Green Tea (Camellia sinensis L.) Phytochemicals in the Regulation of Sleep and Wakefulness

Abstract

녹차(Camellia sinensis L.)는 다양한 생리활성을 가지고 있어, 전통 음료 및기능성 음료로 전 세계적으로 널리 소비되고 있다. 이러한 효능은 주로 폴리페놀과 카페인과 같은 생리활성 물질에 기인한다. 특히 카페인은 아데노신 A2A 수용체를 길항함으로써 각성 효과를 유도하는 주요 성분으로 알려져 있다. 기존 연구에 따르면, 녹차 추출물의 카페인 및 폴리페놀의 상대적 함량은 추출 방법에 따라 크게 달라질 수 있으며, 이러한 조성의 차이는 녹차 추출물의 생리적 효능에도 영향을 미치는 것으로 보고된 바 있다. 특히, 이와 같은 조성 변화는 각성 유도 효과와 진정 또는 수면 유도 효과와 같은 상반된 생리활성에 영향을 미칠 수 있다. 본 연구에서는 추출 조건의 변화가 녹차 에탄올 추출물(green tea ethanol extract, GE)의 각성 및 수면 조절 효능에 미치는 영향을 규명하고자 하였다. 이를 위해 다양한 조성 특성을 가진 GE에 대해 일련의 생체 내 실험을 수행하였으며, 반복 투여 후의 지속적인 효능 또한 함께 평가하였다. 더불어, 수면 유도 효과를 향상시키기 위해 열수 처리(hot water treatment, HT)를 활용한 디카페인 공정을 적용하였다. 우선, GE의 양방향적 기능성, 즉 각성과 수면 유도 효과를 확인하기 위해 반응표면분석법(response surface methodology, RSM)을 적용하여 추출 조건을 체계적으로 조절하였다. 테스트한 조건 중, 95% 에탄올로 195분 추출한 GE는 가장 강한 각성 유도 효과(47.6 ± 4.0분)를 보였고, 이는 25 mg/kg의 순수 카페인과 비슷한 결과였으며, 카페인 함량은 58.9 mg/g이었다. 수면 구조 분석에서 GE(1500 mg/kg)는 투여 후 3시간 동안 각성을 1.4배(p < 0.001) 증가시켰다. 상관관계 분석 결과, 총 폴리페놀 함량(total phenolic content, TPC) 대비 카페인 비율(TPC/caffeine ratio)이 각성 효과와 높은 양의 상관관계(R² = 0.9428)를 보였다. 특히, EGCG와 함께 L-테아닌을 고려한 경우, 수면 지속 시간과의 상관관계가 더욱 높아지는 경향(R² = 0.9464)을 나타냈다. GE의 단일 성분을 이용한 후속 실험에서도 GE의 각성 및 수면 조절 효과는 TPC/caffeine 비율과 밀접하게 관련되어 있음이 확인되었다. TPC/caffeine 비율이 9 미만인 경우 강한 각성 효과를 보였으며, 10 이상인 경우에는 이러한 각성 효과가 약화되거나 억제되는 경향을 보였다. 이러한 결과는 GE의 기능적 특성이 단순한 카페인 함량이 아니라 폴리페놀과 카페인 간의 조화로운 균형에 의해 결정된다는 점을 시사한다. 이 결과를 바탕으로, GE의 각성 유도 효과가 만성 투여 동안 지속될 수 있는지 평가하기 위한 후속 실험이 진행되었다. GE(1500 mg/kg)는 C57BL/6N 쥐에 3주 동안 경구 투여되었으며, 그 효과는 참조 각성제인 카페인(25 mg/kg)과 비교하여 수면 구조 분석에 미치는 영향을 평가하였다. GE는 입면시간을 유의미하게 증가시키고 각성을 연장했으며, 투여 기간 동안 내성이나 금단 증상 없이 각성효과를 유지하였다. 투여하지 않은 기존 그룹(baseline)과 비교했을 때, GE는 1일째와 21일째 첫 3시간 동안 각성을 1.9배(p < 0.001)에서 2.1배(p < 0.001)까지 유의미하게 증가시켰다. 또한, GE는 3시간 동안 각성 유도 효과를 유지했으며, 21일째 4시간까지 각성 증가와 NREMS 감소가 관찰되었다. 이전 연구에서 확인한 것처럼, GE는 카페인과 유사하게 델타 활동 변화가 나타나지 않았다. GE 1500 mg/kg은 95.6 mg/kg에 해당하는 카페인을 포함하고 있었음에도 불구하고, 25 mg/kg 카페인만큼 강한 각성 효과를 유도하지 않아 GE의 다른 생리활성 성분들이 카페인의 자극 효과를 상쇄하거나 조절할 수 있음을 시사한다. 이러한 결과는 GE가 장기적으로 안정된 각성 유도 효과를 유지할 수 있음을 보여주며, 안정적인 식물 기반 자극제로서의 잠재력을 나타낸다. 또한, 녹차의 수면 촉진 효과를 향상시키기 위한 디카페인 처리 방법으로 열수 추출법(hot water treatment, HT)의 가능성을 추가적으로 조사하였다. 다양한 추출 조건을 비교한 결과, HT는 카페인 농도를 줄이면서도 생리활성 화합물을 높은 농도로 유지하는 것을 보여주었다. 특히, HT를 통한 디카페인 처리 후 EGCG 농도가 증가했으며, 이는 수면 촉진 효과에 중요한 역할을 한다. 분석 결과, TPC/caffeine (R² = 0.8709) 및 EGCG/caffeine (R² = 0.7798) 비율과 수면 시간 사이에 유의미한 상관관계를 확인할 수 있었으며, 이는 카페인과 폴리페놀의 균형이 수면 결과에 중요한 역할을 한다는 것을 시사한다. HT-GE는 아무런 디카페인 처리를 하지 않은 GE보다 EGCG 농도가 더 높았으며, 이는 HT를 통한 디카페인 처리로 수면 촉진 화합물의 농도가 증가했음을 나타낸다. 이러한 결과는 HT-GE가 열수 처리로 최적화된 폴리페놀 대 카페인 비율을 통해 아무런 처리하지 않은 GE보다 더 강한 수면 촉진 효과를 보인다는 것을 강조한다. 또한, HT-GE에서 EGCG 농도가 증가한 것은 디카페인 과정이 수면 유도 성질을 강화한다는 것을 더욱 시사한다. 이 결과는 각성과 수면을 모두 조절할 수 있는 녹차가 각성제로서뿐만 아니라 상당한 치료적 잠재력을 가진 천연 수면 보조제로서도 유망한 가능성을 제시하며, 녹차의 추가적인 연구 가능성을 시사한다.|Green tea (Camellia sinensis L.), a widely consumed beverage, contains caffeine, a natural stimulant. However, certain green tea extracts are known to exhibit both hypnotic and arousal effects. This study aimed to identify the components responsible for these dual effects using a GE. Using RSM, it was found that only specific extraction conditions notably induced arousal effects in ICR mice during the pentobarbital-induced sleep test. Among the conditions tested, extracting with 95% ethanol for 195 min produced the strongest arousal effect, corresponding to a caffeine content of 58.9 mg/g, similar to the effect of 25 mg/kg of caffeine. Moreover, the