Material preparation and analytic method
Extraction concentrator was purchased from Seugyung Eng. (Ansan, Korea). Agilent Infinity 1260 was used for qualitative and quantitative analysis of Acorus gramineus extract.
Materials and reagents
Methanol and acetonitrile were purchased from J.T. & Baker (Pennsylvania, USA). Hydrochloride was purchased from JUNSEI (Tokyo, Japan). Phosphoric acid and sodium phosphate dibasic anhydrous (Na2HPO4) were purchased from SAMCHUN (Sung-nam, Korea). Borate buffer (3-mercaptopropionic acid in borate buffer) and OPA (Ortho-Phthal aldehyde) was purchased from Agilent (Santa Clara, (California), USA). GABA standard material was purchased from Sigma-Aldrich (St. Louis, (Missouri), USA). Finasteride was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan).
AG extraction method
Acorus gramineus root was purchased from Umji, cultivated at Jeju Island in Korea and harvested in September 2014. Acorus gramineus was confirmed by BTC R&D center in accordance with the confirmation test method of The Korean Herbal Pharmacopoeia. A voucher specimen was deposited at the Herbarium of the ChonBuk National University, Republic Of Korea. AG was produced as follows: 1 kg of dried Acorus gramineus root and 20 kg of water were placed into the extraction concentrator and extracted for 6 h at 90 °C. Then extract was filtered by 200 mesh net. The filterate was then concentrated using an evaporator at 65 °C and dried using a vacuum dryer (Ilshin Corp., Korea). A total of 215 g of extract powder was obtained. Based on the HPLC analysis, GABA was selected as the indicator material in Acorus gramineus
Sample solution preparation
GABA consists of single bond so it cannot absorb UV/Vis light. Because of this, it is necessary to derivatize a compound that has absorption ability and HCl was used for derivatization. Distilled water 1 L included 8.8 mL of 35% HCl were combined in 1 L volumetric flask and 1.2 g of AG was mixed for 10 min.
Standard solution preparation
GABA standard 250 mg was added to a 250 mL flask and dissolved by solution that made to sample solution. Then, the mixture was shaken for 5 min with an ultrasonic shaker. The resulting solution was diluted to the standard solution concentrations: 50, 80, 100, 150, 200 μg/mL.
Derivatization and HPLC-DAD analyzing conditions
The hydrolyzed sample was automatically derivatized with OPA (Ortho-Phthal aldehyde) in accordance with the agilent autosampler program. An amount of 36.5 μL sample was injected on a Zorbax Eclipse AAA column, 4.6 × 150 mm, particle size 3.5 μm (Agilent, USA), at 40 °C. The mobile phase (A) 40 mM Na2HPO4 in 0.1% H3PO4 in distilled water and (B) acetonitrile was applied as follows: 0.0–0.9 min, 15% B; 1.9–10.0 min, 15–57% B; 10.0–10.5 min, 57–80% B; 10.5–13.0 min, 80% B and re-equilibration of the column over 15 min. The flow rate was 1.5 mL min−1 and diode array detector (DAD) was used for acquiring chromatograms at 338 nm.
Experimental animal treatment
Experimental rats (age 10 weeks, weight 405 ± 10 g) were purchased from Orient-Bio Ltd. (Sung-nam, Korea). Animals were housed at 22 ± 2 °C, with 55 ± 5% humidity and a 12 h dark/light cycle. They were provided access to food and water ad libitum. The institutional Animal Care and Use Committee National University (Kwangju, Korea) approved the protocol for the animal study and the animals were cared for in accordance with the “Guidelines for Animal Experiment” established by the university.
BPH induction and treatment
Rats were randomly divided into a control group (n = 7) and a BPH induced group (n = 35). To induce BPH, 3 mg/kg of testosterone propionate (TP) was injected daily for 4 weeks .
The BPH induced group was further divided into a control group, an AG medicated group and finasteride-treated group. These groups were fed AG (100, 250, or 500 mg/kg daily) and finasteride (10 mg/kg daily) respectively by oral ingestion over 4 weeks.
After maintained 1 week, animals were castrated to remove any internal testosterone influence. The rats were anesthetized with CO2 and ether, both sides of the scrotum were incised to expose the testicles and the epididymis and the spermatic cord, vascular tracts and incised part were sutured.
Castration was executed referring to the OECD Hershberger assay method provided by the Institute of National Toxicity Laboratory. One week after castration, the rats were used for experiments .
Measurement of body weight and feeding efficiency
Initial body weight was recorded 1 week after castration. During the 8 weeks experimental period, body weight was measured every 3 days and the TP injection quantity and feeding quantity were recorded.
Prior to dissection, the rats were fasted for 12 h and a final body weight was recorded. Body weight variation in the experimental animals was described as weight gain. Feeding efficiency was calculated using the food efficiency ratio (FER) based on the feeding quantity of the experimental animals .
Organ weight and storage
Rats were anesthetized with CO2 and the abdomen was cut open to expose the organs. The organs were weighed then stored at -80 °C after being washed once with 1× PBS solution .
Analysis of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
Experimental animal serum was separated from blood after centrifugation at 890×g. ALT and AST were measured at 505 nm wave length using spectrophotometer (MQS200R; BioTek Instruments, Inc., Winooski, VT, USA). The results were converted to Karmen units and compared to each other .
