Compositions containing plant extracts and applications thereof

Abstract

Provided is a composition for reducing the fat content of fat-producing cells and applications thereof. The composition includes plant extracts such as a blueberry extract and a black tea extract.

Claims

1. A method of treating obesity in a human in need thereof comprising administering to the human in need thereof a therapeutically effective amount of a composition comprising at least 0.0625 mg/ml of a red wine extract and at least 0.0625 mg/ml of a Four Seasons spring extract to effectively treat the obesity in said human in need thereof.

2. The method of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

3. The method of claim 2, wherein the composition is in the form of a solution, a powder, a capsule, or a tablet.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings form part of the present specification and are included here to further demonstrate some aspects of the present invention, which can be better understood by reference to one or more of these drawings, in combination with the detailed description of the embodiments presented herein.

(2) FIG. 1 shows the effect of the various compositions according to one embodiment of the invention on the relative fat content of adipocytes.

(3) FIG. 2 shows the effect of the various compositions according to one embodiment of the invention on the relative fat content of adipocytes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(4) The embodiments of the present invention are further described below, in reference to the accompanying drawings. Examples are set forth below to illustrate the features and applications of the present invention, and are not intended to limit the scope of the present invention. Those of ordinary skill in the art will appreciate that various changes and modifications may be made without departing from the spirit or scope of the present disclosure, which is defined in the appended claims.

Definition

(5) Numerical quantities provided herein are approximated values. All experimental values may vary within 20 percent, preferably within 10 percent, and most preferably within 5 percent of the given values.

(6) As used herein, “fat-producing cell” refers to any mammalian cell that functions by synthesizing and storing neutral fats. The fat-producing cells include mature adipocytes that are differentiated and neonatal adipocytes.

(7) As used herein, “pharmaceutically acceptable carrier” refers to one or more solid or liquid vehicles which are not toxic to mammals and which do not affect the biological activity of an active ingredient in a composition.

(8) The present invention provides a composition for reducing the fat content of fat-producing cells. The composition contains either a plurality of plant extracts or a plant extract and plant-derived compounds. The composition is prepared by mixing the extract of black tea, green tea, Pu-erh tea, Four Seasons Spring tea, red wine, green coffee beans, blueberry, citrus, spinach, broccoli (Brassica oleracea var. italica) sprouts, red clover, aloe, rosemary, garlic, pepper, turmeric, wolfberry, or ginseng, or prepared by mixing the aforementioned plant extract with apple polyphenols, beta carotene, or lycopene. The following examples disclose that said composition can greatly reduce the fat content of fat-producing cells.

(9) Materials and Methods

(10) Materials

(11) Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and phosphate buffered saline (PBS) were purchased from Gibco. Oil red O was purchased from Sigma. Formaldehyde and isopropanol were purchased from Echo Chemical. Apple polyphenols were purchased from Giwan Ltd. Beta carotene (β-carotene) was purchased from Goodwin International Trading Co., Ltd. Lycopene was purchased from Hunan Naturalin Bio-Resources Co., Ltd.

(12) Oil Red O Staining and Quantitative Analysis

(13) The neutral fat content of cells was determined by oil red O staining. Prior to staining, the cells were washed twice with PBS and then fixed with 10% formaldehyde at room temperature for 30 minutes. The fixed cells were washed twice with PBS and rinsed with 60% isopropanol for 1 minute. Thereafter, the cells were stained with an oil red O staining solution (1.8 mg/ml oil red O dissolved in 60% isopropanol aqueous solution) for 1 hour, and then treated with 60% isopropanol for 5 seconds to remove excess dye. Lastly, 100% isopropanol was added to the cells and incubated with shaking for 10 minutes to dissolve the intracellular dye, and the absorbance of the cell suspension at 510 nm was measured using an ELISA (enzyme-linked immunosorbent assay) reader (BioTek). The statistical significance of differences between data was determined by Student's t-test using the Excel software.

Example 1 Preparations of Plant Extracts

(14) 1-1 Black Tea Extract

(15) This example exemplifies the method of preparing a black tea extract. Black tea leaves (the fermented leaves of Camellia sinensis) are first washed, dried, and crushed coarsely with a pulverizer. Next, the coarsely crushed black tea leaves are extracted with water as the solvent, wherein the solvent and the coarsely crushed black tea leaves are mixed uniformly at a liquid-solid ratio of 5-20:1-5, and the extraction temperature is between 50° C. and 100° C., preferably between 75° C. and 95° C. The extraction time is about 0.5 to 3 hours. After cooled to room temperatures, the black tea extract obtained from the extraction step is filtered through a 400 mesh filter to remove solid residues. The filtered black tea extract may further be concentrated under reduced pressure at 45° C. to 70° C. to obtain a concentrated product.

