South african ashwagandha in combination with melatonin

Abstract

The present invention relates to the combined use of Withania and melatonin. In the present application, the combined use of Withania and melatonin has a significant synergistic effect in improving sleep and / or improving mood. In particular, the effect of improving sleep and / or improving mood is superior when Withania is administered in the morning in combination with melatonin administered in the evening, compared to the effect of both administered in the evening and the effect of both administered alone.

Description

Technical Field

[0001] This application relates to the combined use of Ashwagandha and melatonin, particularly the use of Ashwagandha and melatonin to improve sleep and / or improve mood. Background Technology

[0002] In today's fast-paced and high-pressure living and working environment, the number of people suffering from insomnia and anxiety is on the rise, seriously affecting their daily work and life, and becoming a disease that cannot be ignored.

[0003] Melatonin (MT), chemically known as N-acetyl-5-methoxytryptamine, plays a physiological role in regulating the human circadian rhythm and sleep. In recent years, melatonin has been widely used in the treatment of various sleep disorders, and numerous clinical studies have been conducted.

[0004] Ashwagandha (Withania somnifera, WS), also known as Indian ginseng, is a traditional medicine in the Ayurvedic medical system of India, often used for its anti-anxiety, sleep-aiding, and anti-aging effects. The British Pharmacopoeia lists the root of Ashwagandha as usable in Traditional Herbal Medicinal Products (THMP); as a dietary supplement or health food, extracts from the root of Ashwagandha are generally used. Summary of the Invention

[0005] Through long-term research, the inventors unexpectedly discovered that combining Ashwagandha with melatonin improves sleep and / or mood, and that the two have a significant synergistic effect in improving sleep and / or mood. In particular, the effect of improving sleep and / or mood when Ashwagandha is applied in the morning combined with melatonin in the evening is superior to that when both are applied simultaneously in the evening or individually. These research findings provide a theoretical basis for clinical application and have significant practical value.

[0006] One aspect of this application provides the use of a combination of Ashwagandha and melatonin in the preparation of a pharmaceutical or health food, wherein Ashwagandha is applied in the morning and melatonin is applied in the evening;

[0007] The combination has the function of improving sleep and / or improving mood. The drug or health food has the function of improving sleep and / or improving mood.

[0008] A second aspect of this application provides a method for administering Ashwagandha and melatonin to a subject in need, wherein the Ashwagandha is administered in the morning and the melatonin is administered in the evening.

[0009] In some embodiments, the Ashwagandha is a medicine or health food. In some embodiments, the melatonin is a medicine or health food.

[0010] In some implementations, the method is used to improve the sleep and / or mood of the subject.

[0011] A third aspect of this application provides a combined package of Ashwagandha and melatonin, wherein Ashwagandha is a medicine or health food, and the Ashwagandha is applied in the morning; and the melatonin is a medicine or health food, and the melatonin is applied in the evening.

[0012] In some embodiments, the combination package includes a carrier containing instructions for use, which recommend that Ashwagandha be applied in the morning and melatonin be applied in the evening. The carrier containing the instructions for use may, exemplarily, be a user manual, exemplarily be a product box, or exemplarily be a label printed or affixed to a product packaging container.

[0013] In some implementations, the combination package is used to improve the sleep and / or mood of the recipient.

[0014] A fourth aspect of this application provides a method for improving sleep and / or improving mood, comprising administering an effective amount of Ashwagandha and melatonin to a subject in need, wherein the Ashwagandha is administered in the morning and the melatonin is administered in the evening.

[0015] In some embodiments, the Ashwagandha is administered in the form of a medicine or health food. In some embodiments, the melatonin is administered in the form of a medicine or health food.

[0016] In some implementations, the improvement of sleep includes, but is not limited to, shortening sleep latency and increasing sleep duration.

[0017] In some implementations, the improvement of mood includes, but is not limited to, relieving anxiety, relieving tension, reducing stress, and increasing mood.

