2022 Volume 45 Issue 7 Pages 919-925
The microscopic test method (microscopic examination) used to identify crude drugs is a common method in the identification of the original plant source by determining the characteristics from a small sample quantity. However, in recent years, the number of examiners who are familiar with the microscopic examination technique has decreased. In recent years, high-resolution X-ray computed tomography (HRXCT) has been used to visualize the internal structure of plants. HRXCT scans an object using X-rays and enables visualization of the internal structure of the crude drug using a computer. Therefore, in this report, HRXCT was used to easily observe the internal morphology of crude drugs using the Ephedra Herb as an example. The same internal morphological characteristics were observed using both, microscopic examination and HRXCT methods. Image analysis using HRXCT did not require specific techniques, such as sectioning, and the same tissue could be observed from any orientation using a single scan. It afforded remarkable technical simplification and reduction in time to inspect the organization's characteristics. Therefore, image analysis using HRXCT is a useful method for crude drug identifications.
The Japanese Pharmacopoeia describes the analytical and test methods for the identification of crude drugs.1) Methods based on genetic information, chemical analysis, sensory tests, and test methods based on morphological observation are used for the identification of crude drugs. The test methods using a microscope (microscopic examination) are a kind of morphological identification. Accurate characterization or identification can be performed even with powdered crude drugs, irrespective of their storage conditions. This is a common method to identify the source from a small amount of sample. However, there are drawbacks to this technique. For example, flower buds with complicated textures require the technique for sectioning, which is time consuming. It is also difficult to grasp the overall shape of large samples and brittle tissues and it takes a lot of time to prepare slides. In addition, when creating a standard for the identification of powdered crude drugs, it is necessary to give an accurate name to the tissue pieces. For this purpose, it is necessary to observe the tissues of all parts of the original plant or animal, which requires considerable time and skill. Therefore, the number of testers involved in microscopic examinations has been decreasing in recent years.
On the other hand, high-resolution X-ray computed tomography (HRXCT) scans an object with X-rays and displays the internal structure of the object using a computer to calculate the difference in transmittance from the X-rays of the substance. In addition to this being a nondestructive measurement technique, a three-dimensional image can be obtained by reconstruction, and an image from any direction can be displayed.2) As for the X-ray computed tomography equipment used for industrial purposes, equipment that can acquire high-quality data with high accuracy and low noise has been developed. In recent years, attempts have been made to visualize the internal structure of plants.3–7) Therefore, the use of HRXCT for the internal morphology of crude drugs is an attractive method as special techniques such as sample pretreatment and section preparation are not required. In addition, even inexperienced testers can easily observe the internal morphology. Therefore, we used this method to easily distinguish the internal morphology of crude drugs, such as the Ephedra Herb.
The Ephedra Herb (Ephedra sinica or a mixture of E. sinica and E. intermedia.) was stored at National Institute of Health Sciences (NIHS). The cuts of sample numbers 1 and 2 are from the Inner Mongolia, sample numbers 3 and 4 are from Gansu, and sample number 5 is the domestic market product. Sample number 6 is the powder of sample number 3 and is “moderately fine powder”,1) sample number 7 is the powder of the domestic market product and is “fine powder”1).
HRXCT TechniqueA high-resolution three dimensional X-ray microscope (nano3DX, Rigaku Corporation, Tokyo, Japan) was used for these studies. An overview of the measurement chamber is shown in Fig. 1.
The sample was placed close to the detector and rotated at the sample stage during the scanning process.
A high-intensity X-ray generator (1.2 kW), close-up photography with parallel beam, anode target: Cu (8.0 kV), acceleration voltage of 40 kV, tube current of 30 mA, 10× lens, and a scientific complementary metal-oxide semiconductor (sCMOS) camera was used for imaging. The resolution, exposure time, and number of projection images varied under the following conditions.
The following software was used for three-dimensional reconstruction and image analysis: Viewer (nano 3DX measurement software), 3D Viewer (nano 3DX rendered image display software), and ImageJ software.
Microscopic AnalysisAn Axio Scope A1 optical microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a digital microscope VHX-970F (Keyence Corporation, Osaka, Japan) was used as an image-magnifying device. Additionally, Crusher (Retsch MM400, Verder Scientific Co., Ltd., Tokyo, Japan) was used to make-powder.
