FUNCTIONALLY REINFORCED DESALTED NUTRITIONAL COMPOSITIONS FROM HALOPHYTES AND PREPARATION METHOD THEREOF
20190142046 ยท 2019-05-16
Inventors
- Deuk Hoi KIM (Goyang-si, Gyeonggi-do, KR)
- Mee Hyang KWEON (Seoul, KR)
- Eun Ah CHO (Seoul, KR)
- Hyun Joo YOON (Suwon-si Gyeonggi-do, KR)
- Seon Yeong Park (Seoul, KR)
Cpc classification
A23L33/105
HUMAN NECESSITIES
A61K2236/51
HUMAN NECESSITIES
A61K31/192
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
A23L33/20
HUMAN NECESSITIES
A61K2236/331
HUMAN NECESSITIES
A61K33/06
HUMAN NECESSITIES
A23L33/30
HUMAN NECESSITIES
A61K36/21
HUMAN NECESSITIES
A61K2236/53
HUMAN NECESSITIES
International classification
A23L33/105
HUMAN NECESSITIES
A61K33/06
HUMAN NECESSITIES
A23L5/20
HUMAN NECESSITIES
A61K36/21
HUMAN NECESSITIES
A23L33/20
HUMAN NECESSITIES
Abstract
Disclosed are a functionally reinforced desalted nutritional composition, a desalted extract and a cold-water-extracted salt substitute, which are derived from halophytes that grow in coastal regions under highly saline conditions and thus retain high salt concentrations, as well as the use of the desalted nutritional composition for combating obesity. More particularly, this invention relates to a functionally reinforced desalted nutritional composition, a desalted extract and a salt substitute cold-water-extracted from halophytes that inhabit extreme environments of high salinity under high salt stress, the halophytes being desalted through a cold water extraction process at a low temperature based on the difference in water solubility of salts with change in temperature to allow only sodium chloride to be selectively removed, and the composition thus having decreased sodium content as well as having increased content of useful minerals such as potassium, as well as nutrients and physiologically active substances, which are naturally contained in halophytes.
Claims
1. A method of preparing a functionally reinforced desalted nutritional composition from a halophyte, comprising the steps of: (a) mixing dried powder of the halophyte with water at 9 C. or lower and stirring a resultant mixture; (b) centrifuging the stirred mixture and removing a supernatant having a high salt content to recover a desalted precipitate; and (c) drying the desalted precipitate.
2. A functionally reinforced nutritional composition from a halophyte, comprising sodium of less than 6.8 wt % and carbohydrates of 61 wt % or greater, based on a dry weight.
3. The functionally reinforced nutritional composition from the halophyte as set forth in claim 2, comprising potassium (K) of 0.1 to 3.0 wt %, calcium (Ca) of 0.1 to 2.0 wt % and magnesium (Mg) of 0.1 to 1.5 wt %, based on the dry weight.
4. The functionally reinforced nutritional composition from the halophyte as set forth in claim 2, comprising polyphenols of 0.1 to 10.0 wt % and flavonoids of 0.1 to 7.0 wt %, based on the dry weight.
5. The functionally reinforced nutritional composition from the halophyte as set forth in claim 2, comprising chlorophylls of 0.3 to 10.0 wt % based on the dry weight.
6. The functionally reinforced nutritional composition from the halophyte as set forth in claim 2, comprising trans-ferulic acid.
7. A method of preparing a functionally reinforced desalted extract from a halophyte, comprising the steps of: (a) mixing dried powder of the halophyte with water at 9 C. or lower and stirring a resultant mixture; (b) centrifuging the stirred mixture and removing a supernatant having a high salt content to recover a desalted precipitate; (c) extracting the desalted precipitate in a liquid phase to obtain an extract; and (d) drying the liquid-phase extract.
8. The method of preparing the functionally reinforced desalted extract from the halophyte as set forth in claim 7, further comprising drying the desalted precipitate before the extracting the desalted precipitate in the liquid phase.
9. A functionally reinforced desalted extract from a halophyte, which is extracted from a desalted product of the halophyte and has a total salt content of less than 11.0 wt % and insoluble dietary fiber of less than 3.2 wt %, based on a dry weight.
10. The functionally reinforced desalted extract from the halophyte as set forth in claim 9, comprising polyphenols of 0.1 to 10.0 wt % and flavonoids of 0.1 to 7.0 wt %, based on the dry weight.
11. The functionally reinforced desalted extract from the halophyte as set forth in claim 9, comprising chlorophylls of 0.3 to 10.0 wt % based on the dry weight.
12. A method of preparing a cold-water-extracted salt substitute from a halophyte, comprising the steps of: (a) mixing dried powder of the halophyte with water at 9 C. or lower and stirring the mixture; (b) centrifuging the stirred mixture to obtain a supernatant; (c) concentrating the supernatant and purifying the concentrate with activated carbon; and (d) spray-drying the purified concentrate.
13. The method of preparing a cold-water-extracted salt substitute from the halophyte as set forth in claim 12, which has a total salt content of 50.0 wt % or more and has a salt composition in which potassium (K) and sodium (Na) are contained at a weight ratio (K:Na) ranging from 1:10.1 to 1:19.0.
14. A cold-water-extracted salt substitute from a halophyte, which has a total salt content of 50.0 wt % or more and has a salt composition in which potassium (K) and sodium (Na) are contained at a weight ratio (K:Na) ranging from 1:10.1 to 1:19.0.
15. The cold-water-extracted salt substitute from the halophyte as set forth in claim 14, comprising glutamic acid in an amount of 0.1 to 50 mg/g.
16. A pharmaceutical composition for combating obesity and for reducing body fat, comprising the halophyte-derived functionally reinforced desalted nutritional composition of claim 2.
17. A functional food for combating obesity and for reducing body fat, comprising the halophyte-derived functionally reinforced desalted nutritional composition of claim 2.
18. A feedstuff for combating obesity and for reducing body fat, comprising the halophyte-derived functionally reinforced desalted nutritional composition of claim 2.
19. A pharmaceutical composition for combating obesity and for reducing body fat, comprising the halophyte-derived trans-ferulic acid of claim 6.
20. A functional food for combating obesity and for reducing body fat, comprising the halophyte-derived trans-ferulic acid of claim 6.
21. A feedstuff for combating obesity and for reducing body fat, comprising the halophyte-derived trans-ferulic acid of claim 6.
Description
DESCRIPTION OF DRAWINGS
[0045] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
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BEST MODE
[0061] Halophytes, which contain various useful substances such as dietary fiber, essential amino acids, vegetable minerals and physiologically active substances, have a limitation in their applicability owing to their high salt content. According to the present invention, based on the difference in water solubility of salts with change in temperature, when dried powder of a halophyte was extracted with stirring in cold water at a low temperature for a short time, compared with the cases of being extracted with room-temperature water and hot water, the elution of useful minerals, except for sodium, and organic soluble components was remarkably reduced, whereas there was no great difference in the elution of sodium salt.
[0062] According to the present invention, to a halophyte were added cold water, room-temperature water and hot water, respectively, followed by stirring and centrifugation to remove a supernatant. The resulting desalted extract was recovered and dried so as to yield a halophyte-derived functionally reinforced desalted nutritional composition. The extraction with cold water was found to be able to effectively remove sodium salts while minimizing the elution of organic substances.
[0063] In one aspect of the present invention, the present invention relates to a method of preparing a functionally reinforced desalted nutritional composition from a halophyte, comprising the steps of (a) mixing dried powder of the halophyte with water at 9 C. or lower and stirring the mixture, (b) centrifuging the stirred mixture and removing a supernatant having a high salt content to recover a desalted precipitate, and (c) drying the desalted precipitate, and a functionally reinforced desalted extract from a halophyte, including sodium of 0.04 to 6.8 wt % and carbohydrates of 61 wt % or greater, based on the dry weight.
[0064] Halophytes are plants that naturally grow in saline habitats, such as in coastal regions and around salt fields. Examples of halophytes include, but are not limited to, glasswort (Salicornia Spp.), Suaeda asparagoides, and Suaeda japonica.
[0065] The halophyte dried powder may be prepared by washing a halophyte to remove impurities followed by drying. A dried product itself can be used, but a powder form is preferred for more effective extraction.
