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Anticonvulsant and sedative–hypnotic activity screening of pearl and nacre (mother of pearl)
Author’s Accepted Manuscript Anticonvulsant and sedative-hypnotic activity screening of pearl and nacre (mother of pearl) Jing-Xian Zhang, Shang-Rong Li, Shuai Yao, QiRui Bi, Jin-Jun Hou, Lu-Ying Cai, Su-Mei Han, Wan-Ying Wu, De-An Guo www.elsevier.com/locate/jep
PII: DOI: Reference:
S0378-8741(16)30039-3 http://dx.doi.org/10.1016/j.jep.2016.01.039 JEP9950
To appear in: Journal of Ethnopharmacology Received date: 24 September 2015 Revised date: 31 December 2015 Accepted date: 26 January 2016 Cite this article as: Jing-Xian Zhang, Shang-Rong Li, Shuai Yao, Qi-Rui Bi, JinJun Hou, Lu-Ying Cai, Su-Mei Han, Wan-Ying Wu and De-An Guo, Anticonvulsant and sedative-hypnotic activity screening of pearl and nacre (mother of pearl), Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.01.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Anticonvulsant and sedative-hypnotic activity screening of pearl and nacre (mother of pearl)
Jing-Xian Zhang1, Shang-Rong Li1, Shuai Yao, Qi-Rui Bi, Jin-Jun Hou, Lu-Ying Cai, Su-Mei Han, Wan-Ying Wu*, De-An Guo
National Engineering Laboratory for TCM Standardization Technology, Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China
1
These authors contributed equally to this work.
*
Corresponding author. Tel.: +86 21 20231000/2221; fax: +86 21 50272789. E-mail address: [email protected] (W.-Y. Wu).
1
ABSTRACT Ethnopharmacological relevance: Pearl and nacre are valuable traditional medicines to treat palpitations, convulsions or epilepsy in China for thousands of years. However, the active ingredients are not clear till now. Aim of the study: The main purpose of the current investigation was to assess the anticonvulsant and sedative-hypnotic activity of pearl powder and nacre powder, including their corresponding 6 protein extracts. Material and methods: Determination of the amino acid composition of the obtained protein was carried out by ultra-performance liquid chromatography (UPLC) combined
with
6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate
(AQC)
pre-column derivatisation. The influence of the tested drugs on locomotor activity and convulsions latency was recorded. The contents of 5-Hydroxytryptamine (5-HT) and γ-aminobutyric
acid
(GABA)
in
brain
were
detected
by
enzyme-linked
immunesorbent assay (ELISA) kits. In addition, immunohistochemistry was carried out to evaluate the changes of 5-HT3 and GABAB. In parallel, the expressions of them were demonstrated by western blot. Results: The obtained data suggested that pearl original powder (1.1g/kg), pearl water-soluble protein (0.2g/kg), pearl acid-soluble protein (0.275g/kg), pearl conchiolin protein (1.1g/kg), nacre original powder (1.1g/kg), nacre water-soluble protein (0.2g/kg), nacre acid-soluble protein (0.7g/kg) and nacre conchiolin protein (1.1g/kg) could down-regulate the expression of 5-HT3 and up-regulate the level of GABAB to varying degrees compared with the control group. Besides, drug 2
administration also reduced the locomotor activity and increased convulsions latency with a certain mortality. Conclusions: These findings correlated with the traditional use of pearl and nacre as sedation and tranquilization agents, thus making them interesting sources for further drug development and also providing critical important evidence for the selection of quality control markers.
Keywords: Pearl, Nacre, Anticonvulsant, Sedative-hypnotic, 5-HT3, GABAB
1. Introduction Pearl, as recorded in the Chinese Pharmacopeia 2015, is formed in oysters, Pteria martensii (Dunker), or mussels, Hyriopsis cumingii (Lea) or Cristaria plicata (Leach) and nacre (mother of pearl) is the conch of these molluscs (China Pharmacopoeia Committee, 2015). As traditional Chinese medicines (TCMs), both pearl and nacre are recorded as anticonvulsant and sedative-hypnotic agents from the ancient times (Dai et al., 2008; Liu et al., 2014a, 2014b). There are at least 57 Chinese patent medicines (CPMs) containing pearl or nacre in Chinese Pharmacopeia 2015 and most of them have sedative efficacy, such as Angong Niuhuang Pill, Xinnaojing Tablet, Xiegan Anshen Pill, and Qingkailing Injection (Guo et al., 2014, Zhang et al., 2015a). Medicinal pearl and nacre are generally used in the form of original powder in pharmaceutical factory or in clinical, through thoroughly smashing and grinding of natural products to make the ingredients more bioavailable. A considerable number of 3
investigations have revealed that pearl and nacre are rich sources of calcium, accounting for over 90% of the weight, followed by conchiolin protein, and a small amount of trace metal elements. Although the chemical compositions and pharmacological efficacy of the two traditional medicines are clear, the exact bioactive ingredients are still far from identification, which lead to a lower use efficiency of the natural products and also hinder the development of the related novel products. 5-HT is an important excitatory neurotransmitter that widely distributed in animals. It binds with multiple receptors to regulate many complex behaviors in vertebrates and invertebrates (Gerhardt et al., 1997; Tierney, 2001). It is well known that 5-HT is engaged in learning, anxiety, emotion, reproduction, pain perception, sleep and locomotion in mammals. However, it is found that 5-HT and its receptors are implicated in neuronal functions including circadian rhythm, feeding, locomotion, memory, parturition and development (Filla et al., 2004; Kawai et al., 2011; Panasophonkul et al., 2009). Inhibitory neurotransmitter GABA is also one of the critical neurotransmitters (NTs), whose metabolites are closely related to the central nervous system (CNS) depressant activity. In addition, previous literature has documented the role of GABA in CNS of mollusks (Gunaratne et al., 2014). However, it still remains unknown that whether pearl powder, nacre powder and their corresponding protein extracts could improve locomotor activity and convulsions latency. Moreover, the exact active ingredients need to be illustrated. Hence, the current experiment screened the anticonvulsant and sedative-hypnotic activity of pearl 4
powder, nacre powder and their corresponding protein extracts in regulating 5-HT and GABA.
2. Materials and methods 2.1. Materials Eighteen batches of pearls and eighteen batches of nacres were purchased from medicinal plant markets, mainly from Shandong and Zhejiang Provinces in China. The pooled samples from different batches of pearls and nacres were prepared as the tested samples. Voucher specimens were deposited in the laboratory of the author. Amino acid standards (purity 99%) were purchased from the China Institute of Pharmaceutical and Biological Products. The internal standard (IS), α-amino butyric acid (AABA), was purchased from Sigma (St. Louis, MO, USA) with a purity of 99%. AccQ·Tag™ Eluent A concentrate (P/N 186003838) and Elution B (P/N 186003839) used for gradient program, as well as the AccQ·Fluor™ pre-column AQC derivatisation kit were from Waters (Milford, MA, USA). Diazepam (2 ml / 10 mg) was purchased from Tianjin Jinyao amino acids CO., Ltd. 5-HT and GABA ELISA kits were purchased from Elabscience Biotechnology Co., Ltd. Primary antibodies against 5-HT and GABA were produced by Cell Signaling Technology Inc (Beverly, MA, USA). All other chemicals and reagents used for study were of analytical grade and were purchased from approved organizations.
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2.2. Sample preparation and amino acid analysis The crude materials were crushed into very fine powder according to the process method recorded in the Chinese Pharmacopeia 2015 (China Pharmacopoeia Committee, 2015). The sample powder of 100 g was immersed with 10 times of water at 4 °C for 24 h and the solution was filtered. The filtrate was freeze-dried to obtain the water-soluble protein. Ten times of diluted hydrochloric acid was then added to the residue and stirred, and allowed to react at 4 °C for 12 h. The solution was centrifuged at 12000 rpm for 20 min. The precipitate was freeze-dried to yield the conchiolin protein and the supernatant was dialyzed with the molecular weight cut off (MWCO) 3000 for 48 h and the solution was then freeze-dried to obtain the acid-soluble protein. Amino acid analysis was carried out by UPLC combined with AQC pre-column derivatisation in accordance with the method described previously (Zhang et al., 2015b). 2.3. Animals 100 male and 100 female mice, weighing 20-22 g, obtained from Shanghai Jiesijie Laboratory Animal Co., Ltd., were housed in a temperature and humidity controlled animal facility with a set of 12 h light-dark cycle. Mice had free access to water and food pellets ad libitum.
