Authentication of the 31 species of Toxic and Potent Chinese Materia Medica (T/PCMM) by microscopic technique, part 2: Three species of seed T/PCMM

Toxic and Potent Chinese Materia Medica (T/PCMM) are being used more and more in the treatment of various diseases. In view of their toxic side effects and to ensure their safe use, accurate and reliable authentication is indispensable. However, identifying characteristics of T/PCMM are seldom reported, even though modern microscopy can provide ample, unique identifying characteristics from cells found in transverse sections and powders. In particular, no systematic authentication studies on seed T/PCMM have been conducted. In the course of our study on 31 T/PCMM originating from plants, animals, minerals, and secreta, an accurate and convenient method, based on microscopic techniques, has been developed and reported for the authentication of animal T/PCMM. The present study deals with detailed investigations on three species of seed T/PCMM, namely Semen Hyoscyami (Hyoscyamus niger L.), Semen Euphorbiae (Euphorbia lathyris L.), and Semen Strychni (Strychnos nux-vomica L.). The macroscopic characters are here described in detail, and the microscopic characters were conclusively determined by common and polarized light microscopy. Results showed that these three T/PCMM can be easily identified by the present method even when powdered and combined. Thus, the microscopic method is applicable for authentication of the earlier three T/PCMM, and the morphological and microscopic characteristics described here are proposed as parameters to establish the authenticity of these three T/PCMM.     

Authentication of the 31 species of toxic and potent Chinese materia medica by microscopic technique assisted by ICP-MS analysis, part 4: four kinds of toxic and potent mineral arsenical CMMs

Toxic and Potent Chinese Materia Medica (T/PCMM) is a special and very important category of Chinese medicines. They have long been used in traditional medical practice and are being used more and more widely throughout the world in recent years. As there may be many fatal toxic effects caused by misusing or confusion of T/PCMM, their quality and safety control arouse increasing attention internationally. Researches on the accurate identification to ensure the safe use of T/PCMM are acquired; however, there are few reports on authentication. We are carrying out a series of studies on 31 T/PCMM originating from plants, animals, minerals, and secreta. In our previous studies, we proved that modern microscopic authentication is a simple, fast, effective, low cost, and less toxic method for identifying animal, seed, and flower T/PCMM. In the present study, we focused on the authentication of four kinds of mineral arsenicals, including orpiment (mainly containing As₂S₃), realgar (mainly containing As₄S₄), arsenolite, and arsenic trioxide (mainly containing As₂O₃). We examined the macroscopic and microscopic characteristics of the above minerals and found that they all can be easily identified and authenticated by using light microscopy coupled with polarized microscopy. Moreover, the authentication results for arsenolite and arsenic trioxide are confirmed by ICP‐MS analysis. We are sure that the morphological and microscopic characteristics indicated here are indispensable to establishing standards for these four mineral T/PCMMs. Microsc. Res. Tech. 74:1‐8, 2011. © 2010 Wiley‐Liss, Inc.     

Authentication of the 31 species of toxic and potent Chinese Materia medica (T/PCMM) by microscopic technique, part 1: three kinds of toxic and potent animal CMM

Microscopic authentication is an effective method for quality control of Chinese Materia Medica (CMM) because of its speed, convenience and low cost. However, the application of modern microscopic technique in quality evaluation of Toxic and Potent Chinese Materia Medica (T/PCMM) of animal origin is seldom reported. This gap in published knowledge is increasingly serious because confusion in T/PCMM has led to serious medical problems in China and other countries in recent years. To ensure the safe and effective use of T/PCMM, an accurate and convenient method, based on macroscopic and microscopic techniques, was developed for the authentication of animal T/PCMM. The color microscopic photos of the crude drug were acquired with the light microscope, and from these their morphological and microscopic characters were described. The present method was successfully applied in the analysis of 31 T/PCMM including 17 samples originating from plants, 3 from animals, 9 from minerals, and the remaining 2 from secreta. The macro- and microscopic characters of three animal T/PCMM, namely Mylabris (Mylabris phalerata or Mylabris cichorii), Huechys (Huechys sanguinea), and Lytta (Lytta caraganae) were conclusively determined and are here presented. The results demonstrated that it was feasible to use the present microscopic characters for the authentication of the above three animal T/PCMM.     