[0066] As seen in
[0067] The dried product of halophyte is preferably used in an amount of 40 to 70 g per 1 L for extraction. When less than 40 g is used, the solvent amount is relatively large, increasing the total amount to be centrifuged. This makes the extraction process ineffective. On the other hand, an amount greater than 70 g does not ensure effective stirring.
[0068] The stirring is preferably carried out for 1 to 5 minutes. Stirring for less than one minute results in a decrease in desalination efficiency for halophytes. When the stirring time exceeds 5 minutes, the elution of soluble organic components as well as sodium salt is increased.
[0069] After stirring, the stirred mixture is centrifuged, and a supernatant having a high salt content is then removed to recover a desalted precipitate.
[0070] The precipitate may be obtained according to a method commonly known in the art, and the method is not particularly limited as long as it can separate the stirred mixture into a supernatant and a precipitate. For example, a filtration method may be used to obtain the precipitate.
[0071] According to the intended need, the desalted precipitate may be further stirred so as to further lower the remaining small amount of salt content.
[0072] The finally desalted precipitate is recovered and dried.
[0073] Since the method of preparing a functionally reinforced desalted nutritional composition from a halophyte according to the present invention enables the effective removal of only sodium chloride with no loss of useful physiologically active substances, the desalted nutritional composition prepared according to the present invention may comprise sodium (Na) of 0.04 to 6.8 wt %, carbohydrates of 61 wt % or greater, potassium (K) of 0.1 to 3.0 wt %, calcium (Ca) of 0.1 to 2.0 wt %, magnesium (Mg) of 0.1 to 1.5 wt %, polyphenols of 0.1 to 10.0 wt %, flavonoids of 0.1 to 7.0 wt %, and chlorophylls of 0.3 to 10.0 wt %, based on the dry weight.
[0074] When the desalted precipitate, obtained by desalting a halophyte with cold water, and its dried powder were extracted with water or ethanol, as compared with an extract from a non-desalted halophyte, they were found to have remarkably reduced salt content as well as having remarkably increased content of functional components and nutrients.
[0075] In another aspect, the present invention provides a method of preparing a functionally reinforced desalted extract from a halophyte, comprising the steps of (a) mixing dried powder of the halophyte with water at 9 C. or lower and stirring the mixture; (b) centrifuging the stirred mixture and removing a supernatant having a high salt content to recover a desalted precipitate; (c) extracting the desalted precipitate in a liquid phase to obtain an extract; and (d) drying the liquid-phase extract, and a functionally reinforced desalted extract from a halophyte, which is characterized in that it is extracted from a desalted product of the halophyte and has a total salt content of less than 11.0 wt % and insoluble dietary fiber of less than 3.2 wt %, based on the dry weight.
[0076] The desalted precipitate from a halophyte is recovered using the same method described above. To elute physiologically active functional components, the desalted precipitate may be extracted with water, or may be dried and then extracted with an organic solvent, such as methanol, ethanol, butanol, ethyl acetate, acetone or diethyl ether, thus giving a desired extract. When the liquid-phase extraction is carried out with an organic solvent, the desalted precipitate is preferably dried before being subjected to liquid-phase extraction.
[0077] The liquid-phase extraction of the desalted halophyte precipitate with an organic solvent may be carried out by reflux extraction at room temperature or near a temperature at which the organic solvent becomes volatile. In this case, the desalted precipitate is preferably used in an amount of 40 to 75 g per liter of extraction solvent. An amount less than 40 g increases the cost of the extraction solvent. When the amount of solvent exceeds 75 g, extraction efficacy is reduced. The additional liquid-phase extraction process has an advantage of increasing the extract yield.
[0078] The halophyte-derived functionally reinforced desalted extract prepared according to the present invention is characterized in that it has a total salt content of less than 11.0 wt % and insoluble dietary fiber of less than 3.2 wt %, and may comprise polyphenols of 0.1 to 10.0 wt %, flavonoids of 0.1 to 7.0 wt % and chlorophylls of 0.3 to 10.0 wt %.
[0079] Since the halophyte-derived functionally reinforced desalted extract has various in vivo physiological activities, such as antioxidant, anti-thrombotic, anti-hypertensive and anti-diabetic activities, it has the potential to be applied as a raw material in foods, cosmetics, medicines, and the like.
[0080] In addition, the halophyte-derived functionally reinforced desalted nutritional composition comprises, as an effective component, trans-ferulic acid, which suppresses the adipocyte differentiation and genes involved in lipid synthesis, as well as containing dietary fiber in an amount greater than before being desalted. Thus, the composition has good anti-obesity and body-fat-reducing effects.
[0081] In yet another aspect, the present invention relates to a pharmaceutical composition and functional food and feedstuff for anti-obesity and body-fat-reducing effects, comprising the above functionally reinforced desalted halophyte dried product.
[0082] Meanwhile, after the halophyte dried powder was stirred in cold water and a desalted precipitate was recovered by centrifugation, the remaining supernatant was found to have a high content of sodium chloride while having a low content of potassium and a high content of glutamic acid, and is thus applicable as a vegetable salt substitute having a clean salty taste with a savory (umami) flavor.
[0083] Thus, in still another aspect, the present invention relates to a method of preparing a cold-water-extracted salt substitute from a halophyte, comprising the steps of (a) mixing dried powder of the halophyte with water at 9 C. or lower and stirring the mixture; (b) centrifuging the stirred mixture to obtain a supernatant; (c) concentrating the supernatant and purifying the concentrate with activated carbon; and (d) spray-drying the purified concentrate, and a halophyte-derived cold-water-extracted salt substitute prepared according to the method, which is characterized in that it has a total salt content of 50.0 wt % or more and a salt composition in which the weight ratio of potassium to sodium (K:Na) ranges from 1:10.1 to 1:19.0.
[0084] The cold-water-stirred supernatant remaining after a desalted precipitated is recovered can be concentrated to a salinity of 15 to 19% and a total solid content of 20% or higher. Thus, the supernatant may be purified using activated carbon of 3-5% based on the total solid content of the concentrate, and then spray-dried to give a halophyte-derived cold-water-extracted salt. The amount of activated carbon used in the purification may be varied to control the content of organic substances and the color of salt.
[0085] The concentration method of the cold-water-stirred supernatant is not particularly limited as long as it can concentrate the supernatant. Preferred is vacuum concentration.
MODE FOR INVENTION
[0086] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.
Example 1: Evaluation of Cold Water Extraction of S. europaea for Desalting Effect
[0087] To 100 g of dried powder of glasswort (Salicornia europaea) was added 2 liters of cold water (4 C. and 9 C.), room-temperature water (20 C.) and hot water (100 C.), respectively. Extraction was carried out with stirring (300 rpm) at low temperatures (4 C. and 9 C.) and at room temperature (20 C.). Hot-water extraction was performed using a 100 C. reflux condenser.
[0088] In order to determine optimal conditions for maximizing desalination, the stirred mixture was centrifuged at a time interval of 5 min at 10,000 rpm for 20 min. Supernatants were vacuum-filtered through a membrane filter (0.45 m pore size) and then analyzed for salinity (ATAGO ES-421, ATAGO Co. LTD. Japan) and Brix (ATAGO PAL-1, ATAGO Co. LTD. Japan). Also, the supernatants were vacuum-concentrated, freeze-dried (EYELA FDU-2200, ETELA, Japan), and then analyzed for total solid content. The measured total solid contents are, along with salt percentage in total solid and content of solids other than salt, given in Table 1, below.
