CC BY
9No. ifl(min) MW(Da) Fragmentions
Identification Formula
glucuronic acid glycosiders
22 4.745 405.2175 225.1488, 179.0974 - -
23 4.799 373.189 193.1218 - -
24 4.927 461.0755 285.038 Kaempferol-3- C21H18O12
beta-O-
glucuronide
25 5.149 338.1955 130.0827 - -
26 5.298 481.17 315.0763, 152.0110, 108.0152 - -
27 5.467 521.2597 503.2419, 389.2181, 371.2088, Alangionoside B C24H42O12
227.1548
28 5.546 301.0394 503.2419, 389.2181, 371.2088, Quercetin C15H10O7
227.1548
29 5.867 389.2169 371.2112, 227.1644, 209.1576, Rehmaionoside A C19H34O8
181.1403
30 6.399 299.0531 284.0296, 255.0330, 227.0333 3-methyl C16H12O6
keampferol
31 7.078 373.1759 355.2752, 275.2171, 96.9665, - -
79.9550
Study on the Mechanism of Sleep Improving Function of Root of Tall Oplopanax Effective Parts Based on the Regulation Mechanism of Cytokine-
theory
Yu Shuang Li Tingli Huang Lili
(College of Pharmacy of Heilongjiang University of Chinese Medicine, Harbin, China)
Abstracts: This experiment based on cytokine regulating mechanism to explore the mechani sm of sleep improving function of the effective parts of Root of Tall Oplopanax. In experiment, mice of ICR specie are chose, and enzyme-linked immunoassay method is applied to determine the sleep related cytokine content in ICR mice serum and brain and to research on the sleep improving mechanism of the effective parts of Root of Tall Oplopanax(64g/kg).
The results show that the mice, after have been given the effective parts of Root of Tall Oplopanax(64 g/kg) for 7 days, express an obvious increase of IL - 1 beta (p < 0.05) and TNF alpha (p < 0.01) content in hypothalamus. An increase of TNF alpha (p < 0.05) and IL - 4 (p < 0.05) in pallium, a significant decrease of IL-10 level in hypothalamus (p<0.01) and a decrease of IL-4 (p<0.05), IL-10 (p<0.05) level in serum are also found. The experimental results prove that the sleep improving function of the effective parts of Root of Tall Oplopanax is associated with cytokines levels in the brain. It plays a role of sleep improving by increasing the content of sleeping cytokines (IL-ip * TNF-a), and reducing the content of sleep inhibiting cytokines (IL-4* IL-10).
Key words: Root of Tall Oplopanax; Sleep; Cytokines
Root of Tall Oplopanax is one of Araliaceae oplopanax miq perennial deciduous shrub[1]. Root of Tall Oplopanax has often been used in the treatment of insomnia in the folk medicine, but its mechanism is not clear. Our lab have been identified in the previous work that Root of Tall Oplopanax water decoction (64 g/kg) has definite effect to improve sleep and also confirmed that the effective parts of Root of Tall Oplopanax to improve sleep is Root of Tall Oplopanax water decoction of 60% ethanol elution parts[2].This experiment is on the basis of the above results, and focus on exploring the sleep improving mechanism of the effective parts of Root of Tall Oplopanax. In recent years, the researchers found that Root of Tall Oplopanax can enhance immune function, and cytokines play an important role in sleep regulation. Lots of experiments confirmed that some cytokines such as IL - 1, IL - 2 and TNF-acan promote sleep, and IL - 4, IL-10, IL - 13, etc. inhibit sleep. Therefore, this Experiment is based on cytokine regulating mechanism to explore the mechanism of the sleep improving function of the effective parts of Root of Tall Oplopanax.
Materials and methods
1. Materials: Mice of ICR specie, weight between 18 to 22 grams and male were chose as experiment objective. Mice were raised in an environment of (24 ± 2)°C temperature, ( 55±15)% humidity and in a 12 hr:12 hr light-dark alternative environment. The mice had free access to food and water.