Combination of normal light and fluorescence microscopy for authentication of five Lonicera species flower buds

The flower buds of five Lonicera species, Lonicera japonica Thunb., L. macranthoides Hand.-Mazz., L. hypoglauca Miq., L. confusa DC. and L. fulvotomentosa Hsu et S.C. Cheng are confusable and usually utilized under the same name "Jinyinhua" in different areas for morphological similarity. Studies found that these five species possess extreme differences in chemical compounds, correspondingly showing different pharmacological activities and clinical applications. To ensure efficacy and safety of these herbal medicines and prevent unknown adverse effect, in this work, a simple, rapid and effective method combining normal light and fluorescence microscopy was developed for authentication. Surface slides and transverse sections of these buds were investigated to reveal their differences. As a routine technique, normal light microscopy which gives detailed microscopic features such as glandular hairs and nonglandular hairs, can easily distinguish four species except L. confusa. Fluorescence technique, which could present different distribution of fluorescence materials, is further employed to identify three species including L. confusa successfully. It is the first report to identify these five Lonicera species by combining normal light and fluorescence microscopy. This work indicated combining normal light and fluorescence microscopy could be a powerful method in authentication of confused species.     

Identification of powdered Chinese herbal medicines by fluorescence microscopy,Part 1: Fluorescent characteristics of mechanical tissues,conducting tissues,and ergastic substances

The light microscope has been successfully used in identification of Chinese herbal medicines (CHMs) for more than a century. However, positive identification is not always possible. Given the popularity of fluorescence microscopy in bioanalysis, researchers dedicated to finding new ways to identify CHMs more effectively are now turning to fluorescence microscopy for authentication purposes. Some studies on distinguishing confused species from the same genus and on exploring distributions of chemicals in tissues of CHMs by fluorescence microscopy have been reported; however, no systematic investigations on fluorescent characteristics of powdered CHMs have been reported. Here, 46 samples of 16 CHMs were investigated. Specifically, the mechanical tissues including stone cells and fibers, the conducting tissues including three types of vessels, and ergastic substances including crystals of calcium oxalate and secretions, in various powdered CHMs were investigated by both light microscope and fluorescence microscope. The results showed many microscopic features emit fluorescence that makes them easily observed, even against complex backgrounds. Under the fluorescence microscope, different microscopic features from the same powdered CHM or some same features from different powdered CHMs emitted the different fluorescence, making this information very helpful for the authentication of CHMs in powder form. Moreover, secretions with unique chemical profiles from different powdered CHMs showed different fluorescent characteristics. Hence, fluorescence microscopy could be a useful additional method for the authentication of powdered CHMs if the fluorescent characteristics of specific CHMs are known. Microsc. Res. Tech. 2010. © 2010 Wiley‐Liss, Inc.     

Identification of Chinese herbal medicines by fluorescence microscopy: fluorescent characteristics of medicinal bark

Medicinal bark refers to structures outside the vascular cambium of stems, branches and roots of gymnospermous and dicotyledonous plants that are used as medicinal materials; bark is an important type of Chinese herbal medicine. However, identification of the species from which the bark comes can be very difficult, especially when the bark is dried and sliced. In our previous studies, we have found that fluorescence microscopy is a powerful tool for the identification of easily confused Chinese herbal medicines, powdered Chinese herbal medicines and decoction dregs. To establish the fluorescent characteristics by which medicinal barks can be identified, for ensuring their safe and effective use, a systematic microscopic investigation by normal light and fluorescence microscope was carried out on transverse section samples of 11 medicinal barks commonly used in China. Specifically, the fluorescent characteristics of mechanical tissues, including stone cells and fibres as well as secretory tissues, have been observed. The microscopic features of medicinal bark are here systematically and comparatively described and illustrated. Under the fluorescence microscope, various tissues emitted fluorescence of different colours, and we found that both the colours and the intensity can be used to distinguish and identify these barks.     

