Effect of borneol, moschus, storax, and acorus tatarinowii on expression levels of four amino acid neurotransmitters in the rat corpus striatum.

The present study collected cerebrospinal fluid samples from the corpus striatum in rats treated with borneol, moschus, storax, and acorus tatarinowii using brain microdialysis technology. Levels of excitatory neurotransmitters aspartic acid and glutamate, as well as inhibitory neurotransmitters glycine and γ-aminobutyric acid, were measured in samples using reversed-phase high-performance liquid chromatography, phosphate gradient elution, and fluorescence detection. Results showed that concentrations of all four amino acid neurotransmitters significantly increased in the corpus striatum following treatment with borneol or moschus, but effects due to borneol were more significant than moschus. Acorus tatarinowii treatment increased γ-aminobutyric acid expression, but decreased glutamate concentrations. Storax increased aspartic acid concentrations and decreased glycine expression. Results demonstrated that borneol and moschus exhibited significant effects on con amino acid neurotransmitter expression; storax exhibited excitatory effects, and acorus tatarinowii resulted in inhibitory effects.

INTRODUCTION

Borneol, moschus, storax, and acorus tatarinowii are used to induce resuscitation, exhibiting properties of fast absorption, wide distribution, and quick elimination in the brain. These drugs remain at high concentrations for long periods of time in the brain and can pass through the blood-brain barrier (BBB), thereby participating in bidirectional modulation of BBB permeability. The primary pharmacological effects to the central nervous system are exerted by sedative effects and the regulation of brain mechanisms to reduce brain injury[1]. Borneol, also called “compound long nao,” is a colorless and transparent crystal. Borneol has both sedative and refreshing effects on the central nerve[2]. Borneol has been shown to enhance BBB permeability[34] and increase brain concentrations of drugs, as well as protect the brain and BBB while inducing no pathological damage[5].

Moschus is a dried secretion from the ripe, male musk cyst of Moschus berezovskii Flerov, M. sifanicus Przewalski, or M. moschiferus Linnaeus[6]. Moschus has been shown to contribute to bidirectional central nervous system modulation: low-dose moschus excites the center, and high doses inhibit the center[7].

Acorus tatarinowii, a dried rhizome from Acorus tatarinowii Schott, has been shown to excite and calm the central nervous system, as well as provide treatment for encephalopathy[8].

Storax is prepared from trunk resin of Lipuidambar orientalis Mill. following processing[9].

The effects of these drugs for inducing resuscitation have been reflected in aspects of sedative and hypnosis, anti-convulsion, anti-epilepsy, cerebral protection, and anti-hypoxia, although differences exist between the resuscitation drugs[10]. However, few studies have compared the effects of resuscitation drugs on brain neurotransmitter expression.

The present study utilized microdialysis technology[11]and high-performance liquid chromatography (HPLC) to assess concentration changes in excitatory neurotransmitters aspartic acid and glutamate, as well as inhibitory neurotransmitters glycine and γ-aminobutyric acid (GABA), in the corpus striatum of rats following treatment with borneol, moschus, storax, and acorus tatarinowii. The central nervous system effects were subsequently compared.

RESULTS

Quantitative analysis of experimental animals

A total of 30 rats were randomly assigned to five groups: control, borneol, moschus, acorus tatarinowii, and storax. Following oral administration, the probes were implanted in the corpus striatum and remained left for 60 minutes; corpus striatum samples were collected and tested at 15, 30, 45, 60, 75, 90, and 105 minutes. All 30 rats were included in the final analysis.

HPLC detection for linear range of amino acid concentration

An Agilent 1200 high-performance liquid chromatographic system was used to detect amino acid concentrations, demonstrating that the four amino acids exhibited a good linear relationship along the plotted standard curve (0.031 25-2.000 mg/L) (Table 1, Figure 1).

