500-M, TPGS-600-M, TPGS-750-M and TPGS-1000-M, or a mixture thereof. In another aspect of the above, the ratio of emulsifying agent to resveratrol or pterostilbene is in the range of 3:1 to 10:1; 1:1 to 3:1; 4:1 to 5:1; or 5:1 to 8:1. In another aspect, the resultant emulsion has a particle size of less than 250 nanometers (nm), less than 100 nm; between 25 and 100 nm, below 50 nm or between 15 and 50 nm. In one aspect of each of the above embodiments and aspects, the composition, formulation or emulsion comprises a mixture of resveratrol and pterostilbene.
In one variation, the mixture of resveratrol to pterostilbene is in a ratio of about 95:5, 90:10, 85:15, 80:20, 75: 25, 70:30, 65:35, 60: 40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90 or 5:95.
[0011] In another embodiment of each of the above embodiments and aspect, there is provided a stable and homogeneous aqueous formulation or composition comprising the PATENT APPLICATION Attorney Docket No.
ISS.000200CA
pterostilbene-caffeine co-crystal complex, a resveratrol-caffeine co-crystal complex and a mixture thereof in water is at a co-crystal complex:water (wt:wt) ratio of 10:90. 20:80, 30:70, 40:
60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5 or more.
DETAILED DESCRIPTION OF THE INVENTION:
ISS.000200CA
pterostilbene-caffeine co-crystal complex, a resveratrol-caffeine co-crystal complex and a mixture thereof in water is at a co-crystal complex:water (wt:wt) ratio of 10:90. 20:80, 30:70, 40:
60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5 or more.
DETAILED DESCRIPTION OF THE INVENTION:
[0012] We have discovered a methodology to solubilize resveratrol or pterostilbene such that it may be incorporated into aqueous based beverages readily at concentrations sufficient to provide doses appropriate for provision of the health benefits of resveratrol or pterostilbene consumption. Such beverages include clear beverages, sodas such as Coke or Pepsi , fruit juices such as orange juice, apple juice, carbonated or non-carbonated water or beverages etc...
[0013] General procedures for preparation of nanoparticulate TPGS based formulations are found in Arbor Therapeutics, LLC Standard Operating Procedures; ART 001 Coarse Emulsion Preparation Rev. 1, and ART 003 Nicomp 380 ZLS Particle Size Analyses Rev. 1.
Exceptions to these SOPs are noted as needed.
Exceptions to these SOPs are noted as needed.
[0014] Abbreviations: TPGS ¨ D-a-Tocopheryl Polyethyleneglycol-1000-succinate, PSB ¨ Pterostilbene; RVT - Resveratrol; IPA ¨ Isopropanol; REM ¨ resultant emulsion; A -percent of total solids; WN ¨ weight to volume; Recovery, A - percent of PSB
recovered in resultant emulsion after sterile 0.22 p.m filtration; mfg ¨ manufacturing; ND
¨ not determined;
BDL ¨ below detection limit; 0/N ¨ overnight.
List of equipment used:
Description Manufacturer Model Number 400 gram balance Denver Instrument SI-403 100 gram balance Denver Instrument APX-100 Magnetic stirring plate Barnstead Thermolyne, Cimarec Particle Sizer Particle Sizing Systems NicompTM 380 ZLS
HPLC Agilent HP 1100 Series List of materials used:
Reagent Vendor Part Number Lot Number D-a-Tocopheryl Polyethyleneglycol-1000- lsochem NA 1101040048 succinate Pterostilbene ChromaDex. ASB-00016996-100 00016996-1206 Resveratrol Sigma-Aldrich R-5010 Sigma NA
Distilled Water Kroger Grocery Distilled Water NA
50 mL 09-741-88 NA
Sterile Filters, 0.22 pin, PES Fisher Scientific 150 mL 09-500 mL 09-761-107 NA
Cuvettes, polystyrene 4.5 mL Fisher Scientific 14 955 125 NA
PATENT APPLICATION
Attorney Docket No. ISS.000200CA
Reagent Vendor Part Number Lot Number Powerade, Lemon Lime DEC3013CCB3B, 13:37 Powerade Lifewater, strawberry SOBE
AUG2613, dragonfruit 0115DL022234 Minute Maid, Pink lemonade Minute Maid LTF7W4P, RTKS4LN
Welch's Tropical Carrot Welch's NE12K16 17 04:46 C
Vitamin water, acai-blueberry- GLACEAU
SEP0913CCC1A, 10:21 pomegranate CT931 Perform 02, G series GATORADE
0CT3013CT564, Pluronic 31R1 Poly (Propylene glycol)-block-Poly (Ethylene glycol)-block- Sigma-Aldrich Poly (Propylene glycol) PPG-PEG-PPG
Pluronic 17R4 Poly (Propylene glycol)-block-Poly (Ethylene glycol)-block- Sigma-Aldrich Poly (Propylene glycol) PPG-PEG-PPG
Pluronic L-64 Poly (Propylene glycol)-block-Poly (Ethylene glycol)-block- Sigma-Aldrich 1001442463 Poly (Propylene glycol) PPG-PEG-PPG
Pterostilbene and Caffeine Co- Chromadex 00016988 Crystal Caffeine, 99% Alfa Aesar A10431 B06Y038 Cremophor ELP Sigma-Aldrich 30906 BCBH0387V
recovered in resultant emulsion after sterile 0.22 p.m filtration; mfg ¨ manufacturing; ND
¨ not determined;
BDL ¨ below detection limit; 0/N ¨ overnight.
List of equipment used:
Description Manufacturer Model Number 400 gram balance Denver Instrument SI-403 100 gram balance Denver Instrument APX-100 Magnetic stirring plate Barnstead Thermolyne, Cimarec Particle Sizer Particle Sizing Systems NicompTM 380 ZLS
HPLC Agilent HP 1100 Series List of materials used:
Reagent Vendor Part Number Lot Number D-a-Tocopheryl Polyethyleneglycol-1000- lsochem NA 1101040048 succinate Pterostilbene ChromaDex. ASB-00016996-100 00016996-1206 Resveratrol Sigma-Aldrich R-5010 Sigma NA
Distilled Water Kroger Grocery Distilled Water NA
50 mL 09-741-88 NA
Sterile Filters, 0.22 pin, PES Fisher Scientific 150 mL 09-500 mL 09-761-107 NA
Cuvettes, polystyrene 4.5 mL Fisher Scientific 14 955 125 NA
PATENT APPLICATION
Attorney Docket No. ISS.000200CA
Reagent Vendor Part Number Lot Number Powerade, Lemon Lime DEC3013CCB3B, 13:37 Powerade Lifewater, strawberry SOBE
AUG2613, dragonfruit 0115DL022234 Minute Maid, Pink lemonade Minute Maid LTF7W4P, RTKS4LN
Welch's Tropical Carrot Welch's NE12K16 17 04:46 C
Vitamin water, acai-blueberry- GLACEAU
SEP0913CCC1A, 10:21 pomegranate CT931 Perform 02, G series GATORADE
0CT3013CT564, Pluronic 31R1 Poly (Propylene glycol)-block-Poly (Ethylene glycol)-block- Sigma-Aldrich Poly (Propylene glycol) PPG-PEG-PPG
Pluronic 17R4 Poly (Propylene glycol)-block-Poly (Ethylene glycol)-block- Sigma-Aldrich Poly (Propylene glycol) PPG-PEG-PPG
Pluronic L-64 Poly (Propylene glycol)-block-Poly (Ethylene glycol)-block- Sigma-Aldrich 1001442463 Poly (Propylene glycol) PPG-PEG-PPG
Pterostilbene and Caffeine Co- Chromadex 00016988 Crystal Caffeine, 99% Alfa Aesar A10431 B06Y038 Cremophor ELP Sigma-Aldrich 30906 BCBH0387V
[0015]
For the preparation of TPGS-750-M and related compounds, see "TPGS-750-M:
A Second-Generation Amphiphile for Metal-Catalyzed Cross-Couplings in Water at Room Temperature" Lipshutz, B. H. et al. J. Org. Chem. 2011, 76, 4379-4391, and Sigma Aldrich.
For the preparation of TPGS-750-M and related compounds, see "TPGS-750-M:
A Second-Generation Amphiphile for Metal-Catalyzed Cross-Couplings in Water at Room Temperature" Lipshutz, B. H. et al. J. Org. Chem. 2011, 76, 4379-4391, and Sigma Aldrich.
[0016]
The following experimental description detailed for the analysis of pterostilbene is also performed similarly with resveratrol, and a mixture of pterostilbene and resveratrol. The analytical results for resveratrol compositions are similar to those obtained for pterostilbene.
Analytical Quantitation of Pterostilbene (PSB) or Resveratrol (RVT), or Mixtures thereof.
The following experimental description detailed for the analysis of pterostilbene is also performed similarly with resveratrol, and a mixture of pterostilbene and resveratrol. The analytical results for resveratrol compositions are similar to those obtained for pterostilbene.
Analytical Quantitation of Pterostilbene (PSB) or Resveratrol (RVT), or Mixtures thereof.
[0017]
Quantitation of pterostilbene in concentrated TPGS emulsions employs the analytical method Pterostilbene.M. or Resveratrol.M. Phenomenex 4.6 x 50 mm Luna 5 .
C18(2) 100A, part number 00B-4252-E0 column, mobile phase: isocratic 40/60 acetonitrile/0.01 M H3PO4 water for 7 minutes followed by a column wash and re-equilibration, flow rate: 1.5 mL/minute, detection: 254 nm, column temperature: 40 C, and injection volume:
5 L. Sample PATENT APPLICATION Attorney Docket No.
ISS.000200CA
preparation is a 1:10 dilution with isopropanol. Pterostilbene elutes at 4.5 minutes. A calibration curve/response linearity was prepared using 0.1, 0.25, 0.5, 1.0 and 2.5 mg/mL
solutions. The response is linear with an R2 of 0.9999. Since the target concentration of pterostilbene in consumer products is expected to be about 0.08 mg/mL (50-100 mg/drink serving), the sensitivity of the method was increased by changing the detection wavelength to 310 nm, the absorbance max of pterostilbene. Quantitation of resveratrol may also employ the above method.
Quantitation of pterostilbene in concentrated TPGS emulsions employs the analytical method Pterostilbene.M. or Resveratrol.M. Phenomenex 4.6 x 50 mm Luna 5 .
C18(2) 100A, part number 00B-4252-E0 column, mobile phase: isocratic 40/60 acetonitrile/0.01 M H3PO4 water for 7 minutes followed by a column wash and re-equilibration, flow rate: 1.5 mL/minute, detection: 254 nm, column temperature: 40 C, and injection volume:
5 L. Sample PATENT APPLICATION Attorney Docket No.
ISS.000200CA
preparation is a 1:10 dilution with isopropanol. Pterostilbene elutes at 4.5 minutes. A calibration curve/response linearity was prepared using 0.1, 0.25, 0.5, 1.0 and 2.5 mg/mL
solutions. The response is linear with an R2 of 0.9999. Since the target concentration of pterostilbene in consumer products is expected to be about 0.08 mg/mL (50-100 mg/drink serving), the sensitivity of the method was increased by changing the detection wavelength to 310 nm, the absorbance max of pterostilbene. Quantitation of resveratrol may also employ the above method.
[0018] Quantitation of pterostilbene in consumer products employs the analytical method Pterostilbene 310nm.M. Method: Phenomenex 4.6 x 50 mm Luna 5 C18(2) 100A, part number 00B-4252-E0 column, mobile phase: isocratic 40/60 acetonitrile/0.01 M
H3PO4 water for 7 minutes followed by a column wash and re-equilibration, flow rate: 1.5 mL/minute, detection:
310 nm, column temperature: 40 C, and injection volume: 5 L. Sample preparation is a 1:1 dilution with isopropanol. Pterostilbene elutes at 4.5 minutes. A calibration curve/response linearity was prepared using 0.01, 0.025, 0.05, and 0.1 mg/mL solutions. The response is linear with an R2 of 0.9997. Quantitation of resveratrol may also employ the above method.
H3PO4 water for 7 minutes followed by a column wash and re-equilibration, flow rate: 1.5 mL/minute, detection:
310 nm, column temperature: 40 C, and injection volume: 5 L. Sample preparation is a 1:1 dilution with isopropanol. Pterostilbene elutes at 4.5 minutes. A calibration curve/response linearity was prepared using 0.01, 0.025, 0.05, and 0.1 mg/mL solutions. The response is linear with an R2 of 0.9997. Quantitation of resveratrol may also employ the above method.
[0019] Quantitation of pterostilbene and caffeine in one method requires a detection wavelength at which both compounds have UV adsorbance and a mobile phase composition change to retain caffeine slightly in the analysis. Quantitation of pterostilbene and caffeine in concentrated TPGS emulsions employs the analytical method Pterostilbene 280.M.
Method:
Phenomenex 4.6 x 50 mm Luna 5 . C18(2) 100A, part number 00B-4252-E0 column, mobile phase: 100% 0.01 M H3PO4 water for 0.5 minutes, 100 to 60 % 0.01 M H3PO4 water/40%
acetonitrile from 0.5 to 2 minutes then hold for 5 minutes 40/60 acetonitrile/0.01 M H3PO4 water followed by a column wash and re-equilibration, flow rate: 1.5 mL/minute, detection: 280 nm, column temperature: 40 C, and injection volume: 1 L. Sample preparation is a 1:10 dilution with isopropanol. Caffeine elutes at 2.4 minutes and pterostilbene elutes at 7.1 minutes.
Quantitation of resveratrol and caffeine may use the above method.
Example 1. Preparation of 5 mg/mL PSB/TPGS, or RVT/TPGS emulsion.
Method:
Phenomenex 4.6 x 50 mm Luna 5 . C18(2) 100A, part number 00B-4252-E0 column, mobile phase: 100% 0.01 M H3PO4 water for 0.5 minutes, 100 to 60 % 0.01 M H3PO4 water/40%
acetonitrile from 0.5 to 2 minutes then hold for 5 minutes 40/60 acetonitrile/0.01 M H3PO4 water followed by a column wash and re-equilibration, flow rate: 1.5 mL/minute, detection: 280 nm, column temperature: 40 C, and injection volume: 1 L. Sample preparation is a 1:10 dilution with isopropanol. Caffeine elutes at 2.4 minutes and pterostilbene elutes at 7.1 minutes.
Quantitation of resveratrol and caffeine may use the above method.
Example 1. Preparation of 5 mg/mL PSB/TPGS, or RVT/TPGS emulsion.
[0020] Emulsion preparation: 5.008 g of TPGS was added to 100 mL of distilled water and mixed on magnetic stirring plate for 1 hr. 1 g of PSB was dissolved in 1.2 mL of 95%
ethanol. 0.6 mL of ethanol solution of PSB was added slowly to 100 mL of clear TPGS solution during mixing. The mixture stirred for 2 hr at RT. Upon clearing, the emulsion was filtered through 0.22 vtm filter, and particle size and PSB content was determined (Table 1).
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Table 1. Particle size, PSB content, and particle stability of resultant emulsion.
Particle size PSB Formulation Stability Manufacturing Lot # by i Date ntensity, Content, Recovery, Days past Particle size nm mg/mL mfg nm 11-Jun-13 005.20.3 23.5 4.08 81.6 14 21.7 [0021] HPLC Analysis. "Pterostilbene.M." method was used for determination of PSB
content. PSB content in resultant emulsion determined by HPLC was 4.08 mg/mL.
Data indicate that 81.6% of the PSB used for preparation of this formulation was incorporated into TPGS
particles (Table 1). Analysis of RVT provides similar results with PSB above.
ethanol. 0.6 mL of ethanol solution of PSB was added slowly to 100 mL of clear TPGS solution during mixing. The mixture stirred for 2 hr at RT. Upon clearing, the emulsion was filtered through 0.22 vtm filter, and particle size and PSB content was determined (Table 1).
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Table 1. Particle size, PSB content, and particle stability of resultant emulsion.
Particle size PSB Formulation Stability Manufacturing Lot # by i Date ntensity, Content, Recovery, Days past Particle size nm mg/mL mfg nm 11-Jun-13 005.20.3 23.5 4.08 81.6 14 21.7 [0021] HPLC Analysis. "Pterostilbene.M." method was used for determination of PSB
content. PSB content in resultant emulsion determined by HPLC was 4.08 mg/mL.
