Identification of “Huoshan shihu” Fengdou: Comparative authentication of the Daodi herb Dendrobium huoshanense and its related species by macroscopic and microscopic features

“Huoshan shihu,” derived from Dendrobium huoshanense, is a well‐known, valuable, and rare Daodi herb. Because of its higher price and scarce resources, its related species D. officinale and D. moniliforme are usually presented as “Huoshan shihu” for sale. Fengdou, the processed form of D. huoshanense, its identification is particularly important. To effectively identify Fengdou of D. huoshanense and protect the Daodi herb, the morphological and microscopic characteristics of the stems of three Dendrobium species were examined. The results showed that macroscopic and microscopic features helpful for identification of the three species were the total lengths and internode numbers of stems, diameter and length of each internode, number of vascular bundles, and presence or absence of silica masses. The key features useful for distinguishing between D. huoshanense and its related species, and also between their Fengdou, were the stem lengths and the trends of change in diameter in different internodes. The results of the study indicated that a combination of macroscopic and microscopic identification techniques might conveniently and effectively be applied for identification of Dendrobium species. “Longtou Fengwei” is the main feature of the Daodi herb “Huoshan shihu,” (D. huoshanense Fengdou) and reflects the wisdom behind the protection of Daodi herbs in ancient times.     

Distinguishing the Chinese materia medica Tiepishihu from similar Dendrobium species of the same genus using histological and microscopic method

The Chinese Materia Medica, Tiepishihu, used as a tonic for over one thousand years, is a well‐known precious medicine in China. According to the Chinese Pharmacopoeia, its source is the species Dendrobium officinale Kimura et Migo, which is distinguished from other species in Dendrobium genus. However, these species from the same genus are similar with Tiepishihu and caused confusion in the market. To find a quick and simple method to distinguish Tiepishihu from other similar species, histologic and microscopic methods were combined together to investigate the transverse section of stem of Tiepishihu and other similar species. Phloroglucinol test solution with hydrochloric acid was used to reveal the lignified tissue by staining the transverse section of Tiepishihu and similar species. Results revealed the unique identification characteristics to distinguish Tiepishihu from similar species, which were difficult to distinguish by other methods. The identification characteristics of Tiepishihu include the cells of vascular bundle sheath were stained red, parenchyma cells were not stained red. What's more, other species can be distinguished from each other with microscopic and histological characteristics. These characteristics proved stable and can be easily observed by normal light microscopic examination. This method is rapid, accurate, stable, and inexpensive.     

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.     

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.     

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.     

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.     

Using microscopic techniques for taxonomic implications of seed and fruits of Delphinium L. (sensu lato) (Ranunculaceae)

In present research, micromorphological characters of the genus Delphinium L. (sensu lato) Ranunculaceae seeds and fruits were studied using microscopic techniques. A total of 37 species were studied using light (LM) and scanning electron microscopy (SEM) to evaluate seeds and fruits micromorphological characteristics for the taxonomic identification and species relationship in the genus. For each taxon, 33 characters were observed and studied. All characters were analyzed using MVSP software and UPGMA method by using dissimilarity index (average taxonomic distance). Considerable differences were observed in trichomes shape of fruit and seed color features. The results revealed that all studied taxa are separated into three clades. The first clade consists of D. pallidiflorum and D. semibarbatum, which have black seed and two types of glandular and crispate trichomes in fruit. The second clade consists of D. speciosum, D. szowitsianum, D. dasystachyson, D. aquilegifolium, D. ursinum, C. trigonelloides, and C. camptocarpa, which had a brown color seeds, two types of glandular and villous trichomes on fruits also have been observed. The remaining species were classified as third clade having various colors including yellow, black, and brown color seeds, the trichome shapes included one of the following forms strigose, villous, trichomes less, glandular, hooked, and crispate trichomes in fruits. The aim of the present study is to use micromorphological characters of seeds and fruits for the taxonomic identification and species delimitation in the genus Delphinium.     

Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) and its taxonomic significance

The Pollen morphology of subfamily Caryophylloideae (Caryophyllaceae) from different phytogeographical region of Pakistan has been evaluated. In this research, 16 species belong to 6 genera of subfamily Caryophylloideae have been studied using light (LM) and scanning electron microscopy (SEM) for both qualitative and quantitative characters. Different palyno‐morphological features were observed including; pollen ornamentation, pore ornamentation, echini arrangement, echinidensity, number of pori, size of polar and equatorial diameter, P/E ratio, exine thickness, and size of pore were studied. The palyno‐morphological characters of subfamily Caryophylloideae have taxonomically significant in identification and delimitation of species. Two pollen types, i.e., subspheroidal (15 species) and prolate (one species Vaccaria hispanica) were observed. Microechinate‐punctate and microechinate‐perforate pollen ornamentation were examined. Two pollen types of pori, i.e., prominent (5 species) and sunken (11 species) ornamentation have been reported. Three types of echini arrangement have been reported irregular, regular and rather regular, while three type of echinidensity (i.e., medium, dense and sparse) were observed. Pori numbers were found different in different species range from 5 in Silene apetala to 19‐35 in Silene vulgaris. Based on different palyno‐morphological characters, taxonomic key was develop for quick and accurate identification. The quantitative data were processed using SPSS software for average, minimum, maximum, and standard error.     

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.     

Microscopic diagnosis of the leaf and stem of Piper solmsianum C.DC

Piper solmsianum C.DC., which is popularly known as pariparoba, is a shrub that measures 1–3 m in height and it inhabits areas with wet tropical soils. The objective of this study was to analyze the leaf and stem anatomy using light microscopy, scanning electron micrographs, and energy‐dispersive X‐ray spectroscopy in order to provide information for species identification. The anatomical profile showed the following main microscopic markers: hypostomatic leaf; hypodermis layer on both sides; pearl glands; biconvex midrib shape; five collateral vascular bundles in open arc with the central bundle larger than the others; circular stem shape; collateral vascular bundles arranged in two rings; sinuous sclerenchymatic sheath in the pith; secretory idioblasts; and starch grains in the mesophyll, in the ground parenchyma of the midrib, petiole, and in the stem; and six morphotypes of calcium oxalate crystals (styloids, cuneiform, tabular crystal rosettes, cuneiform crystal rosettes, elongated square dipyramids, as well as very elongated square dipyramids).     

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.     

Pollen morphological variation of Berberis L. from Pakistan and its systematic importance

Due to overlapping and diverse morphological characters, Berberis is among the most taxonomically complex genera. Palynology is one of the taxonomic tools for delimitation and identification of complex species. In this study, pollens of 10 Berberis species were analyzed through light and scanning electron microscopy. Qualitative as well as quantitative features (pollen shape, size, presence or absence of colpi, colpi length and width, exine thickness, ornamentation, pollen class, aperture, and polar–equatorial ratio) were measured. Five species were observed to have colpate (pantocolpate) with elongated ends, radially symmetrical, isopolar, monads, and psilate‐regulate pollens. In polar view, six pollen were spheroidal, two were ovoid, one spherical, and one oblate. Similarly, variation in pollen length was prominent and the largest pollen on polar view was recorded for B. psodoumbellata 60–65 μm (62.4 ± 0.9), while the smallest one was observed for B. lycium 29–35 μm (32.2 ± 1). The observed variation in both quantitative and qualitative features were important in taxonomic identification. This shows that palynological characters are helpful in identification of Berberis genus at the species level.     

Taxonomic investigation of selected rust fungi using scanning electron microscopy from Khyber Pakhtunkhwa,Pakistan

Rusts comprises the largest natural group of plant pathogens including approximately 8% of all described Fungi. Rust fungi are extremely plant pathogens responsible for great losses to agriculture productivity. Rust species belong to several genera among which more than half are Puccinia species. In Pakistan, rust causes severe damage to agriculture crops. Current study was carried out to identify and characterize different rust species common in the research area through microscopy and Scanning electron microscopy (SEM) in Khyber Pakhtunkhwa, Pakistan. Morpho-anatomical investigation of each collected rust species was carried out using different standard protocols. The dimensions of spores were measured and snapped under a stereomicroscope. SEM was used to examine the shape, size, and ornamentation of the spores of each rust fungus. Results revealed documentation of seven rust fungi, that is, Melampsora euphorbiae, Phragmidium barclayi, Puccinia nepalensis, P. exhausta, P. menthae, Uromyces capitatus, and Uromyces decorates belong to four different genera, were recorded. SEM revealed that spermogonia and Aecia were missing in most of the rust fungus studied. Uredinia was found in a scattered, irregular, lengthy, and epidermis-enclosed form. Urediniospores were found to be ovulating, elongated, echinulate, globose to sub-globose, ellipsoid to ovoid, and globose to sub-globose. Telia was found as sub-epidermal, amphigenous, dispersed, minute, and spherical cells. Teliospores ranged in form from cylindrical to oblong. The germ pores were detected in both apical (top cell) and basal (bottom cell) idiosyncratic and pedicel-attached cells. The techniques used in the current investigation will aid mycologists in rust identification and microscopic characterization.     

