Pharmacological Properties of Jaeumgeonbi-Tang on Redox System and Stress-Related Hormones in Chronic Subjective Dizziness: A Randomized, Double-Blind, Parallel-Group, Placebo-Controlled Trial.

Jaeumgeonbi-Tang (JGT), a traditional herbal medicine, has been used to treat dizziness and vertigo in Korea and China for hundreds of years. The purpose of this study was to evaluate the pharmacological properties of JGT in chronic subjective dizziness (CSD) patients. A randomized, double-blind, parallel-group and placebo-controlled trial was performed with a total of 50 CSD patients. The patients were randomly assigned to one of two groups: JGT or placebo (n = 25 for each). All participants received the treatment (placebo or JGT, 24 g/day) for 4 weeks. We analyzed the serum levels of oxidative stressors, antioxidants, and stress hormones. Serum levels of lipid peroxidation, but not nitric oxide, were significantly decreased in the JGT group. JGT not only prevented the decline of serum total glutathione contents and total antioxidant capacity, but it also increased superoxide dismutase and catalase activities. Serum levels of stress hormones including cortisol, adrenaline, and serotonin were notably normalized by JGT treatment, but noradrenaline levels were not affected. Regarding the safety and tolerability of JGT, we found no allergic, adverse, or side effects in any of the participants. JGT showed beneficial effects on CSD patients by improving redox status and balancing psycho-emotional stress hormones.

Ukgansan Protects Dopaminergic Neurons against MPTP-Induced Neurotoxicity via the Nurr1 Signaling Pathway.

Nuclear receptor-related 1 protein (Nurr1) is a nuclear hormone receptor that protects dopaminergic neurons and is a promising therapeutic target for Parkinson's disease (PD). Parkinson's disease is a neurodegenerative disorder caused by the destruction of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and the long-term use of conventional dopamine replacement therapies causes many side effects, highlighting the need for new treatments such as complementary and alternative medicine. Ukgansan has been used in East Asia to treat neurological disorders, including neurodegenerative diseases, and has been reported to have strong effects in treating patients with PD. In addition, recent studies have reported that Ukgansan has a neuroprotective potential. However, there are no detailed studies on the mechanism of action of Nurr1. Thus, unlike previous studies, we focused on the Nurr1 pathways. We confirmed neurotoxicity and apoptosis signaling in the differentiated PC12 cells. In addition, to confirm the protective effect of Ukgansan, we conducted behavioral tests (motor coordination and postural balance, and bradykinesia) and tyrosine hydroxylase immunohistochemistry in both the SNpc and striatum. Specifically, this study demonstrated the effect of Ukgansan in protecting dopaminergic neurons and increasing Nurr1 involved in maintaining dopamine levels by activating Nurr1 expression in MPTP-induced PC12 cells and a mouse model of PD. In this mechanism, the loss of dopaminergic neurons and dopamine depletion were suppressed, and motor impairment caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity was improved. These results provide evidence that Ukgansan ameliorates PD's motor symptoms and progression.

Pharmacological Effects of Gami-Yukmijihwang-Tang on the Lipopolysaccharide-Induced Hippocampus Oxidation and Inflammation via Regulation of Sirt6.