(16) 1-2 Green Tea Extract

(17) This example exemplifies the method of preparing a green tea extract. Green tea leaves (the unfermented leaves of Camellia sinensis) are first washed, dried, and crushed coarsely with a pulverizer. Next, the coarsely crushed green tea leaves are extracted with water as the solvent, wherein the solvent and the coarsely crushed green tea leaves are mixed uniformly at a liquid-solid ratio of 5-20:1-5, and the extraction temperature is between 50° C. and 100° C., preferably between 75° C. and 95° C. The extraction time is about 0.5 to 3 hours. After cooled to room temperatures, the green tea extract obtained from the extraction step is filtered through a 400 mesh filter to remove solid residues. The filtered green tea extract may further be concentrated under reduced pressure at 45° C. to 70° C. to obtain a concentrated product.

(18) 1-3 Pu-Erh Tea Extract

(19) The Pu-erh tea extract is obtained by extracting Pu-erh tea leaves (post-fermented leaves of Camellia sinensis). The extract may be purchased from Nanjing Zelang Biotechnology Co., Ltd.

(20) 1-4 Four Seasons Spring Tea Extract

(21) This example exemplifies the method of preparing a Four Seasons Spring tea extract. Four Seasons Spring tea leaves (the leaves of the Four Seasons Spring tea plant) are first washed, dried, and crushed coarsely with a pulverizer. Next, the coarsely crushed Four Seasons Spring tea leaves are extracted with water as the solvent, wherein the solvent and the coarsely crushed Four Seasons Spring tea leaves are mixed uniformly at a liquid-solid ratio of 5-20:1-5, and the extraction temperature is between 50° C. and 100° C., preferably between 75° C. and 95° C. The extraction time is about 0.5 to 3 hours. After cooled to room temperatures, the Four Seasons Spring tea extract obtained from the extraction step is filtered through a 400 mesh filter to remove solid residues. The filtered Four Seasons Spring tea extract may further be concentrated under reduced pressure at 45° C. to 70° C. to obtain a concentrated product.

(22) 1-5 Red Wine Extract

(23) The red wine extract is obtained by extracting red wines. The extract may be purchased from Shanghai Boyoutang Biotechnology Co., Ltd.

(24) 1-6 Green Coffee Bean Extract

(25) The green coffee bean extract is obtained by extracting unroasted seeds of Coffea spp. plants. The extract may be purchased from ARJUNA NATURAL EXTRACTS Ltd (India).

(26) 1-7 Blueberry Extract

(27) The blueberry extract is obtained by extracting the fruit of North American blueberry (Vaccinium cyanococcus). The extract may be purchased from Biomed Herbal Research Co., Ltd.

(28) 1-8 Citrus Extract

(29) The citrus extract is obtained by extracting the fruit of mandarin orange (Citrus reticulata). The extract may be purchased from Roterm Trading Co., Ltd.

(30) 1-9 Spinach Extract

(31) The spinach extract is obtained by extracting spinach (Spinacia oleracea). The extract may be purchased from Hong Siang Farm Products Factory.

(32) 1-10 Broccoli Sprout Extract

(33) The broccoli sprout extract is obtained by extracting the sprout of broccoli (Brassica oleracea vat: italica). The extract may be purchased from Chori Co., Ltd (Japan).

(34) 1-11 Red Clover Extract

(35) The red clover extract is obtained by extracting red clover (Trifolium pretense). The extract may be purchased from Material World Industrial Co. Ltd.

(36) 1-12 Aloe Extract

(37) The aloe extract is obtained by extracting Aloe vera. The extract may be purchased from Ambe Phytoextracts Pvt. Ltd (India).

(38) 1-13 Rosemary Extract

(39) The rosemary extract is obtained by extracting rosemary (Rosmarinus officinalis). The extract may be purchased from Jiajing Baica Co., Ltd.

(40) 1-14 Garlic Extract

(41) The garlic extract is obtained by extracting the bulb of garlic (Allium sativum). The extract may be purchased from Changsha Huir Biological Tech Co., Ltd.

(42) 1-15 Pepper Extract

(43) The pepper extract is obtained by extracting the fruit of pepper (Piper nigrum). The extract may be purchased from Material World Industrial Co. Ltd.

(44) 1-16 Turmeric Extract

(45) The turmeric extract is obtained by extracting the rhizome of turmeric (Curcuma longa). The extract may be purchased from ARJUNA NATURAL EXTRACTS Ltd. (India).

(46) 1-17 Wolfberry Extract

(47) The wolfberry extract is obtained by extracting the fruit of wolfberry (Lycium chinense). The extract may be purchased from Hunan Huakang Biotech Inc.

