COMPOSITION FOR IMPROVING SLEEP DISORDERS CAUSED BY CAFFEINE CONTAINING CITRUS RIND EXTRACT

Abstract

A food composition for preventing or improving sleep disorders caused by caffeine containing a citrus rind extract is provided. The citrus is at least one selected from the group consisting of clementine, kumquat, mandarin, tangerine, sweetie, citron, citrus sudachi, cheonhyehyang, calamansi, pomelo, hanrabong, orange, lemon, grapefruit, lime, and yuja.The extract is extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof.

Claims

1. A food composition for preventing or improving sleep disorders caused by caffeine containing a citrus rind extract.

2. The food composition of claim 1, wherein the citrus is at least one selected from the group consisting of clementine, kumquat, mandarin, tangerine, sweetie, citron, citrus sudachi, cheonhyehyang, calamansi, pomelo, hanrabong, orange, lemon, grapefruit, lime, and yuja.

3. The food composition of claim 1, wherein the citrus rind extract contains at least one of hesperidin and narirutin.

4. The food composition of claim 1, wherein the extract is extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixed solvent thereof.

5. The food composition of claim 1, wherein the food composition is coffee.

6. The food composition of claim 1, wherein the food composition is food additives.

7. The food composition of claim 1, wherein the sleep disorders are insomnia or circadian rhythm sleep disorders caused by caffeine.

8. A pharmaceutical composition for preventing and treating sleep disorders caused by caffeine containing a citrus rind extract.

9. A method for preventing, improving, or treating sleep disorders caused by caffeine comprising administering a citrus rind extract to a subject.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0063] FIG. 1 illustrates a result of confirming ingredients contained in a citrus unshiu peel sample through HPLC.

[0064] FIG. 2 illustrates a result of HPLC quantitative analysis of hesperidin contained in the citrus unshiu peel.

[0065] FIG. 3 illustrates a result of HPLC quantitative analysis of narirutin contained in the citrus unshiu peel.

[0066] FIG. 4 illustrates the contents of hesperidin and narirutin contained in 10 mg of a citrus unshiu peel sample.

[0067] FIG. 5 illustrates a hippocampal organotypic slice culture process.

[0068] FIG. 6 illustrates a microelectrode array (MEA) setting process of the hippocampal tissue.

[0069] FIG. 7 illustrates a microelectrode array (MEA) experimental protocol (A) and a spike detection process (B) using a band pass filter.

[0070] FIG. 8 illustrates a result of confirming that the neural activity caused by caffeine is alleviated when a citrus unshiu peel extract is treated (A: neural activity in treatment of caffeine, B: neural activity in treatment of caffeine and citrus unshiu peel extract, C: comparison in neural activity before washing in treatment of caffeine alone and treatment of caffeine and citrus unshiu peel extract, D: comparison in neural activity after washing in treatment of caffeine alone and treatment of caffeine and citrus unshiu peel extract).

[0071] FIG. 9 illustrates a result of confirming that the neural activity caused by caffeine is alleviated when hesperidin, narirutin, or a mixture thereof contained in the citrus unshiu peel extract is treated (A: neural activity in treatment of caffeine, B: neural activity in treatment of caffeine and hesperidin, C: neural activity in treatment of caffeine and narirutin, D: neural activity in treatment of caffeine, hesperidin, and narirutin).

[0072] FIG. 10 illustrates comparison of a result before and after washing of confirming that the neural activity caused by caffeine is alleviated when hesperidin, narirutin, or a mixture thereof contained in the citrus unshiu peel extract is treated (A: before caffeine washing, B: after caffeine washing).

[0073] FIG. 11 illustrates an experimental process of oral administration and recording to an SD rat for measuring sleep duration (A: oral administration process to SD rat, B: recording process for measuring sleep duration).

[0074] FIG. 12 illustrates a result of confirming that the sleep duration is recovered when the citrus unshiu peel extract is treated.

