Carbon dot with nerve soothing effect

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of oyster carbon dots (carbon quantum dots / nano particles / nano particles / nano components) in preparation of a medicine with a nerve soothing effect. Animal experiments show that the oyster carbon dots have obvious sleep-aiding, calming and anxiety-resisting effects.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the preparation and application of oyster carbon dots (carbon quantum dots / nanoparticles / nanoparticles / nano-type components). Background Art

[0002] Anxiety disorder is a common mental illness in clinical practice, which is mainly manifested as uneasiness, worry and even fear, often accompanied by cognitive or behavioral patterns such as avoidance and hesitation. Its onset is often closely related to environmental changes and social pressure. The current treatments for anxiety disorder mainly include drug therapy, psychological behavioral therapy and traditional Chinese medicine treatment. Serotonin reuptake inhibitors and serotonin / norepinephrine reuptake inhibitors are currently commonly used drugs in clinical practice, but they have disadvantages such as long treatment cycles and obvious side effects that affect patient compliance. Therefore, new drugs with high efficacy and low toxicity and side effects are urgently needed in clinical practice.

[0003] Oysters are the shells of the long oyster Ostrea gigas Thunberg, Dalian Bay oyster Ostreatalienwhanensis Crosse or Ostrea rivularis Gould, which are animals of the oyster family. They have the effects of calming the mind, subduing yang and replenishing yin, softening and dispersing lumps. In traditional Chinese medicine, they are used for palpitations, insomnia, dizziness, tinnitus, scrofula, phlegm, and masses. Calcined oysters are astringent and firm. They are used for spontaneous sweating, night sweats, spermatorrhea, metrorrhagia, stomachache and acid regurgitation. However, the material basis of the calming effect of oysters has not yet been clearly revealed, which affects the wide application of oysters in clinical practice. Summary of the invention

[0004] Definition of terminology: Oyster carbon dots (carbon quantum dots / nanoparticles / nanoparticles / nano-like components) are a new type of nanomaterial with a particle size of less than 10 nm that is separated, purified and extracted from oysters.

[0005] In view of this, the purpose of the present invention is to provide a safe and efficient tranquilizer - oyster carbon dots (Oy-CDs), which has good biosafety. Animal experiments have found that oyster carbon dots have obvious sleep-inducing, sedative and anti-anxiety effects.

[0006] Specifically, the present invention is implemented as follows: the use of oyster carbon dots (carbon quantum dots / nanoparticles / nanoparticles / nano-type components) in the preparation of sedative drugs, the preparation of oysters includes the following steps:

[0007] Raw oysters are taken, crushed, and boiled with water to obtain an aqueous solution containing Oy-CDs. The solution is further filtered, the filtrate is concentrated, and the concentrate is purified by dialysis or ultrafiltration to obtain a purified Oy-CDs aqueous solution.

[0008] The particle size of the Oy-CDs is 1-9 nm.

[0009] The Oy-CDs are composed of a carbon skeleton with a lattice spacing of about 0.2 nm, including but not limited to C elements, N elements and O elements.

[0010] The Oy-CDs are used in preparing sedative drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images of Oy-CDs, 1A: morphological characteristics of Oy-CDs under transmission electron microscopy; 1B: frequency distribution of particle size of Oy-CDs; 1C: morphological characteristics of Oy-CDs under high-resolution transmission electron microscopy; 1D: lattice spacing measurement diagram of Oy-CDs.

[0012] Figure 2 :Infrared spectrum of Oy-CDs

[0013] Figure 3 :Oy-CDs UV absorption spectrum

[0014] Figure 4 : Fluorescence spectra of Oy-CDs, 4A: excitation spectrum and emission spectrum; 4B: emission spectrum of Oy-CDs at different excitation wavelengths.

[0015] Figure 5 : XPS spectra of Oy-CDs, 5A: XPS full scan spectrum; 5B: C 1s; 5C: O 1s; 5D: N 1s and 5E: high-resolution spectrum of Ca2p.

[0016] Figure 6 :Effects of Oy-CDs on HPA axis hormones in mice with acute restraint stress model, 6A: serum corticotropin-releasing hormone (CRH); 6B: serum adrenocorticotropic hormone (ACTH); 6C: serum corticosterone (CORT); 6D: serum norepinephrine (NA).

[0017] Figure 7 : Anti-anxiety effect of Oy-CDs on chronic restraint stress model mice in the open field test, 7A: total distance walked by mice in the open field; 7B: frequency of mice entering the central area; 7C: cumulative time mice stayed in the central area; 7D: movement routes and activity heat map of mice in the open field.