[0018] In some embodiments, the Ashwagandha described in this application is an extract from the roots of Ashwagandha.

[0019] In some embodiments, the Ashwagandha described in this application is an extract from the roots of Ashwagandha; the dosage of the Ashwagandha root extract is 100-1500 mg per day.

[0020] In some embodiments, the *Withania somnifera* described in this application is an extract from the roots of *Withania somnifera*; the dosage of the *Withania somnifera* root extract is 120–1250 mg daily. Journal literature (J Funct Morphol Kinesiol, 2021, 6, 20) has disclosed that daily intake of 120 mg to 1250 mg of *Withania somnifera* can improve bodily functions. The British Pharmacopoeia 2009 includes "Withania Somnifera Root for use in THMP" (THMP = Traditional Herbal Medicinal Product), specifying that the *Withania somnifera* root contains not less than 0.01% withaferin A (C). 28 H 38 O6) and not less than 0.01% anolide A (withanolide, C 29 H 42 O7). Ashwagandha root extract is made from the roots of Ashwagandha, with a concentrated and enhanced content of active ingredients. For example, This is a commercially available Ashwagandha root extract, which can be used as a standalone product or as a supplementary ingredient in various herbal and health food formulations, wherein the withanolide content is approximately 5%. In some embodiments, the Ashwagandha root extract described herein is made from Ashwagandha roots, and the withanolide content in the Ashwagandha root extract is 1.5% to 10% (preferably 2.5% to 6%).

[0021] In some embodiments, the melatonin dosage described in this application is 0.3 to 15 mg per day.

[0022] In some embodiments, the melatonin dosage described in this application is 1–10 mg daily. The drug information leaflet approved by the European Medicines Agency (EMA) indicates that daily doses of melatonin up to 300 mg do not cause significant adverse reactions.

[0023] Unless otherwise specified, the application subject in this application refers to humans.

[0024] The ashwagandha or melatonin described in this application can be present in pharmaceuticals or health foods in unit dose form for ease of administration.

[0025] As used in this application, the term "medicine" means any substance that provides therapeutic, preventative, and / or beneficial effects.

[0026] As used in this application, the term "health food" refers to food that claims to have specific health benefits or is intended to supplement vitamins and minerals. In other words, health food is suitable for consumption by specific groups of people, has the function of regulating bodily functions, is not intended to treat diseases, and does not cause any acute, subacute, or chronic harm to the human body.

[0027] In this application, the drug or health food contains ashwagandha or melatonin as the active ingredient, and a pharmaceutically acceptable carrier and / or excipient. The drug or health food can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use.

[0028] In this application, the medicament may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, intrabladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is oral, oral, or sublingual. Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose. In some preferred embodiments, the medicament is administered orally.

[0029] In this application, the health food product can be administered by any suitable method known in the art, including but not limited to oral, oral, and sublingual administration. In some preferred embodiments, the health food product is administered orally.

[0030] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Absorption delaying agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate).

[0031] The term "morning" in this application should be understood in the context of everyday life. Exemplarily, but not exclusively, "morning" can be understood as the period from sunrise to five hours after sunrise.

[0032] The term "evening" in this application should be understood in everyday life. Exemplarily, but not exclusively, "evening" can be understood as the period from sunset to eight hours after sunset.

[0033] As used in this application, the term "anxiety" refers to a specific mental state involving extreme fear or worry. Symptoms include excessive worry, tension, and fear of future events.

[0034] As used in this application, the term "insomnia" refers to a common health problem that affects the quality and quantity of an individual's sleep. Clinical symptoms of insomnia mainly include difficulty falling asleep, frequent awakenings during the night, early awakenings with difficulty falling back asleep, and feeling tired and having difficulty concentrating after waking up.

[0035] The manifestations of "improving sleep" in this application include, but are not limited to, shortening sleep latency and increasing sleep duration.

[0036] The manifestations of "improving mood" in this application include, but are not limited to, relieving anxiety, relieving tension, relieving stress, and increasing mood; it should be noted that these manifestations are not completely parallel, but rather have a certain degree of overlap.

