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Title:
COMPOSITION APPLICABLE FOR ENHANCING WOUND HEALING HAVING POLYSACCHARIDE PEPTIDES OF RICE PARTICLE, A FIRST EXTRACT OF RICE BRAN AND A SECOND EXTRACT OF SESAMIN
Document Type and Number:
WIPO Patent Application WO/2019/164458
Kind Code:
A1
Abstract:
A first embodiment of the invention is a composition applicable for enhancing wound healing comprising a mixture having polysaccharide peptides of rice particle, a first extract of rice bran and a second extract of sesamin. In a first alternative embodiment of the invention, the polysaccharide peptides, the first extract and the second extract are in a ratio of 1: 1: 1 by weight. In a second alternative embodiment of the invention, the polysaccharide peptides is acquired by heating the rice particle at a temperature of 120°C for 20 minutes under a pressure of 15 psi in the presence of an aqueous phase. The rice particles can be pulverized rice powder with a particle size of 160 to 315 microns.

Inventors:
KONGTAWELERT PRACHYA (TH)
POTHACHAROEN PERAPHAN (TH)
Application Number:
PCT/TH2018/000006
Publication Date:
August 29, 2019
Filing Date:
February 23, 2018
Export Citation:
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Assignee:
THAILAND EXCELLENCE CENTER FOR TISSUE ENG AND STEM CELLS (TH)
International Classes:
A61K36/899; A61K31/726; A61K36/185; A61P17/02
Domestic Patent References:
WO2010060362A12010-06-03
Foreign References:
US20160256480A12016-09-08
US20070104858A12007-05-10
Other References:
PHETPORNPAISAN, P. ET AL.: "A local Thai cultivar glutinous blackrice bran: a source of functional compounds in immunomodulation, cell viability and collagen synthesis, and matrix metalloproteinase-2 and -9 inhibition", JOURNAL OF FUNCTIONAL FOODS, vol. 7, 17 January 2014 (2014-01-17) - March 2014 (2014-03-01), pages 650 - 661, XP055631811
SHARIF, M. R. ET AL.: "Evaluation of the wound healing activity of sesame oil extract in rats", WORLD JOURNAL OF MEDICAL SCIENCES, vol. 9, no. 2, 2013, pages 74 - 78, XP055631814
Attorney, Agent or Firm:
BURFORD, Nutthaporn (TH)
Download PDF:
Claims:
CLAIMS

1. A composition applicable for enhancing wound healing comprising a mixture having polysaccharide peptides of rice particle, a first extract of rice bran and a second extract of sesamin.

2. The composition of claim 1, wherein the polysaccharide peptides, the first extract and the second extract are in a ratio of 1 : 1 : 1 by weight.

3. The composition of claim 1, wherein the polysaccharide peptides is acquired by heating the rice particle at a temperature of 120 °C for 20 minutes under a pressure of 15 psi in the presence of an aqueous phase.

4. The composition of claim 3, wherein the rice particles are pulverized rice powder with a particle size of 160 to 315 microns.

5. The composition of claim 1, wherein the first extract of rice bran is acquired by mechanical polishing of rice grains.

6. The composition of claim 5, wherein the second extract of sesamin is an ethanolic extract.

7. The composition of claim 1, wherein the second extract of sesamin is an ethanolic extract and wherein the composition further comprises glucosamine sulfate.

8. The composition of claim 1, wherein the second extract of sesamin is acquired by bringing sesame oil into contact with absolute ethanol solvent, removing the sesamin from the solvent and vaporizing the solvent to yield the second extract.

Description:
COMPOSITION APPLICABLE FOR ENHANCING WOUND HEALING HAVING POLYSACCHARIDE PEPTIDES OF RICE PARTICLE, A FIRST EXTRACT OF RICE BRAN AND A SECOND EXTRACT OF SESAMIN

TECHNICAL FIELD

The present disclosure relates to a composition for wound healing. Particularly, the present disclosure relates a composition applicable for enhancing wound healing comprising a mixture having polysaccharide peptides of rice particle, a first extract of rice bran and a second extract of sesamin.

