2022 Volume 45 Issue 7 Pages 955-961
Rehmannia glutinosa is an important medicinal plant in Asia, and its roots are used as an ingredient in herbal medicine. However, the roots exhibit different medicinal effects depending on the processing conditions. Since the catalpol content differs greatly during the process, the catalpol content is an essential index for quality evaluation. R. glutinosa roots have various weights, diameters, and lengths, and there are differences between individuals and within an individual immediately after harvest. We found that, catalpol content in the roots tended to increase as root diameter increased. Furthermore, it has been reported that catalpol content decreased with drying, and our results also supported this phenomenon. To clarify the reason for the decrease in catalpol content, we investigated the effect of β-glucosidase in R. glutinosa cells. An in situ assay for β-glucosidase activity revealed that the activity in the tissue inside the cambium disappeared one month after drying under natural conditions, and the activity in the tissue outside the cambium completely disappeared after two months. Because catalpol content remained almost unchanged even after drying for two months, it was clarified that β-glucosidase activity had minimal involvement in the decrease in catalpol content in R. glutinosa roots. Based on the above results, we proposed that slicing the roots and rapidly removing water by natural drying is best to obtain dry root with little loss of catalpol content.
The roots of Rehmannia glutinosa (Gaertn.) Libosch. ex Fisch. et C. A. Mey. (Rehmanniaceae) are an important ingredient in herbal medicine used as tonifying blood in China, Japan, and other Asian countries. Catalpol is one of the components responsible for the medicinal effects of R. glutinosa. However, catalpol is unstable and easily decomposed by processing.1) Catalpol content in R. glutinosa roots varies greatly depending on the lot, and some roots do not contain catalpol at all. It is essential to control the contents of active constituents in dried root (Kan-Jio) and steamed & dried root (Juku-Jio) to ensure optimum medicinal effects.2) In the same manner, it is necessary to control catalpol content in R. glutinosa roots to ensure stable quality.
R. glutinosa cultivated in Japan produces roots with weights of up to 35 g, diameters of up to 3.5 cm, and lengths of up to 25 cm. The roots have diverse shapes: string-like without any thick part, sweet potato-like, and string-like with gradually thickening parts. For medicinal purposes, generally, only the thick part is used. Our preliminary examination has revealed that catalpol content varied depending on the part between individuals and the part within an individual. Therefore, clarifying the distribution pattern of catalpol among individual roots and within a root (localization) would lead to the production of R. glutinosa with the same catalpol content.
As catalpol is often used for the quality evaluation of R. glutinosa, there is a need to find a better drying method that would minimize the reduction of catalpol content. In ancient Chinese Materia Medica3) it was described that the best way to dry the roots was to put them outside for natural drying. In our previous study,4) we found that heating and drying methods substantially reduced catalpol content in R. glutinosa roots. Therefore, it is necessary to observe changes in catalpol content during natural drying to clarify the best natural drying method. Previous paper reported that catalpol content in different root diameter of Rehmannia glutinosa at different growth stages were irregular.5) So, we need to investigate the relationship between the root diameter of Rehmannia glutinosa and the content of catalpol during the harvest to season.
It has been reported that catalpol was hydrolyzed by β-glucosidase.6,7) In addition, it has been demonstrated that R. glutinosa roots contained β-glucosidase, which strongly hydrolyzes catalpol.8,9) However, there are no studies of β-glucosidase distribution in R. glutinosa root tissues and changes in β-glucosidase activity during natural drying. It has been reported that intracellular β-glucosidase was involved in the degradation of oleuropein, an iridoid glycoside found in olive fruit (Olea europaea).10) That study also showed the in situ localization of β-glucosidase activity in olive tissue, namely, the cytological localization of β-glucosidase was detected by the hydrolysis of chromogenic synthetic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-Glc), which produced an insoluble blue precipitate in the cells. Therefore, we used this method to observe the distribution of β-glucosidase in R. glutinosa tissues and the changes in β-glucosidase activity as R. glutinosa roots were naturally dried. By comparing the changes in β-glucosidase activity and catalpol content, we determined the effect of β-glucosidase on catalpol degradation during the natural drying process.