Table 1

Aspartic acid (Asp), glutamate (Glu), glycine (Gly), and γ-aminobutyric acid (GABA) standard curves and linear coefficients

Figure 1

Standard curves of aspartic acid (Asp), glutamate (Glu), glycine (Gly), and γ-aminobutyric acid (GABA).

HPLC detection for precision and probe recovery of the amino acids

According to chromatographic conditions, in vitro probe recovery of each amino acid was calculated as follows: aspartic acid = 6.67%, glycine = 6.09%, glutamate = 12.5%, and GABA = 13.7%. The relative standard deviation of measured peak areas and peak migration times of the amino acids is shown in Table 2.

Table 2

Relative standard deviation of aspartic acid (Asp), glutamate (Glu), glycine (Gly), and γ-aminobutyric acid (GABA) peak area and peak migration time (%)

Effect of borneol, moschus, storax, and acorus tatarinowii on amino acid neurotransmitter levels in the rat corpus striatum

HPLC was used to measure amino acid levels in microdialysis samples of the corpus striatum. Comparisons with standard HPLC showed that the amino acids from the brain dialysate samples were sufficiently separated (Figure 2).

Figure 2

High-performance liquid chromatography results of amino acids in brain dialysate samples of borneol, moschus, storax, and acorus tatarinowii groups, as well as standard samples.

I: Control group; II: borneol group; III: moschus group; IV: storax group; V: acorus tatarinowii group. Asp: Aspartic acid; Glu: glutamate; Gly: glycine; GABA: γ-aminobutyric acid.

Aspartic acid concentration

Compared with the control group, aspartic acid levels significantly increased in the borneol, moschus, and storax groups (P < 0.05 or P < 0.01), but no significant differences were measured between the acorus tatarinowii and control groups (P > 0.05; Table 3).

Table 3

Aspartic acid concentrations (×10-4 g/L) in brains of borneol, moschus, storax, and acorus tatarinowii groups

Glutamate concentration

Compared with the control group, glutamate levels significantly increased in the borneol and moschus groups (P < 0.05 or P < 0.01), but glutamate expression in the acorus tatarinowii group was significantly less than control group (P < 0.05). No difference was detected between storax and control groups (P > 0.05; Table 4).

Table 4

Glutamate concentrations (×10-4 g/L) in brains of borneol, moschus, storax, and acorus tatarinowii groups

Glycine concentration

Compared with the control group, glycine levels significantly increased in the borneol, moschus, and acorus tatarinowii groups (P < 0.05 or P < 0.01), in particular the borneol group. However, glycine concentrations decreased in the storax group (P < 0.01; Table 5).

Table 5

Glycine concentrations (×10-4 g/L) in brains of borneol, moschus, storax, and acorus tatarinowii groups

GABA concentration

Compared with the control group, GABA concentrations significantly increased in the borneol, moschus, and acorus tatarinowii groups (P < 0.05 or P < 0.01). GABA concentrations significantly increased during the first 30 minutes in the acorus tatarinowii group, but concentration levels remained similar to the other groups at the other three time points. No differences were detected between the storax and control groups (P > 0.05; Table 6).

Table 6

Gamma-aminobutyric acid concentrations (×10-4 g/L) in brains of borneol, moschus, storax, and acorus tatarinowii groups

DISCUSSION

Neurotransmitters of the central nervous system play a role in a variety of physiological functions. Concentration changes in brain tissue reflect physiological functions of the central nervous system. Drugs can alter these concentrations and influence the regulation of central nervous system functions[12]. Concentrations of excitatory neurotransmitters, such as aspartic acid and glutamate, and inhibitory neurotransmitters, such as glycine and GABA, as well as the ratio between excitatory and inhibitory neurotransmitters, are commonly used as biochemical indicators for diagnosing neurological diseases[13].