(48) 1-18 Ginseng Extract

(49) The ginseng extract is obtained by extracting the root of Panax ginseng. The extract may be purchased from Hunan Huacheng Bio, Inc.

Example 2

(50) Reduction of the Fat Content in Fat-Producing Cells by Compositions Containing Plant Extracts

(51) To examine the effect of the composition of the invention on the fat storage of fat-producing cells, oil red O staining was employed to monitor changes in the fat content of the adipocytes differentiated from OP9 mouse stromal cell line (ATCC CRL-2749) and treated with the indicated plant extracts or combinations thereof. Briefly, OP9 cells were seeded at 8×10.sup.4 cells/well in 24-well culture plates, where each well contained 500 μl of pre-adipocyte expansion medium (90% DMEM, 20% FBS, and 1% penicillin/streptomycin), and cultured at 37° C. for 7 days. The medium was refreshed every 3 days during cell culture with adipocyte differentiation medium (90% DMEM medium, 20% FBS, and 1% penicillin/streptomycin). After 7 days, complete differentiation into adipocytes were confirmed by examining oil droplets formed in the cells using a microscope (ZEISS; at 400× magnification). Thereafter, each of the plant extracts or each of the compositions containing plant extracts, listed in TABLE 1, was added to the cells, which were then cultured at 37° C. for 7-10 days, during which the adipocyte differentiation medium was refreshed every 3 days. Finally, the medium was removed, and the cells of each group were washed with PBS and subjected to oil red O staining for determination of the fat content. The relative fat content is a ratio of the cellular fat content of the experimental group relative to that of the control group (expressed as a percentage). The adipocytes of the control group were treated similarly with the adipocyte differentiation medium free of a plant extract.

(52) TABLE-US-00001 TABLE 1 Groups Treatments Relative fat content Control —   100% Comparative Black tea 94.60% group 1 0.0625 mg/ml Comparative Red wine 102.80%  group 2 0.0625 mg/ml Comparative Green tea 92.40% group 3 0.0625 mg/ml Comparative Pu-erh tea 93.90% group 4 0.0625 mg/ml Comparative Four Seasons Spring tea 98.70% group 5 0.0625 mg/ml Comparative Citrus 123.70%  group 6 0.0625 mg/ml Comparative Spinach 118.60%  group 7 0.0625 mg/ml Comparative Green coffee bean 110.00%  group 8 0.0625 mg/ml Comparative Blueberry 156.40%  group 9 0.0625 mg/ml Comparative Turmeric 94.70% group 10 0.125 mg/ml Comparative Lycopene 73.34% group 11 0.015625 mg/ml Comparative Broccoli sprout 76.63% group 12 0.125 mg/ml Comparative Apple polyphenols 92.00% group 13 0.015625 mg/ml Comparative β-carotene 99.00% group 14 0.5 mg/ml Comparative Ginseng 125.78%  group 15 0.125 mg/ml Comparative Wolfberry 127.13%  group 16 1 mg/ml Comparative Garlic 114.01%  group 17 1 mg/ml Comparative Red clover 81.46% group 18 0.5 mg/ml Comparative Aloe 118.60%  group 19 1 mg/ml Comparative Rosemary 84.64% group 20 0.0625 mg/ml Comparative Pepper 108.83%  group 21 0.5 mg/ml Comparative Spinach + Lycopene 76.28% group 22 0.015625 mg/ml + 0.015625 mg/ml Comparative Spinach + Broccoli sprout 82.17% group 23 0.125 mg/ml + 0.125 mg/ml Comparative Spinach + Aloe 102.24%  group 24 1 mg/ml + 1 mg/ml Comparative Spinach + Pepper 114.07%  group 25 0.5 mg/ml + 0.5 mg/ml Experimental Blueberry + Black tea 67.50% group 1 0.0625 mg/ml + 0.0625 mg/ml Experimental Blueberry + Green tea 70.90% group 2 0.0625 mg/ml + 0.0625 mg/ml Experimental Red wine + Black tea 57.90% group 3 0.0625 mg/ml + 0.0625 mg/ml Experimental Red wine + Green tea 60.10% group 4 0.0625 mg/ml + 0.0625 mg/ml Experimental Red wine + Pu-erh tea 60.00% group 5 0.0625 mg/ml + 0.0625 mg/ml Experimental Red wine + Four Seasons Spring tea 56.90% group 6 0.0625 mg/ml + 0.0625 mg/ml Experimental Red wine + Citrus 48.20% group 7 0.0625 mg/ml + 0.0625 mg/ml Experimental Red wine + Spinach 59.10% group 8 0.0625 mg/ml + 0.0625 mg/ml Experimental Red wine + Green coffee bean 50.80% group 9 0.0625 mg/ml + 0.0625 mg/ml Experimental Citrus + Turmeric 89.05% group 10 0.125 mg/ml + 0.125 mg/ml Experimental Citrus + Lycopene 70.51% group 11 0.015625 mg/ml + 0.015625 mg/ml Experimental Citrus + Broccoli sprout 57.80% group 12 0.125 mg/ml + 0.125 mg/ml Experimental Citrus + Apple polyphenols 64.16% group 13 0.015625 mg/ml + 0.015625 mg/ml Experimental Citrus + β-carotene 66.16% group 14 0.5 mg/ml + 0.5 mg/ml Experimental Citrus + Ginseng 70.16% group 15 0.125 mg/ml + 0.125 mg/ml Experimental Citrus + Wolfberry 69.45% group 16 1 mg/ml + 1 mg/ml Experimental Citrus + Garlic 92.82% group 17 1 mg/ml + 1 mg/ml Experimental Citrus + Red clover 86.87% group 18 0.5 mg/ml + 0.5 mg/ml Experimental Citrus + Aloe 79.16% group 19 1 mg/ml + 1 mg/ml Experimental Citrus + Rosemary 65.21% group 20 0.0625 mg/ml + 0.0625 mg/ml Experimental Citrus + Pepper 76.81% group 21 0.5 mg/ml + 0.5 mg/ml Experimental Spinach + Turmeric 75.75% group 22 0.125 mg/ml + 0.125 mg/ml Experimental Spinach + Apple polyphenols 88.58% group 23 0.015625 mg/ml + 0.015625 mg/ml Experimental Spinach + β-carotene 93.82% group 24 0.5 mg/ml + 0.5 mg/ml Experimental Spinach + Ginseng 82.17% group 25 0.125 mg/ml + 0.125 mg/ml Experimental Spinach + Wolfberry 80.69% group 26 1 mg/ml + 1 mg/ml Experimental Spinach + Garlic 88.64% group 27 1 mg/ml + 1 mg/ml Experimental Spinach + Red clover 67.98% group 28 0.5 mg/ml + 0.5 mg/ml Experimental Spinach + Rosemary 82.58% group 29 0.0625 mg/ml + 0.0625 mg/ml