According to the previous data11,12) the weight of R. glutinosa roots immediately after harvest was set at 100%, and the drying process was considered complete when the weight of the roots was reduced to 25% of the weight immediately after harvest. Then, the dried roots were used as herbal medicine. In this study, we subjected R. glutinosa roots to three different processing methods, recorded the time required to reach the specified moisture content and the change in catalpol content during natural drying for the processed roots, and discussed the best processing method.
The objectives of this study were to clarify the relationship between R. glutinosa root morphology and catalpol content and to investigate the difference in catalpol content between individuals and localization of catalpol content in the individual. We confirmed changes in β-glucosidase activity during natural drying and discussed the best method for natural drying.
Rehmannia glutinosa strain H01 as obtained from the Medicinal Plant Garden, School of Pharmacy, Kanazawa University. The Japanese commercial product used in this study was Jio (Lot. 005018011, Tochimoto Tenkaido, Osaka, Japan), which was derived from R. glutinosa roots. All materials used in this study were stored in the Specimen Room, Kanazawa University, Japan. The samples were identified as Kaikei type by Professor Yohei Sasaki.13)
Preparation of Samples for Quantitative Analysis of CatalpolThe samples were freeze-dried (FDU-1110, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) and then pulverized in a grinder (IFM-800, Iwatani Corporation, Osaka, Japan). The powder that passed through a 300 µm sieve was used as the sample.
HPLCTo 50 mg of powder in a microtube was added 1 mL of CH3CN/H2O solution (1 : 99). The mixture was subjected to ultrasonication (DENTCRAFT® Ultrasonic 3800, Yoshida Dental, Tokyo, Japan) at 20 °C for 30 min and centrifugation (Centrifuge 5417R, Eppendorf Co., Ltd., Tokyo, Japan) for 10 min (13000 rpm). A 0.3 mL aliquot of the supernatant was diluted fivefold with the mobile phase and passed through a 0.45 µm membrane filter for HPLC quantification.
HPLC was performed at 40 °C using a YMC-Triart C18 ϕ4.6 × 250 mm column. The mobile phase14) was 1% acetonitrile in water (containing 0.1% phosphoric acid). The flow rate was 0.6 mL/min. UV detection was performed at λ = 210 nm. The injection volume was 10 µL. The HPLC system consisted of a pump (L-2130, Hitachi High-Tech Corporation, Tokyo, Japan), an autosampler (L-2200, Hitachi High-Tech Corporation), a UV detector (L-2400, Hitachi High-Tech Corporation), a column oven (SSC-2300, Schambeck SFD GmbH, Germany), and Chromato-PRO (Run Time Corporation, Tokyo, Japan) software for data processing. Catalpol content was calculated by converting the concentration of catalpol in the sample solution to dry matter using the loss on drying value.2) In the determination of catalpol, the regression line of the calibration curve (y = 4538225 × (−11637)) was linear (R2 = 0.99994).
Differences in Catalpol Content among Individual RootsSamples of different sizes were selected from R. glutinosa fresh roots, and the diameter of the thickest part of the root, the overall length, and the weight were measured. The catalpol content in each sample was determined (Fig. 1, top).
Each root was divided into seven segments, and the three non-adjacent parts were designated as the upper, middle, and lower parts. Root length and diameter are expressed as L and d, respectively.
Catalpol content in different parts of the root and the relationship between diameter and catalpol content were analyzed.
Fresh roots without mold and with individual weight greater than 10 g were selected as samples from strain H01. Each root was cut vertically into seven segments along the major axis, and three non-adjacent segments were selected. The segment closest to the ground was the upper part, the middle segment was the middle part, and the remaining segment was the lower part. Catalpol content in each segment was determined and the operation was repeated for 14 samples (Fig. 1, bottom).