6
2.4. Locomotor activity test Mice were randomly divided into ten groups (n = 10) with half male and female as follows: control group (A), diazepam (2.2 mg/kg) group (B), pearl original powder (1.1 g/kg) group (C), pearl water-soluble protein (0.2 g/kg) group (D), pearl acid-soluble protein (0.275 g/kg) group (E), pearl conchiolin protein (1.1 g/kg) group (F), nacre original powder (1.1 g/kg) group (G), nacre water-soluble protein (0.2 g/kg) group (H), pearl acid-soluble protein (0.7 g/kg) group (I) and nacre conchiolin protein (1.1 g/kg) group (J). The dosage were selected based on the results in the preliminary experiment and the related reference (Dai et al., 2008). After starvation for 12 h, mice were placed individually in an open container (50 × 20 cm) with a black surface covering the inside walls and allowed to explore freely to acclimatize the environment for 2 min before the test. The container was divided into 12 equal squares and the number of panes was recorded. The number of squares crossed and rearing behaviors were recorded for 4 min. The above experimental procedure was repeated once 1 h after intragastrically treated with tested drugs and diazepam. Then, mice were sacrificed, and the brain was harvested subsequently and set aside at -80 °C for further analysis.
7
2.5. Anticonvulsant experiment Mice were randomly divided into ten groups (n = 10) with half male and female as follows: control group, diazepam group, pearl original powder (1.1 g/kg) group, pearl water-soluble protein (0.2 g/kg) group, pearl acid-soluble protein (0.275 g/kg) group, pearl conchiolin protein (1.1 g/kg) group, nacre original powder (1.1 g/kg) group, nacre water-soluble protein (0.2 g/kg) group, nacre acid-soluble protein (0.7 g/kg) group and nacre conchiolin protein (1.1 g/kg) group. Tested drugs were intragastrically administered for 3 consecutive days. Diazepam (2.2 mg/kg) was orally treated for 3 consecutive days. After intragastric administration 1 h on day 3, mice were intraperitoneally injected with 0.5% of pentylenetetrazol (PTZ, 100 mg/kg). Convulsions latency time was recorded subsequently and mortality of mice were observed 6 h later. 2.6. Measurement of 5-HT3 and GABAB neurotransmitter levels The measurements of 5-HT3 and GABAB in brain of mice were conducted using ELISA kits according to the manufacturer’s instruction. 2.7. Immunohistochemistry The paraffin sample of brain were heated for 1 h in the oven, deparaffinized in xylene, rehydrated by graded ethanol solutions, microwaved in sodium citrate buffer, cooled to room temperature naturally and incubated in 3% hydrogen peroxide. Each section was blocked with 3% BSA at room temperature. Samples were then incubated with primary antibody overnight at 4 °C after removing blocking solution, secondary 8
antibody for 20 min at 37 °C and three antibody for another 20 min at 37 °C, respectively. Then, sections were stained with DAB and restained with hematoxylin. Followed by dehydrating and drying, the sections were mounted with neutral gum and observed under a microscope. 2.8. Western blot Brain tissues were minced and homogenized in ice-cold RIPA lysis buffer, followed by centrifugation at 12000 rpm for 5 min at 4 °C to remove the debris. Based on the concentration measured by BCA assay, equal amounts of 5-HT, GABA and GAPDH per line were subjected to a 10% SDS-polyacrylamide gel electrophoresis for the separation and transferred onto the polyvinylidene difluoride membrane. Target blots were blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4 °C using specific antibodies against 5-HT, GABA and GAPDH. The bands were then incubated for 1 h at room temperature with the corresponding secondary antibodies after washing with TBST three times. Enhanced chemiluminescence detection (ECL) reagents and a gel imaging system (Tanon Science & Technology Co., Ltd., China) were used to visualize the protein sheets. 2.9. Statistical analysis All data were expressed as means ± standards deviation (SDs). Evaluation between the control group and the administered groups respectively were analyzed by One-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. The values were considered statistically significantly different at p < 0.05. 9
3. Results 3.1. Amino acids analysis The yields for the obtained proteins including water-soluble protein, acid-soluble protein and conchiolin protein for pearl and nacre are 0.16%, 0.40%, 1.30% and 0.10%, 0.43%, 1.80%, respectively. The amino acid composition was determined by UPLC combined with AQC pre-column derivatisation and the chromatograms were shown in Fig. 1. It was obvious that different kinds of protein contained similar amino acids, mainly including arginine (Arg), serine (Ser), glycine (Gly), asparagine (Asp), glutamic acid (Glu), threonine (Thr), alanine (Ala), proline (Pro), cystine (Cys), tyrosine (Tyr), valine (Val), lysine (Lys), isoleucine (Ile), leucine (Leu) and phenylalanine (Phe). Conchiolin proteins from both pearl and nacre possessed the most abundant amino acids.
10
Fig. 1. Amino acid compositions of pearl/nacre water-soluble protein, pearl/nacre acid-soluble protein and pearl/nacre conchiolin protein.
3.2. Effect of tested drugs on locomotor activity in mice The effects of pearl powder, nacre powder and their corresponding protein extracts on the locomotor activity of mice were listed in Table 1. Among all the tested drugs, pearl conchiolin protein dramatically reduced the numbers of squares crossed behaviors compared with the control group, as well as diazepam, while the efficacy of nacre conchiolin protein was secondary. However, pearl water-soluble protein, pearl acid-soluble protein and nacre acid-soluble protein decreased rearing behaviors only, while pearl water-soluble protein, pearl conchiolin protein, nacre original powder, nacre acid-soluble protein, and nacre conchiolin protein significantly reduced rearing behaviors. 3.3. Effect of tested drugs on PTZ-induced convulsions The effects of pearl and nacre powder and their corresponding protein extracts on PTZ-induced convulsions were shown in Table 2. The mortality of mice after 6 h was also listed. Diazepam, nacre water-soluble protein and nacre original powder remarkably prolonged the convulsions latency time compared with those in the control group. Meanwhile, pearl original powder, pearl water-soluble protein, pearl acid-soluble protein, pearl conchiolin protein and nacre conchiolin protein also significantly enhanced convulsions latency time, while nacre acid-soluble protein had 11
no significant influence on convulsions latency, which partially illustrated that nacre acid-soluble protein might showed no anticonvulsant activity.
Table 1 Effect of tested drugs on locomotor activity in mice (means ± SD, n = 10). Group
Dose (g/kg)
Crossing
Rearing
Control
——
153.4±24.5
40.5±8.3
Diazepam
2.2×10-3
114.7±9.2**
17.5±6.9**
Pearl original powder
1.1
148.8±31.7
34.1±11.1*
Pearl water-soluble protein
0.2
153.6±31.2
30.7±9.1**
Pearl acid-soluble protein
0.275
159.0±23.3
34.3±7.0
Pearl conchiolin protein
1.1
113.5±24.6**
30.2±9.2**
Nacre original powder
1.1
149.1±23.1
28.8±8.9**
Nacre water-soluble protein
0.2
144.6±22.5
37.3±9.6
Nacre acid-soluble protein
0.7
176.7±36.9
26.0±9.6**
Nacre conchiolin protein
1.1
138.6±28.9**
30.6±5.0**
Table 2 Effect of tested drugs on PTZ-induced convulsions (means ± SD, n = 10). Groups
Dose (g/kg)
Convulsions latency / min
mortality / %
Control
——
2.5±0.8
70.0
Diazepam
2.2×10-3
5.1±1.7**
0
Pearl original powder
1.1
3.4±1.0*
80.0
Pearl water-soluble protein
0.2
4.1±1.5*
80.0
Pearl acid-soluble protein
0.275.
3.6±1.1*
30.0
Pearl conchiolin protein
1.1
3.8±1.3*
50.0
Nacre original powder
1.1
4.6±1.2**
70.0
Nacre water-soluble protein
0.2
5.3±1.1**
80.0
Nacre acid-soluble protein
0.7
3.3±1.0
80.0
Nacre conchiolin protein
1.1
3.8±1.4*
60.0
12
3.4. Effect of tested drugs on 5-HT3 and GABAB in brain As shown in Fig. 2, the concentration of excitatory neurotransmitter 5-HT3 in brain was down-regulated, while the level of inhibitory neurotransmitter GABAB was up-regulated with tested drugs treatment in comparison with the control group. However, pearl original powder (C) and nacre acid-soluble protein (I) reduced 5-HT content without significant difference.