BACKGROUND

Poor wound healing remains a significant cause of morbidity and mortality worldwide. Estimated world prevalence of various wound types in 2011 include: 114,271,000 cases for surgical wounds; 1,627, 000 cases for traumatic wounds; 20,645,000 cases for laceration wounds; 10,221,000 cases for bum wounds; 40,400,000 cases for chronic wounds (pressure, venous/arterial and diabetic); 618,000 cases for carcinomas; 103,000 cases for melanoma; 103,000 cases for complicated skin cancer wounds (MedMarket Diligence, LLC; Report #S190 and Report #S251). Wounds remain difficult to treat, despite advances in wound dressing and care and a better understanding of the cellular and molecular biology of wound healing. Wound healing is a complex but well- orchestrated tissue repair process including: hemostatic/inflammatory response, cell proliferation/ formation of connective tissues, wound contraction and remodeling. Although several therapeutic strategies proposed to accelerate the wound healing of acute and/or chronic wounds over the past few decades, effective treatment of wound healing still remains a challenge.

Stem cells are characterized by their multipotency and capacity for self-renewal and differentiation to various cell types. Stem cells can be categorized as embryonic stem cells (ESCs), tissue specific progenitor stem cells (TSPSCs), mesenchymal stem cells (MSCs), umbilical cord stem cells (UCSCs), bone marrow stem cells (BMSCs), and induced pluripotent stem cells (iPSCs) (Mahla RS. Stem cells applications in regenerative medicine and disease therapeutics. Int J Cell Biol. 20l6;20l6:6940283). Stem cells have potential uses in many different areas of research, biotechnology and medicine such as study human development, testing of new drugs, screening toxins, testing gene therapy methods, and replace damaged tissues. Regarding to the ability in production of proregenerative cytokines, stem cells have therapeutic potential for wound healing. Preclinical of stem cells in various animal models and clinical trials have been studied in wound healing, such as for tissue regeneration. However, translation of the laboratory experiment results into clinical applications has been limited by the dependence of stem cell propagation. Currently, stem cell for clinical application is based on their subsequent large-scale in vitro expansion by conventional static adherent cultures in the presence of fetal bovine serum (FBS) (Jung S et al. Large- scale production of human mesenchymal stem cells for clinical applications. Biotechnol Appl Biochem. 2012;59(2): 106-20).

However, an effective composition for generation of large quantities of stem cell is required to fulfil the biomedical research demand and clinical needs of wound healing are still needed. Therefore there is a need for a low cost, large quantity wound healing composition with regenerative properties for the body that can help in modulating immune responses, reducing inflammation and providing protective effects for cells, tissues, and the organs of the body.

SUMMARY OF INVENTION

A first embodiment of the invention is a composition applicable for enhancing wound healing comprising a mixture having polysaccharide peptides of rice particle, a first extract of rice bran and a second extract of sesamin.

In a first alternative embodiment of the invention, the polysaccharide peptides, the first extract and the second extract are in a ratio of 1 : 1 : 1 by weight.

In a second alternative embodiment of the invention, the polysaccharide peptides is acquired by heating the rice particle at a temperature of 120 °C for 20 minutes under a pressure of 15 psi in the presence of an aqueous phase. The rice particles can be pulverized rice powder with a particle size of 160 to 315 microns.

In a third embodiment of the invention, the first extract of rice bran is acquired by mechanical polishing of rice grains. The second extract of sesamin can be an ethanolic extract.

In a fourth embodiment of the invention, the second extract of sesamin is an ethanolic extract and wherein the composition further comprises glucosamine sulfate.

In a fourth embodiment of the invention, the second extract of sesamin is acquired by bringing sesame oil into contact with absolute ethanol solvent, removing the sesamin from the solvent and vaporizing the solvent to yield the second extract.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure are described herein with reference to the drawings in which:

FIG. 1 is a graph charting the mediated proliferation rate of mesenchymal stem cells (MSC) for an embodiment of the invention.

FIG. 2 is a flowchart of the steps involved in an embodiment of the invention for preparation of polysaccharide peptides of rice particle.

FIG. 3 is a flowchart of the steps involved in an embodiment of the invention for preparation of ethanolic extract of rice bran.

FIG. 4 is a chromatogram of standard of anthocyanins: cyanidin 3-O-glucoside chloride, C3G; peonidin 3-O-glucoside chloride, P3G; cyanidin 3-O-rutinoside chloride, C3R; and cyanidin chloride.

FIG. 5 is a chromatogram of anthocyanin content in purple rice extracts (PREs) from ethanolic extracts.

FIG. 6 is a flowchart of the steps involved in an embodiment of the invention for preparation of ethanolic extract of sesamin.

FIG. 7 is a mass spectrogram and chemical structure of the ethanolic extract of sesamin purified from S. indicum.