Fresh roots without mold were selected from strain H01 and cut into different segments according to their thickness. The diameter of each segment was measured, and segments with the same diameter were mixed and freeze-dried. Then, catalpol content was determined. Each root was divided into nine segments according to diameter (0.0 to 0.7, 0.7 to 1.4, 1.4 to 2.1, 2.1 to 3.0, 3.0 to 5.0, 5.0 to 8.0, 8.0 to 14.0, 14.0 to 23.0, and 23.0 to 30.0 mm) (Fig. 2).
a. 0.0 to 0.7 mm; b. 0.7 to 1.4 mm; c. 1.4 to 2.1 mm; d. 2.1 to 3.0 mm; e. 3.0 to 5.0 mm; f. 5.0 to 8.0 mm; g. 8.0 to 14.0 mm; h. 14.0 to 23.0 mm; i. 23.0 mm to 30.0 mm. a. (n = 3); b to i. (n = 5).
The cambium was visible in the root cross section. The tissue inside and outside the cambium were clearly separated, and catalpol content in those two layers was measured (n = 12) (Fig. 3).
The cambium is clearly visible in the root cross section. The color inside the cambium is different from that outside the cambium. The tissue inside and outside the cambium can be clearly distinguished.
Strain H01 roots were harvested from the Medicinal Plant Garden of Kanazawa University in November 2018. Roots weighing more than 20 g and having approximately the same maximum diameter were selected as samples. The roots were placed in a drying shed in the Medicinal Plant Garden of Kanazawa University to avoid rain and direct sunlight and dried under natural conditions outdoors. Catalpol content was determined and β-glucosidase activity was observed separately once a month. Ten samples were selected every month to determine catalpol and moisture contents (Fig. 4a).
Whole root (a), 10 mm slices (b), and 7 mm dices (c).
Roots were freshly sectioned at 4 °C into 80 µm thickness using a plant microtome (MTH-1, Aperza Catalog). Ten roots were selected, and each root was sectioned at six different places. The sections were immediately placed in screw pipes containing 5 mL of detection buffer according to Mazzuca et al.,10) containing 60 µM X-Glc (FUJIFILM Wako Pure Chemical Corporation, LKF6939), 50 mM phosphate buffer pH 6.5, 1 mM potassium ferricyanide (Lot. SKP4774, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 1 mM potassium ferrocyanide (FUJIFILM Wako Pure Chemical Corporation Lot. SKN6088), and 10 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0. The sections were incubated at 37 °C for 3 h. The reaction was terminated by adding cold phosphate buffer, and the sections were mounted with glycerin solution (glycerin : H2O = 1 : 1) on slides for optical microscopy (GLB-B1500, Shimadzu Rika Corporation, Tokyo, Japan). Photographs were taken with Moticam X3 (Shimadzu Rika Corporation) and analyzed using Motic Images Plus 2.3S (Shimadzu Rika Corporation).
Exploration of Optimal Drying MethodThe same materials as those in the above experiment were used. The roots were cut into 10 mm slices and 7 mm dices. In order to determine the quality of R. glutinosa for use as herbal medicine, the weight of roots immediately after harvest was set at 100%, and the drying process was considered complete when the weight of the roots was reduced to 25% of the weight immediately after harvest.11,12) Then, catalpol content was measured (Figs. 4b, c).