Data indicate that 81.6% of the PSB used for preparation of this formulation was incorporated into TPGS
particles (Table 1). Analysis of RVT provides similar results with PSB above.
[0022] Particle size analysis and stability. The resultant emulsion was stable. Table 1 shows that particle size did not increase over 14 days of monitoring. Particle size for RVT are similar to PSB.
[0023] Example 2. Preparation of 10 mg/mL PSB/TPGS emulsion. 5.004 g of TPGS and 1.003 g of PSB were weighed and placed in 400 mL beaker. 9 mL of 95% ethanol were added to the solids, and the beaker containing ethanol mixture of TPGS/PSB was placed in the water bath, and incubated at 60 C for 20 min until clear. The ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
[0024] Emulsion preparation: The beaker was removed from vacuum oven and 100 mL
of distilled water preheated to 60 C was added to the highly viscous clear film of TPGS/PBS
mixture, the beaker was placed in 60 C water bath and total mixture was incubated for 30 min to dissolve. The water TPGS/PBS mixture was mixed on magnetic stirring plate for 2 hrs at RT.
Upon clearing of the mixture, the emulsion was filtered through 0.22 vim filter, and particle size and PSB content of the resultant emulsion were determined (Table 2).
Table 2. Particle size, PSB content, and particle stability of resultant emulsion.
Particle size PSB Formulation Stability Manufacturing Lot# by intensity,Date Content, Recovery, Days past Particle size nm mg/mL mfg nm 12-Jun-13 005.21.4 26.1 10.57 105.6 40 25.9 [0025] HPLC Analysis."Pterostilbene. 310nm.M." method was used for determination of PSB content. PSB content in resultant emulsion determined by HPLC was 10.57 mg/mL. Data indicate that 105.6% of the PSB used for preparation of this formulation was incorporated into TPGS particles (Table 2). An incorporation value above 100% could result because of water evaporation during 30 min of incubation at 60 C.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
of distilled water preheated to 60 C was added to the highly viscous clear film of TPGS/PBS
mixture, the beaker was placed in 60 C water bath and total mixture was incubated for 30 min to dissolve. The water TPGS/PBS mixture was mixed on magnetic stirring plate for 2 hrs at RT.
Upon clearing of the mixture, the emulsion was filtered through 0.22 vim filter, and particle size and PSB content of the resultant emulsion were determined (Table 2).
Table 2. Particle size, PSB content, and particle stability of resultant emulsion.
Particle size PSB Formulation Stability Manufacturing Lot# by intensity,Date Content, Recovery, Days past Particle size nm mg/mL mfg nm 12-Jun-13 005.21.4 26.1 10.57 105.6 40 25.9 [0025] HPLC Analysis."Pterostilbene. 310nm.M." method was used for determination of PSB content. PSB content in resultant emulsion determined by HPLC was 10.57 mg/mL. Data indicate that 105.6% of the PSB used for preparation of this formulation was incorporated into TPGS particles (Table 2). An incorporation value above 100% could result because of water evaporation during 30 min of incubation at 60 C.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
[0026] Particle size analysis and stability. The resultant emulsion was stable. It can be seen in Table 2 that particle size did not significantly change over 40 days of monitoring.
[0027] Example 3. Dilution of PSB/TPGS emulsion into distilled water.
[0028] Procedure 1: PSB/TPGS / Water solution preparation: 170 ill of TPGS/PSB
emulsion was added to 21 mL of Distilled Water. The water solution of PSB/TPGS
emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Water solution was diluted 1:1 with IPA. HPLC analysis of PSB content in Table 3a.
Table 3a. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated HPLC, Recovery, Days past PSB, mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.083 97.00 3 [0029] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 3a shows that after 3 days of storage at RT the PSB
content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data shows that formulated PSB is stable in water.
emulsion was added to 21 mL of Distilled Water. The water solution of PSB/TPGS
emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Water solution was diluted 1:1 with IPA. HPLC analysis of PSB content in Table 3a.
Table 3a. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated HPLC, Recovery, Days past PSB, mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.083 97.00 3 [0029] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 3a shows that after 3 days of storage at RT the PSB
content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data shows that formulated PSB is stable in water.
[0030] Procedure 2: PSB/TPGS/Water solution preparation: 170111 of TPGS/PSB
emulsion (lot#005.21.4) was added to 21 mL of Distilled Water. The solution of PSB/TPGS
emulsion was mixed and stored for 3 and/or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS / Water solution was diluted 1:1 with IPA, filtered through 0.22 i_tm filter, and HPLC
analysis of PSB content was performed (Table 3b).
Table 3b. PSB content and Stability of resultant solution.
PSB content Preparation HPLC
Calculated PSB, HPLC, Days past Date Date Recovery, %
mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.082 95.83 3 25-Jun-13 0.086 0.084 98.17 11 [0031] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 3b shows that after 11 days of storage at RT the PSB
content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the water. Data shows high stability of formulated PSB in water.
emulsion (lot#005.21.4) was added to 21 mL of Distilled Water. The solution of PSB/TPGS
emulsion was mixed and stored for 3 and/or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS / Water solution was diluted 1:1 with IPA, filtered through 0.22 i_tm filter, and HPLC
analysis of PSB content was performed (Table 3b).
Table 3b. PSB content and Stability of resultant solution.
PSB content Preparation HPLC
Calculated PSB, HPLC, Days past Date Date Recovery, %
mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.082 95.83 3 25-Jun-13 0.086 0.084 98.17 11 [0031] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 3b shows that after 11 days of storage at RT the PSB
content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the water. Data shows high stability of formulated PSB in water.
[0032] Procedure 3: PSB/TPGS / Water solution preparation: 170 IA of TPGS/PSB
emulsion (lot#005.21.4) was added to 21 mL of Distilled Water. The water solution of PATENT APPLICATION Attorney Docket No.
ISS.000200CA
PSB/TPGS emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 1.0 mL aliquot of PSB/TPGS / Water solution was filtered through 0.22 p.m filter, diluted 1:1 with IPA. and HPLC analysis of PSB content was performed (Table 3c).
Table 3c. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated HPLC, Recovery, Days past PSB, mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.073 85.31 3 [0033] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 3c shows that PSB content of the water/PSB/TPGS emulsion when first filtered and then diluted with IPA was significantly lower compared to unfiltered (Procedure 1) and/or dilution with IPA first and then filtered (Procedure 2) solutions.
Considering that no degradation of PSB or precipitate was observed in the water, the data suggest loss of some material on the filter when not diluted with IPA. Procedure 1 and 2 yielded similar PSB values.
emulsion (lot#005.21.4) was added to 21 mL of Distilled Water. The water solution of PATENT APPLICATION Attorney Docket No.
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PSB/TPGS emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 1.0 mL aliquot of PSB/TPGS / Water solution was filtered through 0.22 p.m filter, diluted 1:1 with IPA. and HPLC analysis of PSB content was performed (Table 3c).
Table 3c. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated HPLC, Recovery, Days past PSB, mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.073 85.31 3 [0033] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 3c shows that PSB content of the water/PSB/TPGS emulsion when first filtered and then diluted with IPA was significantly lower compared to unfiltered (Procedure 1) and/or dilution with IPA first and then filtered (Procedure 2) solutions.
Considering that no degradation of PSB or precipitate was observed in the water, the data suggest loss of some material on the filter when not diluted with IPA. Procedure 1 and 2 yielded similar PSB values.
[0034] Particle size analysis and stability. PSB/TPGS/Water solution preparation: 170 IA of PSB/TPGS emulsion (lot#005.21.4) was added to 21 mL of Distilled Water.
The water solution of PSB/TPGS emulsion was mixed and stored for 0, 2, and 6 days at RT.
0, 2, and/or 6 days later 0.3 mL of PSB/TPGS/Water solution was added to the cuvette containing 2.2 mL of water and particle size was measured. To obtain baseline for distilled water 0.3 mL of water were added to cuvette containing 2.2 mL of water. Particle size analysis is noted in Table 3d. Particle size did not change over 6 days at RT, showing that dilution in water does not affect emulsion stability.
Table 3d. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Water N/A 0 19-Jun-13 Water plus Lot# 005.21.4 36.6 0 21-Jun-13 Water plus Lot# 005.21.4 37.7 2 25-Jun-13 Water plus Lot# 005.21.4 38.0 6 [0035] Example 4. Preparation of PSB/TPGS/Reb A emulsion. 100 mg of Reb A
was added directly to 5 mL of PSB/TPGS emulsion lot# 005.21.4 and mixed on magnetic stirring plate at RT until clear (-30 min). Upon clearing of the mixture, the emulsion was filtered through 0.22 tm filter, and particle size and PSB content of the emulsion determined (Table 4).
Table 4. Particle size, PSB content, and particle stability of resultant emulsion.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Particle size PSB Formulation Stability Manufacturing Lot# by Date intensity, Content, Recovery, Days past Particle size nm mg/mL mfg nm 14-Jun-13 005.21.5 16.2 9.60 90.8 4 16.1 [0036] HPLC Analysis. "Pterostilbene.M." method was used for determination of PSB
content. PSB content in resultant emulsion determined by HPLC was 9.6 mg/mL.
The data indicate that 90.8% of the PSB determined in emulsion lot # 005.21.4 was recovered in Reb A
containing emulsion (Table 4). Direct filtration of the aqueous PSB/TPGS
emulsion without first dilution in IPA causes some loss to the filter by adsorption onto the filter membrane.
The water solution of PSB/TPGS emulsion was mixed and stored for 0, 2, and 6 days at RT.
0, 2, and/or 6 days later 0.3 mL of PSB/TPGS/Water solution was added to the cuvette containing 2.2 mL of water and particle size was measured. To obtain baseline for distilled water 0.3 mL of water were added to cuvette containing 2.2 mL of water. Particle size analysis is noted in Table 3d. Particle size did not change over 6 days at RT, showing that dilution in water does not affect emulsion stability.
Table 3d. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Water N/A 0 19-Jun-13 Water plus Lot# 005.21.4 36.6 0 21-Jun-13 Water plus Lot# 005.21.4 37.7 2 25-Jun-13 Water plus Lot# 005.21.4 38.0 6 [0035] Example 4. Preparation of PSB/TPGS/Reb A emulsion. 100 mg of Reb A
was added directly to 5 mL of PSB/TPGS emulsion lot# 005.21.4 and mixed on magnetic stirring plate at RT until clear (-30 min). Upon clearing of the mixture, the emulsion was filtered through 0.22 tm filter, and particle size and PSB content of the emulsion determined (Table 4).
Table 4. Particle size, PSB content, and particle stability of resultant emulsion.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Particle size PSB Formulation Stability Manufacturing Lot# by Date intensity, Content, Recovery, Days past Particle size nm mg/mL mfg nm 14-Jun-13 005.21.5 16.2 9.60 90.8 4 16.1 [0036] HPLC Analysis. "Pterostilbene.M." method was used for determination of PSB
content. PSB content in resultant emulsion determined by HPLC was 9.6 mg/mL.
The data indicate that 90.8% of the PSB determined in emulsion lot # 005.21.4 was recovered in Reb A
containing emulsion (Table 4). Direct filtration of the aqueous PSB/TPGS
emulsion without first dilution in IPA causes some loss to the filter by adsorption onto the filter membrane.
[0037] Particle size analysis and stability. The particle size of the Reb A containing emulsion was significantly smaller than the emulsion with PSB and TPGS only.
The resultant emulsion was stable. As seen in Table 4 particle size did not change over 4 days of monitoring.
The resultant emulsion was stable. As seen in Table 4 particle size did not change over 4 days of monitoring.
[0038] Example 5. Dilution of PSB/TPGS/Reb A emulsion in distilled water.
[0039] Procedure 1: PSB/TPGS/Reb A water solution preparation: 170 1 of PSB/TPGS/Reb A emulsion (lot#005.21.5) was added to 20 mL of distilled water.
The water solution of PSB/TPGS/Reb A emulsion (lot# 005.21.5) was mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Reb A/Water solution was diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 5a).
Table 5a. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated HPLC, Days past Date Date Recovery, %
PSB, mg/mL mg/mL
preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.081 99.26 3 [0040] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 5a shows that after 3 days of storage at RT PSB content of resultant solution determined by HPLC was similar to calculated amount of PSB added to the water. Data shows that TPGS/Reb A formulated PSB is stable in water.
The water solution of PSB/TPGS/Reb A emulsion (lot# 005.21.5) was mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Reb A/Water solution was diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 5a).
Table 5a. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated HPLC, Days past Date Date Recovery, %
PSB, mg/mL mg/mL
preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.081 99.26 3 [0040] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 5a shows that after 3 days of storage at RT PSB content of resultant solution determined by HPLC was similar to calculated amount of PSB added to the water. Data shows that TPGS/Reb A formulated PSB is stable in water.
[0041] Procedure 2: PSB/TPGS/Reb A water solution preparation: 170 tl of PSB/TPGS/Reb A emulsion (lot#005.21.5) was added to 20 mL of distilled water.
The solution of PSB/TPGS/Reb A emulsion was mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/Reb A water solution was diluted 1:1 with IPA, filtered through 0.22 p.m filter, and HPLC analysis of PSB content (Table 5b).
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Table 5b. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated HPLC, Days past Date Date Recovery, %
PSB, mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.081 99.26 3 25-Jun-13 0.082 0.083 101.72 11 [0042] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 5b shows that after 11 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the aqueous medium. Data suggest that TPGS/Reb A formulated PSB is stable in water.
The solution of PSB/TPGS/Reb A emulsion was mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/Reb A water solution was diluted 1:1 with IPA, filtered through 0.22 p.m filter, and HPLC analysis of PSB content (Table 5b).
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Table 5b. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated HPLC, Days past Date Date Recovery, %
PSB, mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.081 99.26 3 25-Jun-13 0.082 0.083 101.72 11 [0042] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 5b shows that after 11 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the aqueous medium. Data suggest that TPGS/Reb A formulated PSB is stable in water.
[0043] Procedure 3: PSB/TPGS/Reb A water solution preparation: 1701_11 of PSB/TPGS
/Reb A emulsion (lot#005.21.5) was added to 20 mL of distilled water. Water solution of PSB/TPGS/Reb A emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 1.0 mL
aliquot of PSB/TPGS/Reb A/Water solution was filtered through 0.22 i_tm filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 5c).
Table 5c. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated HPLC, Days past Date Date Recovery, 0/0 PSB, mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.068 83.33 3 [0044] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 5c shows that PSB content of the emulsion mixture directly filtered and then diluted with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA first and then filtered (Procedure 2) solutions. No degradation of PSB or precipitate was observed in the water, the data shows the loss of some material on the filter when the emulsion preparation is not diluted with IPA. Procedure 1 and 2 yielded similar PSB content.
/Reb A emulsion (lot#005.21.5) was added to 20 mL of distilled water. Water solution of PSB/TPGS/Reb A emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 1.0 mL
aliquot of PSB/TPGS/Reb A/Water solution was filtered through 0.22 i_tm filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 5c).
Table 5c. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated HPLC, Days past Date Date Recovery, 0/0 PSB, mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.068 83.33 3 [0044] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 5c shows that PSB content of the emulsion mixture directly filtered and then diluted with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA first and then filtered (Procedure 2) solutions. No degradation of PSB or precipitate was observed in the water, the data shows the loss of some material on the filter when the emulsion preparation is not diluted with IPA. Procedure 1 and 2 yielded similar PSB content.
[0045] Particle size analysis and stability. PSB/TPGS/Reb A/ Water solution preparation: 170 ,1 of PSB/TPGS/Reb A emulsion (lot#005.21.5) was added to 20 mL of distilled water. Water solution of PSB/TPGS/Reb A emulsion was briefly mixed and stored for 6 days at RT. Immediately after preparation and/or 6 days later 0.3 mL of PSB/TPGS/Reb A/Water solution was added to the cuvette containing 2.2 mL of distilled water and particle size was measured. To obtain baseline for distilled water 0.3 mL of distilled water were added to cuvette containing 2.2 mL of distilled water. See Table 5d.
