RCM-101 is a herbal formula with 18 herbal ingredients, modified from a traditional Chinese medicine formula. Each herb for the formula was supplied in a granulated form produced under Good Manufacturing Practices by Min Tong Pharmaceutical Company (Taichong, Taiwan) which holds certification from the Australian Therapeutic Goods Administration (TGA-GMP No: 1888). Authenticated, quality-certified raw herbs were first tested to ensure that they were free of heavy metals. They were then washed, dried and extracted in boiling water for 1 – 1.5 hour. The aqueous extract was separated by filtration (100 mesh) and the water content was reduced to 60% by heating (50 – 60°C) under reduced pressure (50 – 70 mmHg) for 2 – 5 hours. The concentrated extract of each herb was combined with starch as an excipient and the product was dried and ground into fine granules. For each preparation, 1 g of granulated product was equivalent to 5 g of the raw herb. The granulated herbal preparations were sterilised and sealed in plastic bottles. In our laboratory, the granulated preparations of the herbs were combined in the proportions given in Table 1 to produce the herbal formula. All herbal ingredients of RCM-101 are approved in the Australian Register of Therapeutic Goods as active raw herbs for use in medicines.

The herbal formula was extracted with ethanol (120 mg/mL) at room temperature with continuous agitation for 4 hours. The ethanol extract was collected by centrifugation (5000 rpm for 10 minutes) and vacuum filtration. The extract was dried using a rotary evaporator (Büchi Rotavapor, Brinkman Company, Westbury, NY, USA) and stored below -20°C. It was diluted to the required concentrations on the day of use.

Compound 48/80, histamine hydrochloride, O-phthalaldehide, spermidine hydrochloride, bovine serum albumin (BSA), phosphate buffer saline, heparin, disodium ethylenediaminetetraacetic acid (EDTA), lipopolysaccharide (LPS) E.Coli, calcium ionophore A23187, Hanks' balanced salt solution, RPMI 1640 medium, fetal bovine serum, phenylmethylsulfonyl fluoride, gentamycin, leupeptin, pepstatin A and nordihydroguaiaretic acid (NDGA) were obtained from Sigma Chemical Company (St Louis, MO, USA). Monoclonal mouse anti-rat cyclooxygenase 2 antibodies and mouse macrophage lysate were obtained from Transduction Laboratories (Lexington, KY, USA). Goat anti-rabbit horseradish peroxidase conjugated immunoglobulin G was obtained from Dako Corporation (CA, USA). The Coomassie blue protein assay kit was purchased from Bio-Rad (USA). Polyclonal rabbit anti-mouse cyclooxygenase-1 antibody, immune-enzyme analysis PGE₂ kit, arachidonic acid, prostaglandin B₂, LTB₄, 6-trans LTB₄, 6 trans-12 epi LTB₄, 5-hydroxyeicosatetraenoic acid (5-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). HPLC-grade methanol was supplied by Selby-Biolab (Clayton, Victoria, Australia). All other analytical reagents were obtained from Merck Pty Ltd (Kilsyth, Victoria, Australia).

Rat peritoneal mast cells were collected in Tyrode buffer as previously described 11. Briefly, rats (Sprague-Dawley, 200 – 300 g) of either sex were killed and 10 mL of Tyrode buffer (NaCl, 137 mM; KCl, 2.7 mM; HEPES, 10 mM; MgCl₂, 1 mM; CaCl₂, 1.0 mM; NaH₂PO₄, 0.41 mM), containing 0.3% BSA and 5 units/mL heparin was injected into the peritoneal cavity. The abdomen was gently massaged for about 90 seconds, and then carefully opened and the cell-containing peritoneal fluid collected with a transfer pipette. The cell-containing fluid was centrifuged at 4°C at 800 rpm for 5 minutes. The cells were collected, washed in 10 mL of Tyrode buffer and centrifuged again. This procedure was repeated twice 11. The cells were then suspended in the concentration of 1 × 10⁶ cells/mL in 10 mM HEPES-Tyrode buffer (pH 7.4) containing 0.1% BSA.