Yukmijihwang-Tang is widely used in traditional Korean medicine to treat age-related disorders. In the present study, we re-prescribed Gami-Yukmijihwang-Tang (YJT), which is slightly modified from Yukmijihwang-Tang by adding more medicinal plants to evaluate its pharmacological effects on underlying mechanisms against repeated lipopolysaccharide (LPS)-injection-induced neuroinflammation in the hippocampus regions. C57BL/6J male mice (16-24 weeks old) were divided into six groups: (1) the control group (DW with 0.9% saline injection), (2) LPS group (DW with LPS injection), YJT groups ((3) 100, (4) 200, or (5) 400 mg/kg of YJT with LPS injection), and (6) glutathione (GSH) group (100 mg/kg of GSH with LPS injection), respectively. Mice were orally administrated with various doses of YJT or glutathione (GSH) for the first five days. Neuroinflammation in the hippocampus region was induced by repeated injection of LPS during the last three days. As predicted, LPS not only increased oxidative stress-related markers including malondialdehyde, 4-hydroxynonenal, nitrotryptophan, and hydrogen peroxide, but also drastically enhanced inflammatory reactions including nitric oxide, inducible nitric oxide synthase, p65, and toll-like receptor 4, respectively. YJT administration, on the other hand, notably decreased the above pathological alterations by enhancement of antioxidant capacities such as superoxide dismutase and catalase activities. To explain the underlying pharmacological actions of YJT, we focused on a representative epigenetic regulator, a nicotinamide adenine dinucleotide + (NAD+)-dependent chromatin enzyme, Sirtuin 6 (Sirt6). Neuroinflammation in hippocampus regions depleted Sirt6 at the protein level and this alteration directly affected the nuclear factor erythroid 2-related factor (Nrf2)/hemeoxygenase (HO)-1 signaling pathway in the LPS group; however, YJT significantly recovered the Sirt6 protein levels, and it could recover the abnormal status of Nrf2/HO-1 signaling pathways in the hippocampus regions. Additionally, Sirt6 led to the up-regulation of GSH sub-enzymes of mRNA expression and protein levels of total GSH content. These findings suggest that YJT can protect against LPS-induced neuroinflammation and oxidative stress by regulating the Sirt6-related pathways and normalizing the GSH redox cycle.

Antiviral Effect of Hyunggaeyungyo-Tang on A549 Cells Infected with Human Coronavirus.

BACKGROUND: Herbal medicine is widely recommended to treat viral infectious diseases. Over 123,000,000 individuals have been infected with the coronavirus since a worldwide pandemic was declared in March 2020. We conducted this research to confirm the potential of herbal medicine as a treatment for coronavirus. METHODS: We infected the A549 cell line with betacoronavirus OC43 and then treated it with 100 µg/mL Hyunggaeyungyo-tang (HGYGT) or distilled water with a control of HGYGT. We measured the mRNA expression levels of proinflammatory cytokines and interferon stimulated genes (ISGs) to confirm the effectiveness of HGYGT upon coronavirus infection. RESULTS: We found that the effects of HYGYT decrease the expression level of pPKR, peIF2α, IFI6, IFI44, IFI44L, IFI27, IRF7, OASL, and ISG15 when administered to cells with coronavirus infection. The expressions of IL-1, TNF-α, COX-2, NF-κB, iNOS, and IKK mRNA were also significantly decreased in the HGYGT group than in the control group. CONCLUSION: Through the reduction of the amount of coronavirus RNA, our research indicates that HGYGT has antiviral effects. The reduction of IKK and iNOS mRNA levels indicate that HGYGT reduces coronavirus RNA expression and may inhibit the replication of coronavirus by acting on NF-kB/Rel pathways to protect oxidative injury. In addition, decreases in mRNA expression levels of proinflammatory cytokines indicate that the HGYGT may relieve the symptoms of coronavirus infections.

Effects of Acupuncture on Lowering Blood Pressure in Postmenopausal Women with Prehypertension or Stage 1 Hypertension: A Propensity Score-Matched Analysis.

Postmenopausal women have a higher prevalence of hypertension compared to premenopausal women. Hypertension is a risk factor for cardiovascular diseases, the prevalence of which is ever increasing. This study investigated the effects of long-term acupuncture on lowering the blood pressure of postmenopausal women with prehypertension and stage 1 hypertension. Participants were 122 postmenopausal women aged less than 65 years, diagnosed with prehypertension or stage 1 hypertension (systolic blood pressure 120-159 mmHg or diastolic blood pressure 80-99 mmHg). We used a propensity score-matched design. The experimental group (n = 61) received acupuncture for four weeks every six months over a period of two years. The control group (n = 61) received no intervention. An Analysis of covariance (ANCOVA) was performed for the primary efficacy analysis. Relative risk ratios were used to compare group differences in treatment effects. Acupuncture significantly reduced the participants' diastolic blood pressure (-9.92 mmHg; p < 0.001) and systolic blood pressure (-10.34 mmHg; p < 0.001) from baseline to follow-up. The results indicate that acupuncture alleviates hypertension in postmenopausal women, reducing their risk of developing cardiovascular diseases and improving their health and quality of life.