The present study compared the influence of these four resuscitation drugs on brain amino acid neurotransmitter levels using a microdialysis technique[11]. HPLC results showed that the amino acids were sufficiently separated, and not affected by other impurities in the brain dialysis samples. Compared with the control group, these amino acid levels significantly increased in the borneol and moschus groups. However, glutamate levels significantly decreased, and the inhibitory amino acid GABA increased, in the acorus tatarinowii group, suggesting an inhibitory influence of acorus tatarinowii on the central nervous system. Storax increased aspartic acid and reduced glycine, suggesting that storax resulted in excitatory influences in the central nervous system. Restlessness, delirium, and depression have been shown to be associated with central nervous system imbalances[14]. The present resuscitation drugs could potentially help to balance the central nervous system by regulating neurotransmitter expression in the brain. Borneol and moschus significantly increased amino acid expression and could, therefore, be utilized to refresh and resuscitae. Storax exhibited a mild excitory effect in the brain and could, therefore, be used to improve memory, ameliorate dementia, and treat depression. The inhibitory effect of acorus tatarinowii could be used for anti-convulsion, anti-epileptic, and cerebroprotection[15]. In summary, borneol and moschus significantly influenced excitatory and inhibitory neurotransmitter expression in the brain. Acorus tatarinowii primarily exhibited an inhibitory effect, while storax exhibited a mild excitory effect.

MATERIALS AND METHODS

Design

A randomized, controlled, animal experiment.

Time and setting

The present study was performed at the Pharmacy of Traditional Chinese Medicine, PLA General Hospital, China from April 2010 to May 2011.

Materials

Animals

A total of 30 healthy, specific pathogen-free, male, Sprague Dawley rats, aged 2 months and weighing 250 ± 10 g, were provided by the Laboratory Animal Center of PLA General Hospital (certificate No. SCXX (Jing) 2009-0007). All experimental procedures were performed in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China[16].

Drugs

Borneol was purchased from Beijing Zhicheng Factory, China (batch number: 20091101); moschus was purchased from Beijing Zhicheng Factory (batch number: 20080301); storax was purchased from Beijing Lvye Pharmaceutical, China (batch number: 09031701); acorus tatarinowii was purchased from Beijing Lvye Pharmaceutical (batch number: 09042002). All drugs were identified by a pharmacist from the Pharmacy of Traditional Chinese Medicine, PLA General Hospital, China.

Methods

Preparation of medicinal acorus tatarinowii

Acorus tatarinowii pieces (200 g) were mixed with seven times the amount of water and were boiled for five hours to extract the essential oil. When oil was no longer extracted, boiling was terminated and the solution was allowed to reach room temperature. The piston at the bottom of the extractor was opened, and the water was slowly released, revealing a lower layer of oil. The extraction rate of collecting volatile oil was 1.5%. The dregs were discarded, and the water decoction was condensed to 1 g/mL. The volatile oil and the decoction was mixed[17].

Drug administration

The borneol group was intragastrically perfused with borneol solution (0.27 g/kg daily); the moschus group was intragastrically perfused with a moschus water suspension (0.09 g/kg daily); the storax group was intragastrically perfused with a storax balm solution (0.9 g/kg daily); and the acorus tatarinowii group was intragastrically perfused with an acorus tatarinowii volatile oil extraction solution (7.5 g crude drug/kg daily), for 7 consecutive days, respectively. The medication dose was administered according to the Pharmacopoeia.

Preparation of artificial cerebrospinal fluid

Artificial cerebrospinal fluid comprised 7.363 g NaCl, 0.122 g CaCl2, 2.31 g NaHCO3, 0.172 8 g MgCl2, 0.179 g KCl, 0.071 g Na2SO4, and 0.068 g KH2PO4 dissolved in 1 L distilled water. The pH value was adjusted to 7.38 with phosphoric acid, followed by filtration through a 0.2-μm microporous membrane. The prepared artificial cerebrospinal fluid was stored at 4°C.