The 200 mL peritoneal cell suspensions were incubated with various concentrations of RCM-101 for 10 minutes at 37°C and then exposed to compound 48/80 for 10 minutes. Aliquots of 100 μL of rat peritoneal mast cells in Tyrode buffer were combined with 100 μL aliquots of RCM-101 extract in Tyrode buffer such that 5 × 10⁵ cells/mL were incubated with RCM-101 at concentrations of 1, 10 and 100 μg/mL for 10 minutes immediately prior to stimulation of the cells with compound 48/80. The cell suspensions were then centrifuged at 4°C at 4800 rpm and the supernatant collected. As an internal standard, 10 μL of spermidine (1 mg/mL) was added to 200 mL aliquots of the supernatant, followed by 20 μL of 30% perchloric acid (HCIO₄). The mixture was then filtered and 100 μL was transferred into HPLC vials for histamine determination 11. The Ca²⁺ chelating agent, EDTA, (100 μM) was used as a positive control.

The HPLC system (Shimadzu, Kyoto, Japan), which included a fluorescent detector (Shimadzu RF10XL), C-10ATvp pumps, SIL-10ADvp auto-injector and STRODS-II reversed phase column, equipped with post-column derivatisation was set up as previously described 11. Samples of the peritoneal cell supernatant/internal standard solution were injected into the HPLC system using an autosampler. Histamine and spermidine were detected with excitation and emission wavelengths of 360 nm and 440 nm, respectively. Four-point standard curves for histamine were prepared, ranging from 50 – 2500 ng/mL in 10 mM HEPES-Tyrode buffer.

Synthesis of LTB₄ was induced in neutrophils as previously described 15, with slight modification. Porcine blood was collected from a local abattoir. Neutrophils were isolated using a Percoll gradient and suspended in Hanks' buffer, containing 5 mM HEPES. Suspended neutrophils (2.8 × 10⁶ cells/mL) were incubated (37°C) with RCM-101 extract, NDGA (as a positive control, 0.1, 1 or 10 μg/mL) or vehicle (ethanol), for 5 minutes before the addition of arachidonic acid (2.5 μM) substrate. Porcine neutrophils were suspended in Hanks' buffer in concentration of 2.8 × 10⁶ cells/mL. RCM-101 (0.1, 1, 100 μg/mL) was added 10 minutes before the calcium ionophore A23187 (2.5 μM). After 5 minute incubation, production of LTB₄ was initiated by the addition of the calcium ionophore A23187 (2.5 μM) and 5 minutes later the reaction was terminated by adjusting the pH to 3 with citric acid. PGB₂ (45 ng) and 15-HETE (83 ng) were then added as internal standards. The reaction mixture was extracted with 5 mL of chloroform/methanol (7:3 v/v) and dried under vacuum. The residue was dissolved in 120 μL of HPLC mobile phase (methanol-water-acetic acid, 76/34/0.08, v/v/v, pH 3.0) and leukotriene metabolites were assayed using a Waters HPLC system equipped with an auto sampler, a multi-solvent delivery system and a Waters 996 Photodiode Array Detector. Standard curves were prepared by the addition of LTB₄ (10 – 200 ng) and 5-HETE (500 – 800 ng) to neutrophil suspensions. Data were analysed using Water Millenium Software, Version 3.2, results being expressed as percentage of the vehicle control which was taken as 100%.

Murine macrophages (Raw 264.7 cells, American Type Culture Collection, Rockville, MD, USA) were grown in RPMI 1640 medium, supplemented with 10% heat-inactivated FBS, 100 μg/mL gentamycin, 1.5 g/L sodium bicarbonate and 10 mM HEPES, at 37°C, in an atmosphere containing 5% CO₂. Cells were sub-cultured once a week by harvesting them with trypsin/EDTA and seeding them in 75 cm² flasks. Once confluent, murine macrophages were suspended in serum-free RPMI medium at concentration of 2 × 10⁵ cells/mL, and the cells were seeded in 24-well plates (1 × 10⁵ cells/well), in serum-free RPMI medium. Cells were then treated with RCM-101 (1, 10, or 100 μg/mL) or vehicle 10 minutes before the addition of LPS (1 μg/mL). The supernatant and the cells were separated. PGE₂ was assayed in the supernatant using an immune-enzyme analysis kit. The assay depends on competition between PGE₂ and PGE₂acetylcholinesterase conjugate (PGE₂-tracer) for a limited amount of monoclonal PGE₂antibody. The assays were carried out according to the manufacturer's protocol, in triplicate. PGE₂ release was calculated using software supplied by the kit manufacturer.