Application of microscopy in authentication and distinguishing of 11 Paris species in West Sichuan

In the trade and raw drug market, the medicinal plants of genus Paris are available in the form of rhizomes without any vouchers, thus making it difficult to identify and distinguish the different species of Genus Paris. Recent studies have shown that the species of Paris possess different chemical constituents, pharmacological activities, and efficiencies in clinical application. To distinguish 11 species of Paris collected from the western Sichuan province of China and ensure their safety and efficacy, in the present work, the microscopic characteristics of rhizomes and the crude drug powder of the 11 species of Paris were compared using a light microscope according to the usual microscopic techniques. The results of the microscopic features were systematically described and illustrated. The differences among these species are great enough that the identity of most material can be easily determined. Also, semiquantitative and quantitative micrographic parameter tables were simultaneously presented. Further, the key authentication parameters based on these anatomic characteristics analyzed was drawn up and presented for the Paris species studied. The study indicated that light microscopy and related techniques could provide a method that is convenient, feasible, and can be unambiguously applied in the authentication of species of Paris. This information is required not only for the identification procedures that guarantee the utilization of the appropriate raw material, but also for the quality control standards demanded. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

Light microscopy and scanning electron microscopy: Implications for authentication of misidentified herbal drugs

Plant‐based drugs have reached remarkable acceptability as therapeutic remedy for various diseases due to the adverse effects of contemporary medicines. This increasing popularity of herbal drugs leads to a growing herbal market for the development of plant‐based drugs, nutraceuticals, pharmaceuticals and cosmeceuticals. Herbal drug adulteration is a complex problem which currently has undeniable consequences on health and nutrition. Ambiguities in nomenclature, misidentification and resemblance of colour and texture of the crude herbal drugs are the major causes of adulteration. Three different species commercially marketed under the same trade name Halion are Lepidium apetalum, Asparagus officinalis, and Lepidium didymum. The genuine source of Halion is Lepidium apetalum, which is authenticated by using basic and advanced taxonomic techniques. Morphology, anatomy and palynology of the misidentified sources were done using light and scanning electron microscopic techniques for authentication. This study may help to set microscopic techniques as a tool to achieve quality and standardization of the genuine source of the herbal drug. Phytochemical analysis and biological screening is needed for the further establishment of authenticity and quality of herbal drugs.     

Identification of four Aconitum species used as “Caowu” in herbal markets by 3d reconstruction and microstructural comparison

Authentication is the first priority when evaluating the quality of Chinese herbal medicines, particularly highly toxic medicines. The most commonly used authentication methods are morphological identification and microscopic identification. Unfortunately, these methods could not effectively evaluate some herbs with complex interior structures, such as root of Aconitum species with a circular conical shape and an interior structure with successive changes. Defining the part that should be selected as the standard plays an essential role in accurate microscopic identification. In this study, we first present a visual 3D model of Aconitum carmichaeli Debx. constructed obtained from microscopic analysis of serial sections. Based on this model, we concluded that the point of largest root diameter should be used as the standard for comparison and identification. The interior structure at this point is reproducible and its shape and appearance can easily be used to distinguish among species. We also report details of the interior structures of parts not shown in the 3D model, such as stone cells and cortical thickness. To demonstrate the usefulness of the results from the 3D model, we have distinguished the microscopic structures, at their largest segments, of the other three Aconitum species used for local habitat species of Caowu. This work provides the basis for resolution of some debate regarding the microstructural differences among these species. Thus, we conclude that the 3D model composed of serial sections has enabled the selection of a standard cross‐section that will enable the accurate identification of Aconitum species in Chinese medicine. Microsc. Res. Tech. 78:425–432, 2015. © 2015 Wiley Periodicals, Inc.     