[0018] Figure 8: Anti-anxiety effect of Oy-CDs on chronic restraint stress model mice in elevated plus maze experiment, 8A: total distance walked by mice in elevated plus maze; 8B: frequency of mice entering each area; 8C: cumulative time mice stayed in each area; 8D: proportion of time mice stayed in the open arms of elevated plus maze; 8E: proportion of frequency of mice entering the open arms of elevated plus maze; 8F: movement routes and activity heat maps of mice in elevated plus maze compared with those of control group.

[0019] Fig. 9 :Effects of Oy-CDs on HPA axis hormones in chronic restraint stress model mice, 9A: serum corticotropin-releasing hormone (CRH); 9B: serum adrenocorticotropic hormone (ACTH); 9C: serum corticosterone (CORT); 9D: serum norepinephrine (NA).

[0020] Fig.10 : Condition of neurons in hippocampal tissue of mice with chronic restraint stress model, 10A: HE staining; 10B: Nissl staining, scale bar = 500 μm; 10C: average optical density (AOD) of Nissl bodies in hippocampal tissue.

[0021] Fig.11 : Expression of BDNF and CREB proteins in the hippocampus of mice with chronic restraint stress model, 11A: BDNF positive expression in the hippocampus, CA3 and DG regions; 11B: CREB positive expression in the hippocampus, CA3 and DG regions (scale bar in hippocampus = 500 μm, scale bar in CA3 = 100 μm, scale bar in DG = 100 μm). DETAILED DESCRIPTION

[0022] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0023] Example 1: Extraction and purification of carbon dots from oysters

[0024] Crush the raw oysters, add water, and boil for 1 hour. The cooled solution is filtered with a 0.22μm microporous membrane. The filtrates are combined, concentrated, and centrifuged to remove the precipitate. The supernatant is dialyzed with a 1000Da dialysis bag, and the water outside the bag is replaced every day for at least 3 days. The dialysis solution in the dialysis bag is the purified Oy-CDs aqueous solution. In addition, take the supernatant after centrifugation and ultrafilter it with a 1wDa ultrafiltration tube. The part in the ultrafiltration tube is the purified Oy-CDs aqueous solution.

[0025] Electron microscopy technology (high and low resolution electron microscopy) and optical technology (ultraviolet visible light analysis technology, fluorescence spectroscopy analysis technology, infrared spectroscopy analysis technology) are used to characterize and analyze the morphological characteristics, functional group changes and other physical and chemical characteristics of Oy-CDs.

[0026] Figure 1 TEM and HRTEM of Oy-CDs show uniformly distributed nanoparticles, with a particle size distribution range of 0.8-2.7 nm and a lattice spacing of 0.228 nm.

[0027] Figure 2 :Infrared spectrum of Oy-CDs, oyster nano-components at 3441cm -1 、2900cm -1 、2921cm -1 With 2851cm -1 、1631cm -1 、1384cm -1 and 1122cm -1 A series of characteristic absorption peaks appear at the NH4+ / -OH bond, -CH bond, C=O bond, CN bond, and COC bond, respectively.

[0028] Figure 3 : Oy-CDs UV absorption spectrum, there is a weak absorption peak at 261.4nm.

[0029] Figure 4 : Fluorescence spectrum of Oy-CDs. Oy-CDs has fluorescence properties.

[0030] Figure 5 : XPS spectrum of Oy-CDs, C 1s, O 1s, N 1s and Ca 2p correspond to four peaks, which are detected at 285.2, 532.1, 400.0 and 347.6 eV, respectively.

[0031] Example 2: Experimental study on the synergistic effect of sodium pentobarbital on sleep using oyster nanoparticles

[0032] 1. Modeling and drug administration

[0033] Thirty mice were weighed and randomly divided into model group, diazepam group, Oy-CDs high-dose group, Oy-CDs medium-dose group, and Oy-CDs low-dose group according to the random number table method. After 3 days of adaptive feeding of mice, except for the model group and diazepam group, which were given the same volume of NS, the other groups were given the corresponding dose of Oy-CDs solution for 7 consecutive days.