Gait Disturbance Improvement and Cerebral Cortex Rearrangement by Acupuncture in Parkinson's Disease: A Pilot Assessor-Blinded, Randomized, Controlled, Parallel-Group Trial.

BACKGROUND: Parkinson's disease (PD) leads to impaired mobility and limited independence. OBJECTIVE: We investigated the effects of acupuncture on gait disturbance and analyzed hemodynamic changes caused by acupuncture in the cerebral cortex of patients with PD. METHODS: Participants (n = 26) with gait disturbance due to PD were randomly assigned to the intervention (acupuncture twice a week for 4 weeks + conventional therapy) or control (conventional therapy) groups. We analyzed gait parameters using the GAITRite system and hemodynamic responses in the cerebral cortices using functional near-infrared spectroscopy, Unified Parkinson's Disease Rating Scale (UPDRS) scores, neurotransmitter levels, as well as the immediate effects of acupuncture in patients with PD. RESULTS: The participants tended to walk with hypometric gait (high cadence, short steps) overground. After acupuncture treatment, those in the intervention group showed a significant reduction in cadence and the UPDRS scores involving "walking and balance" compared with those in the control group (P = .004 and P = .020, respectively); the stride, swing, and single support times were significantly increased (P = .006, P = .001, and P = .001, respectively). Oxyhemoglobin levels in the intervention group while walking on a treadmill were significantly increased in the prefrontal and supplementary motor areas. The oxyhemoglobin levels in the prefrontal cortex and swing time revealed significant positive correlations. CONCLUSIONS: Our findings indicated that acupuncture tended to improve hypometric gait and rearranged activation of the cerebral cortex. Thus, acupuncture may be a useful complementary treatment for gait disturbance, including hypometric gait, in patients with PD. Trial Registration Number. Clinical Research Information Service (KCT0002603), https://cris.nih.go.kr/cris/index.jsp.

Traditional Korean herbal formulae, Yuk-Mi-Ji-Hwang-Tang, ameliorates impairment of hippocampal memory ability by chronic restraint stress of mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Yuk-Mi-Jihwang-Tang (YJT) has been popularly prescribed to treat aging related disorders over than hundreds of years in East Asia countries. AIM OF THE STUDY: To investigate possible modulatory actions of YJT on chronic restraint stress (CRS)-induced neurodegeneration on hippocampus neuronal injuries. MATERIALS AND METHODS: Mice were orally administered with YJT (100, 200, or 400 mg/kg) or ascorbic acid (100 mg/kg) before 4 h of stress for 28 days. Morris water maze task was completed from day 24th to 28th, and stress hormones and biochemical analyzes were measured. RESULTS: Four weeks of the CRS abnormally affected memory impairments by measurement of escape latency and time spent in the target quadrant. Additionally, neurotransmitters were also drastically altered in serum or hippocampus protein levels by CRS. Gene expressions for 5-hydroxytryptamine (5-HT) receptor, 5-HT-transport, and tryptophan hydroxylase were also altered, whereas YJT led to normalize the above alterations. Additionally, YJT also beneficially worked on endogenous redox system as well as inflammatory reactions in the hippocampal neurons. We observed that hippocampal excitotoxicity was induced by CRS which were evidenced by depletion of phosphor-cAMP response element-binding protein, brain-derived neurotrophic factor, nuclear factor erythroid-2-related factor 2, heme oxygenase-1 and abnormally increases of acetylcholine esterase activities in hippocampus protein levels; however, YJT considerably improved the above pathological conditions. CONCLUSIONS: Our findings supported YJT enhance memory function via regulation of hippocampal excitotoxicity-derived memory impairment, stress hormone, and endogenous redox, respectively.

Effects of acupuncture in postmenopausal women with prehypertension or stage 1 hypertension: study protocol for a prospective, comparative, interventional cohort study.