Fig. 2. Measurement of 5-HT3 and GABAB neurotransmitter levels. Control group (A), diazepam (2.2 mg/kg) group (B), pearl original powder (1.1 g/kg) group (C), pearl water-soluble protein (0.2 g/kg) group (D), pearl acid-soluble protein (0.275 g/kg) group (E), pearl conchiolin protein (1.1 g/kg) group (F), nacre original powder (1.1 g/kg) group (G), nacre water-soluble protein (0.2 g/kg) group (H), nacre acid-soluble protein (0.7 g/kg) group (I), nacre conchiolin protein (1.1 g/kg) group (J). Values represent the mean ± SD and are representative of two independent experiments. **p＜0.01, significantly different from the values in the control group; *p＜0.05, significantly different from the values in the control group. 13
3.5. Effect of tested drugs on the expressions of 5-HT3 and GABAB receptor Immunohistochemistry analysis further exhibited the alterations of 5-HT3 (Fig. 3a) and GABAB (Fig. 3b). All the tested drugs treatment exhibited notable reduced expression of 5-HT3 receptor, especially pearl original powder (C) and pearl water-soluble protein (D). Moreover, all the tested drugs showed markedly recovered level of GABA receptor, especially pearl acid-soluble protein (E), nacre water-soluble protein (H), nacre acid-soluble protein (I), nacre conchiolin protein (J) and nacre original powder (G). The data depicted in Fig. 4 illustrated the inhibited 5-HT3 receptor and highly expressed GABAB with GAPDH expression as internal control, which further confirmed the involvement of 5-HT/GABA balance in sedation and hypnosis. Specifically, nacre conchiolin protein (J) showed no significant influence on the content of 5-HT3. Nacre acid-soluble protein (I) and nacre conchiolin protein (J) showed no remarkable effects on the level of GABAB. Meanwhile, all the other tested drugs exhibited significant difference of 5-HT and GABA expressions compared with the control group.
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Fig. 3. Effect of tested drugs on the expressions of 5-HT3 (a) and GABAB (b) receptor according to immunohistochemistry. control group (A), diazepam (2.2 mg/kg) group (B), pearl original powder (1.1 g/kg) group (C), pearl water-soluble protein (0.2 g/kg) group (D), pearl acid-soluble protein (0.275 g/kg) group (E), pearl conchiolin protein (1.1 g/kg) group (F), nacre original powder (1.1 g/kg) group (G), nacre water-soluble protein (0.2 g/kg) group (H), nacre acid-soluble protein (0.7 g/kg) group (I), nacre conchiolin protein (1.1 g/kg) group (J). Values represent the mean ± SD and are representative of two independent experiments. **p＜0.01, significantly different from the values in the control group; *p＜0.05, significantly different from the values in the control group.
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Fig. 4. Effect of tested drugs on the expressions of 5-HT3 and GABAB receptor according to western blot. control group (A), diazepam (2.2 mg/kg) group (B), pearl original powder (1.1 g/kg) group (C), pearl water-soluble protein (0.2 g/kg) group (D), pearl acid-soluble protein (0.275 g/kg) group (E), pearl conchiolin protein (1.1 g/kg) group (F), nacre original powder (1.1 g/kg) group (G), nacre water-soluble protein (0.2 g/kg) group (H), nacre acid-soluble protein (0.7 g/kg) group (I), nacre conchiolin protein (1.1 g/kg) group (J). Values represent the mean ± SD and are representative of two independent experiments. **p＜0.01, significantly different from the values in the control group; *p＜0.05,significantly different from the values in the control group.
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4. Discussion Pearls have existed for approximately two hundred million years. It is not only famous as a valuable ornament, but also known as a traditional medicine to treat palpitations, convulsions, epilepsy, eye diseases and promote wound healing (Dai et al., 2010; Mangrulkar et al., 2002; Shen et al., 2006; Xu et al., 2001). Pearls also have antioxidant properties (Shao et al., 2010) and provide high calcium bioavailability (Chen et al., 2008). Besides, nacre is commonly used to treat insomnia and anxiety in the modern clinical practice as well (Liu et al., 2014a and 2014b). The anti-depressant activity of different processed products of nacre in mice provides pharmacological foundation for its sedative and hypnotic effects (Li et al., 2014b). In order to identify the material basis of the anticonvulsant and sedative-hypnotic activity documented, the subject analyzed the role of pearl powder, nacre powder and their corresponding water-soluble protein, acid-soluble protein, conchiolin protein in the CNS. It is widely accepted that the reduced number of squares crossed and rearing behavior in locomotor activity test and increased convulsions latency reflected the activity of the tested agents. Specifically, pearl conchiolin protein significantly reduced the number of squares mice crossed behavior, which was in line with positive control diazepam. However, pearl water-soluble protein, pearl acid-soluble protein and nacre acid-soluble protein only showed obviously decreased number of rearing behavior. Since both of crossing and rearing behavior represent the intensity of locomotor activity, it could be reasonable to judge that pearl acid-soluble protein and nacre 17
water-soluble protein might be absent from sedative-hypnotic activity. The PTZ mouse model of generalized myoclonic seizures has long been used in the discovery and characterization of all marked antiepileptic drugs (Loscher et al., 1991; Loscher and Schmidt, 1988; Li et al., 2014a). This classical animal model was adopted to screen the tested agents in the present study. It is considered to be general by intraperitoneal administration of the PTZ ranging from 80 to 90 mg/kg (Hosseini et al., 2011, Koutroumanidou et al., 2013). By contrast, severe seizure persisted after injection at higher dose of PTZ (100 mg/kg) group, where the relative mortality percentage was higher than those of lower doses (Li et al., 2014). In this study, the dose of 100 mg/kg was applied in the consideration of the purpose to examine the effect of pearl or nacre against severe seizure induced by PTZ. It is well known that there is positive correlation between different types of seizure and doses of PTZ. In the current study, all the candidate drugs, except nacre acid-soluble protein, could effectively enhanced convulsions latency time, which suggested that the possibility of nacre acid-soluble protein selected as an anticonvulsant might need more in-depth investigation. The monitoring of neurotransmitters and their metabolites is an effective tool for illustrating normal and pathological neural system functions. Sedative-hypnotics and anticonvulsant drugs used in clinical are a class of central nervous system depressant drugs. 5-HT and GABA are known to maintain the balance in the nervous system for numerous organisms. The levels of them are tightly linked with neurophysiology, behavior, pathology, disease diagnosis and control (Feng et al., 2001; Harada et al., 18
2006). An alteration in the brain levels of neurotransmitters and their metabolites is the indicator of neurotransmission disorders. It has been reported that therapeutic drugs induce sedative-hypnotic effects via modulating neurotransmission such as in the GABAergic or serotonergic systems in the CNS (Attele et al., 2000). Thus, the analysis of 5-HT and GABA contents is of great importance in monitoring efficacy of the tested drugs treatment. The current study provides important new insight into the 5-HT receptor system in the CNS with the treatment of pearl powder, nacre powder and their corresponding protein extracts. Prior documents demonstrated that excessive extracellular 5-HT metabolite 5-hydroxyindoleacetic acid was accompanied by wakefulness and less sleep in freely moving rats (Imeri et al., 1994), which reflected the important role of 5-HT in sedation and hypnosis. Furthermore, the location of 5-HT receptor and the biological activity of its ligands also indicated the participation in the pathogenesis of convulsions (Yang et al., 2012). As demonstrated from literatures that activation of the subtype of 5-HT3 could increase the seizure threshold induced by PTZ. In addition, GABA is the pivotal inhibitory neurotransmitter in the central nervous system. The GABAergic agents display anticonvulsant activity in various animal models and are recently being evaluated for anti-epileptic activity in humans (Ong et al., 2000). PTZ is defined as a GABAA receptor antagonist. Benzodiazepines and barbiturates are the two main classes of hypnotic-sedative and anticonvulsant agents. Wherein, barbiturates are identified to be bind to the benzodiazepine site of the GABA receptor complex and 19
enhance the binding of its neurotransmitter GABA (Joksimović et al., 2013). GABAA and GABAB receptors are involved in the control of epileptogenesisand neuronal excitability. Furthermore, GABAB receptor function could be expressed as that its molecular structure provides a large number of sites for pharmaceutical intervention in symptoms, including drug addition, absence seizures and nociception. More importantly, a number of studies have reported that the expression or function of GABAB in brain tissues in animal models of resistant epilepsy or patients (Billinton et al., 2001). Vergnes elicited the role for GABAB receptors resided in the neo-cortex and hippocampus in some epileptic disorder (Vergnes et al., 1997). The critical role of GABAA receptors in the control of generalized convulsive seizures was also well elucidated (Mohler H., 1997). Slight deficiencies in GABAergic transmission may lead to hyperexcitability and pathological neuronal discharges leading to epilepsy. Thus, when assessing the alleviation of pearl powder, nacre powder and their corresponding protein extracts on convulsions, researcher should take the GABAergic system, especially GABAB, into consideration. The expressions of 5-HT3 and GABAB were identified by ELISA kits, immunohistochemistry and western blot respectively in the present study. Different detection methods exhibited distinct sensitivity, detection range and quantitative manner. All the candidate drugs showed varying degrees of down-regulation of 5-HT and up-regulation of GABAB.
20
5. Conclusion Taken together, the tested drugs exhibited varying degrees of inhibition of locomotor activity and improvement of PTZ-induced convulsions. Besides, the findings in the study exhibited distinct down-regulated levels of 5-HT3 and up-regulated levels of GABAB with pearl original powder, pearl water-soluble protein, pearl acid-soluble protein, pearl conchiolin protein, nacre original powder, nacre water-soluble protein, nacre acid-soluble protein and nacre conchiolin protein treatment, which reflected the essential involvement of 5-HT/GABA balance in sedation and hypnosis. These results provided critical important evidence for the selection of quality control markers and also for drug development correlated with the traditional use of pearl and nacre as sedation and tranquilization agents.
