Pinellia tuber, the dried tuber of Pinellia ternata Breitenbach, is a crude drug registered in the 18th Edition of the Japanese Pharmacopoeia[1–2] and the Chinese Pharmacopoeia 2020 Edition. Raw Pinellia tuber contains homogentisic acid, 3,4-dihydroxybenzaldehyde, polysaccharides, calcium oxalate, etc, and is widely used in Japanese traditional Kampo medicine, such as Hangekobokuto. The administration of insufficiently boiled unprocessed Pinellia tubers causes very strong acrid irritation in the mouth and throat. Owing to this severe irritation, the Chinese Pharmacopoeia uses the term “toxic” to describe Pinellia tuber. According to recent studies, the acridity of Pinellia tubers is caused by insoluble needle-like crystals stored in idioblasts, called raphides, which are composed of calcium oxalate and proteins[7–8]. The protein isolated from raphides has been demonstrated to cause inflammation in the eyes of rabbits alone, and destruction of the needle-points of the crystals is reported to reduce the acridity of the raphides[7,9]. Therefore, raphide acridity may be indispensible to both the needle-like structure of the crystal and its stored protein.
Pinellia tuber-induced acridity is known to be decreased by being processed with ginger, alum, licorice and lime, or boiling for a certain period of time. According to the textbook of traditional Chinese medicine, Pinellia tubers should be used after the detoxification process. The Chinese Pharmacopoeia lists three types of processed Pinellia tuber: “Fabanxia: prepared Pinellia tuber”, which is the dried tuber of Pinellia ternata after being soaked in the decoction of Glycyrrhiza root and the solution of lime at a pH value higher than 12; “Qingbanxia: Pinellia tuber prepared with alum,” which is the dried tuber of Pinellia ternata after being soaked in 8% alum solution; and “Jiangbanxia: Pinellia tuber prepared with ginger”, which is the dried tuber of Pinellia ternata after boiling the decoction fresh rhizome slices of Zingiber officinale (ginger) with alum. In Japan, raw Pinellia tubers are commonly used instead of processed tubers[1–2]; however, based on specific instructions, it should be decocted for a sufficient period of time to remove its acridity. These methods of detoxifying Pinellia tuber have been developed throughout the history of traditional medicine in eastern Asia; however, the mechanisms of detoxification, except for alum, which was reported to destroy the needle crystal through solubilization of calcium oxalate by aluminum ions, have not been thoroughly revealed.
One of the difficulties associated with the study of Pinellia tuber detoxification is the objective and quantitative evaluation of its acridity in causing irritation. Recently, we reported that the acridity of raphides correlates with their lipophilicity, and the degree of their deactivation could be evaluated by measuring the turbidity of the petroleum ether (PE) layer in the water/PE partition of raphide samples using the raphide denaturation assay (RDA) [Supplementary Figure 1, https://links.lww.com/AHM/A40][13–14]. Using RDA, oxalic acid in ginger was found to specifically denature the raphides to reduce their acridity. Oxalic acid may be the active component in the processing of Pinellia tubers using ginger.
In this study, we assessed the features of detoxification by boiling and processing using Glycyrrhiza root extract and lime.
Materials and methods
All crude drugs adhered to the quality control of the 18th Edition of the Japanese Pharmacopoeia. The dried sliced form (approximately 2 mm wide) of Pinellia tuber (lot number, #009120002) and approximately 5 mm pieces of the cut dried root of Glycyrrhiza uralensis (Glycyrrhiza, lot number, #005819004) were purchased from Tochimototenkaido (Osaka, Japan). Soluble starch (β-form) was purchased from Hayashi Pure Chemicals (Osaka, Japan). The glycyrrhizin ammonium salt, glycyrrhetinic acid, glucuronic acid, and other salts were purchased from Nacalai Tesque (Kyoto, Japan).