administration of this GE sample led to a significant enhancement of wakefulness for a duration of 3 h in C57BL/6N mice, as corroborated by the sleep architecture analysis. A correlation analysis of the total phenolic content (TPC) to caffeine ratio in GE revealed that the strength of the arousal effects was correlated with TPC (R2 = 0.9428). The ratio of EGCG to caffeine, two major components of GE, was also found to be more closely associated with sleep duration (R2 = 0.9034). While L-theanine, known for its sleep-promoting properties, did not independently affect the arousal effects of GE, its combined content with EGCG showed a slightly stronger correlation with sleep duration in relation to the caffeine ratio compared to the EGCG/caffeine ratio (R2 = 0.9464). Therefore, the balance between TPC and caffeine appears to influence the stimulant properties of GE, suggesting its potential as both a stimulant and a mild hypnotic agent. Overall, these findings offer valuable insights into optimizing GE for functional foods tailored to specific polyphenol/caffeine ratios.|Wakefulness is described as a state in which individuals are able to respond to a change in situation. In recent years, there has been an increase in the number of individuals remaining awake to compensate for sleep deprivation. Caffeine, a well-known stimulant, is the most widely consumed psychoactive substance globally, primarily ingested through coffee. While green tea (Camellia sinensis L.) contains caffeine levels comparable to coffee, its arousal-inducing effects have yet to be fully explored. The objective of this study was to examine the arousal-inducing effects of GE during chronic administration (three weeks) using sleep architecture analysis. Administration of GE (1500 mg/kg) significantly increased sleep latency and prolonged wakefulness throughout the treatment period. Chronic administration of GE consistently maintained wakefulness for up to 3 h. Throughout the treatment, the arousal-inducing effect of GE (1500 mg/kg) occurred without evidence of tolerance development or withdrawal symptoms, similar to the effects observed with caffeine (25 mg/kg). Notably, GE (1500 mg/kg), which contained 95.6 mg/kg of caffeine, did not produce a greater arousal-inducing effect compared to caffeine at 25 mg/kg. These findings suggest that the arousal-inducing effects of GE persisted over the three-week period without adverse effects, and that GE may modulate caffeine's arousal-inducing properties due to the presence of other hypnotic compounds.|Green tea (Camellia sinensis L.) has been consumed for centuries due to its health benefits, particularly its antioxidant and anti-inflammatory effects. Recent studies have also explored its potential to modulate sleep and wake cycles, although the interaction between its caffeine content and sleep regulation remains complex. While green tea contains significant caffeine levels, it does not exhibit the same arousal-inducing effects as a standard dose of caffeine, likely due to the ratio of polyphenols to caffeine, which can mitigate the stimulant effects of caffeine. This study aimed to investigate the sleep-promoting properties of hot water-treated green tea ethanol extract (HT-GE) using RSM to compare the composition and sleep/arousal effects under different treatment conditions. Additionally, the mechanism of action of HT-GE was explored to understand its impact on sleep regulation. Caffeine, the primary arousal agent in HT-GE, showed