Preparation of derivative reagent

Orthophthaladehyde (12.5 mg; Sigma, St. Louis, MO, USA) was precisely weighed and dissolved in 0.25 mL methanol, mixed with 2.5 mL borate buffer (0.4 M, pH 9.5), and then added to 30 μL β-mercaptoethanol (Sigma). The resultant reagent was stored at 4°C.

Chromatographic conditions

The HPLC device (Agilent 1200), G1311A series quaternary gradient pump, G1329A automatic sampler, G1329A ALS fluorescence detector, HP Rev.A.0501 ChemStation, and Agilent TC-C18(2) (5 μm, 250 mm × 4.6 mm) chromatographic column were provided by Agilent, Santa Clara, CA, USA. Prior to derivation, the column was tested with a G1329A ALS fluorescence detector at excitation wavelength 340 nm and emission wavelength 450 nm. Mobile phase: phosphoric acid buffer solution (0.1 M, prepared with Na2HPO4, pH 6.86) → methanol (70: 30), gradient elution; velocity 1.0 mL/min; column temperature at 30°C; injection volume 10 μL.

Plotting standard curve

Aspartic acid, glutamate, glycine (National Institute for Food and Drug Control, China, lot number: 624-200104), and GABA (Sigma) standards were precisely weighed and prepared into amino acid standard solutions at different concentrations of 2.000, 1.000, 0.500 0, 0.250 0, 0.125 0, 0.062 5, 0.031 25, and 0.015 63 mg/L, respectively. A 10-μL sample solution at each concentration was detected to plot a standard curve; the concentrations represented the X-axis, and peak area served as the Y-axis.

Determination of precision

Standard solution of mixed amino acids at concentrations of 0.125 0, 0.250 0, and 0.031 25 mg/L were continuously sampled three times using the above-described chromatographic conditions. The relative standard deviation from four amino acid peak areas and peak migration time were measured.

Determination of probe recovery

Prior to sampling, the probes were placed in a 2 mg/L sample solution, and in vitro dialysate was collected with the same perfusion fluid, perfusion speed, and time interval as the brain microdialysis. According to the above-described chromatographic conditions, the in vitro probe recovery rate of each amino acid was measured to calculate in vivo amino acid concentrations.

Microdialysis probe implantation

Rats were intraperitoneally anesthetized with 10% chlorate hydrate (3.45 mL/kg) and fixed in a WDT-stereotaxic instrument (State-operated Northwest Optical Instrument Factory, Xi’an, Shaanxi Province, China). Rats were fixed with ear rods and teeth support, and were placed on a warm blanket. The scalp was cut open and the exposed skull was drilled according to coordinates described in the Rat Brain in Stereotaxic Coordinates[18]. Caution was taken to avoid damage to the cerebral dura mater. Using the spinning vertical arm of the stereotactic instrument, the BR-4 brain microdialysis probe (CMA, Stockholm, Sweden) was implanted into the corpus striatum[19].

Sample collection

A microdialysis perfusion system (CMA) was used to perfuse artificial cerebrospinal fluid. Perfusion velocity was maintained at 2.0 μL/min by controlling the micro-syringe pump (CMA-400 type; CMA). At 60 minutes after probe implantation, the dialysate was collected every 15 minutes for a total of seven tubes, and the samples were immediately stored at –80°C. After dialysis, the rats were sacrificed by decapitation, and the microdialysis sampling location was verified using histological methods. If the probe membrane was mislocated or brain damage was severe, the results were not included in the final analysis[19].

HPLC detection of aspartic acid, glutamate, glycine, and GABA concentrations in the corpus striatum

Based on the above-described analytical methods, aspartic acid, glutamate, glycine, and GABA concentrations in the corpus striatum were measured at different sampling time points.

Statistical analysis

Data were expressed as mean ± SD and were analyzed using SPSS 9.0 statistical software (SPSS, Chicago, IL, USA). Comparisons between groups were performed using analysis of variance, and pairwise comparison was performed using the t-test. P < 0.05 was considered statistically significant.