Conflict of interests None of the authors has any conflict of interests regarding this study.
Acknowledgements This study was financially supported by the National High Technology Research and Development Program of China (No. 2013AA093002). The authors thank Dr. Chun-Hua Ma for his help in the revision of this paper.
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References Attele, A.S., Xie, J.T., Yuan, C.S., 2000. Treatment of insomnia: an alternative approach. Altern. Med. Rev. 5, 249–259. Billinton A., Baird V.H., Thom M., Duncan J.S., Upton N., Bowery N.G., 2001. GABA(B) receptor autoradiography in hippocampal sclerosis associated with human temporal lobe epilepsy. Br. J. Pharmacol. 132, 475–480. Chen H.S., Chang J.H., Wu J.S., 2008. Calcium bioavailability of nanonized pearl powder for adults. J. Food Sci. 246–251. China Pharmacopoeia Committee, 2010. Pharmacopoeia of the People’s Republic of China. China Chemical Industry Press, Beijing. Dai, J., Duan, J.A., Li, Y.B., Liu, R., Peng, Y.R., 2008. Investigation of sedative bioactive components and evaluation of the biological effect of freshwater pearls produced in Suzhou. Chin. J. Biochem. Pharm. 29, 294–298. Dai J.P., Chen J., Bei Y.F., Han B.X., Guo S.B., Jiang L.L., 2010. Effects of pearl powder extract and its fractions on fibroblast function relevant to wound repair. Pharmaceutical. Biology. 48, 122–127. Feng, J., Cai, X., Zhao, J., Yan, Z., 2001. Serotonin receptors modulate GABAA receptor channels through activation of anchored protein kinase C in prefrontal cortical neurons. J. Neurosci. 21, 6502–6511. Filla, A., Hiripi, L., Elekes, K., 2004. Serotonergic and dopaminergic influence of the duration of embryogenesis and intracapsular locomotion of Lymnaea stagnalis L. 22
Acta Biol. Hung. 55, 315–321. Gerhardt,
C.C.,
Heerikhuizen,
H.V.,
1997.
Functional
characteristics
of
heterologously expressed 5-HT receptors. Eur. J. Pharmacol, 334, 1–23. Gunaratne, C.A., Sakurai, A., Katz, P.S., 2014. Comparative mapping of GABA-immunoreactive neurons in the central nervous systems of nudibranch molluscs. J. Comp. Neurol. 522, 794–810. Guo, Y., Yan, S., Xu, L., Zhu, G., Yu, X., Tong, X., 2014. Use of angong niuhuang in treating central nervous system diseases and related research. Evid. Based Complement. Alternat. Med. 346918. Harada, K., Aota, M., Inoue, T., Matsuda, R., Mihara, T., amaji, T., Ishibashi, Y.K., Matsuoka, N., 2006. Anxiolytic activity of a novel potent serotonin 5-HT 2 C receptor antagonist FR260010: a comparison with diazepam and buspirone. Eur. J. Pharmacol. 553, 171–184. Hosseini M, Ghasemzadeh Rahbardar M, Sadeghnia HR, Rakhshandeh H., 2011. Effects of different extracts of Rosa damascena on pentylenetetrazol-induced seizures in mice. J. Chin. Integr. Med. 9, 1118–1124. Imeri, L., De Simoni, M. G., Giglio, R., Clavenna, A., Mancia, M., 1994. Changing in the serotonergic system during the sleep-wake cycle: simultaneous polygraphic and voltammetric recordings in hypothalamus using a telemetry system. Neuroscience 58, 353–358. Joksimović, S., Varagic, Z., Kovačević, J., Van Linn, M., Milić, M., Rallapalli, S., Timić, T., Sieghart, W., Cook, J.M., Savić M.M., 2013. Insights into functional 23
pharmacology of α₁ GABA (A) receptors: how much does partial activation at the benzodiazepine site matter? Psychopharmacology (Berl) 230, 113–123. Kawai, R., Kobayashi, S., Fujito, Y., Ito, E., 2011. Multiple subtypes of serotonin receptors in the feeding circuit of a pond snail. Zool. Sci. 28, 517-525. Koutroumanidou E., Kimbaris A., Kortsaris A., Bezirtzoglou E., Polissiou M., Charalabopoulos K., 2013. Increased seizure latency and decreased severity of pentylenetetrazol-induced seizures in mice after essential oil administration. Epilepsy Res. Treat 2013: 532657. Li B.J., Wang L., Sun Z.H., Zhou Y., Shao D.Y., Zhao J., Song Y.N., Lv J.Y., Dong X., Liu C.H., Wang P., Zhang X.Y., Cui R.J, 2014a. The anticonvulsant effects of SR 57227 on pentylenetetrazole-induced seizure in mice. Plos One 9(4), e93158. Li, Y., Sun, J.M., Zhang, J., Li, J.F., Liu, W.C., Zhang, H., 2014b. Anti-depressant effect of Margaritifera concha on mice. J. Jilin Tradit. Chin. Med. 34, 388–389. Liu, D., Kang, X.Y., Ren, B.Y., Zhang, H.Y., Du, H., Song, L., Zhang, H., Sun, J.M., 2014a. Comparison of effects of sedative-hypnotic action of protein and peptides of Margaritifera concha on rats. Jilin J. Tradit. Chin. Med. 34, 172–173, 176 Liu, D., Dai, T.T., Cha, R.B., Qu, P., Li, Y., Du, H., Zhao, S.Y., Song, L., Zhang, H., Sun, J.M., 2014b. Study on sedative and hypnotic effects of Concha Margaritifera and its different processed products on the content of 5-HT in the brain of mice. Jilin J. Tradit. Chin. Med. 34, 61–63. Loscher W., Schmidt D., 1988. Which animal models should be used in the search for new antiepileptic drugs? A proposal based on experimental and clinical 24
considerations. Epilepsy Res. 2, 145–181. Loscher W., Honack D., Fassbender C.P., Nolting B., 1991. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs, III. Pentylenetetrazole seizure models. Epilepsy Res. 8, 177–189. Mangrulkar, R.S., Saint, S., Chu, S., Tierney, L.M., 2002. What is the role of the clinical "pearl"? Am. J. Med. 113, 617–624. Mohler H., 1997. Genetic approaches to CNS disorders with particular reference to GABAA-receptor mutations. J. Recept. Signal Transduct. Res.17, 1–10. Ong J., Kerr D.I., 2000. Recent advances in GABAB receptors: from pharmacology to molecular biology. Acta Pharmacol. Sin. 21, 111–123. Panasophonkul, S., Apisawetakan, S., Cummins, S.F., York, P.S., Degnan, B.M., Hanna, P.J., Saitongdee, P., Sobhon, P., Sretarugsa, P., 2009. Molecular characterization and analysis of a truncated serotonin receptor gene expressed in neural and reproductive tissues of abalone. Histochem. Cell Biol, 131, 629–642. Shao D.Z., Wang C.K., Hwang H.J., Huang C.H. Chen Y.W., 2010. Comparison of hydration, tyrosinase resistance, and antioxidant activation in three kinds of pearl powders. J. Cosmet. Sci. 61, 133–145. Shen Y.T., Zhu J., Zhang H.B., Zhao F., 2006. In vitro osteogenetic activity of pearl. Biomaterials 27, 281–287. Tierney, A.J., 2001. Structure and function of invertebrate 5-HT receptors: a review. Comp. Biochem. Physiol. A128, 791–804. 25
Vergnes M., Boehrer A., Simler S., Bernasconi R., Marescaux C., 1997. Opposite effects of GABAB receptor antagonists on absences and convulsive seizures. Eur. J. Pharmacol. 332: 245-255 Xu H., Huang K., Gao Q., Gao Z., Han X., 2001. A study on the prevention and treatment of myopia with nacre on chicks. Pharmacol. Res. 44, 1–6. Yang, Z., Liu, X., Yin, Y., Sun, S., Deng, X., 2012. Involvement of 5-HT receptors in the pathogenesis of temporal lobe epilepsy. Eur. J. Pharmacol. 685, 52–58. Zhang, D.S., Liu, Y.L., Zhu, D.Q., Huang, X.J., Luo, C.H., 2015a. Point application with Angong Niuhuang sticker protects hippocampal and cortical neurons in rats with cerebral ischemia. Neural Regen. Res. 10, 286–291. Zhang, J.X., Li, S.R., Yao, S., Si, W., Cai, L.Y., Pan, H.Q., Hou, J.J., Yang, W.Z., Da, J., Jiang, B.H., Liu, X., Wu, W.Y., Guo, D.A., 2015b. Ultra-performance liquid chromatography of amino acids for the quality assessment of pearl powder. J. Sep. Sci. 38, 1552–1560.