DETAILED DESCRIPTION

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. The illustrative embodiments described in the detailed description, drawings and claims are not meant to be limiting. Other embodiments can be utilized, and other changes can be made, without departing from the spirit or scope of the subject matter presented herein. Unless specified otherwise, the terms “comprising,”“comprise,”“including” and“include” used herein, and grammatical variants thereof, are intended to represent“open” or“inclusive” language such that they include recited elements but also permit inclusion of additional, un-recited elements.

The mediated proliferation rate of mesenchymal cell for an embodiment of the invention is illustrated in the graph 1-00 of FIG. 1, comparing percentage of proliferation following stimulating mesenchymal stem cell with the composition applicable for enhancing wound healing, polysaccharide peptides, rice bran, and sesamin, respectively. The composition applicable for enhancing wound healing mediates the highest proliferation rate of mesenchymal stem cells at day 5.

FIG. 2 is a flowchart 2-00 of the steps involved in an embodiment of the invention for preparation of polysaccharide peptides of rice particle, which include:

• 2-01 extract rice bran with phosphate buffer with NaCl

• 2-02 yield the aqueous phase of extract

• 2-03 centrifuge and filter the aqueous phase of extract

• 2-04 fractionate with gel filtration

• 2-05 evaporate and freeze-dry the fractions

• 2-06 yield the rice particle

• 2-07 pulverize the rice particle

• 2-08 heat the rice particle

• 2-09 centrifuge and filter the heated rice particle

• 2-10 yield the polysaccharide peptides

The step 2-04 can be performed with the gel filtration column sold under the trademark SUPERDEX ® 200. FIG. 3 is a flowchart 3-00 of the steps involved in an embodiment of the invention for preparation of ethanolic extract of rice bran, which include:

• 3-01 acquire rice bran by mechanical polishing of rice grains

• 3-02 extract rice bran with ethanol

• 3-03 yield the ethanolic extract of rice bran

• 3-04 freeze-dry the ethanolic extract

• 3-05 yield the first extract of rice bran

The standard chromatogram of anthocyanins is charted in the graph 4-00 of FIG. 4, which are cyanidin 3-O-glucoside chloride 1, C3G; peonidin 3-O-glucoside chloride 2, P3G; cyanidin 3-O-rutinoside chloride 3, C3R; and cyanidin chloride 4.

The peak chromatogram of anthocyanin in purple rice extracts (PREs) is charted in the graph 5-00 of FIG. 5, which demonstrates cyanidin 3-O-glucoside chloride, C3G; and peonidin 3-O-glucoside chloride, P3G, respectively.

FIG. 6 is a flowchart 6-00 of the steps involved in an embodiment of the invention for preparation of ethanolic extract of sesamin, which include:

• 6-01 prepare hexane extract from the air-dried, finely, powder seed from sesame (S. indicum )

• 6-02 evaporate the hexane extract

• 6-03 yield the crude hexane extract

• 6-04 fractionate with silica gel chromatography

• 6-05 tes the fractions with thin layer chromatography (TLC)

• 6-06 collect sesamin fractions

• 6-07 fractionate with silica gel chromatography

• 6-08 combine specific subfractions

• 6-09 purify the specific subfraction with ethanol

• 6-10 yield the ethanolic extract of sesamin The mass spectrogram and chemical structure of ethanolic extract of sesamin purified from S. indicum is charted in the graph 7-00 of FIG. 7.

Rice bran is the outer layer of the rice grain and obtained as a byproduct of rice milling process. Sesame is an oilseed crops widely grown in Africa and Asia. It contains phytochemical compounds including sesamin, sesamol, sesamolactol and anthrasesamone F.

A first embodiment of the invention is a composition applicable for enhancing wound healing comprising a mixture having polysaccharide peptides of rice particle, a first extract of rice bran and a second extract of sesamin.

In a first alternative embodiment of the invention, the polysaccharide peptides, the first extract and the second extract are in a ratio of 1:1:1 by weight.

In a second alternative embodiment of the invention, the polysaccharide peptides is acquired by heating the rice particle at a temperature of 120 °C for 20 minutes under a pressure of 15 psi in the presence of an aqueous phase. The rice particles can be pulverized rice powder with a particle size of 160 to 315 microns.

In a third embodiment of the invention, the first extract of rice bran is acquired by mechanical polishing of rice grains. The second extract of sesamin can be an ethanolic extract. In a fourth embodiment of the invention, the second extract of sesamin is an ethanolic extract and wherein the composition further comprises glucosamine sulfate.