TABLE-US-00001 TABLE 1 Brix Solids Extraction Extraction (solid Salt/Total Total other Sample temp Water time Total content)/ solid salt than salt (g) ( C.) (L) (min) Solid Salinity (%) (g) (g) 100 4 2 1 33.0 1.26 79.4 26.2 6.8 100 4 2 5 35.0 1.25 80.0 28.0 7.0 100 4 2 10 35.6 1.27 78.9 28.1 7.5 100 4 2 15 36.3 1.29 77.7 28.2 8.1 100 4 2 20 36.8 1.30 76.9 28.3 8.5 100 4 2 25 37.2 1.31 76.3 28.4 8.8 100 4 2 30 37.5 1.32 76.0 28.5 9.0 100 9 2 1 34.3 1.30 76.6 26.3 8.0 100 9 2 5 36.4 1.30 76.9 28.0 8.4 100 9 2 10 37.0 1.31 76.2 28.2 8.8 100 9 2 15 37.8 1.33 75.0 28.3 9.5 100 9 2 20 38.3 1.35 74.2 28.4 9.9 100 9 2 25 38.7 1.36 73.7 28.5 10.2 100 9 2 30 39.0 1.36 73.3 28.6 10.4 100 20 2 1 40.3 1.52 65.8 26.5 13.8 100 20 2 5 42.3 1.51 66.2 28.0 14.3 100 20 2 10 45.8 1.63 61.5 28.2 17.6 100 20 2 15 47.3 1.67 59.8 28.3 19.0 100 20 2 20 48.8 1.71 58.4 28.5 20.3 100 20 2 25 50.3 1.76 56.9 28.6 21.7 100 20 2 30 51.0 1.78 56.3 28.7 22.3 100 100 2 1 44.1 1.53 65.3 27.1 14.9 100 100 2 5 43.6 1.56 64.0 27.9 15.7 100 100 2 10 48.0 1.71 58.5 28.1 19.9 100 100 2 15 52.8 1.87 53.6 28.3 24.5 100 100 2 20 55.3 1.94 51.5 28.5 26.8 100 100 2 25 56.7 1.98 50.6 28.7 28.0 100 100 2 30 58.5 2.02 49.4 28.9 29.6
[0089] As shown in Table 1, it was found that there was almost no difference in the elution degree of salts with change in temperature. For reference,
[0090] When 100 g of dried powder of glasswort (S. europaea) was extracted with water (2 L) (4 C., 9 C., 20 C. and 100 C.) for 30 min, the eluted amounts of salts were also found to be almost the same at all test temperatures (28.5 g, 28.6 g, 28.7 g and 28.9 g, respectively). Thus, all salts contained in S. europaea were thought to be eluted within 30 min.
[0091] In contrast, the elution of soluble organic solids other than salts was found to increase greatly with increasing temperature. After extraction for 30 min, the solids were found to be eluted 2.47 times higher at room temperature (20 C.) and 3.28 times higher at 100 C. than when extracted at 4 C.
[0092] As an ideal indicator of the desalting effect, the soluble solid content (Brix, %)/salinity (%) index was measured. That is, the lower the Brix/salinity ratio, the lower the presumed loss of organic solids due to desalination. The index was found to increase gradually over time under all temperature conditions.
[0093] In addition, with regard to the difference in the Brix/salinity index, cold water extraction at 0.1 to 9 C. resulted in low index values ranging from 1.26 to 1.36; i.e. a slight difference was found. In contrast, when the extraction was carried out at 20 C. or over, high index values of over 1.5 were measured while the index ranged from 1.5 to 2.02, and the index was also found to increase gradually with increasing temperature. Thus, it is preferable that the extraction for desalination be carried out at 9 C. or lower for as short a time as possible. That is, these results indicate that, when extraction is conducted with cold water at 4 C. or lower for 4 min or shorter, the elution of organic substances is minimized while salts are effectively removed.
Example 2: Preparation of Desalted Nutritional Compositions from S. europaea, S. asparagoides and S. japonica
[0094] Desalted nutritional compositions were prepared using three plants, Salicornia europaea, Suaeda asparagoides and Suaeda japonica, which are known to be extreme halophytes that naturally grow in South Korea. Fresh plants were washed with tap water and freeze-dried to be powdered. Based on the results from Example 1, in which, when extraction is conducted with cold water at 4 C. or lower for 4 min or shorter, the extraction of organic substances is minimized while salts are effectively removed, 100 g of the dried powder was added to 2 L of cold water (4 C.), stirred at 4 C. for 4 min, and centrifuged at 10,000 rpm for 20 min. Then, a supernatant having a high salt content was removed, and a desalted precipitate was recovered. The recovered precipitate was desalted once more by the same method as above so as to minimize remaining sodium salt. The precipitates thus obtained were freeze-dried to give halophyte-derived desalted nutritional compositions (desalted powder).
Test Example 1: Analysis for Components of Halophyte-Derived Desalted Nutritional Compositions
[0095] Non-desalted halophyte dried powders from S. europaea, S. asparagoides and S. japonica and the desalted nutritional compositions (desalted powder), prepared in Example 2 from the same halophytic species, were analyzed for their contents of sodium, nutrients and functional components, and the results are given in Table 2, below. Analysis for calorie carbohydrate and protein content was performed according to the general analytical method of the Korean Food Standards Codex (Korean Food Industry Association). Sodium, potassium, magnesium, iron and calcium levels were measured through wet analysis using an acidic digestion method using nitric acid and then by Inductively Coupled Plasma Spectrometry (ICPS).
[0096] The amounts of other components, namely polyphenols, flavonoids and chlorophylls, were determined as follows.
1-1: Analysis for Total Polyphenol Content
[0097] The total polyphenol content was determined in a 96-well microplate according to a modified Folin-Davis method. Non-desalted and desalted halophyte powders were extracted with 70% methanol, dried and dissolved in distilled water. 20 l of each sample was mixed with 250 l of 2% sodium carbonate and 15 l of 50% Folin-Ciocalteu reagent (Sigma Co., USA), and the solution was allowed to react at room temperature for 30 min. Then, the absorbance was measured at 725 nm using a microreader (Bio-RAD, x-Mark, USA). As a standard, tannic acid solutions of 0 to 500 g/mL (Sigma Co., USA) were used instead of the samples, and, from the calibration curve thus obtained, the amount of total polyphenols contained in the extraction samples were calculated.
1-2: Analysis for Total Flavonoid Content
[0098] The total flavonoid content was determined in a 96-well microplate according to a modified Abdel-Hameed method. Non-desalted and desalted halophyte powders were extracted with 70% methanol, dried and dissolved in distilled water. To 30 l of each sample were added 200 l of 90% diethylene glycol and 5 l of 1 N NaOH. The solution was allowed to react at 37 C. for 1 hr. Then, the absorbance was measured at 420 nm using a microreader (Bio-RAD, x-Mark, USA). As a standard, rutin solutions of 0 to 500 g/mL (Sigma Co., USA) were used instead of the samples, and, from the calibration curve thus obtained, the amount of total flavonoids contained in the extraction samples were calculated.
1-3: Analysis for Total Chlorophyll Content
[0099] 1 g of each of non-desalted and desalted halophyte powders was extracted with 50 mL of 80% acetone at room temperature until the color disappeared. Then, the supernatant was isolated, and the absorbance was measured at 645 nm and 663 nm using a microreader (Bio-RAD, x-Mark, USA). The concentrations of chlorophyll a, chlorophyll b and total chlorophyll were calculated using the equations below.
[0100] Chlorophyll a (mg/mL)=12.720D6632.580D645
[0101] Chlorophyll b (mg/mL)=25.880D6455.500D663
[0102] Total chlorophyll (mg/mL)=7.220D663+20.30D645
TABLE-US-00002 TABLE 2 Halophyte S. europaea S. asparagoides S. japonica Powder Before After Before After Before After Nutrients desalting desalting desalting desalting desalting desalting Calorie 151.63 224.89 159.84 217.62 143.69 201.11 (Kcal/100 g) Total 37.98 74.44 39.41 73.18 32.25 67.20 carbohydrates (%) Proteins (%) 9.79 13.84 9.12 12.79 8.97 12.71 Sodium (%) 13.96 1.34 12.15 1.26 12.77 1.29 Potassium (%) 2.14 3.56 1.99 3.02 2.03 3.09 Calcium (%) 0.41 0.64 0.51 0.78 0.60 0.91 Magnesium (%) 0.74 0.99 0.63 0.81 1.02 1.33 Iron (%) 0.007 0.012 0.012 0.020 0.061 0.077 Total 6.8 11.8 5.9 10.6 6.6 10.9 Polyphenols (mg/g) Total flavonoid 3.3 5.8 2.5 5.0 3.4 5.3 (mg/g) Total 29.49 54.25 25.63 45.85 15.71 32.09 chlorophyll (mg/g)
[0103] As shown in Table 2, compared with before being desalted, the desalted halophyte samples were found to have increased contents of carbohydrates and crude proteins. The main component removed during desalination was found to be sodium (Na), while the concentrations of other minerals, such as potassium, calcium, magnesium and iron, were increased after desalting.
