Materials

Crude drugs; rhubarb (the rhizome of Rheum palmatum Linneá, Rheum tanguticum Maximowicz, Rheum officinale Baillon, Rheum coreanum Nakai or their interspecific hybrids [Polygonaceae]) and glycyrrhiza (the root and stolon, with [unpeeled] or without [peeled] the periderm, of Glycyrrhiza uralensis Fisher or Glycyrrhiza glabra Linneá [Leguminosae]), which are registered and specified as crude drugs in the JP18, were purchased commercially from Tochimoto Tenkaido Co. (Osaka, Japan). TSUMURA Daiokanzoto Extract Granules for Ethical Use were purchased from Tsumura & Co. The daily dose of daiokanzoto consisted of a combination of rhubarb (4.0 g) and glycyrrhiza (2.0 g) according to regimen #2 in the JP18 [2].

Decoction preparation using the conventional method and sampling

The conventional method of decoction preparation was modified the method previously documented [4, 7, 13] and used an electric Kampo formula decoction heater pot (My New Mycon Torobi-ranran, EKSA-10, Tochimoto-Tenkaido Co.). Commercially available crude drugs were used as it is, without any treatment. The mixture of crude drugs or one of each crude drug (the daily dose: rhubarb 4.00 g, glycyrrhiza 2.00 g) was added to the pot of boiling distilled water (500 mL) (time 0 min). The decoction continued to boil on the electric heater for 30 min, after which the heater was turned off and the decoction was set aside for 30 min. At 2, 5, 10, 20, 30, and 60 min, the decoction solution was collected without adding water. The amount of water that evaporated was calculated by comparing the weights before and after the experiment.

Decoction preparation using the IPCD method and sampling

The IPCD method of decoction preparation has been previously described [4,5,6]. Each crude drug (approximately 40 g) was milled once for 1 min in an electric tabletop mill (Y-308B, Yamamoto Denki, Fukushima, Japan). Without sieving, the powder of milled crude drug was used for the IPCD method.

The powder mixture of crude drugs or that of each of the crude drugs (1/3 of the daily dose: rhubarb 1.33 g, glycyrrhiza 0.667 g) was placed in a wine carafe (0.25 L; Luminarc, Dubai, UAE), with the size and structure as reported by Fuek et al. After adding 150 mL of boiled distilled water, the suspension was vigorously stirred for 20 Sect. (2 strokes/sec). Following the 20-sec stirring (time 0 min), it was incubated for 4 min to allow the murky residue of the crude drug powder to precipitate. The immersion liquid was gently decanted into a beaker using a tea strainer. At 2 and 4 min, the decoction solution was collected.

Extraction from daiokanzoto extract granules

Daiokanzoto extract granules (one pack = 1/3 of the daily dose) were dissolved in 150 mL of 50% methanol in a beaker and homogenized (Smurt: NR-50 M, Microtec Co., Ltd., Chiba, Japan).

Spectrophotometric analysis of decoction solution

The decoction solution was centrifuged at 1,900 g for 30 min to obtain the supernatant for measuring color and transparency.

The spectrophotometer (Nippon Denshoku, Tokyo, Japan) was used to measure the Commission Internationale de L’éclairage (CIE) L*a*b*color parameters of the supernatant sample. L*a*b* is a color space defined by the Commission Internationale de L’éclairage (CIE). The lightness value, L*, assigns 0 to black and 100 to white. The a* axis is relative to the green–red opponent colors, with negative values pointing toward green and positive values pointing toward red. The b* axis represents the blue–yellow opponents, with negative numbers pointing toward blue and positive numbers pointing toward yellow [14].

Quantification of quantitative indicator ingredients

According to JP18, sennoside A and glycyrrhizic acid are quantitative indicator ingredients of rhubarb and glycyrrhiza, respectively. The quantitative indicator ingredients were quantified using high-performance liquid chromatography (HPLC). As standard marker compound reagents, sennoside A (for the Japanese Pharmacopoeia Crude Drugs Test [for TLC]) and glycyrrhizic acid Standard (for HPLC) were purchased from Fujifilm Wako Chemicals Corporation (Tokyo, Japan). The authors should add the company name, city, and country names of these reagents.

The decoction solution samples were centrifuged at 15,780 g for 10 min to obtain the supernatant. The supernatant was subsequently filtrated through a membrane filter (Cosmonice Filter [W] [0.45 μm], Nacalai Tesque, Inc., Kyoto, Japan). The filtrate was then subjected to the HPCL system.

The LC-20 A Modular HPLC System (Shimadzu Corporation, Kyoto, Japan) was used to perform HPLC. It consisted of an LC-20AD liquid pump, an SIL-20 A autoinjector, an SPD-20 A UV spectrophotometric detector, and an OTC-20 A column oven. The HPLC conditions adhered to the JP18 [2] with few modifications, as summarized in Table 1.

Statistical analysis

All data are expressed as means ± SEM, and statistical analysis was performed by using Student’s t-test. A difference between means was considered to be significant when the p-value was less than 0.05.