a strong correlation with sleep duration (R2 = 0.9375), while individual polyphenolic compounds such as TPC, TFC, and EGCG showed weaker associations. However, the ratios of these compounds to caffeine demonstrated stronger correlations with sleep duration, suggesting a synergistic effect between caffeine and polyphenols in enhancing sleep-promoting effects. In silico molecular docking studies and in vivo tests using pentobarbital-induced sleep assays further elucidated the potential sleep-enhancing mechanisms of EGCG and its interaction with the GABAA-BZD receptor. These studies suggest that EGCG may act as an allosteric modulator at the GABAA receptor, enhancing sleep by promoting relaxation, similar to the effects of BZD drugs. The molecular docking simulations revealed that EGCG has a significant binding affinity at the BZD binding site, which is critical for regulating sleep. These findings support the hypothesis that optimizing the polyphenol to caffeine ratio in HT-GE could be a promising strategy for developing natural sleep-promoting formulations. The balance between the arousal effects of caffeine and the sleep-inducing effects of EGCG indicate that green tea decaffeination through HT has the potential to be widely used as a natural sleep aid.|Green tea (Camellia sinensis L.) is widely consumed as both a traditional and functional beverage, valued for its health-promoting properties. These effects are primarily attributed to bioactive compounds such as polyphenols and caffeine. Among them, caffeine is well known for its arousal-promoting effects through antagonism of adenosine A2A receptors. Previous reports have shown that the phytochemical profile of green tea extracts, including the relative amounts of caffeine and polyphenols, can differ significantly depending on the extraction method used. Variations in extraction methods can substantially impact the composition of green tea extracts, which in turn may lead to distinct functional outcomes. Notably, such variation may influence the opposing physiological effects of green tea, such as its arousal-promoting versus sedative or sleep-enhancing properties. This study aimed to elucidate how variations in extraction conditions alter the arousal- and sleep-modulating properties of green tea ethanol extract (GE). In addition, a series of in vivo experiments using were conducted to evaluate not only the immediate effects of GE under different compositional profiles but also their sustained efficacy after repeated administration. First, to enhance the sleep-promoting properties of GE, compositional modulation was explored through caffeine reduction using HT as a decaffeination method. To identify the bidirectional effects of GE on arousal and sleep, a response surface methodology (RSM) approach was applied to systematically vary extraction conditions. Among the tested conditions, GE extracted with 95% ethanol for 195 min exhibited the strongest arousal-inducing effect (47.6 ± 4.0 min), showing a comparable outcome to 25 mg/kg of pure caffeine and containing 58.9 mg/g of caffeine. In analysis of sleep architecture, GE at 1500 mg/kg significantly increased wakefulness by 1.4-fold (p < 0.001) during the 3 h period after administration. Correlation analysis revealed that arousal effects were positively associated with the total phenolic content (TPC) to caffeine ratio (R² = 0.9428). In particular, when L-theanine was considered alongside EGCG, the correlation with sleep duration became even