[0104] In addition, the concentrations of polyphenols, flavonoids and chlorophylls, which are expected to have useful physiological activities in halophytes, were greatly increased after desalting. These results indicate that the desalted halophyte powder is applicable as a functional nutritional composition having increased content of useful physiological active substances.
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[0106] Non-desalted Salicornia powder (SP), cold-water-desalted Salicornia powder (CW-DSP) and hot-water-desalted Salicornia powder (HW-DSP) were compared with each other for chlorophyll content, and the result is shown in
[0107] Since chlorophyll, which is a functional raw material designated in the Health Functional Food Code, is a functional substance for improving antioxidant activity and immunity, cold-water-extracted halophyte powder has the potential to be used as a functionally reinforced nutritional composition. In contrast, since chlorophyll is weakly resistant to heat and thus easily degraded by heat, hot-water-desalted halophyte powder has a remarkably low chlorophyll content.
Example 3: Preparation of Functionally Reinforced Desalted Extract from Halophytes (Hot-Water Extracts and Ethanol Extracts)
[0108] 100 g of the functionally reinforced desalted nutritional compositions (desalted powders) from S. europaea, S. asparagoides and S. japonica, prepared in Example 2, were each added to 2 L of distilled water, reflux-extracted at 100 C. for 2 or 4 hrs, centrifuged, filtered, concentrated under pressure, and freeze-dried, thus giving halophyte-derived functionally reinforced desalted hot-water extracts.
[0109] 100 g of the functionally reinforced desalted nutritional compositions (desalted powders) from S. europaea, S. asparagoides and S. japonica, prepared in Example 2 were each added to 2 L of 95% ethanol, reflux-extracted at 751 C. for 2 or 4 hrs, cooled to room temperature, and centrifuged. The supernatants were filtered and concentrated under pressure and freeze-dried, thus giving halophyte-derived functionally reinforced desalted ethanol extracts.
Comparative Example 1: Preparation of Hot-Water Extracts and Ethanol Extracts from Non-Desalted Halophytes
[0110] Hot-water extracts and ethanol extracts obtained from 2 hrs extraction were prepared according to the same method as in Example 3 except that non-desalted halophytes (S. europaea, S. asparagoides and S. japonica) were used instead of the functionally reinforced desalted nutritional compositions (desalted powders) from S. europaea, S. asparagoides and S. japonica.
Test Example 2: Evaluation for Components of Hot-Water Extracts and Ethanol Extracts
[0111] For the samples extracted for 2 hrs in Example 3 and Comparative Example 1, total sugar (carbohydrates) was measured using a modified phenol-sulfuric acid method (Kweon et. al., 1996. Agric. Chem. Biotech. 39. 15-164), and acidic sugars were measured using an m-hydroxybiphenyl method (Blumenkrantz et. al., 1973. Analytical Biochem. 54. 484-489). Total polyphenols, total flavonoids and total chlorophylls were measured according to the same method as in Test Example 1, and this test was replicated three times. The results of analysis of components of the hot-water extracts and the ethanol extracts after halophytes before and after being desalted are shown in Tables 3 and 4.
TABLE-US-00003 TABLE 3 Hot-water S. europaea S. asparagoides S. japonica extracts from Comparative Example Comparative Example Comparative Example halophytes Example 1 3 Example 1 3 Example 1 3 Total 33.3 63.0 25.8 51.0 26.8 51.4 carbohydrates (%) Insoluble 2.1 3.2 1.7 2.9 1.6 2.8 dietary fiber (%) Total neutral 15.5 30.2 14.2 24.9 13.0 25.6 sugars (%) Total acidic 17.8 32.8 11.6 26.1 12.9 22.2 sugars (%) Total proteins 7.8 15.9 6.9 14.0 5.9 12.1 (%) Total salts (%) 55.8 5.5 62 6.0 58 5.8 Total 23.1 40.8 16.6 32.9 21.2 38.8 polyphenols (mg/g) Total 14.5 27.7 11.2 21.5 17.8 31.0 flavonoids (mg/g) Total 10.6 21.8 9.9 18.5 8.5 15.2 chlorophylls (mg/g)
[0112] As shown in Table 3, the hot-water extracts of Comparative Example 1 from non-desalted halophytes (S. europaea, S. asparagoides and S. japonica) were found to have total salts of 55.8 to 62.0%, total carbohydrates of 25.8 to 33.3% and insoluble dietary fiber of 1.6 to 2.1%. Also, the acidic sugar contents were found to range from 11.6 to 17.8%, which are relatively high compared to other general plants, indicating that acidic sugars were composed mainly of glucuronic acid and galacturonic acid.
[0113] As compared with Comparative Example 1 (the hot-water extracts of dried powder before being desalted), the hot-water extract of the cold-water-desalted dried powder, prepared in Example 3, showed a remarkable decrease of more than about 90% in total salt content while showing remarkable increases in total sugar content (51.0 to 63.0%), and, in particular, in total acidic sugar content (22.2 to 32.8%). Many studies have reported that, among sugars, acidic sugars in particular have good immune-enhancing, anti-coagulant, anti-thrombotic, and anticancer activities. Thus, the hot-water extracts of halophyte powder obtained through desalting with cold water can be used in functionally reinforced nutritional compositions because they contain high concentrations of acidic sugars. Compared with Comparative Example 1 (the hot-water extracts of dried powder before being desalted), the hot-water extracts of Example 3 showed increases of 50 to 100% in concentrations of total polyphenols (up to 40.8 mg/g), total flavonoids (up to 31.0 mg/g) and total proteins (up to 15.9 wt %). In case of 4 hrs of hot water extraction, the hot water extracts obtained from the three desalted halophytes showed higher total salt content (9.6 to 10.8%) than 2 hrs hot water-extracted samples.
TABLE-US-00004 TABLE 4 S. europaea S. asparagoides S. japonica Ethanol extracts Comparative Example Comparative Example Comparative Example from halophytes Example 1 3 Example 1 3 Example 1 3 Total 16.3 32.0 18.0 36.3 17.2 35.8 carbohydrates (%) Insoluble 0.17 0.25 0.14 0.26 0.18 0.31 dietary fiber (%) Total neutral 6.5 14.2 10.2 21.9 10.3 25.6 sugars (%) Total acidic 8.8 16.8 6.6 12.1 5.9 10.8 sugars (%) Total proteins 14.2 21.5 14.9 20.0 15.6 22.5 (%) Total salts (%) 35.4 3.5 30.6 2.9 32.1 3.2 Total 45.1 78.8 38.6 66.7 41.2 70.4 polyphenols (mg/g) Total 33.5 56.4 24.4 42.6 29.5 45.4 flavonoids (mg/g) Total 48.7 80.2 45.1 75.6 41.8 73.3 chlorophylls (mg/g)
[0114] As shown in Table 4, the ethanol extracts of Comparative Example 1 from non-desalted halophytes (S. europaea, S. asparagoides and S. japonica) were found to have significantly high total salts of 30.6 to 35.4%, total carbohydrates of 16.3 to 18% and insoluble dietary fiber of 0.14 to 0.18%. Also, total neutral sugar content was found to range from 6.5 to 10.3% while total acidic sugar content ranged from 5.9 to 8.8%, all of which were lower than those of the hot-water extracts (see, Table 3).
[0115] As compared with Comparative Example 1 (the ethanol extracts of dried powder before being desalted), the ethanol extract of the cold-water-desalted dried powder, prepared in Example 3, showed a remarkable decrease of more than about 90% in total salt content while showing remarkable increases in concentrations of total neutral sugars and total acidic sugars. In particular, the ethanol extracts were found to contain plenty of polyphenols, flavonoids and chlorophylls compared with the hot-water extracts, and, compared with Comparative Example 1 (the ethanol extracts of dried powder before being desalted), showed remarkable increases in the content of total polyphenols (76.7 to 90.8 mg/g), total flavonoids (52.6 to 66.4 mg/g) and total chlorophylls (85.3 to 98.2 mg/g). In case of 4 hrs of ethanol extraction, the ethanol extracts obtained from the three desalted halophytes showed higher total salt content (5.5 to 6.9%) than 2 hrs ethanol-extracted samples.