Integration of a high‐NA light microscope in a scanning electron microscope

We present an integrated light‐electron microscope in which an inverted high‐NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high‐resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub‐10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum‐compatible immersion oil. For a 40‐nm‐diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry.     

A mixed microscopic method for differentiating seven species of “Bixie”‐related Chinese Materia Medica

Confusion in the species associated with the name of “Bixie” in Chinese Materia Medica began centuries ago. In recent decades, diverse medicinal plants from the genera Dioscorea and Smilax, and even minor species from the genus Heterosmilax, have been documented under the name Bixie or a very similar name as folk medicines in different areas of China. However, the traditional efficacies and chemical profiles of these herbs are not exactly the same and even vary wildly. Comprehensive authentication of multiple Bixie herbs has not yet been attempted. To differentiate and ensure the correct use of these Bixie‐related herbs, in this study, seven sorts of representative Bixie herbs (Dioscorea collettii, D. zingiberensis, D. nipponica, D. septemloba, Smilax china, S. glabra, and Heterosmilax japonica) were characterized based on the microscopic examination of their powders and cryotomed transverse sections. This is not only the first attempt to distinguish Bixie herbs by a comprehensive microscopic techniques, including common light microscopy, fluorescence microscopy, and polarized light microscopy, but also it is the first research to observe characteristics of transections of crude drugs under polarized lighting for the purpose of authentication. Polarized light has been found to provide a number of unique characteristics. The results indicate that starch granules, stone cells, vascular bundles, and other significant tissue features can be used to authenticate “Bixie” herbs. The method was proven to be quick, handy, specific, and simple. It should be widely applicable to other herbal materials. Microsc. Res. Tech. 77:57–70, 2014. © 2013 Wiley Periodicals, Inc.     

Determination of flavonoids in Flos Chrysanthemi and Flos Chrysanthemi Indici by capillary electrophoresis

Capillary zone electrophoresis coupled with diode array detection is reported for determination of the pharmacologically active flavonoids in Flos Chrysanthemi and Flos Chrysanthemi Indici. The analytes, including linarin, luteolin-7-O-glucoside, acacetin, epicatechin, catechin, diosmetin, luteolin, apigenin, and kaemferol were baseline separated within 30?min at 15?kV with a running buffer consisting of 20?mmol/L sodium borate-50?mmol/L sodium phosphate at pH 9.6. The reproducibility, linear dynamic range, limits of quantitation, limits of detection, and recovery were satisfactory. Three well-known Flos Chrysanthemi and three Flos Chrysanthemi Indici varieties from Anhui and Hubei provinces of China were analyzed. In Flos Chrysanthemi, the orders of abundance of the analytes were variable. Boju samples included the highest concentrations of luteolin-7-O-glucoside, luteolin, and apigenin. The main constituents in Flos Chrysanthemi Indici were linarin, luteolin-7-O-glucoside, catechin, and luteolin. The concentrations of analytes varied in the samples, indicating that the ecological environment and germplasm impacted these constituents.     

Authentication of herbal drug Tukhm‐e‐balango (Lallemantia royleana Benth.) using microscopic,pharmacognostic, and phytochemical characterization

The study is aimed to provide a comprehensive account on authentication of herbal drug named as Tukhm‐e‐balango (Lallemantia royleana Benth.) from the seeds of Ocimum basilicum by using microscopic, pharmacognostic, and phytochemical characterization. The crude medicinal plants and their parts are often adulterated or substituted in market due to improper identification by the consumers while among herbal plant sellers, taxonomic confusion is caused due to morphological similarities of the plant parts and lack of a standard identification system.In microscopy, both herbarium and fresh specimens were studied using qualitative and quantitative morphological characteristics of leaves, seeds, and pollen. For pharmacognosy, solubility, fluorescence, and physicochemical characterizers were analyzed whereas a total phenolic and flavonoids contents was determined in addition to DPPH radical scavenging activity. In current study, microscopic, pharmacognostic, and phytochemical characterization clearly differentiated L. royleana from O. basilicum. The major problem in herbal drug industry is caused due to confusion and controversy of certain synonyms used for more than one or two drugs. Sometimes, under the same common or local name, entirely different taxa are being sold in herbal markets. It is concluded that correct and proper identification of medicinal plants is very crucial to ensure the safety and efficacy of herbal medicines, as many medicinal plants are intentionally or unintentionally adulterated with similar species or varieties. In herbal market, the seeds of L. royleana are adulterated with seeds of O. basilicum due to their similar morphology.     