DETAILED DESCRIPTION OF THE INVENTION

[0075] Hereinafter, the present invention will be described in detail by Examples. However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

PREPARATION EXAMPLE

Preparation of Citrus Unshiu Peel Extract

[0076] 100 g of a citrus unshiu peel (Gio Herb Co., Yangju, Korea) was added with 10 times (w/v) of distilled water and extracted by hot water at 121° C. for 40 minutes. The extract was filtered using Whatman No. 1 filter paper (Whatman International Ltd., Maidstone, UK), and the filtered extract was concentrated for 5 hours at 40° C. using a concentrator (Eyela Co., Tokyo, Japan).

[0077] The concentrated extract was freeze-dried for 7 days at −70° C. and 5 mTorr with a freeze dryer (Ilsin Bio Co., Yangju., Korea), and then grinded with a grinder (Ilsin Bio Co., Yangju., Korea) and used for an experiment. When the citrus unshiu peel was extracted with hot water through the above process, the yield was 9.8%.

TABLE-US-00001 TABLE 1 Raw material Extract Yield (%; extract/raw (g) (g) material) Citrus unshiu peel 100 9.8 9.8

EXPERIMENTAL EXAMPLE 1

Confirmation of Ingredients Contained in Citrus Unshiu Peel Extract

[0078] Compounds contained in the citrus unshiu peel extract of the present invention were confirmed using HPLC. Specifically, the citrus unshiu peel extract was measured using reverse phase HPLC (Vltimate 3000; Thermo Fisher Scientific, Waltham, USA) consisting of an autosampler, a binary pump, and a PDA detector. The reverse phase HPLC used C18 (Discovery C18, 5 μm, 4.6×250 mm; Sigma, Mo., USA). An injection amount thereof was 10 μL and injected at a rate of 0.8 mL/min.

[0079] In the HPLC, solvents (mobile phase) A and B used 100% tertiary distilled water and 100% acetonitrile, respectively, and the composition of gradient elution was performed at 90%

[0080] A/10% B in 0 to 5 minutes, 75% A/25% B in 5 to 14 minutes, 40% A/60% B in 14 to 25.5 minutes, 20% A/80% B in 25.5 to 30.5 minutes, and 90% A/10% B in 30.5 to 35 minutes. All compounds were detected at a wavelength of 280 nm, and the analysis temperature was maintained at 40° C. The data of a sample test solution collected through HPLC/PDA was confirmed by comparing a retention time and a UV spectrum waveform of a standard form.

[0081] As a result, as illustrated in FIG. 1, it was confirmed that narirutin and hesperidin were contained in the citrus unshiu peel sample used in the present invention.

[0082] In addition, the contents of narirutin and hesperidin in the citrus unshiu peel extract were calculated (FIGS. 2 and 3), and as a result, it was confirmed that the contents of narirutin and hesperidin contained in 400 μg/ml of the citrus unshiu peel extract were 136 μg/ml and 40 μg/ml, respectively (FIG. 4).

EXPERIMENTAL EXAMPLE 2

Measurement of Neural Activity

[0083] 1-1. Hippocampal Organotypic Slice Culture (HOSC)

[0084] A method of organotypic culturing of slices of the brain hippocampus using a thin film used an already commonly used method. Specifically, the brain hippocampus was extracted from a Sprague Dawley white young rat (Orientbio Co., Seungnam, Korea) of 5- to 7-day old, and sliced to a thickness of 350 μm using a tissue slicer (Mickle Laboratory Engineering Co., UK). After 5 to 6 slices were disposed into a thin film insert (Millicell-CM; Millipore, MA) with 0.4-μm holes, put in a 6-well plate (SPL Life Sciences Co., Korea), and then cultured in an incubator kept with 5% CO.sub.2 at 36° C. (Dong-A Co., Seoul, Korea). A culture solution (MEM; Welgene, Gyeongsan, South Korea), 20 mM HEPES (Sigma, St. Louis, Mo., USA), a 25% Hank's balanced salt solution (HBSS; Welgene, Gyeongsan, South Korea), 6 g/L D-glucose (Sigma, St. Louis, Mo., USA), 1 mM L-glutamine (Sigma, St. Louis, Mo., USA), 25% horse serum (Welgene, Gyeongsan, South Korea), and 1% penicillin-streptomycin (Gibco BRL, USA), pH=7.1) were changed once every two days, and the culture period was 12 to 14 days, and then used in a subsequent experiment (FIG. 5).