Preparation of the low-starch Pinellia tuber suspension
Pinellia tubers (50 g) were milled using an electric mill (Y-308B, Yamamoto Denki, Fukushima, Japan) and filtered through a 150 µm mesh. The powder was mixed with H2O (350 mL) and maintained at room temperature for 30 min. The mixture was then vortexed vigorously to obtain a thick suspension. The suspension was centrifuged at 18× g for 10 min to precipitate the bulk starch, and the supernatant containing the raphides was collected. H2O (300 mL) was added to the precipitate and the suspension was vortexed vigorously. After centrifugation at 18 × g for 10 min, the second supernatant was separated and combined with the first supernatant. The combined supernatants were centrifuged at 1.0 × 103 × g for 10 min to precipitate the residue containing raphides. After removal of the supernatant, the residue was washed with H2O (50 mL) and centrifuged twice. Of note, a remarkably small amount of raphides was found in the supernatant using a microscope. The final residue was re-suspended in H2O (50 mL) to prepare low-starch Pinellia tuber suspension (LSPS) for subsequent use in the denaturation assay [Supplementary Figure 1, https://links.lww.com/AHM/A40]. The LSPS was prepared in conveniently sized packets and stored at –20°C until use.
PE extract of the raphides of Pinellia tuber
Powdered Pinellia tuber (30 g) was suspended in H2O (120 mL) and allowed to stand at room temperature for 60 min. PE (40 mL) was then added and mixed vigorously. The suspension was kept at 4°C for 90 min, and three layers appeared. When shaken gently, the raphides in the middle layer moved into the upper PE layer in the same manner performed in the RDA. The cloudy upper layer was immediately collected to avoid contamination of the remaining middle layer. This collection procedure was repeated three times with fresh PE (40 mL), and all PE layers were combined and centrifuged at 1.0 × 103 × g for 5 min. The supernatant was removed, and the precipitated raphides were washed three times with fresh PE (20 mL), and then three times with H2O (10 mL). The raphides were finally suspended in PE (7.0 mL) to create the PE extract (PEX) raphide suspension, and stored at −20°C in glass vials [Supplementary Figure 2, https://links.lww.com/AHM/A40]. An aliquot of the suspension was diluted with PE, and the OD660 was measured. Another aliquot of the suspension was dried and the weight of the raphides was precisely measured. The concentration of the PEX raphide suspension used in this study was 7.9 mg/mL.
Preparation of the α-starch solution
A 10% aqueous suspension of β-form (crystal) soluble starch was sealed in a plastic centrifuge tube (50 mL) and incubated at 100°C for 20 min. The suspension was mixed vigorously during the incubation period to prepare a homogeneous starch solution.
Assays of the denaturation activity against the raphides of Pinellia tuber
Either the PEX raphide suspension (180 µL) was added to an aqueous sample solution (4.0 mL) and then PE was thoroughly evaporated by a stream of air and mixed vigorously, or LSPS (0.50 mL) was added to the aqueous sample solutions (3.5 mL) and mixed. The reaction mixtures were pre-incubated for 30 min at 40°C or 100°C in sealed plastic centrifuge tubes. PE (3.0 mL) was then added at room temperature and vortexed vigorously (Supplementary Figure 3A, https://links.lww.com/AHM/A40). The mixtures were allowed to stand for 15 min until the upper, middle, and lower layers appeared (Supplementary Figure 3B, https://links.lww.com/AHM/A40). The tubes were then gently shaken for 10 s to raise the cloud of raphides from the middle layer to the upper layer. The upper PE layer containing the raphide cloud was collected, and the optical density at 660 nm (OD660) was measured immediately (Supplementary Figure 3C, https://links.lww.com/AHM/A40). The procedure after the addition of PE was named RDA. When the raphides of Pinellia tubers are denatured by samples, their lipophilicities decrease; therefore, the OD660 of the PE layer decreases. This sample effect is referred to as raphide denaturing activity.
Preparation of the Glycyrrhiza decoction
Glycyrrhiza (3.0 g) was mixed with H2O (36 mL) in a sealed plastic centrifuge tube (50 mL) and incubated in boiling water for 30 minutes. The suspension was then filtered through cotton gauze, and the filtrate was centrifuged at 1.0 × 103 × g for 10 min. The supernatant was collected and stored as a Glycyrrhiza decoction at −20°C until use. Part of the decoction was lyophilized to yield the extract, and the concentration of this decoction was 30 mg/mL. The concentration of glycyrrhizin in the extract was measured using the method described in the Japanese Pharmacopoeia 18th Edition; this concentration was 9.6% (w/w). The extract (0.54 mg) was dissolved in methanol and used for fingerprint analysis, as shown in Supplementary Figure 4 (https://links.lww.com/AHM/A40).
