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Graphic abstract Unprocessed dried tuberous root of Pinellia ternata (Pinellia Tuber) has been used as a component of traditional Japanese Kampo medicine, while this crude drug is usually used after processing with Ginger in traditional Chinese medicine (TCM). It is known that the raphides contained in unprocessed Pinellia Tuber can induce severe acrid irritation of the oral and laryngopharynx mucosa when it is boiled insufficiently. However, the mechanism of the inducing irritation by the raphides nor that of the detoxification by heat or processing with Ginger have been left unclear, mainly because of the difficulties in the extraction and the purification of the raphides and in the assays of acridity. In this study, we developed an easy protocol that can be used to collect raphides from unprocessed Pinellia Tuber and an assay protocol that can be used to evaluate the acridity of the raphides in vitro. The raphides of Pinellia Tuber were discovered to have a lipophilic character and to be collected easily by the extraction using petroleum ether. It was also found that the denaturation of the raphides could be assayed by the dispersity of them in petroleum ether layer of the water/petroleum ether partition, and the acridity of the raphides was found to be in correlation with the assayed denaturation. The raphides were denatured by heat, methanol, or Ginger extract and the denaturing activity of Ginger on raphides was found to be attributable to its lipophilic and thermostable components, which may explain the meaning of the processing of Pinellia Tuber with Ginger in TCM, and may lead to the development of an easier and safer protocol to administer Pinellia Tuber. In addition, it was found that rinsing the mouth with salad oil can effectively relieve irritation of the oral mucosa caused by unprocessed Pinellia Tuber, probably due to the lipophilicity of the raphides. Electronic supplementary material The online version of this article (10.1007/s11418-020-01425-6) contains supplementary material, which is available to authorized users.

A correction to this paper has been published: https://doi.org/10.1007/s11418-021-01501-5

Review Article, Three factors are crucial if oriental medicine is to maintain its role in modern medicine and continue to progress. The first is the collection of scientific evidence, which in turn will safeguard the advantages of oriental medicine. The second factor is training clinicians who understand and can practice traditional medicine, which will require a restructured model of continuous education from medical school through specialist training. The third factor is actual participation in clinical medicine, which will contribute to patient care. Physician quality and advanced clinical skills are essential in clinical medicine, and we envision two types of clinicians: one who is highly specialized in western medicine and practices traditional medicine in their specialized field and one who is specialized in traditional medicine who practices comprehensive medicine that includes western medicine. The latter will work alongside specialists in western medicine, to discuss and improve patient care.

Aim The immersing powdered crude drugs (IPCD) method has been developed as a quick, easy and high-yielding method for preparing decoctions. Given that the extraction process is different from the conventional decoction method, the safety of this method should be carefully examined. In the present study, the amounts of the diester alkaloids of aconite root, the tuberous root of Aconitum carmichaeli, and sennoside A of rhubarb, the rhizome of Rheum palmatum, were determined in the IPCD immersion. Both of these compounds have strong physiological activities and have been suggested to be decomposed during the decoction process. Methods The amounts of marker compounds in the IPCD immersion prepared from processed or unprocessed aconite root or rhubarb were measured and compared with those prepared using the conventional decoction method. Results The amount of sennoside A extracted with the IPCD method was 1.1-fold increased compared with that from the conventional method. No diester alkaloids were detected in the IPCD immersion prepared from processed aconite root, whereas for unprocessed aconite root, the amount of diester alkaloids extracted from the IPCD immersion was 8.8-fold higher than that from the conventional decoctions. Conclusion The IPCD method can be safely used on rhubarb or well-processed aconite root under the same conditions as other less-powerful crude drugs, whereas precautions are required for unprocessed aconite root.

Aim The immersing powdered crude drugs (IPCD) method was developed as a quick, easy and high-yield way to prepare decoctions. IPCD may be very useful for decoctions of crude drugs containing volatile compounds because it does not involve boiling. The aim of this study was therefore to test this hypothesis using cinnamon bark (the bark of the trunk of Cinnamomum cassia) and perilla herb (the leaves and the tips of the branches of Perilla frutescens var. acuta). Methods The amounts of the marker compounds in the IPCD immersion prepared from cinnamon bark or perilla herb were measured and compared with those prepared with the conventional decoction method. Results The amounts of cinnamaldehyde and perillaldehyde in the IPCD immersion per 1 g of crude drugs exceeded those in the conventional decoctions by two-fold and 30-fold, respectively. Conclusion The IPCD method is outstanding for the decoction of crude drugs containing volatile compounds.