(53) TABLE 1 shows the relative fat content of adipocytes after different treatments; FIGS. 1 and 2 are histograms corresponding to the values shown in TABLE 1. According to TABLE 1 and FIG. 1, compared to the control group, the sole treatment with the red wine extract, the Four Seasons Spring tea extract, the citrus extract, the spinach extract, the green coffee bean extract, the blueberry extract, the ginseng extract, the wolfberry extract, the garlic extract, the aloe extract, the pepper extract, or β-carotene did not significantly reduce the fat content of adipocytes. Some of these treatments even significantly increased the fat content, such as treatment with the blueberry extract, the citrus extract, or the spinach extract (see the comparative groups). In addition, the sole treatment with the black tea extract, the green tea extract, the Pu-erh tea extract, the turmeric extract, or apple polyphenols only resulted in a slight decrease in the relative fat content to about 90% or more.

(54) However, the combination of the blueberry extract with the black tea extract or the green tea extract significantly reduced the relative fat content of adipocytes to 67.50% and 70.9%, respectively (see the experimental groups). Also, the combination of the red wine extract with the black tea extract, the green tea extract, the Pu-erh tea extract, the Four Seasons Spring tea extract, the citrus extract, the spinach extract, or the green coffee bean extract significantly reduced the fat content of adipocytes. Moreover, the combination of the citrus extract with the turmeric extract, the broccoli sprout extract, the ginseng extract, the wolfberry extract, the garlic extract, the red clover extract, the aloe extract, the rosemary extract, the pepper extract, apple polyphenols, β-carotene, or lycopene also resulted in a significantly lower fat content. Furthermore, the combination of the spinach extract with the turmeric extract, the ginseng extract, the wolfberry extract, the garlic extract, the red clover extract, the rosemary extract, apple polyphenols, or β-carotene significantly reduced the fat content of adipocytes. The compositions having the particular combinations set forth above unexpectedly exhibit higher fat-reducing ability than the sum of the fat-reducing ability for the respective single components.

(55) In conclusion, due to the mix of particular plant extracts or the mix of a particular plant extract and a particular plant-derived compound, the composition of the invention greatly reduces the fat content of fat-producing cells, thereby having the potential to reduce body fat and to prevent obesity associated diseases. Therefore, the composition of the invention, along with a pharmaceutically acceptable carrier, may be used in the manufacture of a pharmaceutical composition for reducing the fat content of fat-producing cells. The pharmaceutical composition may be in the form of a solution, a powder, a capsule, or a tablet, but not limited thereto.