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Graphical Abstract
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PII: DOI: Reference:
S0378-8741(16)30039-3 http://dx.doi.org/10.1016/j.jep.2016.01.039 JEP9950
To appear in: Journal of Ethnopharmacology Received date: 24 September 2015 Revised date: 31 December 2015 Accepted date: 26 January 2016 Cite this article as: Jing-Xian Zhang, Shang-Rong Li, Shuai Yao, Qi-Rui Bi, JinJun Hou, Lu-Ying Cai, Su-Mei Han, Wan-Ying Wu and De-An Guo, Anticonvulsant and sedative-hypnotic activity screening of pearl and nacre (mother of pearl), Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.01.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Anticonvulsant and sedative-hypnotic activity screening of pearl and nacre (mother of pearl)
Jing-Xian Zhang1, Shang-Rong Li1, Shuai Yao, Qi-Rui Bi, Jin-Jun Hou, Lu-Ying Cai, Su-Mei Han, Wan-Ying Wu*, De-An Guo
National Engineering Laboratory for TCM Standardization Technology, Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China
1
These authors contributed equally to this work.
*
Corresponding author. Tel.: +86 21 20231000/2221; fax: +86 21 50272789. E-mail address: [email protected] (W.-Y. Wu).
1
ABSTRACT Ethnopharmacological relevance: Pearl and nacre are valuable traditional medicines to treat palpitations, convulsions or epilepsy in China for thousands of years. However, the active ingredients are not clear till now. Aim of the study: The main purpose of the current investigation was to assess the anticonvulsant and sedative-hypnotic activity of pearl powder and nacre powder, including their corresponding 6 protein extracts. Material and methods: Determination of the amino acid composition of the obtained protein was carried out by ultra-performance liquid chromatography (UPLC) combined
with
6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate
(AQC)
pre-column derivatisation. The influence of the tested drugs on locomotor activity and convulsions latency was recorded. The contents of 5-Hydroxytryptamine (5-HT) and γ-aminobutyric
acid
(GABA)
in
brain
were
detected
by
enzyme-linked
immunesorbent assay (ELISA) kits. In addition, immunohistochemistry was carried out to evaluate the changes of 5-HT3 and GABAB. In parallel, the expressions of them were demonstrated by western blot. Results: The obtained data suggested that pearl original powder (1.1g/kg), pearl water-soluble protein (0.2g/kg), pearl acid-soluble protein (0.275g/kg), pearl conchiolin protein (1.1g/kg), nacre original powder (1.1g/kg), nacre water-soluble protein (0.2g/kg), nacre acid-soluble protein (0.7g/kg) and nacre conchiolin protein (1.1g/kg) could down-regulate the expression of 5-HT3 and up-regulate the level of GABAB to varying degrees compared with the control group. Besides, drug 2
administration also reduced the locomotor activity and increased convulsions latency with a certain mortality. Conclusions: These findings correlated with the traditional use of pearl and nacre as sedation and tranquilization agents, thus making them interesting sources for further drug development and also providing critical important evidence for the selection of quality control markers.
Keywords: Pearl, Nacre, Anticonvulsant, Sedative-hypnotic, 5-HT3, GABAB
1. Introduction Pearl, as recorded in the Chinese Pharmacopeia 2015, is formed in oysters, Pteria martensii (Dunker), or mussels, Hyriopsis cumingii (Lea) or Cristaria plicata (Leach) and nacre (mother of pearl) is the conch of these molluscs (China Pharmacopoeia Committee, 2015). As traditional Chinese medicines (TCMs), both pearl and nacre are recorded as anticonvulsant and sedative-hypnotic agents from the ancient times (Dai et al., 2008; Liu et al., 2014a, 2014b). There are at least 57 Chinese patent medicines (CPMs) containing pearl or nacre in Chinese Pharmacopeia 2015 and most of them have sedative efficacy, such as Angong Niuhuang Pill, Xinnaojing Tablet, Xiegan Anshen Pill, and Qingkailing Injection (Guo et al., 2014, Zhang et al., 2015a). Medicinal pearl and nacre are generally used in the form of original powder in pharmaceutical factory or in clinical, through thoroughly smashing and grinding of natural products to make the ingredients more bioavailable. A considerable number of 3
investigations have revealed that pearl and nacre are rich sources of calcium, accounting for over 90% of the weight, followed by conchiolin protein, and a small amount of trace metal elements. Although the chemical compositions and pharmacological efficacy of the two traditional medicines are clear, the exact bioactive ingredients are still far from identification, which lead to a lower use efficiency of the natural products and also hinder the development of the related novel products. 5-HT is an important excitatory neurotransmitter that widely distributed in animals. It binds with multiple receptors to regulate many complex behaviors in vertebrates and invertebrates (Gerhardt et al., 1997; Tierney, 2001). It is well known that 5-HT is engaged in learning, anxiety, emotion, reproduction, pain perception, sleep and locomotion in mammals. However, it is found that 5-HT and its receptors are implicated in neuronal functions including circadian rhythm, feeding, locomotion, memory, parturition and development (Filla et al., 2004; Kawai et al., 2011; Panasophonkul et al., 2009). Inhibitory neurotransmitter GABA is also one of the critical neurotransmitters (NTs), whose metabolites are closely related to the central nervous system (CNS) depressant activity. In addition, previous literature has documented the role of GABA in CNS of mollusks (Gunaratne et al., 2014). However, it still remains unknown that whether pearl powder, nacre powder and their corresponding protein extracts could improve locomotor activity and convulsions latency. Moreover, the exact active ingredients need to be illustrated. Hence, the current experiment screened the anticonvulsant and sedative-hypnotic activity of pearl 4
powder, nacre powder and their corresponding protein extracts in regulating 5-HT and GABA.
2. Materials and methods 2.1. Materials Eighteen batches of pearls and eighteen batches of nacres were purchased from medicinal plant markets, mainly from Shandong and Zhejiang Provinces in China. The pooled samples from different batches of pearls and nacres were prepared as the tested samples. Voucher specimens were deposited in the laboratory of the author. Amino acid standards (purity 99%) were purchased from the China Institute of Pharmaceutical and Biological Products. The internal standard (IS), α-amino butyric acid (AABA), was purchased from Sigma (St. Louis, MO, USA) with a purity of 99%. AccQ·Tag™ Eluent A concentrate (P/N 186003838) and Elution B (P/N 186003839) used for gradient program, as well as the AccQ·Fluor™ pre-column AQC derivatisation kit were from Waters (Milford, MA, USA). Diazepam (2 ml / 10 mg) was purchased from Tianjin Jinyao amino acids CO., Ltd. 5-HT and GABA ELISA kits were purchased from Elabscience Biotechnology Co., Ltd. Primary antibodies against 5-HT and GABA were produced by Cell Signaling Technology Inc (Beverly, MA, USA). All other chemicals and reagents used for study were of analytical grade and were purchased from approved organizations.
5
2.2. Sample preparation and amino acid analysis The crude materials were crushed into very fine powder according to the process method recorded in the Chinese Pharmacopeia 2015 (China Pharmacopoeia Committee, 2015). The sample powder of 100 g was immersed with 10 times of water at 4 °C for 24 h and the solution was filtered. The filtrate was freeze-dried to obtain the water-soluble protein. Ten times of diluted hydrochloric acid was then added to the residue and stirred, and allowed to react at 4 °C for 12 h. The solution was centrifuged at 12000 rpm for 20 min. The precipitate was freeze-dried to yield the conchiolin protein and the supernatant was dialyzed with the molecular weight cut off (MWCO) 3000 for 48 h and the solution was then freeze-dried to obtain the acid-soluble protein. Amino acid analysis was carried out by UPLC combined with AQC pre-column derivatisation in accordance with the method described previously (Zhang et al., 2015b). 2.3. Animals 100 male and 100 female mice, weighing 20-22 g, obtained from Shanghai Jiesijie Laboratory Animal Co., Ltd., were housed in a temperature and humidity controlled animal facility with a set of 12 h light-dark cycle. Mice had free access to water and food pellets ad libitum.
6
2.4. Locomotor activity test Mice were randomly divided into ten groups (n = 10) with half male and female as follows: control group (A), diazepam (2.2 mg/kg) group (B), pearl original powder (1.1 g/kg) group (C), pearl water-soluble protein (0.2 g/kg) group (D), pearl acid-soluble protein (0.275 g/kg) group (E), pearl conchiolin protein (1.1 g/kg) group (F), nacre original powder (1.1 g/kg) group (G), nacre water-soluble protein (0.2 g/kg) group (H), pearl acid-soluble protein (0.7 g/kg) group (I) and nacre conchiolin protein (1.1 g/kg) group (J). The dosage were selected based on the results in the preliminary experiment and the related reference (Dai et al., 2008). After starvation for 12 h, mice were placed individually in an open container (50 × 20 cm) with a black surface covering the inside walls and allowed to explore freely to acclimatize the environment for 2 min before the test. The container was divided into 12 equal squares and the number of panes was recorded. The number of squares crossed and rearing behaviors were recorded for 4 min. The above experimental procedure was repeated once 1 h after intragastrically treated with tested drugs and diazepam. Then, mice were sacrificed, and the brain was harvested subsequently and set aside at -80 °C for further analysis.