Bindings of glycyrrhizin and glycyrrhetinic acid to the raphides
The PEX raphides suspension in PE (0.50 mL) was mixed with either the mixture of 5.0 mM glycyrrhizin aqueous solution (0.50 mL) and 3.5 mL of saturated aqueous solution of Ca(OH)2 (A) or 8.0 mL of 0.94 mM glycyrrhetinic acid in saturated aqueous solution of Ca(OH)2 (B). Thereafter, PE was evaporated using a stream of air. The reaction mixtures were mixed vigorously before pre-incubation.
LSPS (1.5 mL) was mixed with either the mixture of 5.0 mM glycyrrhizin aqueous solution (0.75 mL), 1.0% Ca(OH)2 suspension (2.0 mL), and 1.75 mL of H2O (C), or a mixture of 5.0 mM glycyrrhetinic acid in saturated aqueous solution of Ca(OH)2 (1.25 mL), 1.0% Ca(OH)2 suspension (2.0 mL), and 1.25 mL of H2O (D). The reaction mixtures (A)–(D) were pre-incubated at 40°C for 30 minutes.
PE (3.0 mL) was added to each reaction mixture and mixed vigorously. The sample mixture was then centrifuged at 1.0 × 102 × g for 5 min to separate the upper, middle, and lower layers. The middle layer was carefully collected and washed thoroughly three times with a saturated aqueous solution of Ca(OH)2. After washing, the residue was re-suspended in 50 µL of a saturated aqueous solution of Ca(OH)2 and centrifuged at 4.3 × 103 × g for 5 min. Finally, the supernatants were collected and mixed with 50 µL of EtOH/2 M HCl (4:1) to serve as the negative control groups (A2–D2).
The residues were finally suspended in 100 µL of EtOH/2 M HCl (4:1), and then centrifuged at 4.3 × 103g × for 5 min. The supernatants were collected as sample groups (A1–D1) for analysis by thin-layer normal phase chromatography (TLC). For the standards, each 5.0 mM aqueous solution of glycyrrhizin or glycyrrhetinic acid in saturated aqueous solution of Ca(OH)2 was diluted with 1.5 times the amount of EtOH/2 M HCl (4:1). Thereafter, the supernatants (10 µL) were spotted onto TLC plates (Silica gel 60 F254, Merck Millipore, Burlington, MA, USA) and eluted with n-BuOH/H2O/acetic acid (7:2:1.4) for glycyrrhizin or with ethyl acetate/PE/acetic acid (4:11:1) for glycyrrhetinic acid. Images of the plates were captured under ultraviolet light at 254 nm.
One-way analysis of variance (ANOVA) followed by Bonferroni’s or Dunnett’s multiple test was used to compare the differences among all pairs among multiple groups or between the normal control group and other groups among multiple groups. Student’s t test was used to compare two independent groups. All analyses were conducted using Mac Statistical Analysis Ver 3.0 (Esumi, Tokyo, Japan). Data are expressed as mean ± standard deviation (SD). P < 0.05 was considered to indicate significance.
Raphide denaturation by boiling
The PEX raphides were pre-incubated for 30 min at 40°C or 100°C with or without starch. According to the RDA, after 30 min of boiling, single PEX raphides maintained 52% OD660 of the PE layer compared with the normal control, which was pre-incubated at 40°C. The addition of 2.5% of α-starch lowered the OD660 of the PE layer to 9% of the normal control value. The result of boiling with 2.5% glucose did not significantly differ from the pre-incubation of single PEX raphides at 100°C. The denaturing activity of α-starch was also observed after pre-incubation at 40°C. Neither β-starch nor glucose exhibited significant denaturing activity at 40°C (Figure 1).