[0037] As used in this application, the term "combination" includes combination packaging of the substance for combined administration, and also includes combination therapy / method of use of the substance.

[0038] As used in this application, the term "effective amount" means an amount sufficient to improve sleep or mood. In this application, the effective amount of Ashwagandha or melatonin may vary depending on factors such as the mood and sleep status of the subject, the overall condition of the subject, general characteristics such as age, weight and sex, the method of administration of the drug or health food, and other concurrent treatments, etc.

[0039] In summary, compared with the prior art, the above-described technical solutions conceived in this application can achieve the following beneficial effects:

[0040] This application demonstrates through experiments that the combined use of morning application of Ashwagandha and evening application of melatonin can significantly improve sleep and / or mood. Furthermore, the combined effect of morning application of Ashwagandha and evening application of melatonin produces better sleep and / or mood-improving effects than simultaneous evening application of either or either application of either alone. The research findings of this application have significant practical application value in improving sleep and / or mood. Attached Figure Description

[0041] The accompanying drawings, which are provided to further illustrate this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application.

[0042] Figure 1 This study shows the effect of combined administration of Ashwagandha (WS) and melatonin (MT) on body weight in mice (days 1–14).

[0043] Figure 2 This study shows the effects of the combined use of ashwagandha (WS) and melatonin (MT) on total distance (A), average speed (B), and resting time (C) in mice during spontaneous activity experiments.

[0044] Figure 3 This study shows the effects of combined use of ashwagandha (WS) and melatonin (MT) on the open-arm time (A) and number of attempts (B) in the elevated maze test in mice.

[0045] Figure 4 This study demonstrates the effects of the combined use of ashwagandha (WS) and melatonin (MT) on sleep latency (A) and sleep duration (B) in a pentobarbital-induced sleep experiment in mice.

[0046] Figure 5 This study demonstrates the effect of the combined use of ashwagandha (WS) and melatonin (MT) on serum corticosterone (CORT) in mice. Detailed Implementation

[0047] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only used to illustrate this application and are not intended to limit the scope of this application.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0050] The SPF-grade male Kunming (KM) mice weighing 22–24 g used in this experiment were purchased from Liaoning Changsheng Biotechnology Co., Ltd. All mice were kept under normal light / dark (12h / 12h) conditions, with an ambient temperature of 22±2℃, relative humidity of 55%±5%, and in a clean, pathogen-free environment. The animals had free access to food and water. This animal experimental protocol complied with the requirements and relevant regulations of the China Animal Ethics Committee and the Animal Ethics Committee of Shenyang Pharmaceutical University.

[0051] It should be noted that, due to their inherent biological characteristics and habits, rodents have the opposite circadian rhythm to humans. Therefore, the timing of drug administration was adjusted accordingly in the experimental study of this application.

[0052] Example 1

[0053] 1. Experimental Materials and Methods

[0054] 1.1 Test substance

[0055] Ashwagandha (Withania Somnifera, WS) root extract, batch number: AskPharma Inc., 0123089; melatonin (MT), batch number: LN12.

[0056] 1.2 Main Instruments and Reagents

[0057] Main instruments: electronic balance, enzyme-linked immunosorbent assay (ELISA) reader, self-activity instrument, elevated cross maze, etc.

[0058] Main reagents: 0.5% CMC-Na (batch number: MB1717; purchased from Sigma-Aldrich), diazepam (product / batch number: 220506; purchased from Shandong Xinyi Pharmaceutical Co., Ltd.), sodium pentobarbital (purchased from Lebo), corticosterone kit (product / batch number: JL11918; purchased from Jonlnbio).