7
2.5. Anticonvulsant experiment Mice were randomly divided into ten groups (n = 10) with half male and female as follows: control group, diazepam group, pearl original powder (1.1 g/kg) group, pearl water-soluble protein (0.2 g/kg) group, pearl acid-soluble protein (0.275 g/kg) group, pearl conchiolin protein (1.1 g/kg) group, nacre original powder (1.1 g/kg) group, nacre water-soluble protein (0.2 g/kg) group, nacre acid-soluble protein (0.7 g/kg) group and nacre conchiolin protein (1.1 g/kg) group. Tested drugs were intragastrically administered for 3 consecutive days. Diazepam (2.2 mg/kg) was orally treated for 3 consecutive days. After intragastric administration 1 h on day 3, mice were intraperitoneally injected with 0.5% of pentylenetetrazol (PTZ, 100 mg/kg). Convulsions latency time was recorded subsequently and mortality of mice were observed 6 h later. 2.6. Measurement of 5-HT3 and GABAB neurotransmitter levels The measurements of 5-HT3 and GABAB in brain of mice were conducted using ELISA kits according to the manufacturer’s instruction. 2.7. Immunohistochemistry The paraffin sample of brain were heated for 1 h in the oven, deparaffinized in xylene, rehydrated by graded ethanol solutions, microwaved in sodium citrate buffer, cooled to room temperature naturally and incubated in 3% hydrogen peroxide. Each section was blocked with 3% BSA at room temperature. Samples were then incubated with primary antibody overnight at 4 °C after removing blocking solution, secondary 8
antibody for 20 min at 37 °C and three antibody for another 20 min at 37 °C, respectively. Then, sections were stained with DAB and restained with hematoxylin. Followed by dehydrating and drying, the sections were mounted with neutral gum and observed under a microscope. 2.8. Western blot Brain tissues were minced and homogenized in ice-cold RIPA lysis buffer, followed by centrifugation at 12000 rpm for 5 min at 4 °C to remove the debris. Based on the concentration measured by BCA assay, equal amounts of 5-HT, GABA and GAPDH per line were subjected to a 10% SDS-polyacrylamide gel electrophoresis for the separation and transferred onto the polyvinylidene difluoride membrane. Target blots were blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4 °C using specific antibodies against 5-HT, GABA and GAPDH. The bands were then incubated for 1 h at room temperature with the corresponding secondary antibodies after washing with TBST three times. Enhanced chemiluminescence detection (ECL) reagents and a gel imaging system (Tanon Science & Technology Co., Ltd., China) were used to visualize the protein sheets. 2.9. Statistical analysis All data were expressed as means ± standards deviation (SDs). Evaluation between the control group and the administered groups respectively were analyzed by One-way analysis of variance (ANOVA) followed by Tukey multiple comparison test. The values were considered statistically significantly different at p < 0.05. 9
3. Results 3.1. Amino acids analysis The yields for the obtained proteins including water-soluble protein, acid-soluble protein and conchiolin protein for pearl and nacre are 0.16%, 0.40%, 1.30% and 0.10%, 0.43%, 1.80%, respectively. The amino acid composition was determined by UPLC combined with AQC pre-column derivatisation and the chromatograms were shown in Fig. 1. It was obvious that different kinds of protein contained similar amino acids, mainly including arginine (Arg), serine (Ser), glycine (Gly), asparagine (Asp), glutamic acid (Glu), threonine (Thr), alanine (Ala), proline (Pro), cystine (Cys), tyrosine (Tyr), valine (Val), lysine (Lys), isoleucine (Ile), leucine (Leu) and phenylalanine (Phe). Conchiolin proteins from both pearl and nacre possessed the most abundant amino acids.
10
Fig. 1. Amino acid compositions of pearl/nacre water-soluble protein, pearl/nacre acid-soluble protein and pearl/nacre conchiolin protein.
3.2. Effect of tested drugs on locomotor activity in mice The effects of pearl powder, nacre powder and their corresponding protein extracts on the locomotor activity of mice were listed in Table 1. Among all the tested drugs, pearl conchiolin protein dramatically reduced the numbers of squares crossed behaviors compared with the control group, as well as diazepam, while the efficacy of nacre conchiolin protein was secondary. However, pearl water-soluble protein, pearl acid-soluble protein and nacre acid-soluble protein decreased rearing behaviors only, while pearl water-soluble protein, pearl conchiolin protein, nacre original powder, nacre acid-soluble protein, and nacre conchiolin protein significantly reduced rearing behaviors. 3.3. Effect of tested drugs on PTZ-induced convulsions The effects of pearl and nacre powder and their corresponding protein extracts on PTZ-induced convulsions were shown in Table 2. The mortality of mice after 6 h was also listed. Diazepam, nacre water-soluble protein and nacre original powder remarkably prolonged the convulsions latency time compared with those in the control group. Meanwhile, pearl original powder, pearl water-soluble protein, pearl acid-soluble protein, pearl conchiolin protein and nacre conchiolin protein also significantly enhanced convulsions latency time, while nacre acid-soluble protein had 11
no significant influence on convulsions latency, which partially illustrated that nacre acid-soluble protein might showed no anticonvulsant activity.
Table 1 Effect of tested drugs on locomotor activity in mice (means ± SD, n = 10). Group
Dose (g/kg)
Crossing
Rearing
Control
——
153.4±24.5
40.5±8.3
Diazepam
2.2×10-3
114.7±9.2**
17.5±6.9**
Pearl original powder
1.1
148.8±31.7
34.1±11.1*
Pearl water-soluble protein
0.2
153.6±31.2
30.7±9.1**
Pearl acid-soluble protein
0.275
159.0±23.3
34.3±7.0
Pearl conchiolin protein
1.1
113.5±24.6**
30.2±9.2**
Nacre original powder
1.1
149.1±23.1
28.8±8.9**
Nacre water-soluble protein
0.2
144.6±22.5
37.3±9.6
Nacre acid-soluble protein
0.7
176.7±36.9
26.0±9.6**
Nacre conchiolin protein
1.1
138.6±28.9**
30.6±5.0**
Table 2 Effect of tested drugs on PTZ-induced convulsions (means ± SD, n = 10). Groups
Dose (g/kg)
Convulsions latency / min
mortality / %
Control
——
2.5±0.8
70.0
Diazepam
2.2×10-3
5.1±1.7**
0
Pearl original powder
1.1
3.4±1.0*
80.0
Pearl water-soluble protein
0.2
4.1±1.5*
80.0
Pearl acid-soluble protein
0.275.
3.6±1.1*
30.0
Pearl conchiolin protein
1.1
3.8±1.3*
50.0
Nacre original powder
1.1
4.6±1.2**
70.0
Nacre water-soluble protein
0.2
5.3±1.1**
80.0
Nacre acid-soluble protein
0.7
3.3±1.0
80.0
Nacre conchiolin protein
1.1
3.8±1.4*
60.0
12
3.4. Effect of tested drugs on 5-HT3 and GABAB in brain As shown in Fig. 2, the concentration of excitatory neurotransmitter 5-HT3 in brain was down-regulated, while the level of inhibitory neurotransmitter GABAB was up-regulated with tested drugs treatment in comparison with the control group. However, pearl original powder (C) and nacre acid-soluble protein (I) reduced 5-HT content without significant difference.
Fig. 2. Measurement of 5-HT3 and GABAB neurotransmitter levels. Control group (A), diazepam (2.2 mg/kg) group (B), pearl original powder (1.1 g/kg) group (C), pearl water-soluble protein (0.2 g/kg) group (D), pearl acid-soluble protein (0.275 g/kg) group (E), pearl conchiolin protein (1.1 g/kg) group (F), nacre original powder (1.1 g/kg) group (G), nacre water-soluble protein (0.2 g/kg) group (H), nacre acid-soluble protein (0.7 g/kg) group (I), nacre conchiolin protein (1.1 g/kg) group (J). Values represent the mean ± SD and are representative of two independent experiments. **p＜0.01, significantly different from the values in the control group; *p＜0.05, significantly different from the values in the control group. 13
3.5. Effect of tested drugs on the expressions of 5-HT3 and GABAB receptor Immunohistochemistry analysis further exhibited the alterations of 5-HT3 (Fig. 3a) and GABAB (Fig. 3b). All the tested drugs treatment exhibited notable reduced expression of 5-HT3 receptor, especially pearl original powder (C) and pearl water-soluble protein (D). Moreover, all the tested drugs showed markedly recovered level of GABA receptor, especially pearl acid-soluble protein (E), nacre water-soluble protein (H), nacre acid-soluble protein (I), nacre conchiolin protein (J) and nacre original powder (G). The data depicted in Fig. 4 illustrated the inhibited 5-HT3 receptor and highly expressed GABAB with GAPDH expression as internal control, which further confirmed the involvement of 5-HT/GABA balance in sedation and hypnosis. Specifically, nacre conchiolin protein (J) showed no significant influence on the content of 5-HT3. Nacre acid-soluble protein (I) and nacre conchiolin protein (J) showed no remarkable effects on the level of GABAB. Meanwhile, all the other tested drugs exhibited significant difference of 5-HT and GABA expressions compared with the control group.