[0085] 1-2. Migration of Brain Hippocampal Tissue Cultured with MEA Probes

[0086] After the cultured brain hippocampal slices were carefully isolated from the cultured thin film insert using a pin, the brain hippocampal slices were placed on a 8×8 microelectrode array (MEA; Multi Channel Systems, Germany) with a diameter of 10 μm and an interval of 100 μm coated with 0.01% polyethyleneimine (Sigma, St. Louis, Mo., USA). The slices were injected with a sterilized artificial spinal cord solution (aCSF; 114 mM NaCl, 26 mM NaHCO.sub.3, 10 mM glucose, 3 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl2, 20 mM HEPES, pH=7.4, Sigma, Louis, Mo., USA) at 5% CO.sub.2 and 95% O.sub.2 mixed gas (Dong-A Co., Seoul, Korea) for 15 minutes and stabilized while kept at 33° C. Thereafter, the MEA containing the brain hippocampus was mounted on an amplification device MEA1060 (Multi Channel Systems Inc., Reutlingen, Germany), and the artificial spinal cord solution in the MEA was grounded with Ag/AgCl. During the subsequent experiment, the artificial spinal cord solution into which the 5% CO.sub.2 and 95% 0.sub.2 mixed gas was added was perfused at a rate of 3 mL/min, and the MEA and the artificial spinal cord solution were maintained at 33° C. (FIG. 6) .

[0087] 1-3. Measurement of Neural Activity

[0088] The stabilized hippocampal tissue was recorded for 3 minutes, and then after an artificial cerebrospinal fluid containing 1 mM of caffeine (Sigma, St. Louis, Mo., USA) and an artificial cerebrospinal fluid containing 1 mM of caffeine and an analysis sample (citrus unshiu peel extract of 400 μg/ml, hesperidin of 40 μg/ml, narirutin 136 of μg/ml) were perfused under the same conditions for 20 minutes, respectively, the hippocampal tissue was recorded for 3 minutes. Thereafter, the hippocampal tissue was washed with only the artificial cerebrospinal fluid for 20 minutes and then recorded for 3 minutes to obtain data for total 9 minutes.

[0089] The MEA with the hippocampal tissue read data at a sampling rate of 25 kHz for each channel through a total of 60 channels, and was recorded through a computer using MC_Rack and MC_Data Tool (Multi Channel Systems, GmbH) software. A 4-stage band pass filter was used to extract meaningful spikes from all recorded signals, and Equation is as follows.

[00001]$\begin{array}{cc}\mathrm{Threshold}=k\times \mathrm{median}\left(\frac{\mathrm{abs}\left(X\right)}{0.6745}\right)& \mathrm{Equation}1\end{array}$

[0090] Wherein, X represents a root mean square (RMS) for noise, and k is a factor that determines each threshold. Here, the threshold consisted of a total of three factors (0.6, 1 and 3). When the k value was 0.6 or higher, a meaningful spike was used, and the threshold level at this time was −21.63 μV. This process was performed using a MATLAB (Mathworks Inc., Natick, Massachusetts, USA) program for all 60 channels (FIG. 7).

[0091] As a result, as illustrated in FIG. 8, it was confirmed that the neural activity caused by caffeine was alleviated when the citrus unshiu peel extract was treated.