[0059] 1.3 Experimental Design and Animal Grouping

[0060] SPF-grade male KM strain mice, weighing 22–24 g, were selected and purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0061] After two days of acclimatization, male mice were randomly divided into 6 groups of 12 mice each. The experimental treatments are shown in Table 1: (1) Blank control group (Control, also abbreviated as Con); (2) Melatonin (administered at 9 am) + Ashwagandha (administered at 7 pm) combination group (MT AM + WS PM); (3) Melatonin (administered at 9 am) group (MT AM); (4) Ashwagandha (administered at 7 pm) group (WS PM); (5) Melatonin (administered at 9 am) + Ashwagandha (administered at 9 am) combination group (MT AM + WS AM); (6) Positive drug diazepam (administered at 9 am) group (DZP). Diazepam is a benzodiazepine. This type of drug can inhibit the central nervous system; it has good anti-anxiety, sedative, hypnotic, anticonvulsant and muscle relaxant effects.

[0062] Table 1 Animal grouping and drug administration information

[0063]

[0064] Note: MT stands for melatonin, and WS stands for Ashwagandha.

[0065] This experiment used normal mice to test anxiolytic and sleep indicators after gavage administration of the test substance. All mice were administered the test substance twice daily (9:00 AM and 7:00 PM), and an equal volume of 0.5% CMC-Na was given at non-test substance time points. The blank control group received 0.5% CMC-Na at 9:00 AM and 7:00 PM. Spontaneous activity, elevated maze, and pentobarbital-induced sleep tests were performed on days 14, 15, and 16 after the first administration. The drug administration lasted for 17 days.

[0066] 1.4 Weight Measurement

[0067] Weigh yourself daily and record and analyze the data.

[0068] 1.5 Behavioral Indicator Testing

[0069] (1) Autonomous activity experiment: 1 hour after administration at 19:00 on the 7th or 14th day after the first administration, the experiment was conducted using the autonomous activity instrument computer real-time detection, analysis and processing system. The experiment was conducted in a quiet environment. The mice were placed in the autonomous activity box and tested after 1 minute of adaptation. The experimental process was recorded using video recording equipment. The total journey, average speed and rest time of each group of mice within 5 minutes were recorded. Between the two experiments, the inner wall and bottom of the test box were sprayed and cleaned with a low concentration of ethanol solution.

[0070] (2) Elevated Cross Maze Experiment: The elevated cross maze was designed with arms 30cm long and 5cm wide, closed arms 15cm high, and the track 60cm above the ground. The walls and bottom of the elevated cross maze were made of white polymethyl methacrylate. On days 8 and 15 after the first administration, 1 hour after administration at 7 PM, mice were placed on the central platform with their heads facing the open arms, allowing them to freely explore the four arms for 5 minutes, and their activity was recorded. Observational indicators included: time spent in open arms and frequency of open arms.

[0071] (3) Pentobarbital-induced sleep experiment: The experiment was conducted in a dark environment with the room temperature maintained at around 30℃. Mice were fasted for 12 hours before the experiment, but allowed free access to water. On the mornings of the 9th and 16th days of the experiment, 30 minutes after administration of the drug, mice were induced to sleep with sodium pentobarbital at 46 mg / kg (intraperitoneal injection). Timing was started after injection, and the time from intraperitoneal injection to the disappearance of the righting reflex was observed and recorded for each mouse, i.e., sleep latency (SL). After the righting reflex disappeared, the mice were placed on a black foam pad with their abdomens facing up, and the time from the disappearance of the righting reflex to its recovery was observed and recorded, i.e., sleep time (ST).

[0072] 1.6 Biochemical Indicator Detection

[0073] Blood samples were collected from the fundus venous plexus on day 17 after the first administration. 0.5 ml of blood was collected, incubated at room temperature for 2 hours, centrifuged according to the instructions, and the serum was directly measured. Serum corticosterone levels were determined according to the instructions of the corticosterone kit (batch number: JL11918).

[0074] 1.7 Experimental Data Statistics

[0075] Experimental results are expressed as mean ± standard deviation (Mean ± SE), and statistical analysis was performed using software. One-way ANOVA was used for comparisons between groups, with P < 0.05 considered statistically significant.