First, we investigated individual differences in R. glutinosa roots and tried to clarify the relationship between catalpol content and morphological features including weight, maximum diameter, and length (Fig. 5). The roots of R. glutinosa varied markedly among individuals, and individuals that did not contain catalpol were observed even though they had just been harvested. Regarding the relationship between catalpol content and root weight, the average catalpol contents were 1.0% for 5 to <10 g root weight, 2.3% for 10 to <25 g root weight, and 1.8% for 25 to ≤35 g root weight, and catalpol content tended to be significantly higher in roots weighing 10 to <25 g than in roots weighing 5 to <10 g (Fig. 5a). Regarding the relationship between catalpol content and root maximum diameter, the average catalpol contents were 0.5% for 1.0 to <1.5 cm root diameter, 2.2% for 1.5 to <2.5 cm root diameter, and 2.3% for 2.5 to ≤3.5 cm root diameter, and a significant difference was observed between roots with a diameter of 1.5 cm or less and those with diameters of 1.5 cm or larger (Fig. 5b). Regarding the relationship between catalpol content and root length, the average catalpol contents were 2.0% for <10 cm root length, 1.7% for 10 to <15 cm root length, and 0.9% for 15 to ≤25 cm root length. Catalpol content tended to be lower for longer roots, although no significant difference was noted (Fig. 5c).
In the box and whisker plots, the horizontal black lines indicate the medians, the fork represents the average value, boxes represent the interquartile range (IQR), whiskers extend to the minimum/maximum of the data, and circles indicate individual roots. w: weight 5 ≤ w1 < 10 g (n = 17) 10 ≤ w2 < 25 g (n = 25) 25 ≤ w3 ≤ 35 g (n = 9) d: diameter 1.0 ≤ d1 < 1.5 cm (n = 12) 1.5 ≤ d2 < 2.5 cm (n = 28) 2.5 ≤ d3 ≤ 3.5 cm (n = 11) L: Length 0 ≤ L1 < 10 cm (n = 25) 10 ≤ L2 < 15 cm (n = 21) 15 ≤ L3 ≤ 25 cm (n = 5) Tukey–Kramer, **: p < 0.01.
It was revealed that roots with small weight and diameter had low catalpol content. In other words, because thick R. glutinosa roots (1.5 cm or more) are currently used as herbal medicine, our results supported the suitability of the current root selection method. On the other hand, with regard to root length, the roots are usually not uniform and mainly thin string-like that gradually enlarges. For this reason, the lack of correlation between catalpol content and length was expected, and length was not used as a selection criterion.
Next, to investigate the localization of catalpol content within the same individual, each root was divided into seven parts, and catalpol contents in the upper, middle, and lower parts were measured (Fig. 6). Catalpol content was lower in the upper part than the middle and lower parts. In order to conduct a more detailed investigation, measurements of catalpol content by diameter were performed (Fig. 2). A positive correlation of R = 0.941 was noted between catalpol content and diameter at 5% significance level determined by the significance test of the correlation coefficient (Fig. 7). The results in Fig. 7 supported the results in Figs. 5b and 6. Catalpol content in the tissue inside the cambium was higher than that in the tissue outside the cambium (Fig. 8). This would account for the difference in the degree of discoloration between the tissue inside and outside the cambium during the root drying process.
Upper, middle, and lower mean the parts of the root shown in Fig. 1. In the box and whisker plots, the horizontal black lines indicate the medians, the fork represents the average value, boxes represent the interquartile range (IQR), whiskers extend to the minimum/maximum of the data, and circles indicate individual roots. Means ± standard deviation (S.D.) (n = 14), Tukey **: p < 0.01.
Student’s t test (n = 12), **: p < 0.01.
We performed an in situ assay for intracellular β-glucosidase activity in R. glutinosa roots immediately after harvest. The blue spots were insoluble products of X-Glc precipitation in the cells, and indicated the reaction of β-glucosidase (Fig. 9). After only one month, β-glucosidase disappeared in large quantities, only a small amount remained, and there were obvious residues in the cork layer. The rest of the sections have almost disappeared (Figs. 10a, b). β-Glucosidase activity disappeared after two months (Figs. 10c, d). Therefore, it was clarified that the effect of β-glucosidase on catalpol disappeared two months after harvest and natural drying.