[0092] Specifically, when caffeine was treated, the neural activity was increased, and even after washing after the caffeine was treated, the action of caffeine continued to increase the neural activity by 200% or more (FIG. 8A). On the other hand, it was confirmed that when the caffeine and the citrus unshiu peel extract were treated together, the degree of neural activity was significantly reduced compared to the case of treatment of caffeine alone, and unlike treatment of caffeine alone, the neural activity was not increased continuously even after washing, and thus, the neural activity was maintained without a significant increase (FIG. 8B). With respect to the case of treating caffeine alone and the case of treating caffeine and the citrus unshiu peel extract in combination, even when comparing before and after washing, in treatment with the citrus unshiu peel extract, it was confirmed that the significant neural activity reduction effect was shown (FIGS. 8C and 8D).

[0093] In particular, even after washing after treatment with caffeine, the activity caused by caffeine in the nerves was increased, and this indicates that the active action of caffeine on the nerves does not appear briefly in a short time, but rather continues. The citrus unshiu peel extract of the present invention exhibited the effect of reducing the neural activity by inhibiting the effect of caffeine which was continuously increased as described above.

[0094] In addition, as a result of treating hesperidin, narirutin, or a mixture thereof, which were ingredients contained in the citrus unshiu peel extract, together with caffeine, it was confirmed that both hesperidin and narirutin may inhibit an increase in neural activity caused by caffeine. Furthermore, it was confirmed that the neural activity was more effectively inhibited when hesperidin and narirutin were treated in combination than when each of hesperidin and narirutin was treated (FIG. 9).

[0095] Specifically, when the caffeine was treated, the neural activity was increased, and even after washing after the caffeine was treated, the action of caffeine continued to increase the neural activity by 200% or more (FIG. 9A). On the other hand, it was confirmed that when the caffeine and 40 μg/ml of hesperidin (amount contained in 400 μg/ml of the citrus unshiu peel) were treated in combination, the degree of neural activity was significantly reduced compared to the case of treatment of caffeine alone, and unlike treatment of caffeine alone, the neural activity was not increased continuously even after washing, and thus, the neural activity was maintained without a significant increase (FIG. 9B). It was confirmed that even when the caffeine and 136 μg/ml of narirutin (amount contained in 400 μg/ml of citrus unshiu peel) were treated in combination, similarly, the degree of neural activity was significantly reduced compared to the case of treatment of caffeine alone, and unlike treatment of caffeine alone, the neural activity was not increased continuously even after washing, and thus, the neural activity was maintained without a significant increase (FIG. 9C). Further, it was confirmed that the neural activity reduction effect was further increased when 40 μg/ml of hesperidin and 136 g/ml of narirutin were treated together with caffeine (FIG. 9D).

[0096] Through the results, it was confirmed that the hesperidin and narirutin ingredients may alleviate the neural activity, and the citrus unshiu peel extract containing both the two ingredients may effectively alleviate the neural activity caused by caffeine.

[0097] Furthermore, as illustrated in FIG. 10, when caffeine is treated, the neural activity is continuously shown even after washing the caffeine, and this indicates that the neural activity action caused by caffeine may continue for a long time rather than a short time and there is a possibility of side effects as the neural activity continues for a long time.

[0098] On the other hand, unlike the case of treatment of caffeine alone, it was confirmed that when hesperidin, narirutin, and a mixture of hesperidin and narirutin were treated in combination, a neural activity inhibitory effect persisted even after washing. This indicates that the neural activity induced by caffeine may be inhibited from continuing even after caffeine is removed (FIG. 10).

EXPERIMENTAL EXAMPLE 3

Confirmation of Effect of Improving Sleep Disorders

[0099] 3-1. Experimental Animals

[0100] A 6-week-old Sprague Dawley rat was selected as an experimental animal, and was purchased from Orient Bio Co., Ltd. (Seongnam, Korea). In order to keep an experimental environment constant, an experimental animal per cage was placed and bred one by one. The experimental animals went through a period of adjustment for one day in an environment where 25° C., humidity 50%, and lighting (12 hours light/dark) were automatically maintained and drinking water and diet were supplied freely. All processes followed the policy of Kyunghee University's Experimental Animal Ethics Committee and the regulations associated to Animal Experiment (IRB approval number: KHUASP(SE)-17-039).