[0076] 2. Experimental Results

[0077] 2.1 Effects of combined use of ashwagandha and melatonin on body weight in different groups of mice

[0078] See weight test results Figure 1 .Depend on Figure 1 It can be seen that during the administration period, the weight of mice in each group increased gradually, and there was no significant difference in weight among the groups. This result indicates that there were no obvious toxic side effects in each group within this dosage range. The collective weight loss on the 9th day was because the mice were fasted but allowed to drink water the night before (the pentobarbital-induced sleep experiment described in 1.5(3) was conducted on the 9th day).

[0079] 2.2 Effects of combined use of ashwagandha and melatonin on total distance, average speed, and resting time in spontaneous activity experiments of mice in each group.

[0080] The spontaneous activity test can detect spontaneous activity and exploratory behavior in mice, and is a method for evaluating the spontaneous behavior, exploratory behavior, and stress level of laboratory animals in novel environments. The results of the spontaneous activity test are shown in Table 2 and... Figure 2 Table 2. Effects of combined use of Ashwagandha (WS) and melatonin (MT) on total distance, average speed, and resting time in spontaneous activity experiments of mice in each group.

[0081] Group Total distance (mm) Average speed (mm / s) Static time (s) Con 11598.02±3260.86 38.66±10.87 41.07±38.23 MT AM+WS PM 11885.49±3089.62 39.62±10.30 39.14±33.71 MT AM 10385.86±3481.77 34.62±11.60 50.04±38.82 WS PM 12819.97±1432.19 42.73±4.77 21.87±9.32 MT AM+WS AM 10974.05±3717.89 36.58±12.39 44.72±46.42 DZP 10897.79±3471.64 36.33±11.57 44.51±47.44

[0082] Table 2 shows (data from day 14) that, compared with the control group, none of the drug-treated groups had a significant effect on the total distance traveled, average speed, and resting time during spontaneous activities. This result indicates that the drug administration at this dose does not affect spontaneous motor behavior. Figure 2 The image shows the effects of the combined use of ashwagandha (WS) and melatonin (MT) on the total distance (A), average speed (B), and rest time (C) of mice in the spontaneous activity experiment.

[0083] 2.3 Effects of combined use of ashwagandha and melatonin on the time and number of times mice entered the open arm phase in the elevated maze experiment.

[0084] The cross-maze test utilizes the exploratory nature of animals towards novel environments and their fear of open arms to create a contradictory conflict, evaluating the anxiolytic or anxiolytic effect of test substances. Normal mice maintain some exploratory activity with open arms, while anxious animals exhibit reduced exploratory activity with open arms. The results of the cross-maze test are shown in Table 3 and... Figure 3 .

[0085] Table 3. Effects of combined use of Ashwagandha (WS) and melatonin (MT) on arm-opening time and number of attempts in the elevated maze experiment in each group of mice.

[0086] Group Arm opening time (s) Number of times the arm is opened Con 18.08±14.61 3.08±2.56 MT AM+WS PM 46.62±32.73* 6.92±3.69** MT AM 30.50±32.06 <![CDATA[3.92±3.04 # ]]> WS PM 28.82±32.85 4.73±3.86 MT AM+WS AM 30.00±26.18 4.40±4.18 DZP 45.08±18.23* 6.42±3.09*

[0087] Note: Compared with the Con group, ** represents p<0.01, * represents p<0.05; compared with the MT AM+WS AM group, # represents p<0.05.

[0088] Table 3 (data from day 15) shows that, compared with the control group, the "MT AM+WS PM" group and the "DZP" group significantly increased the time mice spent in the open arm and the number of times mice entered the open arm (p<0.05), suggesting that the combination of "MT AM+WS PM" and diazepam has an anti-anxiety effect on mice. There were no significant differences between the other groups and the control group.