In a fourth embodiment of the invention, the second extract of sesamin is acquired by bringing sesame oil into contact with absolute ethanol solvent, removing the sesamin from the solvent and vaporizing the solvent to yield the second extract. EXPERIMENTS

Isolation and culture of human mesenchymal stem cells was accomplished as follows. PBMNCs were obtained from 35-40 mL of peripheral venous blood by a previously described density gradient centrifugation method. The venous blood was centrifuged at 1500 rpm for 5 min to separate plasma from other blood components. Afterward, plasma was removed for the measurement of sRAGE, and the remaining blood component was diluted with an equal volume of Dulbecco’s modified Eagle’s medium (DMEM) sold under the trademark GIBCO. The diluted mixture was then carefully overlaid on the density gradient cell separation medium sold under the trademark HISTOPAQUE (specific gravity 1.077 g/mL; SIGMA- ALDRICH) and centrifuged at 4000 rpm for 30 min. The PBMNCs were isolated from the mononuclear cell layer, and were then washed twice with warmed RPMI sold under the trademark GIBCO. The isolated PBMNCs were plated in five wells of 24- well culture plates (3.7 c 10 6 ± 7 c 10 5 cells per well) and cultured in RPMI supplemented with 10% (v/v) fetal bovine serum sold under the trademark GIBCO. After they had been cultured for 3 days, the floating cells were discarded and the adherent cells were further cultured in DMEM supplemented with 10% (v/v) fetal bovine serum sold under the trademark GIBCO. The culture medium was changed every 3 days not only to feed the cells but also to discard the floating cells. The plastic-adhered PBMNCs were cultured for 7-10 days until confluence and used for further analysis.

Characterization and cell growth rate of the human mesenchymal stem cells by cell proliferation assay was accomplished as follows. Cell proliferation was assessed by Alamar blue assay (Alamar blue sold under the trademark SIGMA-ALDRICH) and carried out as follows. The cells were individually plated in 96 well-culture plate (IxlO 3 cells/well) and incubated in 5% C0 2 incubator at 37°C. After incubation for 24 hr., 100 mΐ of 10% (v/v) Alamar Blue (rasazurin) in 10% (v/v) FCS DMEM was replaced. After 4 hr. incubation, 100 mΐ of incubated medium from each well was taken to the new well of 96 well plates and the freshly 10% (v/v) FCS DMEM was added back to the chondrocyte cultured well. The produced resorufin in the 4-hr-incubated medium was detected at 540 and 620 nm. The proliferation assay was measured on day 1, 3, 5, 7, 9 and 11 of the culture period. Effect of the composition applicable for enhancing wound healing to cell growth rate of the human mesenchymal stem cells was measured as follows. The cells were individually plated in 96 well-culture plate (lxl 0 3 cells/well) and incubated in 5% C0 2 incubator at 37°C. Twenty-four hours after plating, the cells were exposed to the composition applicable for enhancing wound healing (12.5 mg/ml) or polysaccharide peptides (0.5 mg/ml) or rice bran (12.5 mg/ml) or sesamin for additional 3, 5, 7, 9 and 11 days. Each treatment was carried out in triplicate. After the end of treatment, cell proliferation assay was performed as described above.

Preparation of polysaccharide peptides of rice particle was accomplished as follows. a. Preparation of rice particle

Sample powder of rice bran was dissolved in in 50 mM Phosphate buffer pH 6.98 with 150 mM NaCl at final concentration 5 mg/ml. The mixture was incubated and mixed by using vigorously shaking machine for 45 minutes at room temperature. The mixture was centrifuged at 3000xg for 5 minutes and filtered with 0.22 pm filter. The aqueous phase of extract was fractionated with gel filtration by the gel sold under the trademark SUPERDEX 200 increase 10/300 GL at flow rate 0.75 ml/min for 1.8 CV. The fractions were evaporated and freeze-dried to yield the rice particle.

b. Preparation of polysaccharide peptides

Rice particles were pulverized to a particle size of 160 to 315 microns. The pulverized rice particles were heated at a temperature of 120 °C for 30 minutes under a pressure of 15 psi in the presence of an aqueous phase. The heated rice particle was centrifuged and filtered with 0.22 pm filter to yield polysaccharide peptides.

Preparation of ethanolic extract of rice bran was accomplished as follows. Rice bran were acquired by mechanical polishing of rice grains and then extracted with ethanol. The ethanolic extract of rice bran was collected, evaporated in vacuo at 45-50 °C and freeze-dried to yield the first extract of rice bran.