The crude drugs we used were from Wild medicinal materials planting base in Jian of Jilin province. Root of Tall Oplopanax picking in the spring, plant is about 1.5 meters high. Cut Root of Tall Oplopanax into sections and washed and dried. According to take a certain amount of Root of Tall Oplopanax, with 10 times the amount of distilled water to immerse 2 hours, heating reflux extracting 3 times, each time 1 hour, the extracted liquid, filter to concentrate 1 g/ml, set aside. Take 2 kg of D - 101 macroporous resin, soaking with 95% ethanol for 24 hours, after fully swelling out bubbles, wet packing column, with a 95% alcohol elution and removal of organic and inorganic impurities until eluent doesn't Appear cloudy when mixed with distilled water (1:5) .Then washed with distilled water to no alcohol taste, then make macroporous resin soaking with 5% hydrochloric acid3 hours, washing with distilled water to neutral, use 3% sodium hydroxide soak for 3 hours, washing with distilled water to neutral too. Use Root of Tall Oplopanax water decoction on macroporous resin column, static adsorption after 3 hours with distilled water to sugar free (molish reagent assay), use 60% ethanol elution. Rush about 8 times of column volume, collection of eluent, recycled ethanol, diluted with distilled water to the equivalent dose of 3.2 g/ml, and set aside. Root of Tall Oplopanax extract effective parts total ash content less than 3%. Acid insoluble ash content is not more than 0.1%.
This experiment mainly used four ELISA kits: Interleukin 1 beta (IL - ip) ELISA test kit , Tumor necrosis factor alpha (TNF-a) ELISA test kit , Interleukin 4 (IL - 4) ELISA test kit and Interleukin 10 (IL-10) ELISA test kit . All the ELISA kits were from R&D companies in the United States. The experiment mainly used multifunctional microplate reader to finish the content determination.
2. Methods : In our experiment 20 ICR mice were randomly divided into two groups, named blank group and drug group with 10 mice in each group. Drug group were lavaged with Root of Tall Oplopanax effective parts (64 g/kg), lavage volume was 0.4 ml / 20 g; blank control group were gave the same volume of distilled water lavage for 7 days. 30 min after the last dose, mice's blood were collected through eye blood abstraction. Serum was separated from blood through 3000r/min centrifuging for 15 min. Mice for determining the cytokines in mice brain were grouped in the same way, 30 min after the end of the last dosing, the brain will be directly removed from mouse, and hypothalamus, hippocampus, cortex were separated. The surface blood of these tissues were flushed with ice normal saline and dried with filter paper. The separated brain tissues were separately manually homogenized with 9-times cold normal saline and next were centrifuged at 3000r/min for 10 min. All the samples above were processed and the OD values were measured according to the operating procedure of the kit and the OD values were substituted in the equation of linear regression to calculate the contents of IL-ip, TNF-a, IL-4 and IL-10 in each sample.
Results and discussion
The influence of the effective parts of Root of Tall Oplopanax on the contents of IL-ip, TNF-a, IL-4 and IL-10 in mice serum is shown in the table 1.
Table 1The influence of the effective parts of Root of Tall Oplopanax on the contents of IL-ip, TNF-a, IL-4 and IL-10 in mice serum ( x ±S,n=10)
IL-ip (pg/ml) TNF-a (pg/ml) IL-4 (pg/ml) IL-10 (pg/ml)
Blank Group 6.77±i.72 72.80±i0.65 8.87±0.77 3i.2±5.47
Drug Group 6.02±0.76 72.20±i0.96 7.38±0.94* 27.47±7.i3*
Compared with the blank group *p<0.05
The result shows, compared with the blank group, the effective parts of Root of Tall Oplopanax (64g/kg) can obviously reduce the content of IL-4 and IL10 in serum(p<0.05), and have no influence on the content of IL-ip * TNF-a(p>0.05).
The influence of the effective parts of Root of Tall Oplopanax on the content of IL-ip, TNF-a, IL-4 and IL-10 in the hypothalamus, cortex and hippocampus of mouse is shown in table 2-5: Table 2The influence of the effective parts of Root of Tall Oplopanax on the content of IL-ipin mouse brain ( x ±S,n=10)_
Cortex (pg/ml) hippocampus (pg/ml) hypothalamus (pg/ml)
Blank Group i0.5i±0.92 9.74±0.87 i0.i6±0.58
Drug Group i0.64±i.6i i0.28±i.83 ii.53±i.62*
Compared with the blank group, *p<0.05
The experimental result shows that compares with the blank group, the effective parts of Root of Tall Oplopanax can obviously increase the content of IL-ipin mouse hypothalamus, however have less influence on the content of IL-ipin cortex and hippocampus.