Measurement of testosterone and DHT in blood
Testosterone content was measured with an assay kit (Enzo Life Sciences, Farmingdale, NY, USA) using the following method. Serum (100 μL) was mixed with testosterone enzyme immunoassay (EIA) antibody (50 μL) and cultured for 1 h. Thereafter, 50 μL of conjugate was added and the solution was cultured for 1 h. After washing with washing solution, 200 μL of pNpp substrate solution was added and allowed to for 1 h. 50 μL of stop solution, solution of trisodium phosphate in water, was then added to stop the reaction and the absorbance was measured at 405 nm wave length. Testosterone content was calculated using a testosterone standard curve.
DHT content was measured using an assay kit (Biovendor, Brno, Czech Republic). Serum (50 μL) was mixed with conjugate (100 μL) and left for 1 h at 25 °C. The mixture was washed three times with washing buffer, then 150 μL of 3,3’,5,5’-Tetramethylbenzidine (TMB) substrate was added and the mixture was allowed to react for 15 min. Thereafter, 50 μL of stop solution was added to stop the reaction and the absorbance was measured at 405 nm wavelength. DHT content was calculated using a DHT standard curve .
Measurement of gene expression rate in the prostate
Gene expression in the prostatic gland was measured using a real-time polymerase chain reaction (RT-PCR) method. After washing the exposed organ with 1× PBS, 0.5 mL of TRIzol reagent (Life technologies, Carlsbad, CA) was used to extract the RNA. cDNA was synthesized as follows: 1 μg of extracted RNA, 2 μL of gDNA buffer, 1 μL of reverse transcriptase, 4 μL of RT buffer and 1 μL of RT primer was mixed and allowed to react at 42 °C for 50 min, then at 70 °C for 15 min.
Relative mRNA levels of prostate genes, including 5-α reductase type I, 5-α reductase type II, AR and the inflammatory genes, IL-1β (Interleukin-1β), IL-6, COX-2 (Cyclooxygenase-2) and iNOS (inducible nitric oxide synthase), were measured using RT-PCR with a Rotor-Gene SYBR Green PCR kit (Qiagen, Hilden, Germany). Finasteride was used as a positive control . Quantitative PCR amplification was performed in triplicate using the QuantiTect Promer assay (QT00070518 for SRD5A2, QT00073451 for AR and QT01680476 for ACTB; Qiagen).
IL-1β, IL-6, COX-2 and iNOS PCR primers were as follows: IL-1β, Forward primer: 5′-TTCGACACATGGGATAACGA-3′, Reverse primer: 5′-TCTTTCAACACGCAGGACAG-3′; IL-6, Forward primer: 5′-TACCCCCAGGAGAAGATTCC-3′, Reverse primer: 5′-TTTTCTGCCAGTGCCTCTTT-3′; COX-2, Forward primer: 5′-TGCTGTGGAGCTGTATCCTG-3′, Reverse primer: 5′-CGGGAAGAACTTGCATTGAT-3′ and iNOS, Forward primer: 5′-CTCACTGGGACTGCACAGAA-3′, Reverse primer: 5′-GCTTGTCTCTGGGTCCTCTG-3′.
Measurement of protein expression rate in the prostate
Protein expression rate in the prostate gland was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After washing the exposed prostate gland with 1× PBS (Phosphate buffered saline), KCl lysis buffer was added, allowed to react at 4 °C for 1 h and centrifuged at 16600×g for 20 min at 4 °C. The supernatant was collected and the protein concentration was measured using a BCA (Bicinchoninate) protein quantification method (Amersham, Waltham, MA, USA).
Thirty micrograms of total protein extract was loaded on 4–10% polyacrylamide gel (Life Technologies, Seoul, Korea) for SDS-PAGE. Separated proteins were transferred to PVDF (Polyvinylidene difluoride) membranes (Life Technologies). Blocking was performed using TBS-T (Tris-buffered saline) buffer (0.1% Tween 20, 5% bovine serum albumin) on a shaker for 1 h at 4 °C, then the membrane was washed with TBS-T buffer. COX-2, phosphorylated NF-κB (Nuclear factor kappa B), NF-κB, 5-α reductase type II and β-actin antibody (Cell Signaling Technology, Beverly, MA, USA) were diluted 1:1000 and attached for 16 h at 4 °C. After washing the membrane, anti-Rabbit IgG secondary antibody (Cell Signaling Technology) was attached and the membrane was re-washed. Finally, Amersham ECL (Enhanced chemiluminescence) prime western blotting detection reagent was used to treat the membrane and results were analyzed using an image reader (Supernova 1800, Lugen Sci So., LTD. Bucheon, Korea) .
Hematoxylin & eosin (H&E) staining
The prostate glands were sliced to an approximately 10 μm thickness, attached to slides and left for 5 min. The slides were then washed with flowing water for 5 min, prepared with 94% ethyl alcohol for 1 min and washed with flowing water for 30 s. The slices were stained with hematoxylin for 1 min, washed in flowing water for 30 s, then stained with eosin for 30 s. The slices were again washed with 94% ethyl alcohol two times for 30 s, treated with absolute alcohol for 30 s, washed with xylene for 30 s and washed with flowing water for 30 s .
Results are expressed as mean ± standard error of the mean (SEM). Data were analyzed using one-way analysis of variance (ANOVA). Differences in each group were considered significant at p < 0.05 by Duncan’s multiple range test.