Sample Preparation and Microscopic ExaminationFrozen sections were prepared using a Retoratome REM-710 (Yamato Kohki Industrial, Saitama, Japan) equipped with Electro freeze MC-802A. The sections were placed on a glass slide, and glycerin water (glycerin: water; 1 : 1) was used as a mounting agent and dropped, as mentioned in the JP18 “Microscopic examination”1), and covered with a coverslip. Microscopic examination was performed using a 2×, 4×, and 10× objective lens, a 10× ocular lens, and a 23-inch monitor. Micrographs were shot at 20× magnification. For powder samples, the powder was moistened with glycerin water, dropped on a glass slide, mounted, and imaged, following the same protocol as the frozen sections.
Under Condition 1, the time required to scan one sample was 4 min. Condition 2 required 8 min for scanning and Condition 3 required 10 min. The two-dimensional projection image of sample number 1 (Figs. 2A1, 2, sample number 1) obtained using the HRXCT technique and the transverse section image after reconstruction are shown in Fig. 2B (Condition 1) and Fig. 2C (Condition 3). Micrographs obtained by the microscopic examination technique are shown in Fig. 2D (sample number 5). Even the data obtained by the 4 min scan (Fig. 2B) was sufficient to grasp the outline of the tissue, however by increasing the resolution and number of images, a more detailed image (Fig. 2C) could be obtained. The use of HRXCT has dramatically reduced the time required to understand the characteristics of the tissue compared with microscopic examination.
A: The state of the measurement sample (A1) and two-dimensional projection image of the portion of the large image that is surrounded by a square (FOV: field of view) (A2). A: Sample number 1. B, C: Transverse CT (computed tomography) images. B: The image was measured at 2.55 µm per pixel (Condition 1). C: The image was measured at 1.28 µm per pixel (Condition 2). B, C: Sample number 1. D: Micrograph of the domestic market Ephedra herb (sample number 5, estimated as E. sinica). Bar = 200 µm. cr: crystal, ep: epidermis, f: fiber, m: medulla, x: xylem.
In the HRXCT image shown in Fig. 2, the epidermis (ep), stoma (sto), fiber (f) of the cortex, xylem (x), crystals (cr), and the pigmented mass in the medulla (m) were observed. These tissues are characteristics of E. sinica8–10) and have a similar form as that which was observed through microscopic examination of the market Ephedra Herb (Fig. 2D, presumed to be E. sinica). It was also revealed that the tissue could be confirmed with all the contents still present using the HRXCT technique. Furthermore, the measured values, such as cell diameter, did not differ significantly between the HRXCT and microscopic examinations (data not shown).
Various HRXCT images of the Ephedra Herb obtained using this measurement (Condition 2, sample numbers 1, 3, 4) are shown in Fig. 3. All of them were different from the morphological characteristics of the general E. sinica plant (outer shape, number of fiber bundles, ratio of cortex to pith, shape of pith, etc.),11–20) therefore the details will be examined again.
These images show different morphological characteristics from the general E. sinica plant. A: Sample number 1. B: Sample number 3. C: Sample number 4. Bar = 200 µm.
A scan with an increased resolution of these samples (Condition 4), as shown in Fig. 4, provided images in which the characteristics of each tissue were visualized clealy. The upper images are transverse images of a young stem (Figs. 4A1–C1, sample number 4) and a stem with well-developed secondary xylem (Figs. 4D1, E1, sample number 2). The green line in these images shows the observed position of the longitudinal HRXCT image. Figures 4A1–C1 and Figs. 4D1–E1 are images of the same sample; however, their positions are different. The lower images (Figs. 4A2–E2) show longitudinal HRXCT images at the green line. The images were observed from the front.
A1–E1: Transverse images. The green lines indicate the observed position of the longitudinal HRXCT image. A1: (sto) shows the position observed of the stomata in longitudinal image. A2–E2: Longitudinal images at the green line position. A2: Tangential image near the surface; therefore, it has the same morphology as the paradermal image. B2: Radial image from the epidermis to the medulla. C2: Tangential image near the primary xylem. D2: Radial image of the secondary xylem (vascular bundle). E2: Tangential image of secondary xylem (vascular bundle). A–C: Sample number 4, young terrestrial stem. D, E: Sample number 2, terrestrial stem with a developed secondary xylem. Condition 4. Bar = 100 µm. cr: crystal, cx: cortex, ep: epidermis, f: fiber, m: medulla, per: perforation, pib: bordered pit, pig: pigment, sto: stoma, ts: spiral tracheid, vp: pitted vessel, vs: spiral vessel, x: xylem.