BACKGROUND: Hypertension is a major cause of cardiovascular disease and associated mortality, and postmenopausal women are at a high risk of hypertension. We aim to investigate the hypotensive effect and safety of acupuncture, focusing on postmenopausal women with prehypertension and stage 1 hypertension. In addition, we aim to investigate whether the effect of acupuncture treatment differed, depending on Sasang Constitution and cold-heat pattern. METHODS: This study is designed as an intervention cohort study. Two hundred postmenopausal women aged <65 years with prehypertension or stage 1 hypertension living in Daejeon city in Korea will be recruited, and randomly assigned to either an acupuncture or no-treatment control group. The intervention will consist of four sessions; one session will include acupuncture performed 10 times for 4 weeks. There will be a 20-week observation period after each session, and the total study duration will be 96 weeks. Acupuncture will be applied at the bilateral Fengchi (GB20), Quchi (LI11), Zusanli (ST36), and Sameumgyo (SP6) acupoints. The effect of acupuncture will be evaluated by comparing the change in systolic and diastolic blood pressure between the acupuncture and control groups every 4 weeks until the end of the study. DISCUSSION: To evaluate the success of blood pressure management, long-term observation is required, but no long-term studies have been conducted to evaluate the effect of acupuncture on blood pressure in postmenopausal women. To our knowledge, this study will be the first long-term study to investigate this issue for more than 6-8 weeks.

A traditional Korean multiple herbal formulae (Yuk-Mi-Jihwang-Tang) attenuates acute restraint stress-induced brain tissue oxidation.

We aimed to evaluate the protective effects of Yuk-Mi-Jihwang-Tang (YJT) against acute restraint stress-induced brain oxidative damage. A water extract of YJT was prepared and subjected to high performance liquid chromatography - diode array detector-mass spectrometry (HPLC-DAD-MS). Thirty-six heads of C57BL/6J male mice (7 weeks) were divided into six groups (n = 6/group). The mice were orally administrated YJT (0, 50, 100, or 200 mg/kg) or vitamin C (100 mg/kg) for 5 consecutive days before 6 h of acute restraint stress. In the brain tissue, lipidperoxidation, antioxidant components, and pro-inflammatory cytokines were measured, and the serum corticosterone level was determined. Acute restraint stress-induced notably increased lipid peroxidation in brain tissues, and pretreatment with YJT showed a significant decreased the lipid peroxidation levels (p< 0.05). The levels of antioxidant components including total glutathione contents, activities of SOD and catalase were remarkably depleted by acute restraint stress, whereas these alterations were significantly restored by treatment with YJT (p< 0.05 or p< 0.01). The restraint stress markedly increased pro-inflammatory cytokines, such as TNF-α and IL-6 in the gene expression and protein levels (p< 0.05 or p< 0.01). Pretreatment with YJT significantly attenuated serum corticosterone (200 mg/kg, p < 0.05). YJT drastically attenuated the levels of 4- HNE, HO-1, Nox 2 and iNOSwhich were elevated during acute restraint stress, whereas the Nrf2 level was increased in brain tissue protein levels. Our data suggest that YJT protects the brain tissue against oxidative damage and regulates stress hormones.

ACE Reduces Metabolic Abnormalities in a High-Fat Diet Mouse Model.

The medicinal plants Artemisia iwayomogi (A. iwayomogi) and Curcuma longa (C. longa) radix have been used to treat metabolic abnormalities in traditional Korean medicine and traditional Chinese medicine (TKM and TCM). In this study we evaluated the effect of the water extract of a mixture of A. iwayomogi and C. longa (ACE) on high-fat diet-induced metabolic syndrome in a mouse model. Four groups of C57BL/6N male mice (except for the naive group) were fed a high-fat diet freely for 10 weeks. Among these, three groups (except the control group) were administered a high-fat diet supplemented with ACE (100 or 200 mg/kg) or curcumin (50 mg/kg). Body weight, accumulation of adipose tissues in abdomen and size of adipocytes, serum lipid profiles, hepatic steatosis, and oxidative stress markers were analyzed. ACE significantly reduced the body and peritoneal adipose tissue weights, serum lipid profiles (total cholesterol and triglycerides), glucose levels, hepatic lipid accumulation, and oxidative stress markers. ACE normalized lipid synthesis-associated gene expressions (peroxisome proliferator-activated receptor gamma, PPARγ; fatty acid synthase, FAS; sterol regulatory element-binding transcription factor-1c, SREBP-1c; and peroxisome proliferator-activated receptor alpha, PPARα). The results from this study suggest that ACE has the pharmaceutical potential reducing the metabolic abnormalities in an animal model.