[0034] On the 8th day, the model was established. In a quiet environment, the diazepam group was given diazepam solution by gavage, the model group was given the same volume of NS by gavage, and the other groups were given the corresponding Oy-CDs solution by gavage. After 30 minutes, each group of mice was intraperitoneally injected with sodium pentobarbital (deionized water as solvent), and the sleeping conditions of the mice were observed and recorded, and the sleeping latency and sleeping time of the mice were counted. (The disappearance of the righting reflex for more than 1 minute was used as the standard for falling asleep)

[0035] 2. Test results

[0036] As shown in Table 1, the sleep-inducing effect of Oy-CDs was verified by the sodium pentobarbital threshold dose synergistic sleep experiment. After intraperitoneal injection of sodium pentobarbital, all mice in each group entered a sleep state. The sleep latency of each group of mice pre-administered with Oy-CDs was shortened to a certain extent compared with the control group. The sleep latency of the high-dose group was significantly shortened, and the sleep latency of the medium-dose group was also significantly shortened. In terms of sleep time, the sleep time of the high-dose and medium-dose groups of Oy-CDs was longer than that of the control group. According to the results of this experiment, the sleep-inducing effect of medium and high doses of Oy-CDs is better than that of low doses of Oy-CDs.

[0037] Table 1 Results of the Oy-CDs sodium pentobarbital threshold dose sleep synergy experiment on the sleep of mice

[0038]

[0039] Note: Compared with the control group, * P<0.05, ** P<0.01, *** P<0.001.

[0040] Example 3: Study on sedation, analgesia and antianxiety of oyster carbon dots in acute restraint stress model

[0041] 1 Animal grouping, modeling and drug administration

[0042] Thirty mice were divided into four groups, with six mice in each group, namely, the control group, the model group, the fluoxetine group, and the Oy-CDs group. After three days of adaptive feeding of mice, the control group and the model group were given deionized water, the fluoxetine group was given fluoxetine solution, and the Oy-CDs group was given Oy-CDs solution, and the pre-administration was continued for 7 days.

[0043] Modeling was performed on the 8th day. Each group was gavaged with the corresponding drugs 30 minutes before modeling. Take a 50mL centrifuge tube, use a heated electric soldering iron to make 3 small holes at the bottom and middle of the centrifuge tube, and make a small hole in the centrifuge tube cap to prevent the mouse from suffocating. Put the mouse into the centrifuge tube, tighten the tube cap and expose the mouse tail through the small hole. Restrict the activity of the mouse so that it cannot move forward, backward, left or right. Put the mouse into the restraint device, tilt the centrifuge tube slightly, and make the mouse head slightly upward to prevent the mouse from aspiration of urine due to anxiety. The modeling time is from 10 to 13 o'clock. After modeling, take the mouse out of the restraint device.

[0044] 2. Serum index detection

[0045] Blood was collected from the eyeballs of mice, and the blood was centrifuged at 3000 rpm for 10 minutes after standing to obtain serum. NA, CRH, ACTH, and CORT ELISA kits were used to detect the content of relevant indicators in the serum of mice.

[0046] Figure 6 A shows the CRH level. Compared with the model group, the level in the fluoxetine group was lower, and there was a downward trend in the Oy-CDs group. Figure 6 C shows CORT levels, which were lower in fluoxetine group and Oy-CDs group than in model group. Figure 6 B shows the ACTH level. There was a significant difference between the model group and the fluoxetine group, and the control group and Oy-CDs group decreased. Figure 6 D is the serum NA level, which is regulated by the HPA axis system. The levels in the control group, fluoxetine group and Oy-CDs group were higher than those in the model group.

[0047] Example 4: Study on sedation and anxiolysis of oyster carbon dots in chronic restraint stress model

[0048] 1 Animal grouping, modeling and drug administration

[0049] 30 mice were divided into 4 groups, 6 mice in each group, namely control group, model group, fluoxetine group, and Oy-CDs group. After 3 days of adaptive feeding of mice, the control group and model group were given the same volume of deionized water, the fluoxetine group was given fluoxetine solution, and the Oy-CDs group was given Oy-CDs solution, and drug administration began on the first day of modeling. An open field test was performed on the 15th day of modeling, and an elevated plus maze test was performed on the 16th day. Fasting but not water was allowed to begin in the afternoon of the 17th day, and fasting for 24 hours was performed. A new environment food intake inhibition experiment was performed on the 18th day. The last modeling was performed on the 19th day, and the modeling time was from 7 to 10 o'clock. The corresponding drugs were given to each group by gavage 30 minutes before modeling.