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Table 5d. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Water N/A 0 19-Jun-13 Water plus Lot# 005.21.5 20.9 0 25-Jun-13 Water plus Lot# 005.21.5 21.7 6 [0046] The particle size did not change over 6 days of storage at RT.
Data suggest that dilution in water does not impair PSB/TPGS/Reb A emulsion stability under these conditions (Table 5d). Data suggest chemical and particle stability of formulated PSB in water.
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Table 5d. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Water N/A 0 19-Jun-13 Water plus Lot# 005.21.5 20.9 0 25-Jun-13 Water plus Lot# 005.21.5 21.7 6 [0046] The particle size did not change over 6 days of storage at RT.
Data suggest that dilution in water does not impair PSB/TPGS/Reb A emulsion stability under these conditions (Table 5d). Data suggest chemical and particle stability of formulated PSB in water.
[0047] Example 6. Preparation of PSB/TPGS/PowerAde solution.
Table 6 a. Formulation composition.
Date mg per 22 ml TPGS PSB Powerade 14-Jun-13 8.5 1.7 22 ml [0048] Procedure 1: PSB/TPGS /PowerAde solution preparation: 170 pi of TPGS/PSB
emulsion (lot#005.21.4) was added to 22 mL of PowerAde beverage. PowerAde solution of PSB/TPGS emulsion was mixed and stored for 3 days at RT. 3 Days later 0.6 mL
aliquot of PSB/TPGS/PowerAde solution was diluted 1:1 with IPA, and HPLC analysis of PSB
content was performed (Table 6b).
Table 6 b. PSB content of resultant solution. Stability.
PSB content Preparation HPLC
Calculated PSB, HPLC, Days past Date Date Recovery, A
mg/ml mg/ml preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.079 96.72 3 [0049] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 6b that after 3 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage. Data suggest that TPGS formulated PSB is stable in PowerAde beverage.
Table 6 a. Formulation composition.
Date mg per 22 ml TPGS PSB Powerade 14-Jun-13 8.5 1.7 22 ml [0048] Procedure 1: PSB/TPGS /PowerAde solution preparation: 170 pi of TPGS/PSB
emulsion (lot#005.21.4) was added to 22 mL of PowerAde beverage. PowerAde solution of PSB/TPGS emulsion was mixed and stored for 3 days at RT. 3 Days later 0.6 mL
aliquot of PSB/TPGS/PowerAde solution was diluted 1:1 with IPA, and HPLC analysis of PSB
content was performed (Table 6b).
Table 6 b. PSB content of resultant solution. Stability.
PSB content Preparation HPLC
Calculated PSB, HPLC, Days past Date Date Recovery, A
mg/ml mg/ml preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.079 96.72 3 [0049] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 6b that after 3 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage. Data suggest that TPGS formulated PSB is stable in PowerAde beverage.
[0050] Procedure 2: PSB/TPGS/ PowerAde solution preparation: 170 [11 of PSB/TPGS
emulsion (lot#005.21.4) was added to 22 mL of PowerAde beverage. PSB/TPGS/
PowerAde solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/PowerAde solution was diluted 1:1 with IPA, filtered through 0.22 pm filter, and HPLC analysis of PSB content was performed (Table 6c).
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Table 6c. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.079 96.72 3 25-Jun-13 0.082 0.081 99.17 11 [0051] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 6c shows that after 11 days of storage at RT PSB content in the resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data shows that TPGS formulated PSB is stable in PowerAde beverage.
emulsion (lot#005.21.4) was added to 22 mL of PowerAde beverage. PSB/TPGS/
PowerAde solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/PowerAde solution was diluted 1:1 with IPA, filtered through 0.22 pm filter, and HPLC analysis of PSB content was performed (Table 6c).
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
Table 6c. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.079 96.72 3 25-Jun-13 0.082 0.081 99.17 11 [0051] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 6c shows that after 11 days of storage at RT PSB content in the resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data shows that TPGS formulated PSB is stable in PowerAde beverage.
[0052] Procedure 3: PSB/TPGS/ PowerAde solution preparation: 170 Ill of PSB/TPGS
emulsion (lot#005.21.4) was added to 22 mL of PowerAde beverage. PSB/TPGS/
PowerAde solution was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL
aliquot of PSB/TPGS/PowerAde solution was filtered through 0.22 pm filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 6d).
Table 6d. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.062 75.91 3 [0053] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 6d (recovery %) shows that PSB content of directly filtered PSB/TPGS/PowerAde solution and then dilution with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB
recovery in filtered PowerAde solution of formulated PSB was significantly lower compared to that of filtered PSB/TPGS water solution (examples 3 and 5). Data shows that part of the formulated PSB was bound to the PowerAde coarse matrix and was retained on the filter during filtration. Dilution of PSB/TPGS/PowerAde solution with IPA prior to filtration releases PSB
from the PowerAde matrix and prevents its filter retention.
emulsion (lot#005.21.4) was added to 22 mL of PowerAde beverage. PSB/TPGS/
PowerAde solution was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL
aliquot of PSB/TPGS/PowerAde solution was filtered through 0.22 pm filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 6d).
Table 6d. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.062 75.91 3 [0053] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 6d (recovery %) shows that PSB content of directly filtered PSB/TPGS/PowerAde solution and then dilution with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB
recovery in filtered PowerAde solution of formulated PSB was significantly lower compared to that of filtered PSB/TPGS water solution (examples 3 and 5). Data shows that part of the formulated PSB was bound to the PowerAde coarse matrix and was retained on the filter during filtration. Dilution of PSB/TPGS/PowerAde solution with IPA prior to filtration releases PSB
from the PowerAde matrix and prevents its filter retention.
[0054] Particle size analysis and stability. PSB/TPGS/ PowerAde solution preparation:
170 [1.1 of PSB/TPGS emulsion (lot#005.21.4) was added to 22 mL of PowerAde beverage.
PSB/TPGS/PowerAde solution was mixed and stored for 2 and 6 days at RT.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
170 [1.1 of PSB/TPGS emulsion (lot#005.21.4) was added to 22 mL of PowerAde beverage.
PSB/TPGS/PowerAde solution was mixed and stored for 2 and 6 days at RT.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
[0055] To avoid contribution of coarse beverage matrix to particle size measurement, the beverage was centrifuged at 13,000 rpm for 15 min in an eppendorf centrifuge.
To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL of distilled water. To determine effect of the beverage on particle size of TPGS/PSP
emulsion, 0.6 mL of emulsion (lot #005.21.4) was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of distilled water. See Table 6e.
Table 6 e. Particle size and stability.
Preparation Material Particle size Days past Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot # 005.21.4 36.6 0 19-Jun-13 Powerade 609.9 0 19-Jun-13 Powerade plus Lot# 005.21.4 40.7 21-Jun-13 Powerade plus Lot# 005.21.4 39.6 25-Jun-13 Powerade plus Lot# 005.21.4 41.6 [0056] There was 4 nm initial increase of the particle size of the TPGS/PSB
emulsion upon dilution in Powerade beverage, the particle size did not change over next six days. Absence of further particle size change suggests stability of formulated PSB in Powerade beverage. Data obtained indicate chemical and particle stability of formulated PSB in Powerade beverage and suggest 100% of PSB consumption with the beverage.
To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL of distilled water. To determine effect of the beverage on particle size of TPGS/PSP
emulsion, 0.6 mL of emulsion (lot #005.21.4) was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of distilled water. See Table 6e.
Table 6 e. Particle size and stability.
Preparation Material Particle size Days past Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot # 005.21.4 36.6 0 19-Jun-13 Powerade 609.9 0 19-Jun-13 Powerade plus Lot# 005.21.4 40.7 21-Jun-13 Powerade plus Lot# 005.21.4 39.6 25-Jun-13 Powerade plus Lot# 005.21.4 41.6 [0056] There was 4 nm initial increase of the particle size of the TPGS/PSB
emulsion upon dilution in Powerade beverage, the particle size did not change over next six days. Absence of further particle size change suggests stability of formulated PSB in Powerade beverage. Data obtained indicate chemical and particle stability of formulated PSB in Powerade beverage and suggest 100% of PSB consumption with the beverage.
[0057] Example 7. Preparation of PSB/TPGS/LifeWater solution.
Table 7a. Formulation composition.
mg per 21 mL
Date TPGS PSB LifeWater 14-Jun-13 8.5 1.7 21 mL
Table 7a. Formulation composition.
mg per 21 mL
Date TPGS PSB LifeWater 14-Jun-13 8.5 1.7 21 mL
[0058] Procedure 1: PSB/TPGS/LifeWater solution preparation: 170 ill of TPGS/PSB
emulsion (lot#005.21.4) was added to 21 mL of LifeWater beverage. LifeWater solution of PSB/TPGS emulsion was mixed and stored for 3 days at RT. 3 Days later 0.6 mL
aliquot of PSB/TPGS/LifeWater solution was diluted 1:1 with IPA, and HPLC analysis of PSB
content was performed (Table 7b).
Table 7b. PSB content of resultant solution.
PSB content Preparation HPLC
Dat Date Calculated PSB, HPLC, Recovery, Days past e mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.082 95.83 3 PATENT APPLICATION Attorney Docket No. ISS.000200CA
emulsion (lot#005.21.4) was added to 21 mL of LifeWater beverage. LifeWater solution of PSB/TPGS emulsion was mixed and stored for 3 days at RT. 3 Days later 0.6 mL
aliquot of PSB/TPGS/LifeWater solution was diluted 1:1 with IPA, and HPLC analysis of PSB
content was performed (Table 7b).
Table 7b. PSB content of resultant solution.
PSB content Preparation HPLC
Dat Date Calculated PSB, HPLC, Recovery, Days past e mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.082 95.83 3 PATENT APPLICATION Attorney Docket No. ISS.000200CA
[0059] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 7b shows that after 3 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
The data shows that TPGS formulated PSB is stable in LifeWater beverage.
The data shows that TPGS formulated PSB is stable in LifeWater beverage.
[0060] Procedure 2: PSB/TPGS/ LifeWater solution preparation: 170 tl of PSB/TPGS
emulsion (lot#005.21.4) was added to 21 mL of LifeWater beverage.
PSB/TPGS/LifeWater solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/LifeWater solution was diluted 1:1 with IPA, filtered through 0.22 1.tm filter, and HPLC analysis of PSB content was performed (Table 7c).
Table 7c. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated PSB, HPLC, Days past Date Date Recovery, %
mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.077 89.99 3 25-Jun-13 0.086 0.091 106.35 11 [0061] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 7c shows that after 11 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in LifeWater beverage.
emulsion (lot#005.21.4) was added to 21 mL of LifeWater beverage.
PSB/TPGS/LifeWater solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/LifeWater solution was diluted 1:1 with IPA, filtered through 0.22 1.tm filter, and HPLC analysis of PSB content was performed (Table 7c).
Table 7c. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated PSB, HPLC, Days past Date Date Recovery, %
mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.077 89.99 3 25-Jun-13 0.086 0.091 106.35 11 [0061] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 7c shows that after 11 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in LifeWater beverage.
[0062] Procedure 3: PSB/TPGS/LifeWater solution preparation: 170 tl of PSB/TPGS
emulsion (lot#005.21.4) was added to 21 mL of LifeWater beverage.
PSB/TPGS/LifeWater solution was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL
aliquot of PSB/TPGS/LifeWater solution was filtered through 0.22 tm filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 7d).
Table 7d. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.069 80.64 3 [0063] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 7d (recovery %) shows that PSB content of directly filtered PSB/TPGS/
LifeWater solution and then dilution with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB recovery in PATENT APPLICATION Attorney Docket No.
ISS.000200CA
=
filtered LifeWater solution of formulated PSB was similar to that of filtered PSB/TPGS water solution (examples 3 and 5). Data shows no significant binding of PSB to LifeWater matrix.
emulsion (lot#005.21.4) was added to 21 mL of LifeWater beverage.
PSB/TPGS/LifeWater solution was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL
aliquot of PSB/TPGS/LifeWater solution was filtered through 0.22 tm filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 7d).
Table 7d. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.069 80.64 3 [0063] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 7d (recovery %) shows that PSB content of directly filtered PSB/TPGS/
LifeWater solution and then dilution with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB recovery in PATENT APPLICATION Attorney Docket No.
ISS.000200CA
=
filtered LifeWater solution of formulated PSB was similar to that of filtered PSB/TPGS water solution (examples 3 and 5). Data shows no significant binding of PSB to LifeWater matrix.
[0064] Particle size analysis and stability. PSB/TPGS/LifeWater solution preparation:
170 p.1 of PSB/TPGS emulsion (1 0005.21.4) was added to 21 mL of LifeWater beverage.
PSB/TPGS/LifeWater solution was briefly mixed and stored for 2 and 6 days at RT. The beverage was centrifuged at 13,000 rpm for 15 min in an eppendorf centrifuge.
To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL of distilled water. To determine effect of the beverage on particle size of TPGS/PSP
emulsion, 0.6 mL of emulsion (lot # 005.21.4) was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of distilled water. See Table 7e.
Table 7e. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot# 005.21.4 36.6 0 19-Jun-13 LifeWater 376.9 0 19-Jun-13 LifeWater plus Lot# 005.21.4 39.2 21-Jun-13 LifeWater plus Lot# 005.21.4 37.9 25-Jun-13 LifeWater plus Lot# 005.21.4 38.9 [0065] Although, there was 3 nm initial increase of the particle size of the TPGS/PSB
emulsion upon dilution in LifeWater beverage, the particle size did not change over next six days of monitoring. Data shows that contribution of beverage only particles into final particle size of the mixture was not significant. Absence of further particle size change suggests stability of formulated PSB in LifeWater beverage. Data indicate chemical and particle stability of formulated PSB in LifeWater beverage and show 100% of PSB consumption with the beverage.
170 p.1 of PSB/TPGS emulsion (1 0005.21.4) was added to 21 mL of LifeWater beverage.
PSB/TPGS/LifeWater solution was briefly mixed and stored for 2 and 6 days at RT. The beverage was centrifuged at 13,000 rpm for 15 min in an eppendorf centrifuge.
To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL of distilled water. To determine effect of the beverage on particle size of TPGS/PSP
emulsion, 0.6 mL of emulsion (lot # 005.21.4) was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of distilled water. See Table 7e.
Table 7e. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot# 005.21.4 36.6 0 19-Jun-13 LifeWater 376.9 0 19-Jun-13 LifeWater plus Lot# 005.21.4 39.2 21-Jun-13 LifeWater plus Lot# 005.21.4 37.9 25-Jun-13 LifeWater plus Lot# 005.21.4 38.9 [0065] Although, there was 3 nm initial increase of the particle size of the TPGS/PSB
emulsion upon dilution in LifeWater beverage, the particle size did not change over next six days of monitoring. Data shows that contribution of beverage only particles into final particle size of the mixture was not significant. Absence of further particle size change suggests stability of formulated PSB in LifeWater beverage. Data indicate chemical and particle stability of formulated PSB in LifeWater beverage and show 100% of PSB consumption with the beverage.
[0066] Example 8. Preparation of PSB/TPGS/Minute Maid solution.
Table 8a. Formulation composition.
Date mg per 22 mL
TPGS PSB Minute Maid 14-Jun-13 8.5 1.7 22 mL
Table 8a. Formulation composition.
Date mg per 22 mL
TPGS PSB Minute Maid 14-Jun-13 8.5 1.7 22 mL
[0067] Procedure 1: PSB/TPGS/Minute Maid solution preparation: 170 I of TPGS/PSB emulsion (lot#005.21.4) was added to 22 mL of Minute Maid beverage.
Minute Maid solution of PSB/TPGS emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Minute Maid solution was diluted 1:1 with IPA. See Table 8b.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Table 8b. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.075 91.82 3 [0068] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 8b shows that after 3 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Minute Maid beverage.
Minute Maid solution of PSB/TPGS emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Minute Maid solution was diluted 1:1 with IPA. See Table 8b.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Table 8b. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.075 91.82 3 [0068] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 8b shows that after 3 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Minute Maid beverage.
[0069] Procedure 2: PSB/TPGS/ Minute Maid solution preparation: 170 p1 of PSB/TPGS emulsion (lot#005.21.4) was added to 22 mL of Minute Maid beverage.