Synergistic effects of Artemisia iwayomogi and Curcuma longa radix on high-fat diet-induced hyperlipidemia in a mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal plants Artemisia iwayomogi and Curcuma longa radix are both used to treat hyperlipidemia in traditional Korean and Chinese medicine. AIM OF THE STUDY: To evaluate the anti-hyperlipidemic effects of the 30% ethanol extracts of A. iwayomogi (AI), C. longa (CL), and the mixture of A. iwayomogi and C. longa (ACE), using a high-fat diet-induced hyperlipidemia model. MATERIALS AND METHODS: Six of seven groups of C57BL/6N male mice (i.e., not including the naïve group) were fed a high-fat diet freely for 10 weeks. Of these six groups, five (i.e., not including the control group) were administered a high-fat diet supplemented with AI (100mg/kg), CL (100mg/kg), ACE (50 or 100mg/kg), or Lipitor (20mg/kg). Serum lipid profiles, obesity-related markers, hepatic steatosis, hepatic gene expression, and oxidative stress markers were analyzed. RESULTS: AI, CL, and ACE were associated with significant effects on serum lipid profiles (total cholesterol [TC] and triglyceride), body, liver and peritoneal adipose tissue weights, hepatic lipid accumulation, and oxidative stress biomarkers. ACE at 100mg/kg was associated with significantly greater improvements in serum TC and triglyceride, hepatic triglyceride, epididymal adipocyte size, and oxidative stress biomarkers, compared with AI and CL. AI, CL and ACE normalized lipid synthesis-associated gene expression (peroxisome proliferator-activated receptor gamma, fatty acid synthase, sterol regulatory element-binding transcription factor-1c, and peroxisome proliferator-activated receptor alpha). CONCLUSION: ACE exhibits anti-hyperlipidemia properties and is associated with partially synergistic effects compared with AI or CL alone.

Anti-atherosclerosis and hyperlipidemia effects of herbal mixture, Artemisia iwayomogi Kitamura and Curcuma longa Linne, in apolipoprotein E-deficient mice.

ETHNOPHARMACOLGICAL RELEVANCE: Artemisiaiwayomogi Kitamura and Curcuma longa Linne. (ACE) has been popularly used to treat atherosclerosis as well as hyperlipidemia in the Asian countries. OBJECTIVE: Antiatherosclerotic and anti-hyperlipidemic effects of ACE were evaluated at protein and gene expression level by using apoE(-/-) mice. METHOD: Apoprotein E deficient (apoE(-/-)) mice were randomly divided into five groups and fed freely Western diet (WD) which contained ACE (50, 100 and 200mg/kg) or curcumin (50mg/kg). The C57/BLJ mice were used as normal and which were fed the WD. After 10 weeks of being fed the WD, the atherosclerosis related mediators and hyperlipidemia induced hepatic steatosis were analyzed in serum, aorta tissue or hepatic tissues. RESULTS: Ten-week feeding of WD considerably increased the serum lipid profiles including total cholesterol (TC), low density lipoprotein, high density lipoprotein (HDL), triglyceride, TC/HDL ratio and glucose, and also elevated the total reactive oxygen species (ROS) and inflammatory cytokines (tumor necrosis factor-α, TNF-α; and interlukin-6, IL-6) in the serum levels. ACE treatment significantly resolved these alterations. The aortic lesion formation was significantly decreased as were lipid formations by ACE treatment. Moreover, ACE not only caused significant decreases of the lipid drops on the hepatic tissues, but also restored the antioxidant components. The gene expression levels including SREBP-1c, FAS, SCD-1, PPAR-α, CPT-1, IL-6, IL-1ß and TNF-α in hepatic tissue were altered by Western diet fed in apoE(-/-) mice, while ACE treatment significantly normalized those alterations. CONCLUSIONS: The ACE treatment is beneficial for atherosclerosis in arterial area and hyperlipidemia induced hepatic tissue steatosis.

Quality control of a herbal medicinal preparation using high-performance liquid chromatographic and capillary electrophoretic methods.