Application of microscopy for discrimination of eight Swertia species utilized in the traditional Chinese medicine “Qingyedan”

Many Swertia species are utilized as a traditional medicine under the name “Qingyedan” in China, but are easily confused with one another. To distinguish eight Swertia species (S. mileensis, S. cincta, S. patens, S. punicea, S. delavayi, S. nervosa, S. macrosperma, and S. yunnanensis) and to ensure their safety and efficacy, the microscopic and macroscopic characteristics of the roots, stems, leaves, and flowers of them were examined. The results showed that microscopic and macroscopic features helpful for authentication of the eight species were the sinuosity of the anticlinal walls of epidermal cells and presence or absence of hairs on the leaf lamina; presence or absence of V‐shaped fibers and fibers with sinuous abaxial wall in the sepals; shape of epidermal cells and pattern of papillae on hairs on the margin of corolla nectary; distribution of stomata in leaf and sepal epidermises, stone cells in cortex and phloem of roots and in cortex and pith of stems, crystals in parenchymatous cells of mesophyll and stem, stomata size, stem diameter, and 4‐ or 5‐merous flowers, and so on. Two keys to the eight Swertia species based on macroscopic and microscopic characteristics are presented. The study indicates that microscopy and related techniques are convenient, practicable, and can be unambiguously applied for authentication of Swertia species. Microsc. Res. Tech. 76:296–310, 2013. © 2013 Wiley Periodicals, Inc.     

Comparative authentication of three “snow lotus” herbs by macroscopic and microscopic features

“Snow lotus” is a famous Chinese Materia Medica derived from species of the genus Saussurea (Compositae). To differentiate three representative easily‐confused snow lotus herbs, namely, Saussurea involucrata (Kar. et Kir.) Sch.‐Bip, Saussurea laniceps Hand.‐Mazz., and Saussurea medusa Maxim., macroscopic features of the three herbs were systemically observed, and microscopic features were compared by using ordinary light microscopy, polarized light microscopy and scanning electron microscopy (SEM). The results indicate that, as for macroscopic identification, capitula situation and arrangement, and as for microscopic identification, pollen grains, nonglandular hairs, glandular hairs, and cells of inner surface of the microdiodange can be used to authenticate the three snow lotus herbs. Comprehensive table comparing the characteristics were presented in this study. SEM has been found to provide a number of unique characteristics of pollen grains. Based on the observation of pollen grains, evolution sequence of the three species was speculated. The present method was proven to be efficient, convenient, simple, and reliable, which was successfully applied to the authentication of three snow lotus herbs. Microsc. Res. Tech.1 77:631–641, 2014. © 2014 Wiley Periodicals, Inc.     

Microscopic investigations and pharmacognostic techniques for the standardization of Caralluma edulis (Edgew.) Benth. ex Hook.f.

Herbal medicines frequently suffer with quality controversies because of similar species or varieties. This often leads to sophistication or admixture of the crude drug as they share various look alike physical features. Commercially, stalks of Caralluma edulis are commonly adulterated with other species because of their analogous morphology. In the present research, the microscopic standardization of the drug was performed by structural, histological, and anatomical features. Pharmacognostical characterization was carried out by physicochemical (moisture content, swelling index, pH value, ash values, and extractive values) and fluorescence analysis. The chemical characterization by UV–Visible, Fourier transform infrared spectroscopy, and high‐performance liquid chromatography was also performed that disclosed the chief phytoconstituents of the herbal drug. The above mentioned constraints, being reported for the first time in C. edulis, are substantial in establishing the microscopic and pharmacopoeial standards for future identification and authentication.     