Foliar epidermal anatomical characteristics of taxa of Iris subg. Scorpiris Spach (Iridaceae) from Turkey

Iris L. is one of the important genus of family Iridaceae, consist of 56 taxa naturally occurred in Turkey. The similarities and variations in the subgenus overlapping the taxonomic positions of the species in the subgenera and needs anatomical assessment especially by microscopic techniques. In this study, the taxonomic significance of leaf anatomical characters of 10 Iris subgenus Scorpiris taxa were studied in detail and the relationship among these taxa were evaluated using microscopy techniques. Fresh leaf samples of species were fixed in 70% alcohol solution for anatomical observation under microscope. Eleven different micromorphological features were statistically analyzed to delimit the species in subgenus. Based on morphological and anatomical similarities, we studied relationships among; (1) ssp. turcica, ssp. caucasica, I. nezahatiae and I. pseudocaucasica; (2) correlation between ssp. turcica and ssp. caucasica; (3) association of I. galatica, I. persica, ssp. margaretiae and ssp. stenophylla with each other; (4) relationship between ssp. stenophylla and ssp. margaretiae; and (5) relevance between I. aucheri and I. peshmeniana. Moreover, the taxonomy of subgenus Scorpiris has been discussed in detail with novel and diagnostic features based on micromorphological physiognomies. We found that four species in this study are endemic to Turkey, while seven are critically endangered geophytes in the country. The leaf anatomical characteristics of 10 taxa were divided into three groups. Main aim of this research was to study the taxonomy of the complex subgenus Scorpiris through microscopic techniques.     

Morphological characterization of Hypnaceae (Bryopsida,Hypnales): Investigating four genera from Western Himalayas by using LM and SEM techniques

The Hypnaceae is one of the largest and diversified family among the pleurocarpous mosses which consists of nearly 60 genera and 1,000 species around the world. In Pakistan, it is represented by 15 species and 8 genera. The current research article provides the detail morphological investigation of four different species (genera) of the family Hypnaceae that is, Gollania clarescens, Hypnum revolutum, Homomallium andoi, and Taxiphyllum taxirameum in the Western Himalayas, Pakistan. The research article reports the first SEM study of the leaf surfaces of the studied taxa, along with comprehensive morphological characters of the four species. The main objective of the research project is to present the comparative light and scanning electron microscopic study to discuss the morphology in detail because previously the family is just reported in different bryophyte checklists of the Western Himalayas. Based on results, morphological characters, micromorphological observations, qualitative and quantitative attributes, taxonomic keys for the studied taxa, distribution, and habitat are described. Stereoscope was used to analyze different qualitative characters, and light microscope was used to observe and measure the laminal and alar cells of the leaf. The SEM study reveals many important surface features like cell shape and cell wall. The laminal cells were mostly linear and elongated with thin cell walls. The specimens used for the SEM were air dried, so the laminal cells were somewhat constricted and concaved. The current study project will help to make the contribution in the taxonomy and morphology of this family.     

Scanning electron microscopy as a tool for authentication of oil yielding seed

Now‐a‐days, plant species are consumed globally for various purposes and this increasing demand leads to adulteration due to gradually exploitation in natural resources. The major causes of adulteration may be confusion in nomenclature, unawareness of authentic sources, unavailability of authentic sources, color resemblances, deficiencies in collection procedures, and misidentification. This study aims to use the microscopic techniques such as scanning electron microscopy for the authentication of the oil yielding seeds of four important and traditionally used species Prunus persica, Prunus domestica, and Eruca sativa and Argemone Mexicana from their adulterants. All of these are versatile in usage. Locally, these four plants are adulterated badly and there is need to provide a criteria and a complete monograph for correct identification. This research may prove to be helpful for quality control and as well for future studies to explore other novel aspects of these plants.     