PSB/TPGS/Minute Maid solution was briefly mixed and stored for 3 or 11 days at room temperature (RT). 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/Minute Maid solution was diluted 1:1 with IPA, filtered through 0.22 1.im filter, and HPLC analysis of PSB content was performed (Table 8c).
Table 8c. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated PSB, HPLC, Recovery, Days past Date Date ing/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.077 94.27 3 25-Jun-13 0.082 0.089 108.97 11 [0070] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 8c shows that after 11 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Minute Maid beverage.
PSB/TPGS/Minute Maid solution was briefly mixed and stored for 3 or 11 days at room temperature (RT). 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/Minute Maid solution was diluted 1:1 with IPA, filtered through 0.22 1.im filter, and HPLC analysis of PSB content was performed (Table 8c).
Table 8c. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated PSB, HPLC, Recovery, Days past Date Date ing/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.077 94.27 3 25-Jun-13 0.082 0.089 108.97 11 [0070] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 8c shows that after 11 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Minute Maid beverage.
[0071] Procedure 3: PSB/TPGS/ Minute Maid solution preparation: 170 ill of PSB/TPGS emulsion (100005.21.4) was added to 22 mL of Minute Maid beverage.
PSB/TPGS/Minute Maid solution was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Minute Maid solution was filtered through 0.22 i_tm filter, diluted 1:1 with IPA and HPLC analysis of PSB content was performed (Table 8d).
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
Table 8d. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL c/0 preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.056 68.56 3 [0072] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 8d (recovery %) shows that PSB content of directly filtered PSB/TPGS/Minute Maid solution and then dilution with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions.
PSB recovery in filtered Minute Maid solution of formulated PSB was notably lower compared to that of filtered water PSB/TPGS solution (examples 3 and 5). Data suggest that a substantial portion of formulated PSB was bound to the Minute Maid coarse matrix and was retained on the filter during filtration. Dilution of PSB/TPGS/ Minute Maid solution with IPA
prior to filtration releases PSB from the Minute Maid matrix and prevents its filter retention.
PSB/TPGS/Minute Maid solution was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Minute Maid solution was filtered through 0.22 i_tm filter, diluted 1:1 with IPA and HPLC analysis of PSB content was performed (Table 8d).
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
Table 8d. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL c/0 preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.082 0.056 68.56 3 [0072] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 8d (recovery %) shows that PSB content of directly filtered PSB/TPGS/Minute Maid solution and then dilution with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions.
PSB recovery in filtered Minute Maid solution of formulated PSB was notably lower compared to that of filtered water PSB/TPGS solution (examples 3 and 5). Data suggest that a substantial portion of formulated PSB was bound to the Minute Maid coarse matrix and was retained on the filter during filtration. Dilution of PSB/TPGS/ Minute Maid solution with IPA
prior to filtration releases PSB from the Minute Maid matrix and prevents its filter retention.
[0073] Particle size analysis and stability. PSB/TPGS/Minute Maid solution preparation: 170 IA of PSB/TPGS emulsion (lot#005.21.4) was added to 22 mL of Minute Maid beverage. PSB/TPGS/Minute Maid solution was briefly mixed and stored for 2 and 6 days at RT.
The beverage was centrifuged at 13,000 rpm for 15 min in an eppendorf centrifuge. To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL of distilled water. Effect of the beverage on particle size of TPGS/PSP
emulsion, 0.6 mL of emulsion (lot #005.21.4) was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of water. Particle size noted in Table 8e.
Table 8e. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot# 005.21.4 36.6 0 19-Jun-13 Minute Maid 392.2 0 19-Jun-13 Minute Maid plus Lot# 005.21.4 47.9 0 21-Jun-13 Minute Maid plus Lot# 005.21.4 47.3 2 25-Jun-13 Minute Maid plus Lot# 005.21.4 48.4 6 [0074] There was a 11 nm increase of the particle size of formulated PSB
upon dilution in Minute Maid beverage. The particle size did not change over the next six days of monitoring.
These results suggest significant contribution of beverage only particles into final particle size of the mixture. The light scattering data are in agreement with HPLC data demonstrating substantial binding of formulated PSB to Minute Maid coarse matrix. Absence of further particle size PATENT APPLICATION Attorney Docket No.
ISS.000200CA
change shows stability of formulation. Data indicate chemical and particle stability of formulated PSB in Minute Maid beverage and suggest 100% of PSB consumption with the beverage.
The beverage was centrifuged at 13,000 rpm for 15 min in an eppendorf centrifuge. To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL of distilled water. Effect of the beverage on particle size of TPGS/PSP
emulsion, 0.6 mL of emulsion (lot #005.21.4) was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of water. Particle size noted in Table 8e.
Table 8e. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot# 005.21.4 36.6 0 19-Jun-13 Minute Maid 392.2 0 19-Jun-13 Minute Maid plus Lot# 005.21.4 47.9 0 21-Jun-13 Minute Maid plus Lot# 005.21.4 47.3 2 25-Jun-13 Minute Maid plus Lot# 005.21.4 48.4 6 [0074] There was a 11 nm increase of the particle size of formulated PSB
upon dilution in Minute Maid beverage. The particle size did not change over the next six days of monitoring.
These results suggest significant contribution of beverage only particles into final particle size of the mixture. The light scattering data are in agreement with HPLC data demonstrating substantial binding of formulated PSB to Minute Maid coarse matrix. Absence of further particle size PATENT APPLICATION Attorney Docket No.
ISS.000200CA
change shows stability of formulation. Data indicate chemical and particle stability of formulated PSB in Minute Maid beverage and suggest 100% of PSB consumption with the beverage.
[0075] Example 9. Preparation of PSB/TPGS/Welch's Tropical Carrot solution.
Table 9a. Formulation composition.
mg per 20 mL
Date TPGS PSB Welch's Tropical Carrot 14-Jun-13 8.5 1.7 20 mL
Table 9a. Formulation composition.
mg per 20 mL
Date TPGS PSB Welch's Tropical Carrot 14-Jun-13 8.5 1.7 20 mL
[0076] Procedure 1: PSB/TPGS/Welch's Tropical Carrot solution preparation: 170 I of TPGS/PSB emulsion (lot#005.21.4) was added to 20 mL of Welch's Tropical Carrot beverage.
Welch's Tropical Carrot solution of PSB/TPGS emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Welch's Tropical Carrot solution was diluted 1:1 with IPA. Precipitation noted in the matrix (Table 9b).
Table 9b. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL % preparation 14-Jun-13 0.090 ND N/A 0 17-Jun-13 0.090 N/A N/A 3 [0077] Procedure 2: PSB/TPGS/ Welch's Tropical Carrot solution preparation: 170 I
of PSB/TPGS emulsion (lot#005.21.4) was added to 20 mL of Welch's Tropical Carrot beverage. PSB/TPGS/Welch's Tropical Carrot solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/Welch's Tropical Carrot solution was diluted 1:1 with IPA, filtered through 0.22 m filter, and HPLC analysis of PSB content was performed (Table 9c).
Table 9c. PSB content of resultant solution.
PSB content Preparation HPLC
D Calculated HPLC, Recovery, Days past ate Date PSB, mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.090 0.085 94.61 3 25-Jun-13 0.090 0.089 99.06 11 [0078] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 9c shows that after 11 days of storage at RT PSB content of resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data shows that TPGS formulated PSB is stable in Welch's Tropical Carrot beverage.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Welch's Tropical Carrot solution of PSB/TPGS emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Welch's Tropical Carrot solution was diluted 1:1 with IPA. Precipitation noted in the matrix (Table 9b).
Table 9b. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL % preparation 14-Jun-13 0.090 ND N/A 0 17-Jun-13 0.090 N/A N/A 3 [0077] Procedure 2: PSB/TPGS/ Welch's Tropical Carrot solution preparation: 170 I
of PSB/TPGS emulsion (lot#005.21.4) was added to 20 mL of Welch's Tropical Carrot beverage. PSB/TPGS/Welch's Tropical Carrot solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/Welch's Tropical Carrot solution was diluted 1:1 with IPA, filtered through 0.22 m filter, and HPLC analysis of PSB content was performed (Table 9c).
Table 9c. PSB content of resultant solution.
PSB content Preparation HPLC
D Calculated HPLC, Recovery, Days past ate Date PSB, mg/mL mg/mL preparation 14-Jun-13 0.082 ND N/A 0 17-Jun-13 0.090 0.085 94.61 3 25-Jun-13 0.090 0.089 99.06 11 [0078] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 9c shows that after 11 days of storage at RT PSB content of resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data shows that TPGS formulated PSB is stable in Welch's Tropical Carrot beverage.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
[0079] Procedure 3: PSB/TPGS/ Welch's Tropical Carrot solution preparation: 170 ul of PSB/TPGS emulsion (lot#005.21.4) was added to 20 mL of Welch's Tropical Carrot beverage. PSB/TPGS/Welch's Tropical Carrot solution was mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Welch's Tropical Carrot solution was filtered through 0.22 um filter, diluted 1:1 with IPA. HPLC analysis of PSB content noted in Table 9d.
Table 9d. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated HPLC, Days past Date Date Recovery, %
PSB, mg/mL mg/mL
preparation 14-Jun-13 0.090 ND _ N/A 0 17-Jun-13 0.090 0.034 37.84 3 [0080] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 9d (recovery %) shows that PSB content of directly filtered PSB/TPGS/Welch's Tropical Carrot solution and then dilution with IPA was dramatically lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB recovery in filtered Welch's Tropical Carrot solution of formulated PSB was more than twice lower compared to that of filtered PSB/TPGS water solution (examples 3 and 5).
Data suggest that a major portion of formulated PSB was bound to Welch's Tropical Carrot coarse matrix and was retained on the filter during filtration. Dilution of PSB/TPGS/Welch's Tropical Carrot solution with IPA prior to filtration releases PSB from Welch's Tropical Carrot matrix and prevents its filter retention. Welch's Tropical Carrot beverage has precipitation of coarse matrix in its original bottle and written manufacturer instruction "Shake well".
Table 9d. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated HPLC, Days past Date Date Recovery, %
PSB, mg/mL mg/mL
preparation 14-Jun-13 0.090 ND _ N/A 0 17-Jun-13 0.090 0.034 37.84 3 [0080] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 9d (recovery %) shows that PSB content of directly filtered PSB/TPGS/Welch's Tropical Carrot solution and then dilution with IPA was dramatically lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB recovery in filtered Welch's Tropical Carrot solution of formulated PSB was more than twice lower compared to that of filtered PSB/TPGS water solution (examples 3 and 5).
Data suggest that a major portion of formulated PSB was bound to Welch's Tropical Carrot coarse matrix and was retained on the filter during filtration. Dilution of PSB/TPGS/Welch's Tropical Carrot solution with IPA prior to filtration releases PSB from Welch's Tropical Carrot matrix and prevents its filter retention. Welch's Tropical Carrot beverage has precipitation of coarse matrix in its original bottle and written manufacturer instruction "Shake well".
[0081] Particle size analysis and stability. PSB/TPGS/Welch's Tropical Carrot solution preparation: 170 ul of PSB/TPGS emulsion was added to 20 mL of Welch's Tropical Carrot beverage. PSB/TPGS/Welch's Tropical Carrot solution was briefly mixed and stored for 2 and 6 days at RT. The beverage was centrifuged at 13,000 rpm for 15 mm. To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL of distilled water. To determine effect of the beverage on particle size of TPGS/PSP
emulsion, 0.6 mL of emulsion was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of distilled water. Particle size analysis is noted in Table 9e.
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
Table 9e. Particle size and stability.
Particle size Days past Preparation Date Material by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot#
005.21.4 36.6 0 19-Jun-13 Welch's Tropical Carrot 328.7 0 19-Jun-13 Welch's Tropical Carrot plus Lot# 005.21.4 108.7 21-Jun-13 Welch's Tropical Carrot plus Lot# 005.21.4 79.0 25-Jun-13 Welch's Tropical Carrot plus Lot# 005.21.4 62.4 [0082] There was increase of the particle size of formulated PSB upon dilution in Welch's Tropical Carrot beverage form 36.6 to 108.7 nm (Table 9 e). These results suggest significant contribution of beverage only particles into final particle size of the mixture. The light scattering data are in a good agreement with HPLC data demonstrating substantial binding of formulated PSB to Welch's Tropical Carrot coarse matrix. Particle size decreased over next six days of monitoring from 108.7 to 62.4 nm (Table 9e). This may indicate possible emulsification of the beverage matrix components in the presence of formulated PSB that potentially could improve drinking quality of Welch's Tropical Carrot beverage. Data indicate chemical stability of formulated PSB in Welch's Tropical Carrot beverage and suggest that "shaking well" will provide 100% of PSB consumption with the beverage.
emulsion, 0.6 mL of emulsion was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of distilled water. Particle size analysis is noted in Table 9e.
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
Table 9e. Particle size and stability.
Particle size Days past Preparation Date Material by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot#
005.21.4 36.6 0 19-Jun-13 Welch's Tropical Carrot 328.7 0 19-Jun-13 Welch's Tropical Carrot plus Lot# 005.21.4 108.7 21-Jun-13 Welch's Tropical Carrot plus Lot# 005.21.4 79.0 25-Jun-13 Welch's Tropical Carrot plus Lot# 005.21.4 62.4 [0082] There was increase of the particle size of formulated PSB upon dilution in Welch's Tropical Carrot beverage form 36.6 to 108.7 nm (Table 9 e). These results suggest significant contribution of beverage only particles into final particle size of the mixture. The light scattering data are in a good agreement with HPLC data demonstrating substantial binding of formulated PSB to Welch's Tropical Carrot coarse matrix. Particle size decreased over next six days of monitoring from 108.7 to 62.4 nm (Table 9e). This may indicate possible emulsification of the beverage matrix components in the presence of formulated PSB that potentially could improve drinking quality of Welch's Tropical Carrot beverage. Data indicate chemical stability of formulated PSB in Welch's Tropical Carrot beverage and suggest that "shaking well" will provide 100% of PSB consumption with the beverage.
[0083] Example 10. Preparation of PSB/TPGS/Vitamin Water solution.
Table 10a. Formulation composition.
mg per 21 mL
Date TPGS PSB Vitamin Water 14-Jun-13 8.5 1.7 21 mL
Table 10a. Formulation composition.
mg per 21 mL
Date TPGS PSB Vitamin Water 14-Jun-13 8.5 1.7 21 mL
[0084]
Procedure 1: PSB/TPGS /Vitamin Water solution preparation: 170 [t1 of TPGS/PSB emulsion (100005.21.4) was added to 21 mL of Vitamin Water beverage.
Vitamin Water solution of PSB/TPGS emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Vitamin Water solution was diluted 1:1 with IPA, and HPLC
analysis of PSB content was performed (Table 10b).
Table 10b. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated PSB, HPLC, Recovery, Days past Date Date mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.082 95.83 3 [0085] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 10b shows that after 3 days of storage at RT PSB content in the resultant PATENT APPLICATION Attorney Docket No. ISS.000200CA
solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Vitamin Water beverage.
Procedure 1: PSB/TPGS /Vitamin Water solution preparation: 170 [t1 of TPGS/PSB emulsion (100005.21.4) was added to 21 mL of Vitamin Water beverage.
Vitamin Water solution of PSB/TPGS emulsion was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Vitamin Water solution was diluted 1:1 with IPA, and HPLC
analysis of PSB content was performed (Table 10b).
Table 10b. PSB content of resultant solution.
PSB content Preparation HPLC
Calculated PSB, HPLC, Recovery, Days past Date Date mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.082 95.83 3 [0085] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 10b shows that after 3 days of storage at RT PSB content in the resultant PATENT APPLICATION Attorney Docket No. ISS.000200CA
solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Vitamin Water beverage.
[0086] Procedure 2: PSB/TPGS/ Vitamin Water solution preparation: 170 vtl of PSB/TPGS emulsion (lot#005.21.4) was added to 21 mL of Vitamin Water beverage.
PSB/TPGS/ Vitamin Water solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later, 0.6 mL aliquot of PSB/TPGS/Vitamin Water solution was diluted 1:1 with IPA, filtered through 0.22 tim filter, and HPLC analysis of PSB content was performed (Table 10c).