[0116] These results demonstrated that the methods of preparing a functionally reinforced desalted nutritional composition and a desalted extract from a halophyte according to the present invention, through a cold water desalination process based on the difference in the water solubility of salts with change in temperature, enables the effective removal of only sodium chloride without causing useful functional plant compounds to be eluted, leading to a remarkable increase in their content compared to the case of not being desalted. Thus, the functionally reinforced desalted nutritional composition and the desalted extract from a halophyte according to the present invention have the potential to be applied as a functionally reinforced good nutritional material.
Test Example 3: Evaluation for Pharmaceutical Activities of Hot-Water Extracts from Halophytes
[0117] The hot-water extracts (samples) of halophytes before and after being desalted, prepared in Comparative Example 1 and Example 3, were evaluated for antioxidant, anti-thrombotic, angiotensin-I-converting enzyme (ACE)-inhibiting and -glucosidase-inhibiting activities. The test was replicated three times, and the results are given in Table 5 and in
3-1: Antioxidant Activity
[0118] Antioxidant activity was assayed using 1,1-diphenyl-2-picryl hydrazyl (DPPH, Sigma Co., USA) based on the method of Blois (Chen, et. al., 1999. J. Agric. Food Chem. 47. 2226-2228).
[0119] In brief, 4 mg of DPPH was dissolved in 50 ml of ethanol, and 180 l of the resulting DPPH solution was added to a 96-well microplate. Then, each sample was added at various concentrations (25, 50 and 100 g/ml), mixed for 5 sec, and allowed to react at room temperature for 20 min. The reduction of the DPPH radical, relative to a control not containing a sample, was determined by reading the absorbance at 517 nm. The free radical scavenging activity was expressed as the inhibition percentage of free radicals by the sample. The IC.sub.50 value is defined as the sample concentration required to scavenge 50% of DPPH free radical.
[0120] When reactive oxygen species (ROS) and free radicals generated in cells by our own metabolism are excessively produced, they cause oxidative stress in respective parts of our bodies and thus make it difficult to maintain intracellular homeostasis, eventually leading to a wide variety of diseases, including cancer, brain diseases, such as stroke and Parkinson's disease, heart diseases, ischemia, arteriosclerosis, skin diseases, digestive diseases, inflammation, rheumatoid arthritis, autoimmune disease, and aging. Thus, antioxidant compounds, which remove reactive oxygen species or inhibit free-radical production, can be used for preventing and/or treating various diseases and for suppressing skin aging, which are caused by intracellular oxidative stress.
[0121] Many anti-oxidative polyphenolic and flavonoid compounds have been already reported to be isolated from halophytic plants including S. europaea. As shown in
3-2: Anti-Thrombotic Activity
[0122] Anti-thrombotic activity was evaluated by assaying anti-coagulant activity using a previously known method (Sohn et al., 2004. Kor. J. Pharmacogn 35. 52-61; Kwon et al., 2004. J. Life Science, 14. 509-513; Ryu et al., 2010. J. Life Science, 20. 922-928), and Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPTT) were measured. Used was commercially available control plasma (MD Pacific Technology Co., Ltd, Huayuan Industrial Area, China), and PT and aPTT levels were measured as follows.
3-2-1: Prothrombin Time (PT)
[0123] 30 l of control plasma (MD Pacific Co., China) and 5 l of concentrations (2.5 and 5.0 mg/ml) of each sample were added into a test cuvette of a Genius Semi-automatic Coagulometer CA 51-52 (Shenzhen, China). The cuvette was allowed to warm at 37 C. for 3 min, and 40 l of PT reagent (Diagon, Hungary) was added. Then, the clotting time was recorded. The mean clotting time of four replicates was calculated. As a positive control, aspirin (Sigma Co., USA) was used, and, instead of a sample, DMSO was used as a solvent control. DMSO exhibited a clotting time of 18.1 sec. Prothrombin inhibitory activity was expressed as the clotting time of a sample (Ts) divided by the clotting time of the solvent control (Tc), that is, Ts/Tc, and the results are given in Table 5, below.
3-2-2: Activated Partial Thromboplastin Time (aPTT)
[0124] 30 l of plasma and 5 l of concentrations (2.5 and 5.0 mg/ml) of each sample were added into a test cuvette of a Genius Semi-automatic Coagulometer CA 51-52 (Shenzhen, China). The cuvette was allowed to warm at 37 C. for 3 min, 20 l of aPTT reagent (Diagon, Hungary) was added, and the cuvette was allowed again to stand at 37 C. for 3 min. 20 l of CaCl.sub.2) (35 mM) was added and the clotting time was recorded. Instead of a sample, DMSO was used as a solvent control, and exhibited a clotting time of 58.0 sec. The mean clotting time of four replicates was calculated. Inhibitory activity against coagulation factors was expressed as the clotting time of a sample (Ts) divided by the clotting time of the solvent control (Tc), that is, Ts/Tc, and the results are given in Table 5, below.
TABLE-US-00005 TABLE 5 PT aPTT Hot-water Clotting Clotting extract from Conc. time time halophyte Samples (mg/mL) (sec) Ts/Tc (sec) Ts/Tc S. europaea Comparative 2.5 18.30 0.05 1.01 0.04 60.80 3.04 1.05 0.03 Example 1 5.0 19.60 0.59 1.08 0.04 70.10 3.51 1.21 0.04 Example 3 2.5 28.70 0.86 1.59 0.06 88.30 4.42 1.52 0.05 5.0 33.50 1.01 1.85 0.07 128.90 6.45 2.22 0.07 S. Comparative 2.5 18.46 0.55 1.02 0.04 58.58 2.93 1.01 0.03 asparagoides Example 1 5.0 20.27 0.61 1.12 0.05 60.9 3.05 1.05 0.04 Example 3 2.5 26.79 0.80 1.48 0.07 81.78 4.09 1.41 0.05 5.0 32.04 0.96 1.77 0.04 106.14 5.31 1.83 0.03 S. japonica Comparative 2.5 17.91 0.54 0.99 0.03 56.84 2.84 0.98 0.03 Example 1 5.0 18.46 0.55 1.02 0.10 64.96 3.25 1.12 0.04 Example 3 2.5 23.89 0.72 1.32 0.05 80.62 4.03 1.39 0.05 5.0 29.68 0.89 1.64 0.07 94.54 4.73 1.63 0.09 Notes: Solvent control: PT = 18.1 sec, aPTT = 58.0 sec Positive control (aspirin (1.5 mg/mL)); PT = 21.18 sec, aPTT = 77.28 sec
[0125] The blood, which is a body constituent, has a wide variety of critical functions, such as transporting oxygen, nutrients and wastes, acting as a buffer, maintaining body temperature, regulating osmotic pressure, maintaining ion balance, keeping water content constant, humoral regulation, maintaining and regulating blood pressure, and protecting the body. Under normal blood circulation, the blood clotting system and the clot dissolution system are regulated in a mutually complementary manner to facilitate blood circulation. The normal blood clotting process occurs as follows. Platelets adhere to the wall of the damaged blood vessels and aggregate, promoting the formation of a platelet plug (primary clot). Then, the blood clotting system is activated, and a fibrin clot is formed around platelet clumps. Substances inhibiting the thrombin activity can be used to prevent and treat various clotting disorders caused by abnormal excessive blood clotting. Also, the intrinsic coagulation pathway lead to the formation of a fibrin clot. The intrinsic pathway is activated by a sequential activation of coagulation factors, XII, XI, IX and X, in stepwise order to convert prothrombin to active thrombin. The specific inhibition of clotting factors is also a major target in developing therapeutic agents for clotting disorders.
[0126] As shown in Table 5, as compared with the control, the S. europaea sample prepared in Comparative Example 1 (the non-desalted hot-water extracts) displayed, at 5 mg/ml, slightly increased PT and aPTT values, 1.08 times and 1.21 times, respectively, whereas the S. europaea sample prepared in Example 3 (the cold-water-desalted hot-water extracts), at the same concentration, was found to have considerably prolonged PT and aPTT, 1.85 times and 2.22 times increased, respectively, indicating excellent anti-thrombotic activity.