[0089] Compared with the "MT AM", "WS PM" and "MT AM+WS AM" groups, the "MT AM+WS PM" group had a significantly increased number of open arm movements, suggesting that the "MT AM+WS PM" group had a better anti-anxiety effect than the "MT AM", "WS PM" and "MT AM+WS AM" groups. Figure 3 The image shows the effect of the combined use of ashwagandha (WS) and melatonin (MT) on the arm-opening time (A) and number of times (B) in the elevated maze experiment in each group of mice.

[0090] 2.4 Effects of combined use of ashwagandha and melatonin on sleep latency and sleep duration in pentobarbital-induced sleep experiments in mice of different groups

[0091] The pentobarbital-induced sleep assay was used to detect the sleep-improving effect of the test substance. If the test substance significantly prolonged pentobarbital-induced sleep duration or significantly shortened sleep latency, it indicated that the test substance had a sleep-improving effect at that dose. The experimental results are shown in Table 4 and... Figure 4 .

[0092] Table 4. Effects of combined use of Ashwagandha (WS) and melatonin (MT) on sleep latency and sleep duration in pentobarbital-induced sleep experiments in mice of different groups.

[0093] Group Sleep latency (min) Sleep duration (min) Con 10.66±7.71 19.45±11.62 MT AM+WS PM 7.80±3.51* 41.18±17.60** MT AM 9.45±3.62 26.33±16.33# WS PM 10.12±3.09 30.69±14.43 MT AM+WS AM 9.49±3.60 34.38±15.75* DZP 7.66±1.66* 47.03±18.86***

[0094] Note: Compared with Con, *** represents p<0.001, ** represents p<0.01, and * represents p<0.05; compared with the MT AM+WS AM group,

[0095] # indicates that p < 0.05.

[0096] Table 4 (data from day 16) shows that, compared with the control group, the "MT AM+WS PM" group and the "DZP" group significantly shortened the sleep latency of mice (p<0.05) and significantly increased the sleep time of mice (p<0.05); the "MT AM+WS AM" group had the effect of prolonging sleep time (p<0.05) but had no effect on sleep latency; the "MT AM" group and the "WS PM" group had no significant effect on sleep latency or sleep time. The sleep time of the "MT AM" group, the "WS PM" group, and the "MT AM+WS AM" group was significantly lower than that of the "MT AM+WS PM" group (p<0.05), suggesting that the "MT AM+WS PM" group had the strongest sleep-improving effect, which was better than that of single drug administration or administration of both drugs at the same time point. Figure 4 The image shows the effects of the combined use of ashwagandha (WS) and melatonin (MT) on sleep latency (A) and sleep duration (B) in pentobarbital-induced sleep experiments in mice of different groups.

[0097] 2.5 Effects of combined use of ashwagandha and melatonin on serum corticosterone levels in mice of different groups

[0098] Corticosterone is a 21-carbon steroid hormone produced by the adrenal cortex, also known as adrenocorticosteroid. Because stress increases the production of corticosteroids, corticosterone is a major indicator of stress levels. In addition to its role in stress, corticosterone is also believed to influence human sleep and wakefulness. Experimental results are available in [link to experimental results]. Figure 5 See Table 5.

[0099] Table 5. Effects of combined use of Ashwagandha (WS) and melatonin (MT) on serum corticosterone (CORT) levels in mice of different groups.

[0100] Group CORT (ng / mL) Con 26.89±17.41 MT AM+WS PM 8.12±5.34** MT AM 13.13±18.40 WS PM 11.17±11.26* MT AM+WS AM 9.88±4.97* DZP 6.72±2.87**

[0101] Note: When comparing with Con, ** represents p<0.01, and * represents p<0.05.

[0102] As shown in Table 5 (data from day 17), Ashwagandha and melatonin significantly reduced serum corticosterone levels in mice. Among them, the "MT AM+WS PM" group had a more significant effect on mouse corticosterone compared to the "MT AM" group, the "WS PM" group, and the "MT AM+WS AM" group. This result suggests that the combined use of "MT AM+WS PM" may have a stronger effect on improving sleep. Figure 5 The effect of combined use of ashwagandha (WS) and melatonin (MT) on serum corticosterone (CORT) levels in mice of different groups is shown.