14
Fig. 3. Effect of tested drugs on the expressions of 5-HT3 (a) and GABAB (b) receptor according to immunohistochemistry. control group (A), diazepam (2.2 mg/kg) group (B), pearl original powder (1.1 g/kg) group (C), pearl water-soluble protein (0.2 g/kg) group (D), pearl acid-soluble protein (0.275 g/kg) group (E), pearl conchiolin protein (1.1 g/kg) group (F), nacre original powder (1.1 g/kg) group (G), nacre water-soluble protein (0.2 g/kg) group (H), nacre acid-soluble protein (0.7 g/kg) group (I), nacre conchiolin protein (1.1 g/kg) group (J). Values represent the mean ± SD and are representative of two independent experiments. **p＜0.01, significantly different from the values in the control group; *p＜0.05, significantly different from the values in the control group.
15
Fig. 4. Effect of tested drugs on the expressions of 5-HT3 and GABAB receptor according to western blot. control group (A), diazepam (2.2 mg/kg) group (B), pearl original powder (1.1 g/kg) group (C), pearl water-soluble protein (0.2 g/kg) group (D), pearl acid-soluble protein (0.275 g/kg) group (E), pearl conchiolin protein (1.1 g/kg) group (F), nacre original powder (1.1 g/kg) group (G), nacre water-soluble protein (0.2 g/kg) group (H), nacre acid-soluble protein (0.7 g/kg) group (I), nacre conchiolin protein (1.1 g/kg) group (J). Values represent the mean ± SD and are representative of two independent experiments. **p＜0.01, significantly different from the values in the control group; *p＜0.05,significantly different from the values in the control group.
16
4. Discussion Pearls have existed for approximately two hundred million years. It is not only famous as a valuable ornament, but also known as a traditional medicine to treat palpitations, convulsions, epilepsy, eye diseases and promote wound healing (Dai et al., 2010; Mangrulkar et al., 2002; Shen et al., 2006; Xu et al., 2001). Pearls also have antioxidant properties (Shao et al., 2010) and provide high calcium bioavailability (Chen et al., 2008). Besides, nacre is commonly used to treat insomnia and anxiety in the modern clinical practice as well (Liu et al., 2014a and 2014b). The anti-depressant activity of different processed products of nacre in mice provides pharmacological foundation for its sedative and hypnotic effects (Li et al., 2014b). In order to identify the material basis of the anticonvulsant and sedative-hypnotic activity documented, the subject analyzed the role of pearl powder, nacre powder and their corresponding water-soluble protein, acid-soluble protein, conchiolin protein in the CNS. It is widely accepted that the reduced number of squares crossed and rearing behavior in locomotor activity test and increased convulsions latency reflected the activity of the tested agents. Specifically, pearl conchiolin protein significantly reduced the number of squares mice crossed behavior, which was in line with positive control diazepam. However, pearl water-soluble protein, pearl acid-soluble protein and nacre acid-soluble protein only showed obviously decreased number of rearing behavior. Since both of crossing and rearing behavior represent the intensity of locomotor activity, it could be reasonable to judge that pearl acid-soluble protein and nacre 17
water-soluble protein might be absent from sedative-hypnotic activity. The PTZ mouse model of generalized myoclonic seizures has long been used in the discovery and characterization of all marked antiepileptic drugs (Loscher et al., 1991; Loscher and Schmidt, 1988; Li et al., 2014a). This classical animal model was adopted to screen the tested agents in the present study. It is considered to be general by intraperitoneal administration of the PTZ ranging from 80 to 90 mg/kg (Hosseini et al., 2011, Koutroumanidou et al., 2013). By contrast, severe seizure persisted after injection at higher dose of PTZ (100 mg/kg) group, where the relative mortality percentage was higher than those of lower doses (Li et al., 2014). In this study, the dose of 100 mg/kg was applied in the consideration of the purpose to examine the effect of pearl or nacre against severe seizure induced by PTZ. It is well known that there is positive correlation between different types of seizure and doses of PTZ. In the current study, all the candidate drugs, except nacre acid-soluble protein, could effectively enhanced convulsions latency time, which suggested that the possibility of nacre acid-soluble protein selected as an anticonvulsant might need more in-depth investigation. The monitoring of neurotransmitters and their metabolites is an effective tool for illustrating normal and pathological neural system functions. Sedative-hypnotics and anticonvulsant drugs used in clinical are a class of central nervous system depressant drugs. 5-HT and GABA are known to maintain the balance in the nervous system for numerous organisms. The levels of them are tightly linked with neurophysiology, behavior, pathology, disease diagnosis and control (Feng et al., 2001; Harada et al., 18
2006). An alteration in the brain levels of neurotransmitters and their metabolites is the indicator of neurotransmission disorders. It has been reported that therapeutic drugs induce sedative-hypnotic effects via modulating neurotransmission such as in the GABAergic or serotonergic systems in the CNS (Attele et al., 2000). Thus, the analysis of 5-HT and GABA contents is of great importance in monitoring efficacy of the tested drugs treatment. The current study provides important new insight into the 5-HT receptor system in the CNS with the treatment of pearl powder, nacre powder and their corresponding protein extracts. Prior documents demonstrated that excessive extracellular 5-HT metabolite 5-hydroxyindoleacetic acid was accompanied by wakefulness and less sleep in freely moving rats (Imeri et al., 1994), which reflected the important role of 5-HT in sedation and hypnosis. Furthermore, the location of 5-HT receptor and the biological activity of its ligands also indicated the participation in the pathogenesis of convulsions (Yang et al., 2012). As demonstrated from literatures that activation of the subtype of 5-HT3 could increase the seizure threshold induced by PTZ. In addition, GABA is the pivotal inhibitory neurotransmitter in the central nervous system. The GABAergic agents display anticonvulsant activity in various animal models and are recently being evaluated for anti-epileptic activity in humans (Ong et al., 2000). PTZ is defined as a GABAA receptor antagonist. Benzodiazepines and barbiturates are the two main classes of hypnotic-sedative and anticonvulsant agents. Wherein, barbiturates are identified to be bind to the benzodiazepine site of the GABA receptor complex and 19
enhance the binding of its neurotransmitter GABA (Joksimović et al., 2013). GABAA and GABAB receptors are involved in the control of epileptogenesisand neuronal excitability. Furthermore, GABAB receptor function could be expressed as that its molecular structure provides a large number of sites for pharmaceutical intervention in symptoms, including drug addition, absence seizures and nociception. More importantly, a number of studies have reported that the expression or function of GABAB in brain tissues in animal models of resistant epilepsy or patients (Billinton et al., 2001). Vergnes elicited the role for GABAB receptors resided in the neo-cortex and hippocampus in some epileptic disorder (Vergnes et al., 1997). The critical role of GABAA receptors in the control of generalized convulsive seizures was also well elucidated (Mohler H., 1997). Slight deficiencies in GABAergic transmission may lead to hyperexcitability and pathological neuronal discharges leading to epilepsy. Thus, when assessing the alleviation of pearl powder, nacre powder and their corresponding protein extracts on convulsions, researcher should take the GABAergic system, especially GABAB, into consideration. The expressions of 5-HT3 and GABAB were identified by ELISA kits, immunohistochemistry and western blot respectively in the present study. Different detection methods exhibited distinct sensitivity, detection range and quantitative manner. All the candidate drugs showed varying degrees of down-regulation of 5-HT and up-regulation of GABAB.
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5. Conclusion Taken together, the tested drugs exhibited varying degrees of inhibition of locomotor activity and improvement of PTZ-induced convulsions. Besides, the findings in the study exhibited distinct down-regulated levels of 5-HT3 and up-regulated levels of GABAB with pearl original powder, pearl water-soluble protein, pearl acid-soluble protein, pearl conchiolin protein, nacre original powder, nacre water-soluble protein, nacre acid-soluble protein and nacre conchiolin protein treatment, which reflected the essential involvement of 5-HT/GABA balance in sedation and hypnosis. These results provided critical important evidence for the selection of quality control markers and also for drug development correlated with the traditional use of pearl and nacre as sedation and tranquilization agents.