3-3: ACE Inhibitory Activity
[0127] Inhibitory activity on Angiotensin-I-Converting Enzyme (ACE) was measured by a slightly modified Cushman and Cheung method. 25 l of various concentrations (0.25, 0.5 and 1.0 mg/ml) of each sample was mixed with 25 l of an ACE (2.5 unit) supernatant, which was prepared by dissolving 1 g of rabbit lung acetone powder (Sigma Co., USA) in 10 ml of 0.1 M sodium borate buffer containing 0.3 M NaCl, and 50 l of 0.1 M sodium borate buffer (pH 8.3) containing 0.3 M NaCl. Then, the mixture was preincubated at 37 C. for 10 min.
[0128] Subsequently, 50 l of Hip-His-Leu was added as a substrate, and the mixture was allowed to react at 37 C. for 30 min. The reaction was terminated by adding 100 l of 1 N HCl. Then, 1 ml of ethyl acetate was added, and the reaction mixture was vortexed for 1 min and centrifuged at 3,000 g for 15 min. The separated ethyl acetate supernatant (extract) was recovered in an amount of 0.8 ml. The supernatant was allowed to warm under a hood and was evaporated until dry. When completely dried, it was dissolved in 1 ml of sodium borate buffer. Then, the absorbance was measured at 228 nm, and ACE inhibitory activity was determined. The results are given in
[0129] Angiotensin-I-Converting Enzyme (ACE) cleaves the C-terminal dipeptide His-Leu from the decapeptide angiotensin I and thus converts angiotensin I to active angiotensin II, which stimulates blood vessel constriction. The elevated angiotensin II level due to ACE results in a sharp increase in blood pressure. Angiotensin II also stimulates the secretion of the anti-diuretic hormone aldosterone and promotes water and sodium retention by the kidneys, thereby increasing blood volume and blood pressure. In addition, ACE degrades and thereby inactivates bradykinin, which causes blood vessels to relax and thus causes blood pressure to fall, resulting in an increase in blood pressure. The inhibition of ACE activity may prevent vasoconstriction and thus lower blood pressure. Thus, compounds having inhibitory activity against ACE can be developed as a preventive and/or therapeutic agent for hypertension.
[0130] As shown in
3-4: Alpha-Glucosidase Inhibitory Activity
[0131] The carbohydrate digestive enzymes maltase, sucrase and glucoamylase are present in the brush border of the small intestine, which are also known as alpha-glucosidases. The inhibition of excessive activities of these enzymes blocks the breakdown of disaccharides and polysaccharides into monosaccharides and thereby delays excessive elevation of blood sugar levels. The inhibition of alpha-glucosidase activity has been used as a tool for measuring anti-diabetic efficacy.
[0132] Alpha-glucosidase activity was determined using a slightly modified Ove method (Ove, N.; Cowell, G. M.; Tranum-Jenser, J. Hansen, O.; Welinder, K. G. J. Biol. Chem. 261:12306-12309, 1986).
[0133] To a 96-well microplate were added 20 l of various concentrations (0.25, 0.5 and 1.0 mg/ml) of each sample, 20 l of alpha-glucosidase (Sigma Co., USA, 2 Unit/ml) and 180 l of 100 mM phosphate buffer (pH 7.0), followed by preincubation at 37 C. for 10 min. Then, 30 l of a substrate solution of 20 mM p-nitrophenyl--D-glucopyranose was added, and the mixture was allowed to react at 37 C. for 30 min. To evaluate the inhibition of alpha-glucosidase activity, glucose oxidase-peroxidase reagent was added to 180 l of the reaction mixture in the 96-well plate to generate hydrogen peroxide, which reacts with o-dianisidine to form a colored product. The color intensity was measured at 540 nm, and the absorbance of the reaction mixture was compared with that of a control not containing a sample. As a positive control was used 0 to 10 g/ml of acarbose (Sigma Co., USA).
[0134] Inhibition of alpha-Glucosidase Activity (%)=(1As/Ac)100(%)
[0135] Ac: 540 nm absorbance of control
[0136] As: 540 nm absorbance of sample
[0137] Mammalian alpha-glucosidases are digestive enzymes that are present along the brush border membrane of the differentiated enterocytes lining the villi of the small intestine. Alpha-glucosidases stimulate the hydrolysis of dietary carbohydrates in the form of oligosaccharides and polysaccharides into monosaccharides to allow them to be absorbed. Elevated activity of alpha-glucosidase increases such digestion and thus increase the rate of glucose absorption, causing hyperglycemia. Alpha-glucosidase inhibitors delay the digestion of carbohydrates in the small intestine and thereby lower postprandial blood sugar levels while delaying the insulin secretion induced by high blood sugar levels.
[0138] Commercially available alpha-glucosidase inhibitors include acarbose, miglitol and voglibose, which have been used for treating type 2 diabetes. Isorhamnetin--D-glucopyranoside, which is an antioxidant flavonoid glucoside isolated from an extract of Salicornia europaea, has been reported to have anti-diabetic efficacy.
[0139] As shown in
Example 4: Preparation of Salt Substitute Cold-Water-Extracted from Halophytes
[0140] 100 g of halophyte dried powder (S. europaea, S. asparagoides and S. japonica) was added to 2 liters of cold water (4 C.), stirred at 300 rpm for 4 min, and centrifuged at 10,000 rpm for 20 min. The supernatant having a high salt content was separated while a desalted precipitate was recovered therefrom. The precipitate was further desalted in cold water once more according to the same method as described above, and the second desalted precipitate was recovered. The second supernatant was pooled together with the first supernatant and vacuum-concentrated at 90 C. to achieve a salinity of 18 to 19% and a total solid content of about 26 to 28%. After that, the concentrate was purified using activated carbon in an amount of 5% based on the total solid content, and spray-dried using a spray dryer (EYELA Spray Dryer SD1-1000, Japan), thereby yielding a halophyte-derived cold-water-extracted salt substitute. The cold-water-extracted salt substitute was then evaluated for total salt, cations and glutamic acid content, and the results are given in Table 6 below (the analysis was performed in the research institute of the Korean Food Industry Association).
[0141] The cold-water-extracted salt substitute was found, compared to the hot water-extracted salt of Korean Pat. No. 10-0784229, to have low organic content while having high contents of sodium chloride and glutamic acid, which contribute to a clean salty taste having a savory (umami) flavor. In particular, among cations, the sodium to potassium ratio was greater than 10:1 (Na:K), and thus the present cold-water-extracted salt substitute was found to have a high sodium/potassium ratio. These results demonstrated that the cold water extraction for a short time facilitates only sodium, the water solubility of which is not affected by water temperature, unlike other salts, while allowing other cations to remain in the desalted powder (refer to Table 2). Also, the glutamic acid content was found to be remarkably increased compared to that of the conventional hot-water-extracted salt. This result comes from its nature whereby, since glutamic acid is an acidic amino acid that is highly water soluble, it is, unlike other organic substances including other amino acids, easily eluted even under conditions of stirring in cold water for a short time. In addition to this result, it is also a polar compound that is not adsorbed onto activated carbon during purification. Thus, the present invention enables only the sodium chloride component to be effectively removed from halophytes to obtain a functionally reinforced desalted nutritional composition from a halophyte, as well as enabling the residue of desalination to produce a pure vegetable salt having a clean salty taste with a savory (umami) flavor. Therefore, this invention is an innovative method capable of fully (100%) utilizing halophytes.
TABLE-US-00006 TABLE 6 S. europaea S. asparagoides S. japonica Total salts (%) 88.6 87.4 83.8 Na (mg/100 g) 320,990 288,347 277,324 K (mg/100 g) 25,536 25,791 27.103 Na:K ratio 12.57:1 11.18:1 10.23:1 Ca (mg/100 g) 4.5 3.9 3.7 Mg (mg/100 g) 120 108 125 Fe (mg/100 g) 0.07 0.09 0.11 Glutamic acid 28.50 23.18 25.30 (mg/g)
Test Example 4: Evaluation of Anti-Obesity Effect of the Functionally Reinforced Desalted Nutritional Composition from Halophytes
[0142] Referring to Table 2, the total carbohydrate content was found to be increased about 1.85- to 2.06-fold in the desalted nutritional compositions (desalted powder) through desalination of halophytes. These carbohydrates were analyzed and found to be composed of about 95% or greater dietary fiber in S. europaea, S. asparagoides and S. japonica powders. Table 7, below, shows the dietary fiber contents of halophyte powders before and after being desalted. The dietary fiber content includes both soluble and insoluble dietary fiber (the analysis was performed in the research institute of the Korean Food Industry Association).