Table 3The influence of the effective parts of Root of Tall Oplopanax on the content of TNF-a in mouse brain ( x ±S, n=10)_
Cortex (pg/ml) hippocampus (pg/ml) hypothalamus (pg/ml)
Blank group 90.8±i7.i4 94.20±i3.63 97.30±i9.3i
Drug group i07.60±i3.30* i00.30±i4.27 i22.5±i0.8**
Compared with blank group, *p<0.05, **p<0.01
The experimental result shows that compared with blank group, the effective parts of Root of Tall Oplopanax can significantly increase the content of TNF-a in mouse cortex (p<0.05) and hypothalamus (P<0.01), and have less influence on the content of TNF-ain hippocampus. Table 4The influence of the effective parts of Root of Tall Oplopanax on the content of IL-4 in mouse brain (x ±S,n=i0)_
Cortex (pg/ml) hippocampus (pg/ml) hypothalamus (pg/ml)
Blank group i2.ii±2.52 i3.37±2.89 i5.46±2.i9
Drug group i4.68±2.08* ii.57±i.72 i4.88±i.8i
Compared with blank group, p<0.05
The experimental result shows that compared with blank group, the effective parts of Root of Tall Oplopanax can increase the content of IL-4 in cortex (p<0.05), and has less influence on the content of IL-4 in hippocampus and hypothalamus.
Table 5The influence of the effective parts of Root of Tall Oplopanax on the IL-10 in mouse brain (x ±S,n=i0)_
Cortex (pg/ml) Hippocampus (pg/ml) Hypothalamus (pg/ml)
Blank group 39.33±3.66 36.63±7.74 44.40±6.94
Drug group 43.57±9.47 38.83±5.i0 35.57±3.97**
Compared with the blank group, **p<0.01
The experimental result shows that compared with the blank group, the effective parts of Root of Tall Oplopanax can significantly reduce the content of IL-10 in mouse hypothalamus (p<0.01), and have less influence on the content of IL-10 in the cortex and hippocampus.
Cytokines are the small molecular proteins secreted cells, which have mediating, immunity regulating, inflammation and hematopoiesis process. Cytokines can be generally divided into: interleukin (IL), tumor necrosis factor (TNF), interferon (IFN), colony stimulating factor (CSF), growth factor (GF) etc. most of the cytokines were discovered in the peripheral immune system, however some of the cytokines and its receptor exists in every parts of the central nervous system[3]. As the axis of the nerve - endocrine - immune network adjustment, the cytokines play comprehensive regulating roles in neural development, sleep, body temperature regulation, feeding, neuroendocrine, behavior, learning and memory, pain control, and other activities of the central nervous system function. It is generally believed that microbial infection, tissue damage etc. will cause the body metabolism, immune, endocrine, and central nervous system into the stress state with the performance of fever and immune function enhanced, drowsiness etc. So, many immune enhancer and immune modulators have the function of sleep regulating and sleep quality improving [4].
Besides the cytokines in peripheral blood which could enter the central nervous system, some of the neurons and glial cells can synthesise and release cytokines like IL-ip, TNF - a, IL-6 etc. Neurons which could be influenced by IL-ipand TNF - a exist in the sleep-awake behavior regulating area in brain, such as hypothalamus, hippocampus, cortex and brainstem etc. As it is reported in many literatures, during sleep, the level of IL-ipand TNF - ain blood, hippocampus and hypothalamus are obviously increased, which means IL-ipand TNF - ahave the sleep improving function[5]. Both encephalocoele injection and peripheral vein injection of IL-ipand TNF - acan extend the NREM period sleep time of experimental animals of different species[6]. Some of the immune inhabitors can shorten the sleep time to varying degrees. IL-4 and IL-10 are similar in the biological characteristics, and they are both immune cytokines. Therefore, IL-4 and IL10 are chose as the sleep inhibiting cytokines to be researched in this experiment.