Table 10c. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.082 95.83 3 25-Jun-13 0.086 0.087 101.68 11 [0087] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 10c shows that after 11 days of storage at RT PSB
content in the resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Vitamin Water beverage.
PSB/TPGS/ Vitamin Water solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later, 0.6 mL aliquot of PSB/TPGS/Vitamin Water solution was diluted 1:1 with IPA, filtered through 0.22 tim filter, and HPLC analysis of PSB content was performed (Table 10c).
Table 10c. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.082 95.83 3 25-Jun-13 0.086 0.087 101.68 11 [0087] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 10c shows that after 11 days of storage at RT PSB
content in the resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Vitamin Water beverage.
[0088] Procedure 3: PSB/TPGS/Vitamin Water solution preparation: 170 IA of PSB/TPGS emulsion (lot#005.21.4) was added to 21 mL of Vitamin Water beverage.
PSB/TPGS/Vitamin Water solution was briefly mixed and stored for 3 days at RT.
3 Days later 0.6 mL aliquot of PSB/TPGS/Vitamin Water solution was filtered through 0.22 vim filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 10d).
Table 10 d. PSB content of resultant solution. Stability.
PSB content Preparation HPLC
Calculated PSB, HPLC, Recovery, Days past Date Date mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.070 81.81 3 [0089] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 10d (recovery %) shows that PSB content of filtered PSB/TPGS/Vitamin Water solution and then diluted with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB recovery in filtered Vitamin Water solution of formulated PSB was similar to that of filtered PSB/TPGS
PATENT APPLICATION Attorney Docket No. ISS.000200CA
water solution (examples 3 and 5). The data provide no evidence of significant binding of formulated PSB to Vitamin Water matrix.
PSB/TPGS/Vitamin Water solution was briefly mixed and stored for 3 days at RT.
3 Days later 0.6 mL aliquot of PSB/TPGS/Vitamin Water solution was filtered through 0.22 vim filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 10d).
Table 10 d. PSB content of resultant solution. Stability.
PSB content Preparation HPLC
Calculated PSB, HPLC, Recovery, Days past Date Date mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.070 81.81 3 [0089] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 10d (recovery %) shows that PSB content of filtered PSB/TPGS/Vitamin Water solution and then diluted with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB recovery in filtered Vitamin Water solution of formulated PSB was similar to that of filtered PSB/TPGS
PATENT APPLICATION Attorney Docket No. ISS.000200CA
water solution (examples 3 and 5). The data provide no evidence of significant binding of formulated PSB to Vitamin Water matrix.
[0090] Particle size analysis and stability. PSB/TPGSNitamin Water solution preparation: 170 Ill of PSB/TPGS emulsion was added to 21 mL of Vitamin Water beverage.
PSB/TPGSNitamin Water solution was mixed and stored for 2 and 6 days at RT. To avoid contribution of coarse beverage matrix to particle size measurement, the beverage was centrifuged at 13,000 rpm for 15 min in an eppendorf centrifuge. To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL
of water. To determine effect of the beverage on particle size of TPGS/PSP emulsion, 0.6 mL
of emulsion was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of water. Particle size analysis is noted in Table 10e.
Table 10e. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot # 005.21.4 36.6 0 19-Jun-13 Vitamin Water BDL 0 19-Jun-13 Vitamin Water plus Lot# 005.21.4 41.4 0 21-Jun-13 Vitamin Water plus Lot# 005.21.4 39.4 2 25-Jun-13 Vitamin Water plus Lot# 005.21.4 [0091] Although, there was ¨5 nm initial increase of the particle size of the TPGS/PSB
emulsion upon dilution in Vitamin Water beverage, the particle size did not change over next six days of monitoring. Data suggest that the contribution of beverage only particles into final particle size of the mixture was not significant. Absence of further particle size change suggests stability of formulated PSB in Vitamin Water beverage. Data obtained indicate chemical and particle stability of formulated PSB in Vitamin Water beverage and suggest 100% of PSB
consumption with the beverage.
PSB/TPGSNitamin Water solution was mixed and stored for 2 and 6 days at RT. To avoid contribution of coarse beverage matrix to particle size measurement, the beverage was centrifuged at 13,000 rpm for 15 min in an eppendorf centrifuge. To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL
of water. To determine effect of the beverage on particle size of TPGS/PSP emulsion, 0.6 mL
of emulsion was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of water. Particle size analysis is noted in Table 10e.
Table 10e. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot # 005.21.4 36.6 0 19-Jun-13 Vitamin Water BDL 0 19-Jun-13 Vitamin Water plus Lot# 005.21.4 41.4 0 21-Jun-13 Vitamin Water plus Lot# 005.21.4 39.4 2 25-Jun-13 Vitamin Water plus Lot# 005.21.4 [0091] Although, there was ¨5 nm initial increase of the particle size of the TPGS/PSB
emulsion upon dilution in Vitamin Water beverage, the particle size did not change over next six days of monitoring. Data suggest that the contribution of beverage only particles into final particle size of the mixture was not significant. Absence of further particle size change suggests stability of formulated PSB in Vitamin Water beverage. Data obtained indicate chemical and particle stability of formulated PSB in Vitamin Water beverage and suggest 100% of PSB
consumption with the beverage.
[0092] Example 11. Preparation of PSB/TPGS/Gatorade Perform 02 solution.
Table ha. Formulation composition.
mg per 21 mL
Date TPGS PSB Gatorade Perform 02 14-Jun-I3 8.5 1.7 21 mL
Table ha. Formulation composition.
mg per 21 mL
Date TPGS PSB Gatorade Perform 02 14-Jun-I3 8.5 1.7 21 mL
[0093] Procedure 1: PSB/TPGS/Gatorade Perform 02 solution preparation:
170 1 of TPGS/PSB emulsion (lot#005.21.4) was added to 21 mL of Gatorade Perform 02 beverage.
Gatorade Perform 02 solution of PSB/TPGS emulsion was briefly mixed and stored for 3 days at PATENT APPLICATION Attorney Docket No. ISS.000200CA
RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Gatorade Perform 02 solution was diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 11b).
Table 11b. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated HPLC, Recovery, Days past PSB, mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.081 94.66 3 [0094] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 4b shows that after 3 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Gatorade Perform 02 beverage.
170 1 of TPGS/PSB emulsion (lot#005.21.4) was added to 21 mL of Gatorade Perform 02 beverage.
Gatorade Perform 02 solution of PSB/TPGS emulsion was briefly mixed and stored for 3 days at PATENT APPLICATION Attorney Docket No. ISS.000200CA
RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Gatorade Perform 02 solution was diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 11b).
Table 11b. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated HPLC, Recovery, Days past PSB, mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.081 94.66 3 [0094] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 4b shows that after 3 days of storage at RT PSB content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Gatorade Perform 02 beverage.
[0095] Procedure 2: PSB/TPGS/Gatorade Perform 02 solution preparation:
170 1 of PSB/TPGS emulsion (lot#005.21.4) was added to 21 mL of Gatorade Perform 02 beverage.
PSB/TPGS/Gatorade Perform 02 solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/Gatorade Perform 02 solution was diluted 1:1 with IPA, filtered through 0.22 p.m filter; HPLC analysis of PSB content was performed (Table 11c).
Table 11c. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated HPLC, Recovery, Days past PSB, mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.085 99.34 3 25-Jun-13 0.086 0.085 99.34 11 [0096] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 11c shows that after 11 days of storage at RT PSB
content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Gatorade Perform 02 beverage.
170 1 of PSB/TPGS emulsion (lot#005.21.4) was added to 21 mL of Gatorade Perform 02 beverage.
PSB/TPGS/Gatorade Perform 02 solution was briefly mixed and stored for 3 or 11 days at RT. 3 or 11 Days later 0.6 mL aliquot of PSB/TPGS/Gatorade Perform 02 solution was diluted 1:1 with IPA, filtered through 0.22 p.m filter; HPLC analysis of PSB content was performed (Table 11c).
Table 11c. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated HPLC, Recovery, Days past PSB, mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.085 99.34 3 25-Jun-13 0.086 0.085 99.34 11 [0096] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 11c shows that after 11 days of storage at RT PSB
content in resultant solution determined by HPLC was similar to calculated amount of PSB added to the beverage.
Data suggest that TPGS formulated PSB is stable in Gatorade Perform 02 beverage.
[0097] Procedure 3: PSB/TPGS/ Gatorade Perform 02 solution preparation:
170 1 of PSB/TPGS emulsion (lot#005.21.4) was added to 21 mL of Gatorade Perform 02 beverage.
PSB/TPGS/Gatorade Perform 02 solution was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Gatorade Perform 02 solution was filtered through 0.22 m filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 11d).
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Table 11d. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.071 82.98 3 [0098] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table lid (recovery %) shows that PSB content of directly filtered PSB/TPGS/Gatorade Perform 02 solution and then dilution with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB recovery in filtered Gatorade Perform 02 solution of formulated PSB was similar to that of filtered PSB/TPGS water solution (examples 3 and 5). No evidence of significant binding of formulated PSB to Gatorade Perform 02 matrix.
170 1 of PSB/TPGS emulsion (lot#005.21.4) was added to 21 mL of Gatorade Perform 02 beverage.
PSB/TPGS/Gatorade Perform 02 solution was briefly mixed and stored for 3 days at RT. 3 Days later 0.6 mL aliquot of PSB/TPGS/Gatorade Perform 02 solution was filtered through 0.22 m filter, diluted 1:1 with IPA, and HPLC analysis of PSB content was performed (Table 11d).
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Table 11d. PSB content of resultant solution.
PSB content Preparation HPLC
Date Date Calculated PSB, HPLC, Recovery, Days past mg/mL mg/mL
preparation 14-Jun-13 0.086 ND N/A 0 17-Jun-13 0.086 0.071 82.98 3 [0098] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table lid (recovery %) shows that PSB content of directly filtered PSB/TPGS/Gatorade Perform 02 solution and then dilution with IPA was significantly lower compared to unfiltered (Procedure 1) and/or diluted with IPA and then filtered (Procedure 2) solutions. PSB recovery in filtered Gatorade Perform 02 solution of formulated PSB was similar to that of filtered PSB/TPGS water solution (examples 3 and 5). No evidence of significant binding of formulated PSB to Gatorade Perform 02 matrix.
[0099] Particle size analysis and stability. PSB/TPGS/ Gatorade Perform 02 solution preparation: 170 tl of PSB/TPGS emulsion (lot#005.21.4) was added to 21 mL of Gatorade Perform 02 beverage. PSB/TPGS/Gatorade Perform 02 solution was briefly mixed and stored for 2 and 6 days at RT. To avoid contribution of coarse beverage matrix to particle size measurement, the beverage was centrifuged at 13,000 rpm for 15 min in an eppendorf centrifuge.
To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL of water. To determine effect of the beverage on particle size of TPGS/PSP
emulsion, 0.6 mL of emulsion (lot #005.21.4) was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of distilled water. Particle size analysis is presented in the Table lie.
Table lie. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot# 005.21.4 36.6 0 19-Jun-13 Gatorade Perform 02 512.8 0 19-Jun-13 Gatorade Perform 02 plus Lot#
005.21.4 41 0 21-Jun-13 Gatorade Perform 02 plus Lot#
005.21.4 39.4 2 25-Jun-13 Gatorade Perform 02 plus Lot#
005.21.4 40.9 6 [00100] There was 4.4 nm initial increase of the particle size of the TPGS/PSB
emulsion upon dilution in Gatorade Perform 02 beverage. The particle size did not change over next 6 days. Absence of further particle size change suggests stability of formulated PSB in Gatorade PATENT APPLICATION Attorney Docket No.
ISS.000200CA
Perform 02 beverage. Data shows chemical and particle stability of PSB in Gatorade Perform 02 beverage and suggest 100% of PSB consumption with the beverage.
To measure particle size of the beverage alone, 0.3 mL of supernatant were added to cuvette containing 2.2 mL of water. To determine effect of the beverage on particle size of TPGS/PSP
emulsion, 0.6 mL of emulsion (lot #005.21.4) was mixed with 0.6 mL of supernatant, and 0.6 mL of 1:1 mixture was added to the cuvette containing 1.9 mL of distilled water. Particle size analysis is presented in the Table lie.
Table lie. Particle size and stability.
PreparationParticle size Days past Material Date by intensity, nm preparation 19-Jun-13 Distilled Water plus Lot# 005.21.4 36.6 0 19-Jun-13 Gatorade Perform 02 512.8 0 19-Jun-13 Gatorade Perform 02 plus Lot#
005.21.4 41 0 21-Jun-13 Gatorade Perform 02 plus Lot#
005.21.4 39.4 2 25-Jun-13 Gatorade Perform 02 plus Lot#
005.21.4 40.9 6 [00100] There was 4.4 nm initial increase of the particle size of the TPGS/PSB
emulsion upon dilution in Gatorade Perform 02 beverage. The particle size did not change over next 6 days. Absence of further particle size change suggests stability of formulated PSB in Gatorade PATENT APPLICATION Attorney Docket No.
ISS.000200CA
Perform 02 beverage. Data shows chemical and particle stability of PSB in Gatorade Perform 02 beverage and suggest 100% of PSB consumption with the beverage.
[00101] Example 12. Preparation of PSB/TPGS aqueous emulsion. To determine the lowest TPGS/PSB ratio producing stable emulsion containing 10 mg/mL of PSB in aqueous media. PSB/TPGS ethanol mixtures lot# 005.30.3 and 05.30.4 were prepared on 01 Jul 2013.
Table 12 a. Formulation composition.
Date Lot# Material Weighed, mg (per 100 ml) TPGS/PSB
TPGS PSB Ratio 01-Jul-13 005.30.3 Mixture 3117 1022 3.05 01-Jul-13 005.30.4 Mixture 4040 1044 3.87 [00102] Table 12a: Amounts of TPGS and PSB were weighed and placed in 400 mL
beaker. 9 mL of 95% ethanol were added to the solids, and the beaker containing ethanol mixture of TPGS/PSB was placed in the water bath and incubated at 60 C for 20 min until clear. The beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
Table 12 a. Formulation composition.
Date Lot# Material Weighed, mg (per 100 ml) TPGS/PSB
TPGS PSB Ratio 01-Jul-13 005.30.3 Mixture 3117 1022 3.05 01-Jul-13 005.30.4 Mixture 4040 1044 3.87 [00102] Table 12a: Amounts of TPGS and PSB were weighed and placed in 400 mL
beaker. 9 mL of 95% ethanol were added to the solids, and the beaker containing ethanol mixture of TPGS/PSB was placed in the water bath and incubated at 60 C for 20 min until clear. The beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
[00103] PSB/TPGS emulsions lot# 005.30.7 and lot# 005.30.8 were prepared on 03 Jul 2013. Emulsion preparation: The beaker was removed from vacuum oven and 100 mL
of distilled water preheated to 60 C was added to the highly viscous clear film of TPGS/PBS
mixture, the beaker was placed in a 60 C water bath and total mixture was incubated for 30 min to facilitate dissolution. The water TPGS/PBS mixture was mixed for 2 hrs at RT. Upon clearing of the mixture, the emulsion was filtered through 0.22 lam filter, and particle size and PSB
content of the resultant emulsions were determined (Table 12b).
Table 12b. Particle size, PSB content, and particle stability of resultant emulsions.
Particle size PSB Formulation Stability Manufacturing Lot # by intensi Date ty, Content, Recovery, Days past Particle size nm mg/mL mfg nm 03-Jul-13 005.30.7 180.4 8.7 85.3 19 216.4 03-Jul-13 005.30.8 58.0 10.10 96.7 19 60.6 [00104] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 12b shows that 4/1 (TPGS/PSB) ratio was the lowest ratio that allowed formulation of 10 mg/mL of PSB into aqueous media.
of distilled water preheated to 60 C was added to the highly viscous clear film of TPGS/PBS
mixture, the beaker was placed in a 60 C water bath and total mixture was incubated for 30 min to facilitate dissolution. The water TPGS/PBS mixture was mixed for 2 hrs at RT. Upon clearing of the mixture, the emulsion was filtered through 0.22 lam filter, and particle size and PSB
content of the resultant emulsions were determined (Table 12b).