TABLE-US-00007 TABLE 7 S. europaea S. asparagoides S. japonica Before After Before After Before After Halophyte being being being being being being powder desalted desalted desalted desalted desalted desalted Dietary 36.23 74.15 37.68 72.24 31.79 66.42 fiber content (%)
[0143] Anti-obesity efficacy was investigated for the S. europaea-derived desalted nutritional composition (desalted powder), which has plenty of dietary fiber as well as polyphenols and flavonoids.
Test Example 4-1: Evaluation for Body-Weight-Reducing Effect of the S. Europaea-Derived Desalted Nutritional Composition in Sprague-Dawley Rats with Obesity Induced by High-Fat Diet
[0144] The desalted powder of Salicornia europaea (the desalted nutritional composition), from which 95% or more sodium was removed and which is made rich in dietary fiber, polyphenols and flavonoids through a cold water-desalting process, was evaluated for anti-obesity efficacy. The evaluation was performed in high-fat diet-induced obese Sprague-Dawley (SD) rats using the Desalted Salicornia Powder (DSP) prepared in Example 2, the non-desalted Salicornia Powder (SP) as a comparative control and, as a positive control, the commonly available natural anti-obesity material Garcinia Extract (GE), which is an extract from the roots of Garcinia cambogia. SD rats were randomly divided into five groups each consisting of ten rats, as follows: G1: normal control group, G2: obesity control group induced by high-fat diet, G3: administered with 200 mg/kg of Salicornia Powder (SP), G4: administered with 200 mg/kg of Desalted Salicornia Powder (DSP), and G5: positive control group administered with 200 mg/kg of Garcinia Extract (GE).
[0145]
[0146] At 8, 9 and 10 weeks after administration, the obesity control group showed a remarkable increase in body weight compared to the normal control group (p<0.001), and the body weight of the group that received the Desalted Salicornia Powder (DSP) was kept at a significantly low level compared to the obesity control group (p<0.001), while the body weight of the positive control group that received Garcinia Extract (GE) was also significantly low (p<0.05). In contrast, the body weight of rats in the group that received the non-desalted Salicornia Powder (SP) was found to be significantly high compared to the normal control group, in which obesity was not induced (p<0.01), while being reduced compared to that of the obesity-induced control group. At 11 and 12 weeks after administration, the body weight of rats in the obesity control group, the group administered with 200 mg/kg of the non-desalted Salicornia Powder (SP) and the positive control group was significantly high compared to the normal control group (p<0.01 or p<0.05), whereas the body weight of rats in the group administered with 200 mg/kg of Desalted Salicornia Powder (DSP) and the positive control group was significantly low compared to that of the obesity-induced control group (p<0.001 and p<0.01, respectively). Taken together, the body-weight-reducing effect of the test samples in the high-fat-diet-induced obese SD rats, the Desalted Salicornia Powder (DSP) exhibited the most excellent body-weight-reducing effect, which was statistically significantly higher than that of the positive Garcinia Extract (GE) control (p<0.001). In contrast, the non-desalted Salicornia Powder (SP) showed a slight decrease in body weight in the obese rats, but this decrease was remarkably lower than that of DSP. These results are thought to come from the fact that, compared to the desalted Salicornia powder, the non-desalted Salicornia powder was found to have low content of dietary fiber as well as polyphenols and flavonoids, which inhibit lipid synthesis, while having a high content of sodium chloride, and such a composition may act as a potential factor to induce obesity. Therefore, the desalted Salicornia powder, from which sodium chloride was removed and which was enriched in dietary fiber and functional compounds, may be a functional material highly effective for suppressing obesity.
Test Example 4-2: Evaluation for Body-Fat-Reducing Effect of the S. europaea-Derived Desalted Nutritional Composition in SD Rats Having Obesity Induced by High-Fat Diet
4-2-1. Biochemical Blood Test and Body Fat Analysis
[0147] 12 weeks after obesity was induced, blood samples of about 1 ml were collected from the jugular vein of all rats, injected into a vacutainer tube containing a clot activator, and kept at room temperature for 15 to 20 min to allow coagulation. The blood samples were then centrifuged at 3,000 rpm for 10 min to obtain sera. Thereafter, the sera were analyzed using a blood biochemical analyzer (7020 Hitachi, Japan) for alanine transaminase (ALT), aspartate transaminase (AST), total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) and atherosclerosis index (AI). The results are given in Table 8, below.
TABLE-US-00008 TABLE 8 NC HFD HFD + SP200 HFD + DSP200 HFD + GE200 TC (mg/dL) 71.3 2.9 151.9 3.5*** 134.8 5.1**.sup.# 101.6 5.6.sup.### 117.6 4.4*.sup.## TG (mg/dL) 117 7 171.2 8.5*** 139.7 4.6**.sup.# 120.5 6.6.sup.### 125.7 5.8*.sup.## HDL (mg/dL) 28.6 0.7 29.1 0.8 29.3 1.2 30.7 1.2 29.7 0.7 LDL (mg/dL) 8.9 0.9 16.6 0.9*** 13.5 1.0** 10.9 0.8*.sup.## 12.8 0.6**.sup.# VLDL 17.6 1.5 34.2 1.7** 26.9 0.9*.sup.# 20.1 1.3.sup.### 24.1 1.2.sup.## (mg/dL) AI 2.49 0.05 5.22 0.07*** 3.97 0.10**.sup.# 2.90 0.08*.sup.### 3.62 0.08*.sup.## AST (U/L) 129.1 7.2 181.8 12.3** 157.9 9.9* 139.0 8.0.sup.## 152.3 9.5.sup.# ALT (U/L) 60.1 3.8 137.6 4.8*** 118.7 4**.sup.# 100.1 3.9*.sup.## 114.4 4.6**.sup.#
Data are represented as meanSD (n=10). Significance of difference between NC group and HFD group and between HFD+SP200, HFD+DSP200 and HFD+GE200 were analyzed by One-way ANOVA and Dunnett's multiple comparisons.
[0148] * p<0.05, ** p<0.01, *** p<0.001, .sup.# p<0.05, .sup.## p<0.01, .sup.### p<0.001
[0149] As shown in Table 8, at 12 weeks after administration, AST levels of the obesity control group (HFD) induced with the high-fat diet (HFD) and the HFD+SP200 group, which was administered with HFD plus 200 mg/kg of the non-desalted Salicornia Powder (SP) were significantly higher than that of the normal control group (NC) (p<0.01 and p<0.001). The HFD+DSP200 group, which was administered with HFD plus 200 mg/kg of the Desalted Salicornia Powder (DSP), showed a significantly low AST level compared to the HFD group (p<0.01). In the serum levels of TG and TC, the HFD group, the HFD+SP200 group and the positive control group (HFD+GE200) showed a significant increase compared to the normal control group (NC) (p<0.001, p<0.01 and p<0.05), while the HFD+DSP200 group and the positive control group (HFD+GE200) exhibited a significant decrease compared to the HFD group (p<0.001 and p<0.01). In LDL levels, the HFD group, the HFD+SP200 group and the positive control group (HFD+GE200) showed a significant increase compared to the normal control group (NC) (p<0.001 and p<0.01). This tendency was also similarly observed in VLDL and ALT levels. That is, in high-fat-diet-induced obese rats, the administration of the Desalted Salicornia Powder (DSP) was found to markedly reduce the highly increased serum levels of triglyceride (TG), total cholesterol (TC) and LDL, caused by the high-fat diet, as well as the ALT and AST levels elevated by hepatic steatosis, and these decreases were higher than those in the positive control Garcinia Extract (GE). This effect of reducing fat level and ALT and AST levels in blood was also observed in the group administered with the non-desalted Salicornia Powder (SP), but the effect was remarkably low compared to that of the Desalted Salicornia Powder (DSP).