[0103] In summary, the combination of evening administration of *Ashta prostrata* and morning administration of melatonin, after continuous administration, significantly increased the time mice spent in the open arm and the number of times they entered the open arm, suggesting an anti-anxiety effect in mice. It also significantly shortened sleep latency, significantly increased sleep duration, and significantly reduced serum corticosterone levels, indicating a sleep-improving effect in mice. For mice, the sleep-improving and / or mood-improving effects of evening administration of *Ashta prostrata* combined with morning administration of melatonin were superior to those of simultaneous morning administration of either drug or administration of either drug alone. While simultaneous morning administration of *Ashta prostrata* combined with morning administration of melatonin also showed sleep-improving and / or mood-improving effects in mice, and the effect was superior to that of either drug alone, it was significantly less effective than the combination of evening administration of *Ashta prostrata* combined with morning administration of melatonin, suggesting that evening administration of *Ashta prostrata* combined with morning administration of melatonin may be the optimal method for improving sleep and / or mood in mice.

[0104] The findings of this application provide a theoretical basis for improving sleep and / or mood. Considering that the circadian rhythms of rodents are opposite to those of humans, this study suggests that for humans, morning application of Ashwagandha combined with evening application of melatonin is the optimal method for improving sleep and / or mood.

[0105] Example 2: Sample combination package product for oral administration

[0106] This is a combination packaged product intended to improve sleep and / or mood. It is a health food product. The combination package contains one or more bottles of Ashwagandha supplement and one or more bottles of melatonin supplement. The instructions for use state that Ashwagandha should be applied in the morning in conjunction with melatonin in the evening.

[0107] Example 3: Sample Combination Packaging Product for Oral Use

[0108] This is a combination packaged product intended to improve sleep and / or mood. It is a health food product. The combination package is in blister packaging, with each blister pack containing one or more capsules (or one or more tablets) of Ashwagandha and one or more capsules (or one or more tablets) of melatonin. The instructions for use state that Ashwagandha should be used in the morning in conjunction with melatonin in the evening.

[0109] It should be noted that typical preferred embodiments of this application are disclosed in this specification. The above examples are only used to illustrate the technical solutions of this application and are not intended to limit it. Although this application has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of this application as needed, without departing from the spirit and scope of the technical solutions of this application.

Claims

1. Use of a combination of Ashwagandha and melatonin in the preparation of a medicine or health food, wherein Ashwagandha is applied in the morning and melatonin is applied in the evening.

2. The use as described in claim 1, wherein the Ashwagandha is present in the form of a medicine or health food, and the melatonin is present in the form of a medicine or health food.

3. A combination package containing Ashwagandha and melatonin, wherein the Ashwagandha is present in the form of a medicine or health food, and the Ashwagandha is applied in the morning; and The melatonin is present in the form of a drug or health food, and is applied at night.

4. A combination package containing Ashwagandha and melatonin, wherein the Ashwagandha is present in the form of a medicine or health food, and the melatonin is present in the form of a medicine or health food; The combination package contains a carrier with instructions for use, which recommend that Ashwagandha be applied in the morning and melatonin be applied in the evening.

5. The combination package as described in claim 3 or 4, wherein the combination package is used to improve the sleep and / or mood of the recipient.

6. The combination package as described in claim 3 or 4, wherein the Ashwagandha is an Ashwagandha root extract.

7. The combination package as described in claim 6, wherein the dosage of the Ashwagandha root extract is 100-1500 mg daily.

8. The combination package of claim 7, wherein the dosage of the Ashwagandha root extract is 120-1250 mg daily.

9. The combination package as described in claim 3 or 4, wherein the melatonin dosage is 0.3 to 15 mg per day.

10. The combination package of claim 9, wherein the melatonin dosage is 1 to 10 mg per day.

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