[0101] 2-2. Measurement of Sleep Duration

[0102] The experiment was performed by dividing 15 SD rats into 3 groups (n=5). Water was orally administered to Group 1, caffeine (20 mg/Kg) was orally administered to Group 2, and a citrus unshiu peel extract (100 mg/Kg) and caffeine (20 mg/Kg) were orally administered to Group 3 together.

[0103] Focusing on the fact that rodents were nocturnal in nature, the sleep duration was measured during daytime when SD rats took sleep originally. After the oral administration, the cage was recorded for 5 hours using a camcorder (Sony Co., Tokyo, Japan) from 10 a.m. to measure the sleep duration, and the measurement time was referred to in previous studies. A total of 3 subjects were recorded one by one for each group per day, and only one subject for each cage was placed so that the subjects were not affected by each other. In addition, the illuminance of a space to be measured was kept constant, and humidity 50%, temperature 25° C., and sound insulation were maintained. The sleep pattern and duration were measured using a Multi-Channel Stopwatch (Techsys Co. Seoul, Korea) by determining as a sleep condition when the rat closed eyes and continued for 5 minutes or more in a prone position on the side and summing hours from the start of sleep to the end of sleep (FIG. 11).

[0104] As a result, as illustrated in FIG. 12, when the caffeine was treated, the sleep duration was reduced to about 20 to 30% compared to a normal control, but when the caffeine and the citrus unshiu peel extract were treated together, the sleep duration was restored by about 4 times or higher.

[0105] The results described above confirmed that the citrus unshiu peel extract of the present invention had an effect of improving sleep disorders while sedating the excessive neural activity caused by caffeine, and particularly, it was shown that the citrus unshiu peel extract may inhibit the continuous action of caffeine beyond the effect of inhibiting the neural activity caused by caffeine in a short time. Accordingly, the citrus unshiu peel extract of the present invention may be widely used for improving sleep disorders while stabilizing neural activity induced by caffeine.

[0106] The results of the experiment were expressed as mean±standard error (S.E.M). After converting the calculated spike values into ratios thereof in each group, an ANOVA Tukey post-test was performed to examine differences between groups. At p<0.05, p<0.01, and p<0.001, there were statistically significant differences between groups.

EXPERIMENTAL EXAMPLE 4

Confirmation of Anti-Insomnia Activity of Citrus Rind Extract

[0107] In order to confirm whether the citrus rind extract has an anti-insomnia effect, with respect to mandarin, tangerine, sweetie, citron, citrus sudachi, cheonhyehyang, calamansi, pomelo, hanrabong, orange, lemon, grapefruit, lime, and yuja rind extracts, the experiments of Experimental Examples 2 and 3 were repeated.

[0108] As a result of the experiments, each of the rind extracts had some differences from the citrus unshiu peel extract of Example 1, but a similar or equivalent level of sleep disorder improvement effect was implemented. Through this, it was confirmed that the citrus rind extracts had similar properties and exhibited an anti-insomnia effect in common.

[0109] The aforementioned description of the present invention is to be exemplified, and it will be understood to those skilled in the art that the technical spirit or required features of the present invention can be easily modified in other detailed forms without changing. Therefore, it should be appreciated that the aforementioned embodiments described above are illustrative in all aspects and are not restricted. For example, respective components described as single types can be distributed and implemented, and similarly, components described to be distributed can also be implemented in a coupled form.

[0110] The scope of the present invention is represented by claims to be described below, and it is to be interpreted that the meaning and the scope of the appended claims and all changed or modified forms derived from equivalents thereof come within the scope of the present invention.