Dose-dependence of α-starch on raphide denaturation by boiling
After the α-starch solution was diluted with H2O and pre-incubated with PEX raphides for 30 min at 100°C, RDA was performed. α-Starch had a dose-dependent decrease in OD660 based on RDA, and its IC50 value was approximately 0.53% (Figure 2).
Raphide denaturing activity of glycyrrhiza decoction
The LSPS was pre-incubated with the Glycyrrhiza decoction for 30 min at 40°C. The RDA revealed that a 17% lower OD660 of the PE layer was observed relative to the normal control without Glycyrrhiza decoction with statistical significance (P < 0.001) (Figure 3A). When Ca(OH)2, NaOH, or CaCl2 was mixed with the reaction mixture of LSPS and Glycyrrhiza decoction, strong denaturing activity was observed when combined with Ca(OH)2. This interaction between Glycyrrhiza decoction and Ca(OH)2 was not observed in NaOH or CaCl2. Single Ca(OH)2 displayed significant raphide denaturing activity at approximately 50% of the normal control group value. In contrast, single NaOH or CaCl2 exhibited a significant increase in OD660 in the RDA (Figure 3B).
Dose-dependence of the Glycyrrhiza extract and glycyrrhizin on the raphide denaturing activity
After the Glycyrrhiza decoction was diluted with H2O and pre-incubated with LSPS and Ca(OH)2 for 30 min at 40°C, RDA was conducted. Glycyrrhiza decoction exhibited a significant decrease in OD660 in RDA at more than 1.9 mg extract/mL (Figure 4A). We proceeded to focus on glycyrrhizin as the active ingredient of the raphide denaturing factor in the Glycyrrhiza decoction, as glycyrrhizin is registered as the marker compound in both the Japanese and Chinese Pharmacopoeias[1,3]. Glycyrrhizin exhibited significant raphide denaturing activity in the presence of Ca(OH)2 in a concentration-dependent manner, with an IC50 value of 0.33 mM (Figure 4B).
pH dependence of the raphide denaturing activity of glycyrrhizin
The pH dependence of the raphide denaturing activity of glycyrrhizin with calcium ions was investigated. LSPS was pre-incubated with CaCl2 in the reaction mixtures at pH 8.0–12, with adjustments performed using borate-Na buffers. The raphide denaturing activity was observed to increase as the pH of the reaction mixture increased. Notably, the activity significantly decreased at pH 10 and then increased in accordance with the pH value of the reaction mixture (Figure 5). Glycyrrhizin alone, without CaCl2, did not exhibit raphide denaturing activity at pH 12 (data not shown).
Raphide denaturing activity of glycyrrhizin and glycyrrhetinic acid
LSPS was pre-incubated with glycyrrhizin, glycyrrhetinic acid, or glucuronic acid in the presence of Ca(OH)2, followed by RDA. Glycyrrhizin displayed significant raphide denaturing activity. Glycyrrhetic acid, the aglycone of glycyrrhizin, also exhibited statistically equal activity as glycyrrhizin, whereas glucuronic acid did not exhibit any activity (Figure 6).
Bindings of glycyrrhizin and glycyrrhetinic acid to the raphides
Raphides denatured by Ca(OH)2 with glycyrrhizin or glycyrrhetinic acid were collected, washed, and dissolved in EtOH/2 M HCl (4:1). Glycyrrhizin and glycyrrhetinic acid were detected in the solution of the denatured raphides in HCl, whereas the negative control using the supernatant of Ca(OH)2 suspension did not contain these compounds. The spot at the starting point on the plate was derived from impurities in the HCl reagent (Figure 7).
Calcium ion dependence of the raphide denaturing activity of glycyrrhizin
When LSPS was pre-incubated with glycyrrhizin and either CaCl2 or SrCl2 at pH 12, SrCl2 did not exhibit any significant raphide denaturation activity (Figure 8).
In our previous study using RDA, oxalic acid was identified as the main active constituent in the ginger extract for the detoxification of the Pinellia tuber in the processing of Jiangbanxia. RDA was demonstrated to be highly reproducible, and its result aligned with the sensitivity of the gustatory tests to acrid irritation. In this study, using RDA, we investigated the features of the detoxification of Pinellia tuber by boiling and the traditional processing of Fabanxia.