Aim For the purpose of reducing both the consumption of crude drug resources and patient burden in preparing the decoctions, we developed a new preparation method for the decoctions of Kampo formula: immersion of powdered crude drugs in hot water (IPCD method), inspired by a method of decoction that was widely used in the Song period of China. Methods Ephedra herb (terrestrial stem of Ephedra sinica), scutellaria root (root of Scutellaria baicalensis), and Kampo formulas that contain several crude drugs including ephedra herb and scutellaria root were powdered, and then immersed in hot water to extract the ingredients. The residue was filtered out using a commercially available tea server. The contents of marker compounds in the decoctions were measured. Results Using the IPCD method, the amounts of marker compounds from ephedra herb and scutellaria root transferred to hot water exceeded by 1.3–1.4-fold that obtained by the standard method of decocting the same amounts of cut crude drugs for >30 min. The amount of transferred marker compounds reached a plateau within 4 min after the beginning of immersion, suggesting that only 4 min of immersion is sufficient for the IPCD method. The sludge residue of powdered crude drug produced by immersion could be easily filtered with a commercially available tea server. Conclusion Using this method, the same or greater amounts of marker compounds were transferred in the decoctions in a much shorter time with lower consumption of crude drugs than with the usual decoction method.

Plasmid pSC101 encodes a 37.5 kDa Rep (RepA) protein, which binds to three 21-base repeats (DR-1, DR-2, and DR-3) in the replication origin region (ori) of the plasmid to initiate replication. Rep also binds to two palindromic sequences (IR-1 and IR-2) which overlap the rep promoter. The binding of Rep to IR-2 represses the production of Rep itself. It is highly likely that the balance of these functions of Rep plays a major role in controlling the copy number of pSC101. In this study, we developed a positive-selection system for replication-deficient mutants of the initiator protein. This system can be applied to the study of other replication systems by changing ori and rep of pSC101 to the corresponding genes. Thirty-four replication-deficient (Ini(-)) mutants were isolated with this system, and analyzed as to the relation between the structure and function of the Rep protein. Seventeen of these 34 Ini(-) mutants were found to lack auto-repressor activity as well as initiator activity. DNA sequence analysis showed that one-third (from the C-terminus) of Rep is dispensable for the auto-repressor activity, while the initiator activity seems to require the whole protein.

Purified replication initiator protein (Rep) of plasmid pSC101 binds preferentially to two inverted repeats (IR) overlapping the promoter of its own structure gene, rep. However, the protein has much lower binding affinity for directly repeated (DR) sequences in the replication origin (ori) that are similar to the symmetric sequences. Exonuclease III (exo III) promotes in vitro binding of Rep to the origin repeats. In the present studies, DNA containing the DR sequences was degraded unidirectionally by exo III and then formed a complex with Rep. Analyses of DNA from the complex revealed that Rep bound to the DR sequences only when the degradation proceeded from the 3' end proximal to IR to the DR sequences, resulting in conversion of the duplex structure in a specific downstream region of DR into the single-stranded form. The degradation in the opposite direction had no effect on binding of Rep. These results suggest that a localized structural change of DNA adjacent to DR is required for Rep binding to double-stranded DR sequences. By contrast, exo III strikingly inhibited binding of Rep to DNA containing the IR sequences by introducing a single-stranded moiety into duplex IR sequences.

A novel method for the introduction of an oxy functionality at the C 12 -position of the benzo[c]phenanthridine skeleton was developed. This method was successfully applied to a biomimetic synthesis of macarpine (3) from oxychelirubine (15), which was easily derived from the corresponding protoberberine (9)

The purified dimeric form of the Rep protein, a replication initiator protein of the plasmid pSC101, has a low affinity for repeated sequences, iterons, in the replication origin of the plasmid, and higher affinities for two inverted repeats in the operator region of the rep gene resulting in its functioning as an autorepressor. Studies of binding to various synthetic DNA have established that Rep can bind to duplex iteron-sequence carrying open (non-complementary) strands at one end proximal to the rep gene. Open strands at the opposite end of the iteron have no effect on Rep-binding. One open strand seems to be required in a sequence-specific fashion. A randomly sequenced duplex DNA with the open strands cannot bind to Rep but can function as a significant competitor. This suggests that Rep has some affinity for the open strands and forms a stable complex with the adjacent iteron. The mutated Rep protein, Rep1, which causes an increase in the plasmid copy number in vivo, has equally high affinity for the iteron with the open strands as wild type Rep, though it has a lower affinity for the inverted repeats than the wild type. The Rep dimer might bind to these DNA sequences with different modes.

Purified Rep protein, a replication initiator protein of plasmid pSC101, has less binding affinity for the direct repeats (DR) in the replication origin region (ori) than that for the inverted repeats (IR) in the promoter region of the structure gene of Rep (rep) (Sugiura, S. et al. (1990) J. Biochem. 107, 369-376). We found a protein factor that promotes binding of purified Rep to the DR sequence in the cell extract of Escherichia coli. In the presence of the factor, DNA fragments containing the DR sequence can form a specific DNA-protein complex by the addition of low concentrations of Rep. On the contrary, IR-containing DNA loses its binding activity for Rep by preincubation with the factor. We purified extensively the factor and identified it as exonuclease III (exo III). Enzymatic action of the factor or authentic exo III at 37 degrees C is necessary for binding of Rep to DR-DNA. This binding of Rep to duplex DNA treated with exo III is DR-sequence specific. Since Rep cannot bind to the single stranded DR sequence, the present finding suggests that partial single-stranded regions around the DR sequence are required for binding of Rep.