High performance liquid chromatography (HPLC) analysis demonstrating enhanced wound healing was accomplished as follows. Anthocyanin content in ethanolic extract of purple rice and white rice were analyzed by using the HPLC sold under the trademark ALIGENT 1260 SERIES (Agilent Technologies Ltd.), equipped with a binary pumping system. Modified from Chromadex method, samples (10 mΐ) were injected into the Cl 8 column sold under the trademark ZORBAX ECLIPSE PLUS (4.6 x 100 mm, particle size 3.5 m). The mobile phase consisted of water/formic acid (90:10, V/v) (A) and acetonitrile (B). The gradient program was accomplished at 35 °C: initial time, 4% solution B; 8 min, 15% solution B; 24 min, 80% solution B; 30 min, 4% solution B at a flow rate 1.0 ml/min. Major individual compounds of extracts were analyzed at a wavelength of 530 nm. Four selected pure anthocyanin compound includes cyaniding-3-O-glucoside (C3G), cyaniding-3-O-rutinoside (C3R), peonidin-3- O-glucoside (P3Gj and cyariidin chloride (CC) were sold under the trademark SIGMA. The peak area from the extracts were calculated using calibration curve which constructed by injecting the reference standards concentration range 0-50 pg/ml, these results are represented in FIG. 4.

Determination of anthocyanin content in purple rice extracts was accomplished as follows. To determine the amount of anthocyanin content in ethanolic extracts of purple rice by HPLC, 100 pg/ml of C3G, P3G, C3R and cyanidin chloride were used as the standard , these results are represented in FIG. 4. In the purple rice extracts (PREs) concentrate, two anthocyanins include C3G and P3G were detected, these results are represented in FIG. 5.

Preparation of ethanolic extract of sesamin was accomplished as follows. Seeds of S. indicum Linn were collected from the Lampang Province of Thailand and voucher specimens (BKF No. 138181) were deposited at the National Park, Wildlife and Plant Conservation Department, Ministry of Natural Resources and Environment, Bangkok, Thailand. Air-dried and finely powdered seed (460 g) from S. indicum was percolated 6 times with 4 liters of hexane for 3 days at room temperature. The liquid extract was evaporated under reduced pressure to prepare a crude hexane extract. The latter was used for purification of sesamin. The. crude extract was fractionated by the silica gel column chromatography (CC) sold under the trademark MERCK (MERCK No. 7734, 500 g). Elution of subcomponents began using hexane, and then gradually enriched with EtOAc in hexane up to 20% v/v. Fractions were collected, analyzed by thin layer chromatography (TLC) and subcomponents combined. The fraction containing sesamin which contained mainly colorless crystals, was re-applied to the silica gel column sold under the trademark MERCK (MERCK No. 7734, 120 g). The specific subfraction was further purified by crystallization with EtOH to yield colorless needle crystals, this being identified as sesamin using nuclear magnetic resonance spectroscopy and mass spectrometry (430 mg, 0.11% yield) and confirmed with comparison to the an authentic standard sold under the trademark SIGMA-ALDRICH by co-chromatography using high performance liquid chromatography (the column sold under the trademark HYPERSIL ODS-25, 250 c 4.6 mm by ThermoHypersil Co.) and eluted with linear gradient system comprising CH3CN and H2O (50:50 to 70:50 v/v) at the flow rate of 1.0 ml/min. Eluent was monitored at wavelength of 280 nm.

Chemical characterization of sesamin extract was accomplished as follows. The structure of the purified material was examined and identified by nuclear magnetic resonance spectroscopy and mass spectrometry. Furthermore, co-chromatographic analysis was carried out with the authentic sesamin sold under the trademark SIGMA- ALDRICH for its purity. The mass spectrogram of the purified preparation and chemical structure of sesamin are shown in FIG. 7.

Preparation of the composition was accomplished as follows. A composition applicable for enhancing wound healing which comprises a mixture having (a) polysaccharide peptides of rice particle; (b) a first extract of rice bran and (c) a second extract of sesamin, wherein the polysaccharide peptides, the first extract and the second extract are in a ratio of 1:1 :1 by weight.

The present disclosure serves to address at least some of the mentioned problems and issues associated with the prior art. Although only some embodiments of the present disclosure are disclosed herein, it will be apparent to a person having ordinary skill in the art in view of the present disclosure that a variety of changes and/or modifications can be made to the disclosed embodiments without departing from the scope of the present disclosure. The scope of the present disclosure as well as the scope of the following claims is not limited to embodiments described herein.