Table 12b. Particle size, PSB content, and particle stability of resultant emulsions.
Particle size PSB Formulation Stability Manufacturing Lot # by intensi Date ty, Content, Recovery, Days past Particle size nm mg/mL mfg nm 03-Jul-13 005.30.7 180.4 8.7 85.3 19 216.4 03-Jul-13 005.30.8 58.0 10.10 96.7 19 60.6 [00104] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 12b shows that 4/1 (TPGS/PSB) ratio was the lowest ratio that allowed formulation of 10 mg/mL of PSB into aqueous media.
[00105]
Particle size analysis and stability. The resultant emulsion lot # 005.30.8 was stable. It can be seen in Table 12b that particle size did not significantly change over 19 days of monitoring. Rmulsion lot# 005.30.7 was not stable (Table 12b) and formed a precipitate.
PATENT APPLICATION
Attorney Docket No. ISS.000200CA
Particle size analysis and stability. The resultant emulsion lot # 005.30.8 was stable. It can be seen in Table 12b that particle size did not significantly change over 19 days of monitoring. Rmulsion lot# 005.30.7 was not stable (Table 12b) and formed a precipitate.
PATENT APPLICATION
Attorney Docket No. ISS.000200CA
[00106] Example 13. Preparation of PSB/ PPG-PEG-PPG, Pluronic emulsions. To investigate the possibility of using alternative emulsifying agents, PPG-PEG-PPG. Pluronic block polymers with 2-7, 7-12. and 12-18 HLB (Hydrophilic-lipophilic balance) range to generate PSB containing stable emulsions containing ¨10 mg/mL of PSB. See Table 13a.
Table 13a. Formulation composition.
Weighed, mg Block HLB(per 100 mL) polymer/PSB
Date Lot# Block polymers Material Range Block PSB Ratio polymer 11-Jul-13 005.32.1.27 PPG-PEG-PPG, 2-7 Mixture 2003 1058 1.89 Pluronic 31R1 11-Jul-13 005.32.2.27 PPG-PEG-PPG, 2-7 Mixture 4013 1014 3.96 Pluronic 31R1 11-Jul-13 005.32.3.712 PPG-PEG-PPG, 7-12 Mixture 2036 1017 2.0 Pluronic 17R4 11-Jul-13 005.32.4.712 PPG-PEG-PPG, 7-12 Mixture 3996 1037 3.85 Pluronic 17R4 11-Jul-13 005.32.5.1218 PPG-PEG-PPG, 12-18 Mixture 1994 1014 1.97 Pluronic L-64 11-Jul-13 005.32.6.1218 12-18 Mixture 4007 1013 3.96 PluroniPPG-PEG-PPG, c L-64 [00107] Table 13a shows amounts of block polymers and PSB were weighed and placed in 400 mL beaker. 9 mL of 95% ethanol were added to the solids, and the beaker containing ethanol mixture of TPGS/PSB was placed in the water bath and incubated at 60 C for 20 min until clear. The beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
Table 13a. Formulation composition.
Weighed, mg Block HLB(per 100 mL) polymer/PSB
Date Lot# Block polymers Material Range Block PSB Ratio polymer 11-Jul-13 005.32.1.27 PPG-PEG-PPG, 2-7 Mixture 2003 1058 1.89 Pluronic 31R1 11-Jul-13 005.32.2.27 PPG-PEG-PPG, 2-7 Mixture 4013 1014 3.96 Pluronic 31R1 11-Jul-13 005.32.3.712 PPG-PEG-PPG, 7-12 Mixture 2036 1017 2.0 Pluronic 17R4 11-Jul-13 005.32.4.712 PPG-PEG-PPG, 7-12 Mixture 3996 1037 3.85 Pluronic 17R4 11-Jul-13 005.32.5.1218 PPG-PEG-PPG, 12-18 Mixture 1994 1014 1.97 Pluronic L-64 11-Jul-13 005.32.6.1218 12-18 Mixture 4007 1013 3.96 PluroniPPG-PEG-PPG, c L-64 [00107] Table 13a shows amounts of block polymers and PSB were weighed and placed in 400 mL beaker. 9 mL of 95% ethanol were added to the solids, and the beaker containing ethanol mixture of TPGS/PSB was placed in the water bath and incubated at 60 C for 20 min until clear. The beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
[00108] Emulsion preparation: The beaker was removed from vacuum oven and 100 mL
of water preheated to 60 C was added to the highly viscous clear film of Block polymer /PBS
mixture; the beaker was placed in a 60 C water bath and total mixture was incubated for 30 min to facilitate dissolution. The water Block polymer /PBS mixture was mixed on magnetic stirring plate for 2 hrs at RT. Two hours later the emulsion was filtered through 0.22 }.im filter, and particle size and PSB content of the resultant emulsions were determined (Table 13b).
Table 13b. Particle size, PSB content, and particle stability of resultant emulsions.
Particle size PSB
Formulation Stability Manufacturing Lot# by intensity, Content, Recovery, Days Particle nm mg/mL past mfg size nm 12-Jul-13 005.32.1.27 Not BDL NA NA NA
Detectable 12-Jul-13 005.32.2.27 Not BDL NA NA NA
Detectable 12-Jul-13 005.32.3.712 Not BDL NA NA NA
PATENT APPLICATION
Attorney Docket No. ISS.000200CA
Particle size PSB
Formulation Stability Manufacturing Date Lot# by intensity, Content, Recovery, Days Particle nm mg/mL % past mfg size nm Detectable 12-Jul-13 005.32.4.712 Not BDL NA NA
.. NA
Detectable 12-Jul-13 005.32.5.1218 Not BDL NA NA
.. NA
Detectable 12-Jul-13 005.32.6.1218 6907.9 0.23 2.3 NA
.. NA
of water preheated to 60 C was added to the highly viscous clear film of Block polymer /PBS
mixture; the beaker was placed in a 60 C water bath and total mixture was incubated for 30 min to facilitate dissolution. The water Block polymer /PBS mixture was mixed on magnetic stirring plate for 2 hrs at RT. Two hours later the emulsion was filtered through 0.22 }.im filter, and particle size and PSB content of the resultant emulsions were determined (Table 13b).
Table 13b. Particle size, PSB content, and particle stability of resultant emulsions.
Particle size PSB
Formulation Stability Manufacturing Lot# by intensity, Content, Recovery, Days Particle nm mg/mL past mfg size nm 12-Jul-13 005.32.1.27 Not BDL NA NA NA
Detectable 12-Jul-13 005.32.2.27 Not BDL NA NA NA
Detectable 12-Jul-13 005.32.3.712 Not BDL NA NA NA
PATENT APPLICATION
Attorney Docket No. ISS.000200CA
Particle size PSB
Formulation Stability Manufacturing Date Lot# by intensity, Content, Recovery, Days Particle nm mg/mL % past mfg size nm Detectable 12-Jul-13 005.32.4.712 Not BDL NA NA
.. NA
Detectable 12-Jul-13 005.32.5.1218 Not BDL NA NA
.. NA
Detectable 12-Jul-13 005.32.6.1218 6907.9 0.23 2.3 NA
.. NA
[00109] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 13b shows that PSB was only detected in Lot#
005.32.6.1218 where PPG-PEG-PPG, Pluronic L-64 (HLB Range 12-18) was used for formulation of PSB.
However, the measured PSB content (0.23mg/mL) was far from the target concentration of 10 mg/mL.
005.32.6.1218 where PPG-PEG-PPG, Pluronic L-64 (HLB Range 12-18) was used for formulation of PSB.
However, the measured PSB content (0.23mg/mL) was far from the target concentration of 10 mg/mL.
[00110]
Particle size analysis and stability. Table 13b shows that particle size could be measured only in lot# 005.32.6.1218 where PPG-PEG-PPG, Pluronic L-64 (HLB
Range 12-18) was used for formulation of PSB. Emulsion was unstable and formed a precipitate.
Particle size analysis and stability. Table 13b shows that particle size could be measured only in lot# 005.32.6.1218 where PPG-PEG-PPG, Pluronic L-64 (HLB
Range 12-18) was used for formulation of PSB. Emulsion was unstable and formed a precipitate.
[00111] Example 14. Preparation of PSB/ PPG-PEG-PPG, Pluronic emulsions. Using different PPG-PEG-PPG, Pluronic block polymers with 2-7, 7-12, and 12-18 HLB
(Hydrophilic-lipophilic balance) range at higher block polymer/PSB ratio to generate PSB
containing stable emulsions containing ¨10 mg/mL of PSB.
(Hydrophilic-lipophilic balance) range at higher block polymer/PSB ratio to generate PSB
containing stable emulsions containing ¨10 mg/mL of PSB.
[00112] PSB/ PPG-PEG-PPG, Pluronic block polymers ethanol mixtures were prepared on 16 Jul 2013. Formulation composition is in the Table 14a.
Table 14a. Formulation composition.
Weighed, mg (per Block HLB100 mL) polymer/PSB
Date Lot# Block polymers Material Range Block PSB
Ratio polymer 16-Jul-13 005.33.7.27 PPG-PEG-PPG, 2-7 Mixture 10205 1005 10.15 Pluronic 31R1 16-Jul-13 005.33.8.712 PPG-PEG-PPG, 2-7 Mixture 10191 1006 10.13 Pluronic 17R4 16-Jul-13 005.33.9.1218 PPG-PEG- PPG, 7-12 Mixture 10731 1003 10.70 Pluronic L-64 [00113] Table 14a shows the amount of block polymers and PSB were weighed and placed in 400 mL beaker. 9 mL of 95% ethanol were added, and the beaker containing ethanol mixture of TPGS/PSB was placed in the water bath and incubated at 60 C for 20 min until clear.
The beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
Table 14a. Formulation composition.
Weighed, mg (per Block HLB100 mL) polymer/PSB
Date Lot# Block polymers Material Range Block PSB
Ratio polymer 16-Jul-13 005.33.7.27 PPG-PEG-PPG, 2-7 Mixture 10205 1005 10.15 Pluronic 31R1 16-Jul-13 005.33.8.712 PPG-PEG-PPG, 2-7 Mixture 10191 1006 10.13 Pluronic 17R4 16-Jul-13 005.33.9.1218 PPG-PEG- PPG, 7-12 Mixture 10731 1003 10.70 Pluronic L-64 [00113] Table 14a shows the amount of block polymers and PSB were weighed and placed in 400 mL beaker. 9 mL of 95% ethanol were added, and the beaker containing ethanol mixture of TPGS/PSB was placed in the water bath and incubated at 60 C for 20 min until clear.
The beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
[00114] Emulsion preparation: The beaker was removed from vacuum oven and 100 mL
of distilled water preheated to 60 C was added to highly viscous clear film of Block polymer /PBS mixture, beaker was placed to 60 C water bath and total mixture was incubated for 30 min to facilitate the dissolution. Water Block polymer /PBS mixture was mixed on magnetic stirring plate for 2 hrs at RT. Two hours later the emulsion was filtered through 0.22 [im filter, and particle size and PSB content of the resultant emulsions were determined (Table 14b).
Table 14b. Particle size, PSB content, and particle stability of resultant emulsions.
Manufacturing Particle size Lot#Content, Date by intensity, nm mg/mL Recovery, %
17-Jul-13 005.33.7.27 20576 BDL NA
17-Jul-13 005.33.8.712 420 0.13 1.3 17-Jul-13 005.33.9.1218 113 9.47 94.4 [00115] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 14b shows that PSB was detected in Lot# 005.33.8.712 (0.13 mg/mL) where PPG-PEG-PPG, Pluronic 17R4 (HLB Range 7-12) and Lot# 005.33.9.1218 (9.47 mg/mL) where PPG-PEG-PPG, Pluronic L-64 (HLB Range 12-18) were used for formulation of PSB.
of distilled water preheated to 60 C was added to highly viscous clear film of Block polymer /PBS mixture, beaker was placed to 60 C water bath and total mixture was incubated for 30 min to facilitate the dissolution. Water Block polymer /PBS mixture was mixed on magnetic stirring plate for 2 hrs at RT. Two hours later the emulsion was filtered through 0.22 [im filter, and particle size and PSB content of the resultant emulsions were determined (Table 14b).
Table 14b. Particle size, PSB content, and particle stability of resultant emulsions.
Manufacturing Particle size Lot#Content, Date by intensity, nm mg/mL Recovery, %
17-Jul-13 005.33.7.27 20576 BDL NA
17-Jul-13 005.33.8.712 420 0.13 1.3 17-Jul-13 005.33.9.1218 113 9.47 94.4 [00115] HPLC Analysis. "Pterostilbene 310nm.M" method was used for determination of PSB content. Table 14b shows that PSB was detected in Lot# 005.33.8.712 (0.13 mg/mL) where PPG-PEG-PPG, Pluronic 17R4 (HLB Range 7-12) and Lot# 005.33.9.1218 (9.47 mg/mL) where PPG-PEG-PPG, Pluronic L-64 (HLB Range 12-18) were used for formulation of PSB.
[00116] Particle size analysis and stability. Table 14b shows that particle size decreased from 20576 to 113 nm with increasing of HLB range. Emulsion Lot# 005.33.9.1218 was stable only at 40-50 C and formed a precipitate at RT. Data obtained demonstrate uniqueness of TPGS
(HLB = 14.3) as an efficient formulating agent for generating stable emulsions containing 10 mg/mL of PSB in aqueous solutions. Block polymers, including PPG-PEG-PPG, Pluronic L-64 with similar HLB properties to TPGS, were used in an attempt to formulate PSB.
None of these formulating agents were able to produce stable 10 mg/mL PSB emulsion at similar or even higher than TPGS/PSB ratios.
(HLB = 14.3) as an efficient formulating agent for generating stable emulsions containing 10 mg/mL of PSB in aqueous solutions. Block polymers, including PPG-PEG-PPG, Pluronic L-64 with similar HLB properties to TPGS, were used in an attempt to formulate PSB.
None of these formulating agents were able to produce stable 10 mg/mL PSB emulsion at similar or even higher than TPGS/PSB ratios.
[00117] Experiment 15. Preparation of PSB/Caffeine/TPGS emulsion. To prepare emulsion containing 10 mg/ml of PSB/Caffeine (56.2% w/w PSB and 45.0% w/w Caffeine).
Table 15a. Formulation composition.
Date Lot# Material Weighed, mg (per 100 Calculated Weight, mg based on ml) C of A
TPGS PSB/caffeine PSB Caffeine 30-Jul-13 005.35.1 Mixture 5073 1022 574 460 [00118] 5.073 g of TPGS and 1.022 g of PSB/caffeine were weighed and placed in 400 ml beaker. 10 ml of 95% ethanol were added to the solids, and the beaker containing ethanol PATENT APPLICATION
Attorney Docket No. ISS.000200CA
mixture of TPGS/PSB/caffeine was placed in the water bath, and incubated at 60 C for 20 min until clear. The beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
Table 15a. Formulation composition.
Date Lot# Material Weighed, mg (per 100 Calculated Weight, mg based on ml) C of A
TPGS PSB/caffeine PSB Caffeine 30-Jul-13 005.35.1 Mixture 5073 1022 574 460 [00118] 5.073 g of TPGS and 1.022 g of PSB/caffeine were weighed and placed in 400 ml beaker. 10 ml of 95% ethanol were added to the solids, and the beaker containing ethanol PATENT APPLICATION
Attorney Docket No. ISS.000200CA
mixture of TPGS/PSB/caffeine was placed in the water bath, and incubated at 60 C for 20 min until clear. The beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
[00119] Emulsion preparation: The beaker was removed from oven and 100 ml of water was added to viscous cloudy film of TPGS/PSB/Caffeine mixture, placed into a 60 C water bath and incubated for 30 min to facilitate dissolution. The water TPGS/PSB/Caffeine mixture was mixed on magnetic stirring plate for 2 hrs at RT. Upon clearing of the mixture, particle size and PSB and caffeine content of the resultant emulsion was determined (Table 15b).
Table 15b. Particle size, PSB content, and Caffeine content of resultant emulsion.