Characterization of microscope objective lenses from 1,400 to 1,650 nm to evaluate performance for long-wavelength nonlinear microscopy applications

We have demonstrated a simple method for characterization of objective lens performance at longer wavelengths for 3PLSM and THG imaging. We investigated a range of air and oil-immersion objective lenses across a wavelength range of 1,400-1,650 nm using a synchronously pumped optical parametric oscillator laser source. In the first instance, we investigated the percentage light transmission across this spectral range. Second, we used a simple second harmonic autocorrelation pulse measurement technique to study the dispersion properties of these lenses at the range of input wavelengths. For the objective lenses investigated, we observed pulse broadening on the order of around 4%-7% for air immersion lenses and 9%-12% for oil immersion lenses. Even for the greater dispersion incurred by the application of the oil immersion lenses, these objectives are suitable for longer wavelength application in conjunction with a suitable light source. The same techniques could easily be applied for a larger range of objective lenses and adapted for alternative spectral windows and pulse durations.     

Systematic approach to the correct identification of Asplenium dalhousiae (Aspleniaceae) with their medicinal uses

In present study, multiple microscope techniques were used for the systematics identification of the species Asplenium dalhousiae. The plant was collected from different phytogeographical and its natural habitat of Pakistan, where it shows higher diversity. Morphology, foliar epidermal anatomy, and spore morphological characters of the species were studied in detailed using multiple microscopic techniques through light microscopy (LM) and scanning electron microscopy (SEM). LM and SEM were used for the systematics identification of the species. Traditionally, the species is used in the ailment of many diseases, so the spore morphology, anatomical features, and morphological characters are relevant to describe the species taxonomy. The importance of multiple methods of taxonomic study (e.g., documentation and morphological characteristics) for characterizing herbs are important step in systematic certification to maintain the efficacy of herbal medicines. The aim of the present study is to examine the morphological, anatomical, and spore morphology of the species A. dalhousiae in more detailed for the correct taxonomic identification and their medicinal validation from Pakistan.     

显微系统油浸物镜前片粘接用胶的改进途径

本文介绍了自行研制的显微系统油镜物镜前片粘接固定用、环氧树脂/低分子聚酰胺体系光学结构胶的改进途径、配方组成,以及它在显微系统油浸物镜前片、大视场目镜、分划目镜等光学件与金属框座粘接固定方面的应用。     

Authentication of Chinese Materia Medica decoction dregs. Part II: comparison before and after decoction of four Chinese Materia Medica that mainly comprise storage tissue

Authentication of Chinese Materia Medica (CMM) decoction dregs is important for ensuring the efficacy and safety of CMM when they are used in decoction. If someone got worse or poisoned after taking a decoction while the formula is appropriate, the authentication of CMM dregs is the effective method to explore the reasons. Therefore, a systematic study on the authentication of CMM dregs was carried out. In this study, two pairs of easily confused CMM dregs, Fenge (Puerariae Thomsonii Radix) and Shanyao (Dioscoreae Rhizoma), Dihuang (Rehmanniae Radix) and Huangjing (Polygonati Rhizoma), which mostly comprise storage tissues, were investigated by comparing the morphological and microscopic characteristics. Fenge and Shanyao contain abundant starch granules. After decoction their dregs were hard, nonstarchy and horn-like. Fully gelatinized starch granules were found in the powder of Fenge dregs while incompletely gelatinized starch granules were occasionally found in the Shanyao dregs. In contrast, Dihuang and Huangjing contain water-soluble sugars. After decoction, their dregs were hard and brittle. Their parenchymatous cells were shrunken and unknown crystals were found under microscope. Hence, the morphological and microscopic characteristics of CMM before and after decoction were different, and different changes in the CMM dregs can be illustrated by the different nature of their ergastic substance. These differences could be used to authenticate CMM dregs.