In the HRXCT image shown in Fig. 4A1, the observed position was set to a position close to the outermost layer of the tissue such that the longitudinal image as in Fig. 4A2 has the same characteristics of the surface view. The stomatal shape was similar to that observed in the microscopic examination. The longitudinal shape of the stomata is shown in Fig. 4B2. A micrograph of the tissues of the Ephedra Herb is shown in Supplementary Fig. 1.
A tracheid was confirmed in the vascular bundle shown in Fig. 4C2. Bordered pit (pib) and spiral thickening (ts) were observed in the tracheid and were confirmed by the HRXCT images. Crystals (Supplementary Fig. 1D), which are difficult to observe under the visible light of the microscope, were observed as white and bright on HRXCT images. The vessels are shown in Figs. 4D2 and E2. Vessels are distinguished from the tracheid as the vessels have perforations (per) at the cell boundaries. Many perforations consist of a series of circular holes, which are characteristic of Ephedra vessels and were confirmed using HRXCT images. Spiral thickening (vs) was noted on the wall of the vessels.21–30)
Analyzing Powdered SamplesNext, we measured the powder fixed to the tip of the sample holder with adhesive tape (Condition 2, Fig. 5, Sample number 6). Figure 5A shows an HRXCT image of the powder viewed from the side as shown in the schematic. Figures 5B1 and B2 are HRXCT images in positions 1 and 2 of Fig. 5A, viewed from the above. Tissue fragments of the epidermis, cortex, vascular bundles, and parenchyma cells of the medulla were confirmed.19,31,32) It was possible to easily confirm the characteristics of the tissue pieces by moving the observation position, even if powders of various sizes were mixed. Some fragments of fiber bundles or vascular bundles consist of several cells, and it may be difficult to focus on and observe the details of the tissue morphology when analyzing these areas (Supplementary Fig. 2), especially at high magnification on the microscope. On the other hand, HRXCT obtains images reconstructed approximately every 1 µm (in the case of Condition 2); therefore, it is expected that the characteristics of tissue fragment morphology will become easier to distinguish than with microscopic examination. A micrograph of the tissue fragments from the Ephedra Herb powder is shown in Supplementary Fig. 2.
A: HRXCT image of the powdered Ephedra Herb viewed from the side as shown in the schematic. B1, 2: HRXCT images of positions 1 and 2 of Fig. 5A, viewed from above as shown in the schematic. Sample number 6. Condition 2. Bar = 200 µm. cr: crystal, cx: cortex, ep: epidermis, f: fiber, m: medulla, p: parenchyma, pig: pigment, sto: stoma, x: xylem.
The reconstructed three-dimensional image can be pseudo-colored. As an example, Fig. 6 shows the colored three-dimensional graphics of Ephedra Herb, which is colored in yellow, red, and blue based on the difference in X-ray transmittance. The cuticle wen (cuw) was visible clearly in the young stem. The outermost epidermal cells and the fiber bundles below them (subepidermal fiber) were colored red. Both tissues have the same red color, however, their positions are different. Also, the fiber outside of the phloem and some vessels were colored red. Crystals scattered in the cortex were colored blue (Fig. 6A, Sample number 1). The secondary xylem was visualized in the developed stem (Fig. 6B, Sample number 2). Colored three-dimensional graphics were effective for easy observation of the internal morphology.
Three-dimensional graphics of whole and cut Ephedra Herb colored with yellow, red, and blue. A: Young terrestrial stem. The cuticle wen is visible clearly. Epidermal cells and fibers are colored red. Some vessels are also colored red. Crystals scattered in the cortex are colored blue. B: Terrestrial stem with a developed secondary xylem. The xylem was visualized. A: Sample number 1. B: Sample number 2. cr: crystal, cuw: cuticle wen,cx: cortex, ep: epidermis, f: fiber, m: medulla, pig: pigment, x: xylem.
We first applied HRXCT to evaluate the internal morphology of crude drugs, and it became clear that there were many advantages. Based on the results obtained in the present study, HRXCT can provide insight into the morphology of crude drugs and has various advantages compared to traditional microscopic examination, as summarized in Table 1.