Foliar epidermal anatomy of some selected wild edible fruits of Pakistan using light microscopy and scanning electron microscopy

The present study is focused on the detailed foliar epidermal anatomy of some selected wild edible fruits (WEFs) from Pakistan using light microscopy (LM) and scanning electron microscopy (SEM). The studied species are Ficus racemosa L., Solanum nigrum L., Capparis spinosa L., Physalis divaricata D.Don, Rosa moschata Herrm. and Ribes orientale Desf. collected from various localities of Pakistan. The objective of the present study is to investigate qualitative and quantitative anatomical characters for the identification and differentiation of collected wild edible fruits. The characters studied are shape and size of epidermal cells, anticlinal wall pattern, trichome type and shape, average number of stomata, length and width of stomata and pore. The detailed microscopic investigation and variations in the characters recorded have a key role in the determination and authentication of wild edible fruits. This study possesses great potential for plant taxonomists to further evaluate the species at molecular and genetic levels.     

Identification of Daqingye and Banlangen including crude drugs and decoction dregs from three plant species by normal light and fluorescence microscopy

Daqingye and Banlangen are commonly used Chinese medicinal materials derived from the leaves and roots of Isatis indigotica Fort., respectively, which clinical effects have been confirmed by many studies in recent years. However, many problems have arisen concerning the quality and identity of materials sold in the market under these two names. Thus, the identification of Daqingye and Banlangen has drawn public attention. In this work, transverse sections of Daqingye and Banlangen from I. indigotica Fort. and two easily confused species, namely Baphicacanthus cusia (Nees) Bremek. and Clerodendrum cyrtophyllum Turcz., were investigated with normal light and fluorescence microscopy. The distinguishing features were 7–9 vascular bundles, cystoliths and nonglandular hairs in the leaves of I. indigotica, B. cusia, and C. cyrtophyllum, respectively. The Banlangen could be distinguished according to the characteristics of parenchymous cells, cystoliths, and stone cells. Moreover, the fluorescence features of Daqingye and Banlangen investigated in this study can provide direct points for differentiating those samples. Importantly, whether the crude drugs were decocted could be easily identified by their different fluorescence features, which can ensure their quality in clinical application. This is the first report to distinguish the three species that are commonly found in the market sold as Daqingye and Banlangen by normal light and fluorescence microscopy. This work indicates that the combination of normal light and fluorescence microscopy could be powerful, convenient, and economical for authenticating Daqingye and Banlangen from the three species, including crude drugs and decoction dregs. Microsc. Res. Tech. 76:774–782, 2013. © 2013 Wiley Periodicals, Inc.     

Systematics study through scanning electron microscopy; a tool for the authentication of herbal drug Mentha suaveolens Ehrh

In all over the world, herbal drugs are usually adulterated with similar species or varieties due to incorrect identification. Most of herbal products devoid purity and quality, therefore an attempt was carried out to identify plant species and authenticate its herbal drug products from Mentha suaveolens. Microscopy tools provide an excellent platform to identify plants at species level. In this study, microscopic and pharmacokinetic parameters of M. suaveolens were observed. Plant species were collected from high diverse areas of Northern Pakistan. Macro and micro‐morphology including palynology and anatomical features were analyzed to study M. suaveolens. Species characteristics were studied, while implementing microscopic techniques for the delimitation and identification of the species. Traditionally Mentha species are used to cure several diseases that is, digestive disorders, respiratory disorders. Micromorphology (stem, leaves, flowers structure, length etc.), palynology (shape, size of pollen etc.), and anatomical characters (types of stomata, epidermal cell shape, and trichomes) were studied. Micromorphology and anatomical characters were of great interest and significance to discuss the taxonomy of the species. Taxonomic characters were studied to characterize and authenticate the species. The aim of the present study is to observe in detail the taxonomic identification of the species in term of morphology, palynology, and foliar epidermal anatomy for the correct identification along with their medicinal uses in the area.