Two methods based on high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) were developed for the quality control of "samgiumgagambang" (SGMX), a new herbal medicinal preparation containing 14 herbs. Nine components from SGMX were selected as markers: 5-hydroxymethylfuraldehyde, geniposidic acid, chlorogenic acid, paeoniflorin, 20-hydroxyecdysone, coptisine, berberine, luteolin, and glycyrrhizic acid. The markers were identified and analyzed using HPLC coupled with a UV-diode-array detector and monitored at 250nm with a gradient elution of acetonitrile and water containing formic acid on a C(18) analytical column or using CE with a 70mM borate buffer (pH 9.5) containing 10% methanol on a 60-cm fused silica capillary monitored at 230nm. The marker components in SGMX were well separated using both methods and were readily determined within 60min using HPLC or 13min using CE with good precision and accuracy.

Shengmai-san-mediated enhancement of regenerative responses of spinal cord axons after injury in rats.

Shengmai-san (SMS) is a traditional Chinese medicine used to treat diverse symptoms including cardiovascular and neurological disorders. Here we investigated the effects of SMS on regenerative responses of spinal cord axons in rats that were given contusion injury at the lower thoracic level. The injury cavity was confined to a restricted area by SMS treatment, and the signals of glial scar protein chondroitin sulphate proteoglycan (CSPG) and inflammatory cell marker protein CD11beta were heavily observed within the injury cavity in SMS-treated animals. Anterograde tracing of DiI-labeled corticospinal tract (CST) axons revealed increases in collateral arborization around and within the injury cavity and caudal elongation by SMS treatment. Furthermore, SMS treatment facilitated neurite elongation of dorsal root ganglion (DRG) sensory neurons that were co-cultured with non-neuronal cells prepared from injured spinal cord. Phospho-Erk1/2 was strongly induced in both spinal cord and motor cortical areas after spinal cord injury (SCI), and it was further unregulated in the motor cortex by SMS treatment. In contrast, upregulation of cell division cycle 2 (Cdc2) production by SMS treatment was limited to a local, SCI area. These data suggest that SMS may play an active role in regenerative responses and facilitate axonal regrowth after SCI.

Effects of ursolic acid, betulin and sulfur-containing compounds on mucin release from airway goblet cells.

In this study, we investigated whether ursolic acid, betulin and 2 kinds of sulfur-containing compounds--NAC and MESNA--affect mucin release from airway goblet cells and compared the possible activities of these agents with the inhibitory action on mucin release by PLL and the stimulatory action by ATP. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled using 3H-glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of each agent to assess the effects on 3H-mucin release. The results were as follows: ursolic acid, betulin, MESNA and NAC increased mucin release (40-50 % above control) at the highest concentrations (10(-5) M-10(-3) M). We conclude that ursolic acid and betulin can stimulate mucin release by directly acting on airway mucin-secreting cells and suggest that these agents be further investigated for the possible use as mucoregulators in the treatment of chronic airway diseases.

Effects of baicalein, berberine, curcumin and hesperidin on mucin release from airway goblet cells.

Baicalein, berberine, curcumin and hesperidin are the major components derived from Scutellaria baicalensis, Coptis japonica, Curcuma longa and Poncirus trifoliata, respectively. These plants have been used for the treatment of diverse chronic inflammatory diseases including respiratory disease in oriental medicine and their respective major components were reported to have various biological effects including anti-inflammatory activity. In the present study, we investigated whether these four natural products affect mucin release from airway goblet cells and compared the possible activities of these agents with the inhibitory action on mucin release by PLL and the stimulatory action by ATP. Confluent primary hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled using 3H-glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of each agent to assess the effects on 3H-mucin release. The results were as follows: (i) baicalein did not affect mucin release significantly; (ii) berberine, curcumin and hesperidin increased mucin release at the highest concentration (10 - 4 M); (iii) PLL inhibited and ATP increased mucin release. We conclude that berberine, curcumin and hesperidin can increase mucin release by directly acting on airway mucin-secreting cells and suggest that these agents be further studied for possible use as mild expectorants during the treatment of chronic airway diseases. Abbreviations. PLL:poly- L-lysine ATP:adenosine triphosphate HTSE:hamster tracheal surface epithelial DMSO:dimethylsulfoxide IL-12:interleukin-12 PBS:phosphate-buffered saline