| X-Ray CT scan | Microscopic examination | |
|---|---|---|
| Sectioning | Non-destructive measurement | Requires sectioning of each tissue being observed |
| No sectioning required | Requires skill for sectioning | |
| Easy to visualize fragile samples | Difficult to section fragile samples | |
| Observation and imaging | Hundreds internal morphology images in a few minutes can be get | Requires micrographs of characteristics of each tissue |
| Images from various directions can be observed with a single scan | Requires micrographs from various directions of tissue | |
| Target tissues are easily observed | Requires multiple sections for observation | |
| Intracellular contents can be visualized without damage | Intracellular contents easily flow out of the cells | |
| Monochrome images | Color images | |
| Visualized by the differences in X-ray transmittance | Tissue color and color of intracellular contents can be observed | |
| Based on the difference in X-ray transmittance can be colored an arbitrary color | Sections can be dyed with various staining solutions | |
| Other features | No special techniques required for observation of internal morphology | Microscopic examination listed in JP18 |
| Applicable to powder samples | Commonly used method for distinguishing powdered crude drugs | |
| Easy to observe different sized powder particles | ||
| Expected to be applied to discrimination of crude drugs |
HRXCT is a nondestructive measurement that does not require sectioning and increases the speed of distinguishing tissue characteristics. The advantages of observing the internal morphology of the crude drugs using HRXCT are presented in this table.
HRXCT is capable of taking nondestructive measurements of samples. Therefore, it is not necessary to prepare the sections, as required for microscopic examination and can be used as a means to quickly observe the morphology for the examiner who is not familiar with the microscopic examination technique. This is a particularly useful technique for fragile tissues and samples that are difficult to section, such as buds. It was also possible to measure the same sample multiple times.
Observation of the internal morphology of crude drugs requires sectioning, observation, and photography in the case of microscopic examination, and a long time is taken to prepare standard micrograph especially. As shown in Fig. 2, the HRXCT technique could be used to obtain an overview of the tissue at a minimum measurement time of 4 min. To increase the resolution, the number of projections acquired was increased and the measurement time was increased. As a result, by measuring 1000 projections, using an exposure time of 20 min, the details of the characteristic structure of the Ephedra Herb could be confirmed (Condition 4; Fig. 4). This condition was suitable for observing the tissue of the Ephedra Herb.
In the microscopic examination, it was necessary to identify the part of the specimen and to analyze several sections in the transverse and longitudinal directions near that part. On the other hand, in HRXCT, it is possible to obtain hundreds of projected images in one measurement as shown in “Conditions 1–4.” Using the three-dimensional images obtained by reconstructing the data, it was possible to observe the morphology of any part, as shown in Figs. 2–4. This makes it possible to select only the target parts that are necessary for discrimination and to analyze the morphology of those parts from various directions. In this measurement, a 10× lens was used; however, we were able to confirm almost the same characteristics as those observed by microscopic examination using a 20× objective lens (Figs. 2–6). This suggests that by using a higher magnification lens in HRXCT, it may be possible to obtain more detailed characteristics of the tissue.
In the microscopic examination, the intracellular contents easily flow out from the cells. HRXCT can be used to observe intracellular contents, such as a pigment mass found in the tissue of the medulla (Fig. 2). This suggests that it may reveal features that were previously unobservable by the microscopy technique.
The image obtained by HRXCT was monochromatic. Therefore, the color and content of the tissue cannot be identified; however, the characteristics of the tissue can be distinguished by the difference in transmittance. In addition, it is possible to create an image such as that shown in Fig. 6, in which it is easy to judge the difference in transmittance visually, using an arbitrary color.
Microscopic examination is the best method for distinguishing powdered crude drugs; however, the particle size of the powder is not always uniform, and it is always necessary to adjust the focus to observe such powder. This is a factor that causes eye fatigue and is not suitable for long working hours. In this study, it was revealed that HRXCT could be applied to powder samples. As samples of different particle sizes can be observed at the same time, it leads to a reduction in fatigue and significantly reduces the time required to understand the characteristics of the tissue.
Because technology has advanced and HRXCT become observe even the submicron region the same internal morphological characteristics could be observed on both methods, microscopic examination and HRXCT. The use of HRXCT is very costly so it is difficult to immediately apply it to the pharmacopoeia examination. In order to use HRXCT in the future in pharmacopoeia examinations, the standard image for discrimination obtained by HRXCT will be required, as with microscopic examination. Visualizing the internal morphology of crude drugs using the HRXCT technique helps provide a large amount of information quickly and is expected to be easy means for the discrimination of crude drugs, even for inexperienced testers. By gathering more information and preparing for the standards, it will be possible to use HRXCT to discriminate crude drugs. We believe that further studying and taking advantage of HRXCT in the future can be a means of morphological observation of crude drugs.
This study was supported by the Japan Agency for Medical Research and Development (AMED).
The authors declare no conflict of interest.
This article contains supplementary materials.