Particle size PSB Caffeine Manufacturing Date Lot# by intensity, Content, Recovery, Content, Recovery, nm mg/ml mg/ml 31-Jul-13 005.35.3 13.1 5.66 99 4.53 [00120] HPLC Analysis. "Pterostilbene 280.M." method was used for determination of PSB and caffeine content. PSB content and Caffeine content determined by HPLC
("Pterostilbene 280 NM" method) was PSB was 5.66 mg/mL and caffeine 4.53 mg/mL.
Recovery is based on the weighed amount of the pterostilbene/caffeine co-crystal and the weight % provided by the Chromadex C of A. Data indicate that 99% of the PSB used for preparation of this formulation was incorporated into TPGS particles (Table 15b).
Table 15c. Particle size, PSB content, and stability of resultant emulsion.
Days Particle size PSB Caffeine Manufacturing Lot# past by intensity, Content, Recovery, Content, Recovery, Date mfg nm mg/ml mg/ml 31-Jul-13 005.35.3 6 13.1 5.85 102 4.68 102 [00121] Particle size analysis and stability. The ratio of TPGS to PSB in this emulsion is 8.8:1 and this large ratio produces the small particle size of 13.1 nm. The resultant emulsion was stable. Tables 15b and 15c show that particle size did not change over 6 days of monitoring.
After 30 days the emulsion is visually unchanged.
Table 15b. Particle size, PSB content, and Caffeine content of resultant emulsion.
Particle size PSB Caffeine Manufacturing Date Lot# by intensity, Content, Recovery, Content, Recovery, nm mg/ml mg/ml 31-Jul-13 005.35.3 13.1 5.66 99 4.53 [00120] HPLC Analysis. "Pterostilbene 280.M." method was used for determination of PSB and caffeine content. PSB content and Caffeine content determined by HPLC
("Pterostilbene 280 NM" method) was PSB was 5.66 mg/mL and caffeine 4.53 mg/mL.
Recovery is based on the weighed amount of the pterostilbene/caffeine co-crystal and the weight % provided by the Chromadex C of A. Data indicate that 99% of the PSB used for preparation of this formulation was incorporated into TPGS particles (Table 15b).
Table 15c. Particle size, PSB content, and stability of resultant emulsion.
Days Particle size PSB Caffeine Manufacturing Lot# past by intensity, Content, Recovery, Content, Recovery, Date mfg nm mg/ml mg/ml 31-Jul-13 005.35.3 6 13.1 5.85 102 4.68 102 [00121] Particle size analysis and stability. The ratio of TPGS to PSB in this emulsion is 8.8:1 and this large ratio produces the small particle size of 13.1 nm. The resultant emulsion was stable. Tables 15b and 15c show that particle size did not change over 6 days of monitoring.
After 30 days the emulsion is visually unchanged.
[00122] Experiment 16. Preparation of PSB/Caffeine/TPGS emulsion. To prepare emulsion containing 10 mg/ml of PSB from the PSB/Caffeine co-crystal (56.2%
w/w PSB and 45.0% w/w Caffeine).
Table 16a. Formulation composition.
Date Lot# Material Weighed, mg (per 100 Calculated Weight, mg based ml) on C of A
TPGS PSB/caffeine PSB Caffeine 1-Aug-13 005.36.2 Mixture 5018 1779 1000 801 PATENT APPLICATION Attorney Docket No.
ISS.000200CA
w/w PSB and 45.0% w/w Caffeine).
Table 16a. Formulation composition.
Date Lot# Material Weighed, mg (per 100 Calculated Weight, mg based ml) on C of A
TPGS PSB/caffeine PSB Caffeine 1-Aug-13 005.36.2 Mixture 5018 1779 1000 801 PATENT APPLICATION Attorney Docket No.
ISS.000200CA
[00123] 5.018 g of TPGS and 1.779 g of PSB/caffeine were weighed and placed in 400 ml beaker. 10 ml of 95% ethanol were added to the solids, and the ethanol mixture of TPGS/PSB/caffeine was placed in the water bath, and incubated at 60 C for 20 min until clear.
Beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
Beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
[00124] Emulsion preparation: The beaker was removed from vacuum oven and 100 ml of water was added to highly viscous cloudy film of TPGS/PSB/Caffeine mixture, beaker was placed to 60 C water bath and total mixture was incubated for 30 min to facilitate the dissolution. Then water TPGS/PSB/Caffeine mixture was mixed for 2 hrs at RT.
Upon clearing of the mixture, particle size, PSB and caffeine content of the resultant emulsion was determined (Table 16b). The emulsion is transparent but hazy. The emulsion was filtered through 0.22 p.m filter and particle size, PSB and caffeine content were measured again. The haziness did not change after filtration.
Table 16b. Particle size, PSB content, and Caffeine content of resultant emulsion.
Particle size PSB Caffeine Manufacturing Date Lot# by intensity, Content, Recovery, Content, Recovery, nm mg/ml mg/ml 2-Aug-13 005.36.3 33.4 9.65 97 7.67 96 336.
2-Aug-13 005. 33.5 9.75 98 7.73 97 filtered [00125] HPLC Analysis. "Pterostilbene 280.M." method was used for determination of PSB and caffeine content. PSB content and Caffeine content in resultant emulsion determined by HPLC ("Pterostilbene 280 NM" method) was PSB was 9.65 mg/mL and caffeine 7.67 mg/mL.
Particle size, PSB and caffeine content are not significantly different in the unfiltered emulsion as compared to the filtered emulsion. Recovery is based on the weighed amount of the pterostilbene/caffeine co-crystal and the weight % provided by the Chromadex C
of A. The data indicate that 97% of the PSB used for preparation of this formulation was incorporated into TPGS particles (Table 16b).
Upon clearing of the mixture, particle size, PSB and caffeine content of the resultant emulsion was determined (Table 16b). The emulsion is transparent but hazy. The emulsion was filtered through 0.22 p.m filter and particle size, PSB and caffeine content were measured again. The haziness did not change after filtration.
Table 16b. Particle size, PSB content, and Caffeine content of resultant emulsion.
Particle size PSB Caffeine Manufacturing Date Lot# by intensity, Content, Recovery, Content, Recovery, nm mg/ml mg/ml 2-Aug-13 005.36.3 33.4 9.65 97 7.67 96 336.
2-Aug-13 005. 33.5 9.75 98 7.73 97 filtered [00125] HPLC Analysis. "Pterostilbene 280.M." method was used for determination of PSB and caffeine content. PSB content and Caffeine content in resultant emulsion determined by HPLC ("Pterostilbene 280 NM" method) was PSB was 9.65 mg/mL and caffeine 7.67 mg/mL.
Particle size, PSB and caffeine content are not significantly different in the unfiltered emulsion as compared to the filtered emulsion. Recovery is based on the weighed amount of the pterostilbene/caffeine co-crystal and the weight % provided by the Chromadex C
of A. The data indicate that 97% of the PSB used for preparation of this formulation was incorporated into TPGS particles (Table 16b).
[00126] Particle size analysis. The ratio of TPGS to PSB in this emulsion is 5:1 and this ratio resulted in a particle size of 33.4 nm which is larger than the particle size in Experiment 15 and shows that a lower ratio of PSB and TPGS results in larger particle sizes.
The presence of caffeine does not appear to influence the emulsification of PSB. Three days after manufacture, the filtered emulsion was found to have crystallized. The analytical data of the supernatant is shown in Table 16c below. Both PSB and Caffeine have precipitated from solution. The ratio of PATENT APPLICATION
Attorney Docket No. ISS.000200CA
TPGS to PSB in the supernatant or mother liquors is 9.1:1. The results for particle size and PSB
and caffeine content of the supernatant suggest the maximum stable concentrations of PSB and caffeine and minimum ratio of PSB to TPGS in an emulsion for a given concentration of TPGS
(50 mg/mL). The ratio of TPGS to PSB in the supernatant is 9.1:1.
Table 16c. Particle size, PSB content, and stability of resultant emulsion.
Days PSB Caffeine Manufacturing Particle size by Lot# past Content, Recovery, Content, Recovery Date intensity, nm mfg mg/ml mg/ml ,%
336.
2-Aug-13 005. 3* 13.8 5.49 55 4.58 57 filtered *the emulsion crystallized, analysis is of the supernatant [00127] Dilutions. On 9-Aug-2013 (one week after preparation) Lot# 005.36.3 was warmed to 65 C for 1.5 hours. The crystals had dissolved and the solution was cloudy. Upon cooling and setting for 2 hours, the solution cleared to two phases. Vigorous hand agitation for 15 seconds resulted in a homogenous solution. The particle size analysis result of the solution with the crystals re-dissolved was 31.5 nm. Dilutions of the preparation were made to evaluate the effect of concentration of the PSB and caffeine on emulsion stability.
Dilutions: none, 1:1, 1:10, 1:20 and 1:100 were prepared in 50 mL centrifuge tubes and allowed to set for three days.
On day three the no dilution sample had crystallized and the 1:1 dilution sample had a few crystals. The other dilutions had no crystals. These results suggest that when the PSB
concentration is below 0.6 mg/mL and the caffeine concentration similarly low (below 0.5 mg/mL), a stable emulsion can be readily generated.
The presence of caffeine does not appear to influence the emulsification of PSB. Three days after manufacture, the filtered emulsion was found to have crystallized. The analytical data of the supernatant is shown in Table 16c below. Both PSB and Caffeine have precipitated from solution. The ratio of PATENT APPLICATION
Attorney Docket No. ISS.000200CA
TPGS to PSB in the supernatant or mother liquors is 9.1:1. The results for particle size and PSB
and caffeine content of the supernatant suggest the maximum stable concentrations of PSB and caffeine and minimum ratio of PSB to TPGS in an emulsion for a given concentration of TPGS
(50 mg/mL). The ratio of TPGS to PSB in the supernatant is 9.1:1.
Table 16c. Particle size, PSB content, and stability of resultant emulsion.
Days PSB Caffeine Manufacturing Particle size by Lot# past Content, Recovery, Content, Recovery Date intensity, nm mfg mg/ml mg/ml ,%
336.
2-Aug-13 005. 3* 13.8 5.49 55 4.58 57 filtered *the emulsion crystallized, analysis is of the supernatant [00127] Dilutions. On 9-Aug-2013 (one week after preparation) Lot# 005.36.3 was warmed to 65 C for 1.5 hours. The crystals had dissolved and the solution was cloudy. Upon cooling and setting for 2 hours, the solution cleared to two phases. Vigorous hand agitation for 15 seconds resulted in a homogenous solution. The particle size analysis result of the solution with the crystals re-dissolved was 31.5 nm. Dilutions of the preparation were made to evaluate the effect of concentration of the PSB and caffeine on emulsion stability.
Dilutions: none, 1:1, 1:10, 1:20 and 1:100 were prepared in 50 mL centrifuge tubes and allowed to set for three days.
On day three the no dilution sample had crystallized and the 1:1 dilution sample had a few crystals. The other dilutions had no crystals. These results suggest that when the PSB
concentration is below 0.6 mg/mL and the caffeine concentration similarly low (below 0.5 mg/mL), a stable emulsion can be readily generated.
[00128] Experiment 17. Preparation of PSB/Caffeine/TPGS emulsion. To prepare emulsion containing 11.2 mg/ml of PSB from the PSB/Caffeine co-crystal (56.2%
w/w PSB and 45.0% w/w Caffeine).
Table 17a. Formulation composition.
Date Lot # Material Weighed, mg (per 100 ml) Calculated Weight, mg based on C
of A
TPGS PSB/caffeine PSB Caffeine 1-Aug-13 005.36.1 Mixture 5003 2.008 1128 904 [00129] 5.003 g of TPGS and 2.008 g of PSB/caffeine were weighed and placed in 400 ml beaker. 10 ml of 95% ethanol were added to the solids, and the beaker containing ethanol mixture of TPGS/PSB/caffeine was placed in the water bath, and incubated at 60 C for 20 min until clear. Then beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
w/w PSB and 45.0% w/w Caffeine).
Table 17a. Formulation composition.
Date Lot # Material Weighed, mg (per 100 ml) Calculated Weight, mg based on C
of A
TPGS PSB/caffeine PSB Caffeine 1-Aug-13 005.36.1 Mixture 5003 2.008 1128 904 [00129] 5.003 g of TPGS and 2.008 g of PSB/caffeine were weighed and placed in 400 ml beaker. 10 ml of 95% ethanol were added to the solids, and the beaker containing ethanol mixture of TPGS/PSB/caffeine was placed in the water bath, and incubated at 60 C for 20 min until clear. Then beaker with ethanol TPGS/PSB solution was placed into vacuum oven 0/N.
PATENT APPLICATION Attorney Docket No.
ISS.000200CA
[00130] Emulsion preparation: The beaker was removed from vacuum oven and 100 ml of water was added to highly viscous cloudy film of TPGS/PSB/Caffeine mixture, beaker was placed to 60 C water bath and incubated for 30 min. Water TPGS/PSB/Caffeine mixture was mixed for 2 hrs at RT. Upon clearing of the mixture, particle size, PSB and caffeine content of the resultant emulsion was determined (Table 17b). The emulsion is transparent but hazy.
Table 17b. Particle size, PSB content, and Caffeine content of resultant emulsion.
Particle size PSB Caffeine Manufacturing Date Lot# by intensity, Content, Recovery, Content, Recovery, nm mg/m1 mg/ml 2-Aug-13 005.36.4 42.8 10.79 95 8.56 96 [00131] HPLC Analysis. "Pterostilbene 280.M." method was used for determination of PSB and caffeine content. PSB content and Caffeine content in resultant emulsion determined by HPLC ("Pterostilbene 280 NM" method) was PSB was 10.79 mg/mL and caffeine 8.56 mg/mL.
Recovery is based on the weighed amount of the PSB/caffeine co-crystal and the weight %
provided by the Chromadex C of A. Data indicate that 95% of the PSB used for preparation of this formulation was incorporated into TPGS particles (Table 17b).
Table 17b. Particle size, PSB content, and Caffeine content of resultant emulsion.
Particle size PSB Caffeine Manufacturing Date Lot# by intensity, Content, Recovery, Content, Recovery, nm mg/m1 mg/ml 2-Aug-13 005.36.4 42.8 10.79 95 8.56 96 [00131] HPLC Analysis. "Pterostilbene 280.M." method was used for determination of PSB and caffeine content. PSB content and Caffeine content in resultant emulsion determined by HPLC ("Pterostilbene 280 NM" method) was PSB was 10.79 mg/mL and caffeine 8.56 mg/mL.
Recovery is based on the weighed amount of the PSB/caffeine co-crystal and the weight %
provided by the Chromadex C of A. Data indicate that 95% of the PSB used for preparation of this formulation was incorporated into TPGS particles (Table 17b).
[00132] Particle size analysis. The ratio of TPGS to PSB in this emulsion is 4.4:1 and this ratio results in a particle size of 42.8 nm which is larger than the particle size in Experiment 16 and again shows that a lower ratio of PSB and TPGS result in larger particle sizes. The presence of caffeine does not influence the emulsification of PSB. Three days after manufacture, the filtered emulsion was found to have crystallized. Data of the supernatant or mother liquors is shown in Table 17 c. Both PSB and caffeine have come out of solution. The ratio of TPGS to PSB in the supernatant or mother liquors is 9.1:1. The results for particle size and PSB and caffeine content of the supernatant or mother liquors suggests the maximum stable concentrations of PSB and caffeine and minimum ratio of PSB to TPGS in an emulsion for a given concentration of TPGS (50 mg/mL). The ratio of TPGS to PSB in the supernatant is 9.1:1.
The emulsion of experiment 17 gives similar results to that of experiment 16;
a ratio of 9 to 1 of TPGS to PSB in the presence of caffeine is necessary to form a stable emulsion of PSB at greater than 0.6 mg/mL PSB.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Table 17 c. Particle size, PSB content, and particle stability of resultant emulsion.
Days Particle size PSB Caffeine Manufacturing Lott/ past by intensity, Content, Recovery, Content, Recovery, Date mfg Nm mg/ml mg/m1 2-Aug-13 005.36.4 3* 13.5 5.51 49 4.63 51 *the emulsion crystallized, analysis is of the supernatant [00133] Experiment 18. Preparation of a Cremophor ELP emulsion. To prepare emulsion containing 5% v/v of Cremophor in water and measure particle size.