[0050] The restraint device was prepared in accordance with the device of the acute restraint stress model, and the activity of the mice was restricted so that they could not move forward, backward, left, or right. The mice were placed in the restraint device, and the centrifuge tube was slightly tilted so that the head of the mice was slightly upward to prevent urine reflux and aspiration due to anxiety. Three hours after modeling every day, from 13:00 to 16:00 in the afternoon, no food or water was allowed during the modeling period. After the modeling was completed, the mice were removed from the restraint device.

[0051] The mice were placed in an opaque container for 60 minutes of adaptation before the open field behavioral test. The parameters such as the time the mice stayed in the center of the open field and the total movement distance were evaluated to reflect the anxiety level. The results were analyzed using EthoVision XT 7 software.

[0052] The mice were placed in a container consisting of two opposite non-transparent open arms and two opposite closed arms in a cross shape for an elevated plus maze behavioral experiment. The time the mice spent in the open arms was evaluated to reflect the anxiety level of the mice. The results were analyzed using EthoVision XT 7 software.

[0053] In a quiet and bright environment, mice were placed in a container for a novel environment food inhibition experiment, with food placed in the center of the container. The mice were fasted for 24 hours, and after 3 hours of restraint stress modeling, the mice were placed in the corner of the container, and the time when the mice approached the food and started to eat was recorded, which was an indicator of measuring the anxiety level of the mice.

[0054] Blood was collected from the eyeballs of mice, and the blood was centrifuged at 3000 rpm for 10 minutes after standing to obtain serum. NA, CRH, ACTH, and CORT ELISA kits were used to detect the content of relevant indicators in the serum of mice.

[0055] The mice were killed and decapitated after blood was drawn, and the brain tissue was completely removed and fixed in 4% paraformaldehyde. The neuronal cells in the hippocampus of the mouse brain tissue were observed through H&E sections; the number and structure of the mouse neuronal cells were observed through Nissl chromosomes; and the expression of BDNF and CREB proteins was observed by immunohistochemistry.

[0056] 2Test results

[0057] 2.1 Behavioral observation of mice in the chronic restraint stress model

[0058] like Figure 7As shown in A, chronic restraint stress affected the exploratory ability of mice in the open field test. In terms of the total distance traveled by mice in the open field, compared with the model group, the total distance traveled by mice in the fluoxetine group (1931.99±371.90cm, P<0.01) and the Oy-CDs group (2171.36±396.93cm) decreased, which was similar to the control group. Measuring the frequency of mice entering the central area in the open field test is one of the important indicators for measuring the anxiety state of mice. Figure 7 As shown in B, the crossing frequency of mice in each drug-treated group increased, with 37.0±5.8 times in the fluoxetine group and 40.8±11.6 times in the Oy-CDs group (P<0.05). Figure 7 C shows that after treatment with fluoxetine (44.57±6.44s, P<0.05) and Oy-CDs (39.55±9.43s), the mice's desire to explore increased and they stayed in the central area longer. Figure 7 D shows the movement trajectory of mice in each group in the open field (red is the movement route of mice, and the inner gray area is the central area) and the activity heat map (red indicates that the proportion of mice staying here is higher, blue indicates that the proportion of mice staying here is lower, and the inner white frame line is the central area). The activity range of mice in the fluoxetine group and the Oy-CDs group is close to the surrounding area, and the number of crossings in the central area is more than that in the model group.

[0059] like Figure 8 As shown in A, the total distance traveled by the chronic restraint stress model mice in the elevated plus maze was 1634.76±133.37cm. Compared with the model group, fluoxetine could significantly increase the activity distance of mice in the elevated plus maze (2113.77±176.72cm, P<0.001). The intervention of Oy-CDs (2069.82±153.49cm, P<0.01) also significantly increased the activity distance of mice. In terms of the number of times mice entered the open arms on the left and right sides, compared with the model group, the frequency of entering the open arms in the fluoxetine group (29.5±8.2 times) increased significantly; the frequency of entering the open arms in the Oy-CDs group (22.0±5.9 times) showed an increasing trend compared with the model group. In addition, the frequency of mice in the fluoxetine group entering each area (open arms, central area, closed arms) increased significantly, indicating that the mice in the fluoxetine group had a stronger desire to explore. As shown in Figure 1 Figure 8As shown in C, the time the model group mice stayed in the open arms and central area was significantly reduced, while the time they stayed in the closed arms was significantly increased. Fluoxetine significantly reduced the time the mice stayed in the closed arms. Oy-CDs had a certain pharmacodynamic trend, but there was no statistical difference. OT% [(Open arm stay time OT% = open arm stay time OT / (open arm stay time OT + closed arm stay time CT) × 100%] and OE% [(Open arm entry OE% = open arm entry OE / (open arm entry OE + open arm entry CE) × 100%] are one of the main indicators of the elevated plus maze experiment. Figure 8 As shown in D and 8E, compared with the model group, the OT% in the fluoxetine group was 21.90%±6.41%; the OT% in the Oy-CDs group (15.85%±6.27%, P>0.05) increased to a certain extent. The OE% values ​​in the fluoxetine group (41.72%±9.72%) and the Oy-CDs group (45.43±10.70%) showed an increasing trend compared with the model group, with no statistical difference. Figure 8 F is the activity route map and heat map of mice in the elevated plus maze. The combined analysis of the two shows that the mice in the fluoxetine group and the Oy-CDs group have a tendency to explore the open arms compared with the model group. This shows that pre-medication can improve the exploratory behavior of mice in the elevated plus maze.