Cultured Raw 264.7 cells prepared as described above for determination of PGE₂ production, with and without incubation with RCM-101, were washed twice with ice-cold phosphate buffer saline then lysed with 100 μL/well of lysis buffer (50 mM Tris base, pH 7.6, 2 mM MgCl₂, 1 mM EGTA, 1% TritonX, 1 mM phenyl PMSF, 1 mM pepstatin, 1 mM aprotinin, 1 mM leupeptin) for 5 minutes. The cells and the supernatant were collected and centrifuged for 5 minutes at 14000 rpm. The cell debris was discarded and the supernatant was assayed for protein concentration using Coomassie Protein Assay Kit (Bio-Rad Laboratories Pty Ltd, California, USA) and the UV-visible spectrophotometer (Cintra 5, GBC Scientific Equipment Pty Ltd, Illinois, USA)

COX-1 and COX-2 protein was measured by Western blotting as previously described 16 with a slight modification. Aliquots of 20 μg of total protein were loaded to each lane of 7.5% SDS-polyacrylamide gels. The proteins were then electrically transferred to nitrocellulose membranes which were incubated overnight with a polyclonal anti-rabbit COX-1 antibody or a monoclonal mouse anti-rat COX-2 antibody (diluted 1:500 and 1:2500 respectively) in 5% non-fat milk in Tris-buffered saline (Tris base 25 mM, glycine 19 mM, methanol 20%). On the next day, membranes were washed with Tris-buffered saline (Tris base 20 mM, NaCl 137 mM, Tween-20 0.1%, pH 7.5) for 40 minutes with constant agitation, during which time the buffer was changed every 5 minutes. The membranes were then incubated with swine anti-rabbit or goat anti-mouse secondary conjugated to horseradish peroxidase diluted 1:5000 with blocking buffer (Tris base 20 mM, NaCl 137 mM, Tween-20 0.1%, pH 7.5 and 5% non-fat milk). The results were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Data are expressed as means ± standard deviation (SD). The statistical significance of differences between means was determined by unpaired, two-tailed Student's t-test or, for more than two groups, by first testing for global differences by one- or two-way analysis of variance (ANOVA) and then testing for differences between predetermined pairs of means by Dunnet's test. The differences with probability levels less than 0.05 (P < 0.05) were considered to be statistically significant.

The amount of histamine released from rat unstimulated peritoneal mast cell preparations was 46.3 ± 37.1 ng/mL (n = 14). When the cells were stimulated with compound 48/80 (1 μg/mL) histamine release increased markedly to 638.3 ± 308 ng/mL (n = 14). As shown in Figure 1, compound 48/80-induced histamine release was inhibited by RCM-101 (1 – 100 μg/mL) in a concentration dependent manner. Compound 48/80-induced histamine release was also reduced by 10μM EDTA (Figure 1).

In the absence of calcium ionophore A23187 incubation, the production of LTB₄ by porcine neutrophils was 9.58 ± 4.6 ng/mL. In vehicle (ethanol) control experiments, A23187 incubation increased LTB₄ production to 167.77 ± 70.4 ng/mL (n = 8).

As shown in Figure 2, LTB₄ production in A23187-incubated neutrophils was inhibited by RCM-101 at concentrations of 1 and 10 μg/mL. NDGA (1 and 10 μg/mL) also inhibited A23187-induced LTB₄ production.

Unstimulated Raw 267.4 cells incubated in serum-free RPMI medium for 24 hours produced a baseline concentration of PGE₂ of 52 ± 24.4 pg/mL (n = 6). Incubating the cells with LPS (1 μg/mL) increased the PGE₂ level to 3874 ± 818.13 pg/mL (n = 6). This induced production of PGE₂ was reduced in a concentration-dependent manner by RCM-101 (1 – 100 μg/mL), when present during incubation with LPS. Indomethacin (1, 10 and 100 μM), when present during LPS incubation, completely blocked PGE₂ production. The data are shown in Figure 3.

As shown in Figure 4, immunoreactivity bands corresponding to COX-1 and COX-2 (70 kDa) were detected by Western blot analysis of the supernatant of lysed Raw 264.7 cells. Densiometeric analysis of the marker chemiluminescence indicated that expression of COX-1 protein was unaffected by RCM-101 (10 and 100 μg/mL). Similarly, dexamethasone (10 and 100 μM) also did not alter COX-1 protein expression. In contrast, the expression of COX-2 protein was significantly (P < 0.05, one-way ANOVA, Dunnet's test) reduced by 100 μg/mL RCM-101 and also by 100 μM dexamethasone. Figure 4 shows examples of the visualised bands on nitrocellulose membranes corresponding to COX-1 and COX-2 proteins.