Conflict of interests None of the authors has any conflict of interests regarding this study.
Acknowledgements This study was financially supported by the National High Technology Research and Development Program of China (No. 2013AA093002). The authors thank Dr. Chun-Hua Ma for his help in the revision of this paper.
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References Attele, A.S., Xie, J.T., Yuan, C.S., 2000. Treatment of insomnia: an alternative approach. Altern. Med. Rev. 5, 249–259. Billinton A., Baird V.H., Thom M., Duncan J.S., Upton N., Bowery N.G., 2001. GABA(B) receptor autoradiography in hippocampal sclerosis associated with human temporal lobe epilepsy. Br. J. Pharmacol. 132, 475–480. Chen H.S., Chang J.H., Wu J.S., 2008. Calcium bioavailability of nanonized pearl powder for adults. J. Food Sci. 246–251. China Pharmacopoeia Committee, 2010. Pharmacopoeia of the People’s Republic of China. China Chemical Industry Press, Beijing. Dai, J., Duan, J.A., Li, Y.B., Liu, R., Peng, Y.R., 2008. Investigation of sedative bioactive components and evaluation of the biological effect of freshwater pearls produced in Suzhou. Chin. J. Biochem. Pharm. 29, 294–298. Dai J.P., Chen J., Bei Y.F., Han B.X., Guo S.B., Jiang L.L., 2010. Effects of pearl powder extract and its fractions on fibroblast function relevant to wound repair. Pharmaceutical. Biology. 48, 122–127. Feng, J., Cai, X., Zhao, J., Yan, Z., 2001. Serotonin receptors modulate GABAA receptor channels through activation of anchored protein kinase C in prefrontal cortical neurons. J. Neurosci. 21, 6502–6511. Filla, A., Hiripi, L., Elekes, K., 2004. Serotonergic and dopaminergic influence of the duration of embryogenesis and intracapsular locomotion of Lymnaea stagnalis L. 22
Acta Biol. Hung. 55, 315–321. Gerhardt,
C.C.,
Heerikhuizen,
H.V.,
1997.
Functional
characteristics
of
heterologously expressed 5-HT receptors. Eur. J. Pharmacol, 334, 1–23. Gunaratne, C.A., Sakurai, A., Katz, P.S., 2014. Comparative mapping of GABA-immunoreactive neurons in the central nervous systems of nudibranch molluscs. J. Comp. Neurol. 522, 794–810. Guo, Y., Yan, S., Xu, L., Zhu, G., Yu, X., Tong, X., 2014. Use of angong niuhuang in treating central nervous system diseases and related research. Evid. Based Complement. Alternat. Med. 346918. Harada, K., Aota, M., Inoue, T., Matsuda, R., Mihara, T., amaji, T., Ishibashi, Y.K., Matsuoka, N., 2006. Anxiolytic activity of a novel potent serotonin 5-HT 2 C receptor antagonist FR260010: a comparison with diazepam and buspirone. Eur. J. Pharmacol. 553, 171–184. Hosseini M, Ghasemzadeh Rahbardar M, Sadeghnia HR, Rakhshandeh H., 2011. Effects of different extracts of Rosa damascena on pentylenetetrazol-induced seizures in mice. J. Chin. Integr. Med. 9, 1118–1124. Imeri, L., De Simoni, M. G., Giglio, R., Clavenna, A., Mancia, M., 1994. Changing in the serotonergic system during the sleep-wake cycle: simultaneous polygraphic and voltammetric recordings in hypothalamus using a telemetry system. Neuroscience 58, 353–358. Joksimović, S., Varagic, Z., Kovačević, J., Van Linn, M., Milić, M., Rallapalli, S., Timić, T., Sieghart, W., Cook, J.M., Savić M.M., 2013. Insights into functional 23
pharmacology of α₁ GABA (A) receptors: how much does partial activation at the benzodiazepine site matter? Psychopharmacology (Berl) 230, 113–123. Kawai, R., Kobayashi, S., Fujito, Y., Ito, E., 2011. Multiple subtypes of serotonin receptors in the feeding circuit of a pond snail. Zool. Sci. 28, 517-525. Koutroumanidou E., Kimbaris A., Kortsaris A., Bezirtzoglou E., Polissiou M., Charalabopoulos K., 2013. Increased seizure latency and decreased severity of pentylenetetrazol-induced seizures in mice after essential oil administration. Epilepsy Res. Treat 2013: 532657. Li B.J., Wang L., Sun Z.H., Zhou Y., Shao D.Y., Zhao J., Song Y.N., Lv J.Y., Dong X., Liu C.H., Wang P., Zhang X.Y., Cui R.J, 2014a. The anticonvulsant effects of SR 57227 on pentylenetetrazole-induced seizure in mice. Plos One 9(4), e93158. Li, Y., Sun, J.M., Zhang, J., Li, J.F., Liu, W.C., Zhang, H., 2014b. Anti-depressant effect of Margaritifera concha on mice. J. Jilin Tradit. Chin. Med. 34, 388–389. Liu, D., Kang, X.Y., Ren, B.Y., Zhang, H.Y., Du, H., Song, L., Zhang, H., Sun, J.M., 2014a. Comparison of effects of sedative-hypnotic action of protein and peptides of Margaritifera concha on rats. Jilin J. Tradit. Chin. Med. 34, 172–173, 176 Liu, D., Dai, T.T., Cha, R.B., Qu, P., Li, Y., Du, H., Zhao, S.Y., Song, L., Zhang, H., Sun, J.M., 2014b. Study on sedative and hypnotic effects of Concha Margaritifera and its different processed products on the content of 5-HT in the brain of mice. Jilin J. Tradit. Chin. Med. 34, 61–63. Loscher W., Schmidt D., 1988. Which animal models should be used in the search for new antiepileptic drugs? A proposal based on experimental and clinical 24
considerations. Epilepsy Res. 2, 145–181. Loscher W., Honack D., Fassbender C.P., Nolting B., 1991. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs, III. Pentylenetetrazole seizure models. Epilepsy Res. 8, 177–189. Mangrulkar, R.S., Saint, S., Chu, S., Tierney, L.M., 2002. What is the role of the clinical "pearl"? Am. J. Med. 113, 617–624. Mohler H., 1997. Genetic approaches to CNS disorders with particular reference to GABAA-receptor mutations. J. Recept. Signal Transduct. Res.17, 1–10. Ong J., Kerr D.I., 2000. Recent advances in GABAB receptors: from pharmacology to molecular biology. Acta Pharmacol. Sin. 21, 111–123. Panasophonkul, S., Apisawetakan, S., Cummins, S.F., York, P.S., Degnan, B.M., Hanna, P.J., Saitongdee, P., Sobhon, P., Sretarugsa, P., 2009. Molecular characterization and analysis of a truncated serotonin receptor gene expressed in neural and reproductive tissues of abalone. Histochem. Cell Biol, 131, 629–642. Shao D.Z., Wang C.K., Hwang H.J., Huang C.H. Chen Y.W., 2010. Comparison of hydration, tyrosinase resistance, and antioxidant activation in three kinds of pearl powders. J. Cosmet. Sci. 61, 133–145. Shen Y.T., Zhu J., Zhang H.B., Zhao F., 2006. In vitro osteogenetic activity of pearl. Biomaterials 27, 281–287. Tierney, A.J., 2001. Structure and function of invertebrate 5-HT receptors: a review. Comp. Biochem. Physiol. A128, 791–804. 25
Vergnes M., Boehrer A., Simler S., Bernasconi R., Marescaux C., 1997. Opposite effects of GABAB receptor antagonists on absences and convulsive seizures. Eur. J. Pharmacol. 332: 245-255 Xu H., Huang K., Gao Q., Gao Z., Han X., 2001. A study on the prevention and treatment of myopia with nacre on chicks. Pharmacol. Res. 44, 1–6. Yang, Z., Liu, X., Yin, Y., Sun, S., Deng, X., 2012. Involvement of 5-HT receptors in the pathogenesis of temporal lobe epilepsy. Eur. J. Pharmacol. 685, 52–58. Zhang, D.S., Liu, Y.L., Zhu, D.Q., Huang, X.J., Luo, C.H., 2015a. Point application with Angong Niuhuang sticker protects hippocampal and cortical neurons in rats with cerebral ischemia. Neural Regen. Res. 10, 286–291. Zhang, J.X., Li, S.R., Yao, S., Si, W., Cai, L.Y., Pan, H.Q., Hou, J.J., Yang, W.Z., Da, J., Jiang, B.H., Liu, X., Wu, W.Y., Guo, D.A., 2015b. Ultra-performance liquid chromatography of amino acids for the quality assessment of pearl powder. J. Sep. Sci. 38, 1552–1560.
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