Water, 19 mL, was added to 1 mL of Cremophor in a 20 mL vial, capped and heated to 60 C
then mixed to form a nearly clear solution.
Table 18 a. Formulation composition.
Date Lot# Material Volume, mL
Cremophor Water 27-Aug-13 005.39.1 Mixture 1 mL 19 mL
Table 18 b. Particle size of resultant emulsion.
Manufacturing Date Lot# Particle size by intensity, nm 27-Aug-13 005.39.1 12.6 28-Aug-13 005.39.1 12.8 [00134] Experiment 19. Preparation of PSB/Cremophor ELP emulsion. To prepare emulsion containing 1.0 mg/ml of PSB and 5 mg/mL Cremophor.
Table 19 a. Formulation composition.
Date Lot# Material Weighed, mg (per 100 ml) Cremophor PSB
25-Aug-13 005.38.1 Mixture 5038 1007 [00135] 5.038 g of Cremophor and 1.007 g of PSB were weighed and placed in 200 ml beaker. Cremophor is a viscous oil at RT. The beaker was placed in a 65 C
water bath and over 30 minutes the Cremophor dissolved the PSB based on visual observation. 10 ml of 95% ethanol were added to the oily mixture, and the ethanol mixture of Cremophor/PSB was incubated at 60 C for 20 min until the oil dissolved, and ethanol was evaporated with a Nitrogen stream. The residual ethanol Cremophor/PSB mixture was placed into vacuum oven overnight.
The emulsion of experiment 17 gives similar results to that of experiment 16;
a ratio of 9 to 1 of TPGS to PSB in the presence of caffeine is necessary to form a stable emulsion of PSB at greater than 0.6 mg/mL PSB.
PATENT APPLICATION Attorney Docket No. ISS.000200CA
Table 17 c. Particle size, PSB content, and particle stability of resultant emulsion.
Days Particle size PSB Caffeine Manufacturing Lott/ past by intensity, Content, Recovery, Content, Recovery, Date mfg Nm mg/ml mg/m1 2-Aug-13 005.36.4 3* 13.5 5.51 49 4.63 51 *the emulsion crystallized, analysis is of the supernatant [00133] Experiment 18. Preparation of a Cremophor ELP emulsion. To prepare emulsion containing 5% v/v of Cremophor in water and measure particle size.
Water, 19 mL, was added to 1 mL of Cremophor in a 20 mL vial, capped and heated to 60 C
then mixed to form a nearly clear solution.
Table 18 a. Formulation composition.
Date Lot# Material Volume, mL
Cremophor Water 27-Aug-13 005.39.1 Mixture 1 mL 19 mL
Table 18 b. Particle size of resultant emulsion.
Manufacturing Date Lot# Particle size by intensity, nm 27-Aug-13 005.39.1 12.6 28-Aug-13 005.39.1 12.8 [00134] Experiment 19. Preparation of PSB/Cremophor ELP emulsion. To prepare emulsion containing 1.0 mg/ml of PSB and 5 mg/mL Cremophor.
Table 19 a. Formulation composition.
Date Lot# Material Weighed, mg (per 100 ml) Cremophor PSB
25-Aug-13 005.38.1 Mixture 5038 1007 [00135] 5.038 g of Cremophor and 1.007 g of PSB were weighed and placed in 200 ml beaker. Cremophor is a viscous oil at RT. The beaker was placed in a 65 C
water bath and over 30 minutes the Cremophor dissolved the PSB based on visual observation. 10 ml of 95% ethanol were added to the oily mixture, and the ethanol mixture of Cremophor/PSB was incubated at 60 C for 20 min until the oil dissolved, and ethanol was evaporated with a Nitrogen stream. The residual ethanol Cremophor/PSB mixture was placed into vacuum oven overnight.
[00136] Emulsion preparation: 100 ml of water was added to the viscous clear film of Cremophor/PSB mixture, the beaker was placed to 60 C water bath and incubated for 30 min.
The water Cremophor/PSB mixture was mixed for 1 hr at RT. The mixture never cleared and PATENT APPLICATION Attorney Docket No.
ISS.000200CA
retained a milky appearance. Particle size of the suspension was noted in Table 19b. No settling observed over 3 days.
Table 19 b. Particle size of resultant suspension.
Particle size by intensity, Manufacturing Date Lot#
nm 27-Aug-13 005.38.1 286.9 28-Aug-13 005.38.1 339.3 [00137] Particle size analysis. The ratio of Cremophor to PSB in this emulsion is 5:1.
This ratio resulted in a particle size of approximately 300 nm.
The water Cremophor/PSB mixture was mixed for 1 hr at RT. The mixture never cleared and PATENT APPLICATION Attorney Docket No.
ISS.000200CA
retained a milky appearance. Particle size of the suspension was noted in Table 19b. No settling observed over 3 days.
Table 19 b. Particle size of resultant suspension.
Particle size by intensity, Manufacturing Date Lot#
nm 27-Aug-13 005.38.1 286.9 28-Aug-13 005.38.1 339.3 [00137] Particle size analysis. The ratio of Cremophor to PSB in this emulsion is 5:1.
This ratio resulted in a particle size of approximately 300 nm.
[00138] Experiment 20. Preparation of PSB/Caffeine/Cremophor emulsion. To prepare emulsion containing 10 mg/ml of PSB from the PSB/Caffeine co-crystal (56.2%
w/w PSB and 45.0% w/w Caffeine) with Cremophor.
Table 20 a. Formulation composition.
Date Lot# Material Weighed, mg Calculated Weight, (per 100 ml) mg based on C of A
Cremophor PSB/caffeine PSB Caffeine 25-Aug-13 005.38.2 Mixture 5000* 1773 996 798 * Actual weight not recorded (5 mL) [00139] Cremophor (5 mL) and 1.773 g of PSB/caffeine were weighed and placed in 200 ml beaker. 10 ml of 95% ethanol were added to the solids, and the beaker containing ethanol mixture of Cremophor/PSB/caffeine was placed in the water bath, and incubated at 60 C for 30 min. The solution did not clear and crystallized upon cooling. 10 mL of 95%
ethanol was added and incubated at 60 C for an additional 30 min until the solution cleared.
During evaporation with a nitrogen stream the solution crystallized. The ethanol/Cremophor/PSB/caffeine solids was placed into vacuum oven 0/N.
w/w PSB and 45.0% w/w Caffeine) with Cremophor.
Table 20 a. Formulation composition.
Date Lot# Material Weighed, mg Calculated Weight, (per 100 ml) mg based on C of A
Cremophor PSB/caffeine PSB Caffeine 25-Aug-13 005.38.2 Mixture 5000* 1773 996 798 * Actual weight not recorded (5 mL) [00139] Cremophor (5 mL) and 1.773 g of PSB/caffeine were weighed and placed in 200 ml beaker. 10 ml of 95% ethanol were added to the solids, and the beaker containing ethanol mixture of Cremophor/PSB/caffeine was placed in the water bath, and incubated at 60 C for 30 min. The solution did not clear and crystallized upon cooling. 10 mL of 95%
ethanol was added and incubated at 60 C for an additional 30 min until the solution cleared.
During evaporation with a nitrogen stream the solution crystallized. The ethanol/Cremophor/PSB/caffeine solids was placed into vacuum oven 0/N.
[00140] Emulsion preparation: The beaker was removed from vacuum oven and 100 ml of distilled water was added to solids of Cremophor/PSB/Caffeine and was placed to 60 C water bath and incubated for 30 min to facilitate dissolution. The water/Cremophor/PSB/Caffeine mixture was mixed for 1 hr at RT. The mixture remained milky. Particle size of the resultant suspension was tested the following day (Table 20b). No settling or crystallization after 3 days.
Table 20 b. Particle size of resultant suspension.
Manufacturing Date Lot# Particle size by intensity, nm 27-Aug-13 005.38.2 375.6 28-Aug-13 005.38.2 371.2 PATENT APPLICATION Attorney Docket No.
ISS.000200CA
Table 20 b. Particle size of resultant suspension.
Manufacturing Date Lot# Particle size by intensity, nm 27-Aug-13 005.38.2 375.6 28-Aug-13 005.38.2 371.2 PATENT APPLICATION Attorney Docket No.
ISS.000200CA
[00141] Particle size analysis. The ratio of Cremophor to PSB in this emulsion is approximately 5:1. This ratio resulted in a particle size of approximately 375 nm and due to its appearance and large particle size, it is not likely to be an emulsion of interest.
[00142] The present application discloses unique compositions with specific ratios of formulation components that produce stable RVT- or PSB-containing nanoparticles in the range from 20 to 40 nm. The nanoparticulate emulsion is stable in a variety of beverages providing the calculated beneficial dose of resveratrol or pterostilbene, or a mixture thereof, per serving. The above compositions are prepared with a RVT- and PSB-containing nanoparticles where the stable, soluble compositions of RVT- and PSB-containing nanoparticles resulted in similar stable and clear solutions where a mixture of RVT- and PST-containing nanoparticles were prepared.
[00143] The foregoing examples of the related art and limitations are intended to be illustrative and not exclusive. While a number of exemplary embodiments, aspects and variations have been provided herein, those of skill in the art will recognize certain modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations. It is intended that the following claims are interpreted to include all such modifications, permutations, additions and combinations and certain sub-combinations of the embodiments, aspects and variations are within their scope. The entire disclosures of all documents cited throughout this application are incorporated herein by reference.
Claims (20)
1. A stable and homogeneous aqueous formulation comprising:
a) a composition selected from the group consisting of a pterostilbene-caffeine co-crystal complex, a resveratrol-caffeine co-crystal complex and a mixture thereof; and b) an emulsifying agent in an amount sufficient to solubilize the pterostilbene-caffeine co-crystal complex, a resveratrol-caffeine co-crystal complex and a mixture thereof, to form the stable and homogeneous formulation.
a) a composition selected from the group consisting of a pterostilbene-caffeine co-crystal complex, a resveratrol-caffeine co-crystal complex and a mixture thereof; and b) an emulsifying agent in an amount sufficient to solubilize the pterostilbene-caffeine co-crystal complex, a resveratrol-caffeine co-crystal complex and a mixture thereof, to form the stable and homogeneous formulation.
2. The stable and homogeneous aqueous formulation of claim 1, wherein the emulsifying agent is selected from the group consisting of TPGS, TPGS-300, TPGS-500, TPGS-600, TPGS-750, TPGS-1000, TPGS-M, TPGS-300-M, TPGS-500-M, TPGS-600-M, TPGS-750-M and TPGS-1000-M, or a mixture thereof.
3. The stable and homogeneous aqueous formulation of claims 1 or 2, wherein the emulsifying agent is TPGS.
4. The stable and homogeneous aqueous formulation of any one of claims 1 to 3, wherein the formulation is an emulsion or a clear solution.
5. The stable and homogeneous aqueous formulation of any one of claims 1 to 4, wherein the solution is in water.
6. The stable and homogeneous aqueous formulation of any one of claims 1 to 4, wherein the formulation comprising the pterostilbene-caffeine co-crystal complex, a resveratrol-caffeine co-crystal complex and a mixture thereof in water is at a co-crystal complex:water (wt:wt) ratio of 10:90, 20:80, 30:70, 40: 60, 50:50, 60:40, 70:30, 80:20 or 90:10.
7. A method of solubilizing pterostilbene or resveratrol, or a mixture thereof; in an aqueous media comprising:
a) dissolving the pterostilbene or resveratrol, or a mixture thereof, and an emulsifying agent in a suitable solvent to form a homogeneous solution;
b) evaporating the solvent to form a mixture of pterostilbene or resveratrol, or a mixture thereof, and the emulsifying agent;
c) dissolving the pterostilbene mixture or resveratrol mixture, or a mixture thereof, in the aqueous medium by stirring the mixture with a warm medium to form a stable emulsion of pterostilbene or resveratrol, or a mixture thereof, and emulsifying agent.
a) dissolving the pterostilbene or resveratrol, or a mixture thereof, and an emulsifying agent in a suitable solvent to form a homogeneous solution;
b) evaporating the solvent to form a mixture of pterostilbene or resveratrol, or a mixture thereof, and the emulsifying agent;
c) dissolving the pterostilbene mixture or resveratrol mixture, or a mixture thereof, in the aqueous medium by stirring the mixture with a warm medium to form a stable emulsion of pterostilbene or resveratrol, or a mixture thereof, and emulsifying agent.
8. The method of claim 7, wherein the emulsifying agent has an HLB of greater than 9.
9. The method of claim 7 or 8, wherein the emulsifying agent is selected from the group consisting of TPGS, TPGS-300, TPGS-500, TPGS-600, TPGS-750, TPGS-1000, TPGS-M, TPGS-300-M, TPGS-500-M, TPGS-600-M, TPGS-750-M and TPGS-1000-M, or a mixture thereof.
10. The method of any one of claims 7 to 9, wherein the ratio of emulsifying agent to pterostilbene or resveratrol, or a mixture thereof, is in the range of 3:1 to 10:1.
11. The method of any one of claims 7 to 10, wherein the resultant emulsion comprises a particle size of less than 250 nanometers.
12. An aqueous composition comprising solubilized pterostilbene or solubilized resveratrol, or a mixture thereof, prepared by the method of any one of claims 7 to 11.
13. A method for solubilizing pterostilbene or resveratrol, or a mixture thereof, from a co-crystal complex of pterostilbene and caffeine or resveratrol and caffeine, or a mixture thereof, in aqueous media comprising:
a) dissolving the pterostilbene complex or resveratrol complex, or a mixture thereof, and an emulsifying agent in a suitable solvent to form a homogeneous solution;
b) evaporating the solvent to form a mixture of the pterostilbene complex or resveratrol complex, or a mixture thereof, and the emulsifying agent; and c) dissolving of the pterostilbene mixture or resveratrol mixture, or a mixture thereof, in the aqueous medium by stirring the mixture with a warm medium to form a stable emulsion of pterostilbene and emulsifying agent or resveratrol and emulsifying agent, or a mixture thereof.
a) dissolving the pterostilbene complex or resveratrol complex, or a mixture thereof, and an emulsifying agent in a suitable solvent to form a homogeneous solution;
b) evaporating the solvent to form a mixture of the pterostilbene complex or resveratrol complex, or a mixture thereof, and the emulsifying agent; and c) dissolving of the pterostilbene mixture or resveratrol mixture, or a mixture thereof, in the aqueous medium by stirring the mixture with a warm medium to form a stable emulsion of pterostilbene and emulsifying agent or resveratrol and emulsifying agent, or a mixture thereof.
14. The method of claim 13, wherein the emulsifying agents are GRAS
(Generally Recognized as Safe).
(Generally Recognized as Safe).
15. The method of claim 13 or 14, wherein the emulsifying agent is selected from the group consisting of TPGS, TPGS-300, TPGS-500, TPGS-600, TPGS-750, TPGS-1000, TPGS-M, TPGS-300-M, TPGS-500-M, TPGS-600-M, TPGS-750-M and TPGS-1000-M, or a mixture thereof.
16. The method of any one of claims 13 to 15, wherein the ratio of emulsifying agent to pterostilbene or resveratrol, or a mixture thereof, is in the range of 3:1 to 10:1.
17. The method of claim 16, wherein the resultant emulsion has a particle size of less than 250 nanometers.
18. The method of claim 17, wherein the resultant emulsion comprises a particle size of less than 100 nanometers.
19. The method of claim 17, wherein the resultant emulsion comprises a particle size of between 25 and 100 nanometers.
20. A solubilized composition comprising pterostilbene or resveratrol, or mixture thereof, prepared by the method of any one of claims 13 to 19.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201361872971P | 2013-09-03 | 2013-09-03 | |
| US61/872,971 | 2013-09-03 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2860256A1 true CA2860256A1 (en) | 2015-03-03 |
Family
ID=52629152
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2860256A Abandoned CA2860256A1 (en) | 2013-09-03 | 2014-08-22 | Solubilization of pterostilbene and resveratrol in aqueous beverages |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2860256A1 (en) |
-
2014
- 2014-08-22 CA CA2860256A patent/CA2860256A1/en not_active Abandoned
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Effective date: 20190822 |