4-2-2. Measurement of Abdominal Fat Volume Using Micro-CT
[0150] At 12 weeks after obesity was induced, before autopsy, all rats were scanned by Micro-CT (vivaCT 80, SCANCO Medical, Switzerland) to determine abdominal fat volume (
[0151] As shown in
4-2-3. Statistical Analysis
[0152] For the results of the present experiments, parametric One-way ANOVA test was applied under the assumption of the normality of data. The homogeneity of variance was tested by Levene's test. The significant difference between test groups was determined using Duncan's multiple range test where the ANOVA results were significant and variances were equal and using Dunnett's T3 test where variances were unequal. Statistical analysis was done through SPSS Statistics 18.0 K, and values of P<0.05 were considered statistically significant.
[0153] Taken together the results of the experiments in high-fat-diet-induced obese SD rats, a) the Desalted Salicornia Powder (DSP) remarkably reduced body weight compared with the obesity-induced control group; b) effectively reduced blood lipid levels (TG, TC, LDL and VLDL), blood ALT and AST levels and atherosclerosis index (AI); and c) as apparent in the results of Micro-CT scanning of tested animals, remarkably reduced the body fat, abdominal fat and subcutaneous fat compared with the obesity-induced control group. As such, the desalted Salicornia powder (DSP) was found to have a remarkably excellent effect of reducing body weight and suppressing body fat accumulation compared with the non-desalted Salicornia powder (SP), and this effect was higher than that of the positive control Garcinia Extract (GE).
Test Example 5: Determination of a Marker Compound Effective in Suppressing Adipocyte Differentiation in the S. europaea-Derived Desalted Nutritional Composition
[0154] 5-1. Isolation of a Major Effective Marker Compound from the S. europaea-Derived Desalted Nutritional Composition
[0155] To 100 g of the desalted nutritional composition from Salicornia europaea (desalted powder), prepared according to the method of Example 2, was added 1 L of distilled water and two digestive enzymes, namely amylase and protease. After incubation at 37 C. for 6 hrs, the composition was centrifuged at 10,000 g for 25 min. The supernatant was vacuum-concentrated and freeze-dried. 15.9 g of the sample (DSP-EW) thus obtained through the treatment of the desalted Salicornia powder with digestive enzymes was dissolved in methanol, and methanol-soluble components were then analyzed by high performance liquid chromatography (Agilent HPLC, USA). As shown in
[0156] After that, from 1 g of the methanol-soluble components of the DSP-EW sample, trans-ferulic acid was purified by preparative high performance liquid chromatography (YMC-HPLC, Japan), and used in an experiment with 3T3-L1 cells. The analytical HPLC system used in this experiment was a model 1260 (Infinity, Agilent, USA) equipped with Zorbax Eclipse C18 column (5 m, 4.5250 mm, Agilent) and 1200 DAD detector. In the preparative HPLC was used a model (Multiple Preparative HPLC (LC-forte/R, YMC, Japan) equipped with a prep column (Triart C18, 20 mm150 mm, 5 m, YMC, Japan). Preparative HPLC was performed under a gradient condition of methanol and triple-distilled water as a mobile phase at a flow rate of 15 ml/min using an YMC UV-3400 UV detector set at three wavelengths, namely 210, 254 and 320 nm, and four fractions were purified. As shown in
5-2. The Adipocyte Differentiation-Inhibiting Effect of Trans-Ferulic Acid Purified from the S. Europaea-Derived Desalted Nutritional Composition
[0157] 5-2-1. Differentiation of 3T3-L1 Preadipocytes
[0158] The major marker component of the desalted Salicornia powder, trans-ferulic acid (TFA), was evaluated for its inhibitory effect on adipocyte differentiation using 3T3-L1 cells as an in-vitro model of adipocyte differentiation. 3T3-L1 preadipocytes were checked for contamination every eight hours to improve confidence in the experiment. Primary preadipocytes were propagated, and were then induced to differentiate with a culture medium containing 3-isobutyl-1-methylxanthine (IBMX), dexamethasone and insulin. During the differentiation induction, the medium was changed twice every three days.
[0159] 5-2-2. Oil Red O Staining and Detection for Intracellular Triglyceride Formation
[0160] After being induced to differentiate, 3T3-L1 cells were subjected to Oil Red O staining to detect the presence of intracellular lipid droplets. First, the medium was discarded from each well, and cells were fixed with 4% paraformaldehyde. Subsequently, cells were washed with 100% 1,2-propanediol dehydration solution for 5 min and then stained with Oil Red O stain solution. After Oil Red O staining, 85% 1,2-propanediol stain differential solution was added to each well for cell washing. Finally, distilled water was added to each well to prevent stained cells from drying, and cells were observed under a microscope to determine lipid accumulation.
[0161]
[0162] In addition, as a marker for adipocyte differentiation, intracellular triglyceride formation was investigated. As shown in
[0163] 5-2-3. Real-Time RT-PCR for Detection of Transcription Factors Involved in Lipid Metabolism
[0164] PPAR, FAS, SREBP-1 and C/EBP are transcription factors involved in lipid metabolism, and are produced when 3T3-L1 preadipocytes differentiate into mature adipocytes. C/EBP and PPAR cooperate to accelerate adipogenesis. When preadipocytes proliferate to an early differentiation stage, C/EBP is induced and stimulates PPAR to induce a mature stage of differentiation. PPAR is present mainly in adipose tissue and regulates overall lipid formation, and its capacity in adipocyte differentiation is much better than other transcription factors. FAS can be used as a marker gene when adipocyte differentiation reaches a late stage, and it is a lipid synthesis enzyme involved in lipid metabolism. FAS is most strongly expressed in adipose tissues, and is the last factor of adipose differentiation. FAS is thus a representative marker for anti-obesity effect, and is induced by SREBP-1, which is a transcription factor at an earlier stage.
[0165] Real-Time RT-PCR was performed to investigate the effect of trans-ferulic acid (TFA) on transcription factors involved in lipid metabolism at mRNA expression levels. Total RNA was isolated from a control group and each test group treated with various concentrations of TFA (easy-Blue, iNtRon, INC, Daejeon, Korea) and diluted at the same concentration, and cDNA synthesis was carried out using the diluted RNA samples (cDNA reverse transcription kits, Applied Biosystems, CA, USA). The synthesized cDNA was analyzed for gene expression by Real-Time RT-PCR using the primers listed in Table 9, below.
TABLE-US-00009 TABLE9 PrimerSequence Gene Sense Anti-sense C/EBP SEQIDNO1: SEQIDNO2: 5CAACGCAACGTG 5GTCATTGTCACT GAGA-3 GGTC-3 FAS SEQIDNO3: SEQIDNO4: 5GCTGTTGGAAGT 5GTTCGTTCCTCG CAGC-3 GCGTG-3 PPAR SEQIDNO5: SEQIDNO6: 5GCCCTTTACCAC 5GTTCTACTTTGA AGTTGA-3 TCGCACTT-3 SREBP-1 SEQIDNO7: SEQIDNO8: 5CAGAAGCTCAAG 5CATGCCCTCCAT CAGGA-3 AGACA-3 GAPDH SEQIDNO9: SEQIDNO10: 5GGCCTTCCGTGT 5GCTTCACCACCT TCCTA-3 TCTTGAT-3
[0166] In brief, after 3T3-L1 preadipocytes were treated with TFA at various concentrations, namely 1, 2, 5 and 10 M), Real-time qRT-PCR was performed using the primers of Table 9 (CFX96 real-time PCR detection system, Bio-Rad Laboratories, Hercules, Calif., USA). The PCR conditions included denaturation at 5 C. for 3 min, 45 cycles of 95 C. for 5 sec and 60 C. for 20 sec, and heating up to 95 C. (by increasing the temperature at a rate of 0.2 C./15 sec) to terminate the reaction. As shown in
[0167] 5-2-4. Statistical Analysis
[0168] Data were analyzed using one-way ANOVA analysis, and values of P<0.05 were considered statistically significant.
[0169] Hereinbefore, the present invention has been described in detail and with reference to specific examples thereof. Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Thus, the actual scope of the present invention will be defined by the appended claims and equivalents thereof.
INDUSTRIAL APPLICABILITY
[0170] The halophyte-derived functionally reinforced desalted nutritional composition can be developed as a pharmaceutical composition and functional food and feedstuff for combating obesity and for reducing body fat.