Sections after the in situ assay for β-glucosidase activity. Insoluble products of X-Glc precipitation in cells. Red arrows indicate reaction sites that were visible as blue spots. (a) In R. glutinosa fresh roots, β-glucosidase was distributed in all areas of sliced tissue. (b) The xylem was enlarged for observation, and the distribution of β-glucosidase was observed. (c) β-Glucosidase was widely distributed in the cortex and cork layer. (d) β-Glucosidase was clearly visible in the cells. 40 × (a); 100 × (b and c); 400 × (d).
Sections after the in situ assay for β-glucosidase activity. After one month, only a small amount of β-glucosidase in the xylem remained, and almost all disappeared (a). β-Glucosidase residues were detected near the cork layer. (b). After two months, β-glucosidase was no longer detected in all cross sections (c and d). 100 × (a–b and d); 40 × (c).
The optimum drying method that would suppress the decrease in catalpol content was investigated. When the whole roots and the 10 mm slices and 7 mm dices were air-dried, the slices dried quickly and their weights rapidly decreased to 30% of the weight after harvest after one month. In contrast, the whole roots required nine months to achieve the weight decrease to 25% of the weight after harvest. For root slices that were naturally dried, it was better to complete the natural drying process in April or May (Figs. 11, 12). Catalpol content was significantly higher in both 10 mm slices and 7 mm dices than the whole roots. It was clarified that a long drying period was required for whole roots subjected to natural drying, and catalpol content was greatly reduced during that period. In other words, catalpol content was better preserved in sliced roots air-dried for five months than whole roots dried for nine months, and it was better to complete the air-drying process in April or May.
0 month means Nov. 2018, 1 month means Dec. 2018, 2 month means Jan. 2019. (n = 10, Means ± standard error (S.E.)) Dunnett, **: p < 0.01 (vs. 0 month).
Whole: whole root, 10 mm: 10 mm slices, 7 mm: 7 mm dices. Weight was measured every month, and weight change was calculated by setting the weight of roots immediately after harvest at 100%.
R. glutinosa roots were naturally dried immediately after harvest, and catalpol content was analyzed every month for up to 10 months to examine changes in catalpol content. It was clarified that catalpol content changed significantly around eight months. In other words, it was not appropriate to air-dry for more than eight months to maintain root quality. In Japan, the rainy season in July could have resulted in catalpol degradation. In particular, in Hokuriku, both temperature and humidity are high during the rainy season (the rainy season in Hokuriku is around June).15) It was predicted that the decrease in catalpol content, which continued after August, was due to the high average temperatures in July (25.9 °C) and August (28.2 °C)16) (Fig. 13). From the above results, it was clarified that the natural drying period should be shortened to around seven months. Water content was 25% or less nine months after harvest, and we judged that drying was complete at this point.11) In the case of whole roots, our results suggested that natural drying significantly reduced catalpol content before the drying process was completed (Fig. 14).
Residual rate means catalpol content after drying (n = 10). Means ± S.E. Different letters (a, b) indicate significant differences between groups. Bonferroni multiple test. p < 0.05.
We investigated R. glutinosa roots from the perspective of catalpol content and morphological features including weight, diameter, and length, and found a positive correlation between catalpol content and root diameter, namely, catalpol content tended to increase with increasing root diameter. In addition, our in situ assay for β-glucosidase activity contradicted the previous suggestion that β-glucosidase was involved in the decrease in catalpol content. In other words, β-glucosidase activity in the cells disappeared after approximately two months, and catalpol content remained almost unchanged in this period. We found that the decrease of catalpol content was due to the water content, although the detailed mechanisms are unknown. In addition, we clarified that slicing the roots and rapidly removing water by natural drying was best methods to obtain dried root with little loss of catalpol content.
This work was supported by a Grant-in-Aid for Scientific Research (C) No. 18K06730 for 2018 to 2020 from the Japan Society for the Promotion of Science.
The authors declare no conflict of interest.