This experiment result shows that after given drug, the IL-ipand TNF -acontent in cortex and the hypothalamus of mouse brain increase to varying degrees, and IL-4 and IL-10 content reduce in serum and hypothalamus. The preliminary experiment pvoves that the effective parts of Root of Tall Oplopanax have sleep improving function, so it could be concluded that Root of Tall Oplopanax's sleep improving function is associated with the increasing of the content of sleep improving cytokines IL-ipand TNF - a, and the reducing of the content of sleep inhibiting cytokines IL-4 and IL-10 in serum and hypothalamus. This is also consistent with previous literature reports.
Conclusion
The sleep improving function of the effective parts of Root of Tall Oplopanax is associated with the cytokines content in brain. It plays sleep improving role by increasing sleep improving cytokines (IL-ip4 TNF-a) content, and reducing the sleep inhibiting cytokines (IL-4, IL-10).
References
1. Jiang Yugui, Mi Heming, Ye Hongling. Resources and medicinal of Oplopanax elatus Nakai. China's wild plants. 1992 ; 1: 24~25
2. Xu Lei. Study on Preparation Process and Quality Standard for Improve Sleep Effective Parts of Root of Tall Oplopanax. Heilongjiang University of Chinese Medicine.2012.6
3. LI Li Hua, KU Bao Shan .Regulation of SWS by Hormones and Cytokines. Progress in physiological sciences. 2000,(01):32-36
4. Churchill L. TSP, Wang M.F., et al.Brain distribution of cytokine mRNA induced by systemic administration of interleukin-lßor tumor necrosis factor.Brain research.2006,20(11):64-73
5. Ganz FD.Sleep and immune function.Critical care nurse.2012,32(2):19-25
6. Chen Rui, Zhao Zhongxin. The impacts and regulating mechanism of non rapid eye movement sleep by IL- 1ß and TNF - a. Medical Journal of Chinese People's Liberation Army. 2010,(08):1032-1034
Simultaneously determination of five components in Folium Syringae by ultrahigh performance liquid chromatography
Guan Qingxia Wang Yanhong Yang Zhixin Feng Yufei Li Yongji* College of Pharmacy, Heilongjiang University of Chinese medicine, Harbin City150040, China
Abstracts. A rapid and sensitive method was developed for the simultaneous determination of five bioactive components in folium Syringaeby ultra-performance liquid chromatography. The analytes were separated with a Waters BEH column (C18 1.7 ^m, 50 x 2.1 mm) and a gradient elutionsystem using water and methanol as the mobile phases. The detection wavelength was set at 280nm and flow rate was set at 0.3 mlmin-1. The column compartment was kept atthe temperature of 30° C. Theseparation was achieved within 25 minutes. The linear response range was from 0.500 ^g to 5.015^g for five components and the correlation coefficients were all higher than 0.9995. The method was validated with respect to precision, repeatability, accuracy, recovery, and was successfully applied to quality control for folium Syringae and the preparations.
Key words: ultra-high performance liquid chromatography; Folium Syringae;
determination
1.Introduction
Folium Syringae is dry leaves of Syringa oblate Lindl. in Oleaceae plant[1]. The genus of deciduous shrub distributes widelyand is abundant resources in northeast of China, and it is the main courtyard greening tree species. The water decoction of folium Syringae is used to treat excellently diarrhea and eye disease in folk. Folium Syringae was found to possess therapeutict effects on antiviral[2],anti-inflammatory[3], antibacterial,antitussive, antioxidant and hypothermia[4'5], and also could enhance immune function[6]. It is well known that traditional Chinese medicine is a complex mixturecontaining hundreds of chemically different constituents with therapeutic effects. Therefore, itseems necessary to determine multi-component as muchas possible to ensure the quality of TCM.In the prior reseach , a total 9 compounds of the dry folium Syringae were separated andidentified from folium Syringae [7], and6 compounds of them the were obtained for the first time from folium Syringae.
The aims of the present study are to develop aappropriate analyticalmethod to simultaneously determinefive bioactive components with higher content in folium Syringae(Structure as shown infig.1), and be readily utilizedas a quality control for the medicinal herbs and preparations.