stronger (R² = 0.9464). Additional experiments using single compounds of GE confirmed that the arousal- and sleep-modulating effects of GE were closely associated with TPC/caffeine ratio. Extracts or fractions with a lower TPC/caffeine ratio (< 9) exhibited strong arousal effects, while higher ratios (≥ 10) appeared to diminish or suppress such effects. These results suggest that the balance between polyphenols and caffeine, rather than caffeine content alone, critically influences the functional properties of GE. Based on these results, a follow-up experiment was conducted to assess whether the arousal-inducing effects of GE could be sustained during chronic administration. GE (1500 mg/kg) was orally administered to C57BL/6N mice for three weeks, and its effects on sleep architecture were evaluated in comparison to caffeine (25 mg/kg) as a reference stimulant. GE significantly increased sleep latency and prolonged wakefulness without signs of tolerance or withdrawal throughout the treatment period. Compared to baseline, GE significantly elevated wakefulness amount by 1.9- (p < 0.001) to 2.1-fold (p < 0.001) during the first 3 h on both Day 1 and Day 21. In addition, GE continued the arousal-inducing effects for 3 h post-administration throughout the three weeks, with a significant increase in wakefulness and a reduction in NREMS observed up to the 4th hour on Day 21. As confirmed in previous studies, GE did not show changes in delta activity, similar to that of caffeine. Although GE 1500 mg/kg contained an amount of caffeine equivalent to a 95.6 mg/kg dose, it did not induce stronger arousal effects than 25 mg/kg of caffeine alone, suggesting that other bioactive components in GE may counteract or modulate the stimulating effects of caffeine. These results demonstrate that GE can maintain its arousal-inducing efficacy over time without adverse effects, supporting its potential use as a stable, plant-based stimulant. The study further investigates the potential of hot water treatment (HT) as a decaffeination method for enhancing the sleep-promoting properties of green tea. By comparing various extraction conditions, including the effect of HT on the caffeine and polyphenol composition, this research demonstrates that HT-GE retains high concentrations of bioactive compounds while reducing caffeine content. Notably, decaffeination through HT resulted in increased EGCG levels, which are known to contribute to sleep-enhancing effects. The analysis revealed a significant correlation between the TPC/caffeine (R2 = 0.8709) and EGCG/caffeine (R2 = 0.7798) ratios and sleep duration, suggesting that the balance between caffeine and polyphenols plays a crucial role in modulating sleep outcomes. In addition, EGCG content was higher in HT-GE compared to untreated GE, indicating that the decaffeination process through HT increased the concentration of compounds closely related to sleep-promoting effects. These findings highlight that HT-GE, through the optimal polyphenol to caffeine ratio achieved by HT, shows a stronger sleep-promoting effect compared to untreated GE. In addition, the increased EGCG content in HT-GE further suggests that the decaffeination process enhances its sleep-inducing properties. These results highlight that GE, with its ability to modulate both wakefulness and sleep, presents a promising avenue not only as a stimulant but also as a natural sleep aid with substantial therapeutic potential, suggesting its potential for further exploration.