For RDA, we prepared two types of raphide samples, LSPS and PEX raphides. LSPS was not exposed to the low polar solvent, and was stored under intact conditions compared with PEX raphides. As starch was not completely removed from LSPS, the influence of starch in the experiments performed at high temperature could not be negligible. On the other hand PEX raphides were more purified than LSPS and starch was not identified as an impurity in PEX raphides. However, the surface of PEX raphides easily became lipophilic owing to exposure to PE. We adopted these complementary raphide samples to increase the reliability of the results.
Detoxification was first examined by boiling. To observe the effect of heating alone, purified PEX raphides were employed for this experiment. When the purified raphides were boiled in water for 30 minutes, which is a common process for preparing decoctions in Japanese Kampo medicine, the raphides were found to be significantly denatured. However, approximately half of the raphides remained intact. On the other hand, when α-starch was added to the purified raphides and boiled in water, denaturation was significantly enhanced in a concentration-dependent manner. It was reported that when powdered Pinellia tuber was boiled in water for 30 min and centrifuged, the precipitate retained strong acridity, which implied that the acrid ingredient in Pinellia tuber would originally be thermo-stable to a certain extent. On the other hand, the decoction of raw Pinellia tuber prepared using the common Japanese decocting procedure is known to have almost no acridity. It may be because the raphides stored in the intact idioblasts cannot contact and interact with the starch in the tuber during the decoction procedure and preserve the acridity until the end of boiling, while the raphides released from the destroyed idioblasts freely interact with the starch dissolved in the decoction and are denatured to lose their acridity. In addition, lightly roasted dried raw Pinellia tuber powder was found to preserve its acridity, whereas the acridity was removed when the powder was roasted after being mixed with 5% (w/w) of water (data not shown). Based on these results, we suggest that heat also plays a role in gelatinizing the starch in the Pinellia tuber in the presence of water during the boiling detoxification process, as well as directly affecting the raphides to denature. However, the detailed mechanism of the interaction between raphides and starch remains unclear. The α-starch solution displayed significant denaturing activity at 40°C, whereas neither insoluble β-starch nor glucose displayed significant denaturing activity, suggesting that the starch molecule specifically interacts with raphides to denature them.
The irritative protein in raphides is reported to be a plant lectin. Indeed, we isolated a protein from the purified raphides, which was identified as a c-type lectin encoded in the P. ternata gene; whose details will be provided elsewhere. The P. ternata lectin was reported to be denatured by being heated for 60 min to decrease water solubility by approximately half, which aligned with our result of raphide denaturation by heating without starch in this study. We suggest the denaturation of the raphides could be related to some changes in the lectin in the raphides. Lectin binds to sugar chains. In contrast with starch, glucose did not significantly enhance the denaturation of raphides. Sugar chains of a certain length may be required to denature the raphides. Indeed, soluble dextrin exhibited a similar activity to starch (data not shown). P. ternata lectin may have a certain level of affinity for-with starch, which is enhanced by heating.
We succeedingly examined the raphide denaturing activity of Glycyrrhiza decoction and an aqueous solution of Ca(OH)2, which are components of the traditional processing of Fabanxia. A single Glycyrrhiza decoction displayed significant but weak raphide denaturing activity; however, the activity was markedly enhanced by the addition of Ca(OH)2. Neither the addition of NaOH nor CaCl2 caused denature-enhancing activity (Figure 3), implying that a high pH value and calcium ions are required to enhance the raphide denaturing activity of Glycyrrhiza extract, which aligned with the process of Fabanxia. Glycyrrhiza extract exhibited significant and dose-dependent raphide denaturing activity at concentration greater than 1.9 mg/mL in the presence of Ca(OH)2. At a low concentration (0.94 mg/mL), the Glycyrrhiza extract was found to increase OD660 in RDA (Figure 4A). However, the reason for this phenomenon remains unknown. LSPS contains raphides bound to idioblasts, which exist in the water layer as precipitates in RDA. The Glycyrrhiza extract may free raphides from binding to idioblasts and increase the concentration of free raphides.