[0060] In the 300-second experiment, all mice in the model group did not eat in the new environment. The feeding latency of mice in each drug-treated group was shortened to varying degrees compared with the control group, which was significantly different from the model group. The feeding latency of mice in the fluoxetine group was 83.25±90.50s, and the feeding latency of mice in the Oy-CDs group was 85.63±39.37s.

[0061] 2.2 Effects of oyster carbon dots on HPA axis hormones in chronic restraint stress model mice

[0062] Chronic stress is closely related to the HPA axis. Fig. 9 As shown in A, after treatment with fluoxetine and Oy-CDs, the serum CRH level of mice was reduced, with statistical significance (P < 0.01). Fig. 9 B shows the serum ACTH level, which has the same trend as the CRH level. ACTH in the fluoxetine group and Oy-CDs group was significantly lower than that in the model group.

[0063] CORT is closely related to anxiety and can be used as a modeling drug that directly induces anxiety. In this experiment, it was significantly reduced after fluoxetine treatment, while the Oy-CDs group only showed a downward trend. Compared with the control group, serum NA in the model group decreased, Oy-CDs caused serum NA to increase, and fluoxetine showed an upward trend.

[0064] 2.3 Status of neurons in the hippocampus of the brain tissue of mice with chronic restraint stress model

[0065] like Fig.10 A shows that HE staining was used to observe the neuronal cells in the hippocampus of mouse brain tissue. The neuronal cells in the control group were neatly arranged, while the neuronal nuclei in the model group were condensed, the cytoplasm was concentrated, and the arrangement was disordered. The other groups were in between. Fig.10 As shown in B, Nissl bodies show the number and structure of mouse neuron cells. The number of Nissl bodies in the hippocampus tissue of the model group mice was reduced compared with the control group, the color became lighter, and the volume of neurons was reduced. The average optical density of Nissl bodies (AOD = IOD / Area) was counted using ImageJ software, and the number of Nissl bodies in the hippocampus tissue of the fluoxetine group mice was significantly higher than that of the model group.

[0066] 2.4 Expression of BDNF and CREB proteins in chronic restraint stress model mice

[0067] Immunohistochemistry was used to observe the expression of BDNF and CREB proteins. Fig.11 As shown in the figure, the cumulative optical density (IOD) of BDNF and CREB expression in the model group hippocampus was significantly lower than that in the control group (P < 0.01), especially the protein expression in CA3 region was significantly different. The expression of BDNF and CREB in the fluoxetine group was significantly higher than that in the model group (P < 0.01), and Oy-CDs significantly increased the expression of BDNF and CREB in the hippocampus of mice (P < 0.01, P < 0.05), indicating that the anxiolytic effect of Oy-CDs was positively correlated with the expression of BDNF / CREB in the hippocampus of mice.

Claims

1. Application of oyster carbon dots in the preparation of sedative drugs.

2. The use according to claim 1, characterized in that The tranquilizing effect includes sleep-aiding, sedative and anti-anxiety effects.

3. The use according to claim 1, characterized in that The oyster carbon dots and pharmaceutically acceptable excipients are prepared into clinical preparations; the clinical preparations include but are not limited to injections, oral liquid preparations, hard capsules, soft capsules, tablets, suppositories, water pills, honey pills, concentrated pills, ointments, powders, emulsions, pastes, and smears.

4. The use according to claim 1, characterized in that Use with other drugs or alone.

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