Among the constituents of the Glycyrrhiza extract, we opted to first focus on glycyrrhizin. Glycyrrhizin showed significant raphide denaturing activity in the presence of Ca(OH)2 in a concentration-dependent manner, with an IC50 value of 0.33 mM (Figure 4B). Glycyrrhizin also displayed the strongest denaturing activity at pH 12 (Figure 5), which was in accordance with the instructions for processing Fabanxia to maintain a pH greater than 12 in the reaction mixture.
Glycyrrhiza extract (1.9 mg/mL) exhibited raphide denaturing activity with approximately 57% of the control in RDA. As the concentration of glycyrrhizin in the Glycyrrhiza extract was 9.6%, the concentration of glycyrrhizin in the reaction mixture of the Glycyrrhiza extract (1.9 mg/mL) was estimated to be approximately 0.2 mM. From the concentration-dependent profile, 0.2 mM glycyrrhizin may exhibit approximately 71% of the control in RDA. Therefore, glycyrrhizin was estimated to have an approximately 80% contribution to the denaturing activity of the Glycyrrhiza extract. In addition, the estimated contribution of glycyrrhizin in 3.8 mg/mL of the Glycyrrhiza extract was 36%, using the formula applied above (Figure 4). Therefore, glycyrrhizin must be the main constituent in Glycyrrhiza to denature the raphides; however, besides glycyrrhizin, other active ingredients in the Glycyrrhiza extract can denature the raphides.
Glycyrrhizin is a glycoside that consists of glycyrrhetinic acid and glucuronic acid in its chemical structure. Glycyrrhetinic acid, the aglycone, displayed raphide denaturing activity to an equal degree as glycyrrhizin, whereas glucuronic acid displayed no activity (Figure 6). Glycyrrhetinic acid may serve as the site of the denaturing action of glycyrrhizin in the chemical structure. To further investigate the denaturing mechanism, we collected raphides denatured by glycyrrhizin or glycyrrhetinic acid with Ca(OH)2 from the middle layer of RDA, and then extracted them under acidic conditions. TLC analysis revealed the detection of glycyrrhizin or glycyrrhetinic acid in the denatured raphides, implying that denaturation was induced by the binding of glycyrrhizin or glycyrrhetinic acid to the raphides (Figure 7). The pH profile of the denaturing activity of glycyrrhizin also displayed a small peak at pH 9 (Figure 5). This phenomenon could be related to the pH-dependent conformational changes of the lectin to interact with glycyrrhizin, and will be further assessed in future investigations. The interaction between raphides and glycyrrhizin was found to depend on calcium ions, specifically dicationic metallic ions (Figure 8). C-type lectin binds mannose in a calcium-dependent manner. The calcium dependency of the interaction between raphides and glycyrrhizin may be related to the nature of lectin.
In summary, during the processing of Fabanxia, glycyrrhizin binds to the raphides via the interaction between lectin and glycyrrhetinic acid motif in the basic condition of pH 12 to denature the raphides and detoxify the Pinellia tuber. The detailed mechanisms by which the interaction with starch or the binding of glycyrrhizin denature raphides to lose their lipophilicity remain unclear. Investigations on the mechanisms of raphide denaturation could reveal how raphides cause acrid irritation. Further investigations are anticipated.
Conflict of interest statement
Toshiaki Makino is the editorial board member of this journal. Toshiaki Makino received grant support from Tsumura & Co., Kracie Pharmaceuticals, JPS Pharmaceuticals, Taisho Holdings, and Kobayashi Pharmaceuticals.
This study was supported by a grant from the Oriental Medicine Research Foundation (2022).
Tsukasa Fueki, Takao Namiki, Koichiro Tanaka, and Toshiaki Makino designed this study. Tsukasa Fueki, Itsuki Nose, and Yan Liu performed the experiments. Tsukasa Fueki wrote the draft article, and Toshiaki Makino finalized the article. All authors have read and approved the final manuscript. All data were generated in-house and no paper mill was used. All authors agree to be accountable for all aspects of the work and ensure its integrity and accuracy.
Ethical approval of studies and informed consent
We thank Oriental Medicine Research Foundation for supporting our research.
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