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.     

Distinguishing Foeniculum vulgare fruit from two adulterants by combination of microscopy and GC–MS analysis

Foeniculum vulgare fruit (FVF) is a widely used traditional medicine. However, two adulterants, namely Anethum graveolens fruit (AGF) and Cuminum cyminum fruit (CCF), have been found in use as FVF in China owing to similar appearance and odor. For the purpose of accurate differentiation of the three herbal medicines, extensive anatomical examination and chemical profiling were conducted. Using light microscopy and fluorescence microscopy, the macroscopic and microscopic features of the three species were compared. It was found that some microscopic characteristics, including transverse shape of mericarp, presence or absence of reticulate cells and non‐glandular hairs, as well as fluorescence of endocarp, were of diagnostic significance. Moreover, essential oils were qualitatively and semi‐quantitatively analyzed using GC–MS. The analytical results indicated significant chemical variations in different species: in FVF, trans‐anethole (83.20%) was the predominant volatile compound followed by estragole (5.03%) and limonene (3.45%), while in AGF, the first, second and third compounds with highest content were carvone (42.58%), apiol (20.76%) and limonene (20.32%), and in CCF were cuminlaldehyde (36.00%), 2‐caren‐10‐al (23.25%) and γ‐terpinene (9.65%), respectively. In conclusion, the proposed light microscopy coupled with fluorescence microscopy and/or GC–MS analysis allowed successful distinguishing FVF from AGF and CCF. Microsc. Res. Tech. 78:633–641, 2015. © 2015 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.     

Quality assurance and authentication of herbal drug (Argyrolobium roseum and Viola stocksii) through comparative light and scanning electron microscopy

The quality assurance and authentication of crude herbal drugs play important role in the effective therapeutic effect of herbal drug and their products. There are many reported problems in quality assurance of herbal crude drugs concerning to their correct identification. The present study was designed with the aim to document the authentication and quality assurance of the herbal crude drugs (Argyrolobium roseum and Viola stocksii) thorough light microscopy (LM) and scanning electron microscopy (SEM). The detailed foliar anatomical studies showed polygonal epidermal cells having anticlinal walls in Argyrolobium roseum while rounded epidermal cells were observed in Viola stocksii. The anomocytic stomata type was observed in Argrolobium roseum while actinocytic was noticed in Viola stocksii. The pollen of studied species appeared as tricolporate showing reticulate exine sculpturing in Argrolobium roseum while fine perforations were recorded in Viola stocksii. Furthermore, quantitative analysis of phenols, flavonoids, and antioxidant activity showed high flavonoid and phenol content in Argyrolobium roseum as compared with Viola stocksii. It was observed that Argyrolobium roseum was discriminated from the Viola stocksii based on the leaf and pollen micromorphological traits by using LM and SEM techniques. It was concluded that LM and SEM techniques were found useful for the quality assurance of botanicals and their authentication.     

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.     

Comparative pollen and foliar micromorphological studies using light microscopy and scanning electron microscopy of some selected species of Lamiaceae from Alpine Zone of Deosai Plateau,Western Himalayas

The study was conducted to highlight a detailed account of morphology of pollen chosen species of Lamiaceae through scanning electron microscopy, and the anatomical characteristics of leaf epidermis of seven species using simple light microscopy. In results, Anisomeles indica and Otostegia aucheri belong to subfamily Lamioideae because it has tricolpate pollen while the rest eight species belong to subfamily Nepetoideae (hexacolpate pollen). The exine sculpturing of pollen of studied species was found to be reticulate. In the family Lamiaceae, four kinds of stomata were found anomocytic, anisocytic, diacytic, and actinocytic, respectively. The cell wall patterns of epidermal cells were irregular or polygonal with straight or undulate walls. It was noted that the variety of the epidermal trichomes seems of taxonomically important for the identification of species of Lamiaceae. Both nonglandular and glandular trichomes were analyzed. The nonglandular trichomes were characterized with long, thin, and pointed apical unicellular cells. The nonglandular trichomes were A‐shaped in Thymus linearis. In Perovskia abrotanoides, stellate glandular trichomes were observed whereas in A. indica and Mentha royleana both glandular and nonglandular trichomes were found. In A. indica, the nonglandular trichomes were sessile and peltate in M. royleana. For the first time in this study, pollen and foliar micromorphological features of selected species of this area are carried out. These taxonomic characters were found to be important in discrimination of species from each other. In future, the detailed study with comprehensive morphology coupled with other important characters is required for delimitation of taxa at various levels.     

Identification of monocot flora using pollen features through scanning electron microscopy

Pollen used to track structural and functional evolution in plants as well as to investigate the problems relative to plant classification. Pollen characters including ornamentation, shape, apertural pattern, pollen symmetry, colpus length, width, and margins used to detect the similarities and dissimilarities between genera and also species of the same genus. In this study pollen features of 20 monocot species belonging to 15 genera of the Amaryllidaceae, Asparagaceae, Iridaceae, Ixioliriaceae, Liliaceae, and Xanthorrhoeaceae were studied using scanning electron microscopy (SEM) and light microscopy (LM). In this study two species that is Zephyranthes citrina and Tulbaghia violacea were reported for the first time from Pakistan. Pollen grains were visualized with LM. Non‐acetolyzed and acetolyzed pollen were examined using SEM. A taxonomic key was developed to highlight the variation in pollen features in order to make their systematic application for correct species identification.     

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.     

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.     

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.     

Morphological and microscopic identification of three major medicinal Dendrobium species in Ta‐pieh Mountains area

Dendrobium is an important medicinal material in China. It has the effect of nourishing the stomach, nourishing yin and clearing heat. In China, there are many types of Dendrobium, and different Dendrobium species have different efficacy. The present study is aimed at distinguishing three major Dendrobium species from morphological and microscopic identification in Ta‐pieh mountains area. In this article, the roots, stems and leaves of Dendrobium huoshanense, Dendrobium officinale, and Dendrobium moniliforme are used as materials to compare the differences of tissues of these three Dendrobium species by morphological indexes and microscopic identification of different Dendrobium. The stem morphology of these three Dendrobium species was significantly different except for stem internodes number and the middle part of the stem diameter by measuring the stems of Dendrobium. To ensure the safe use of Dendrobium, we built a fast and convenient method combining normal and fluorescence microscopy was applied in the present study to distinguish D. huoshanense, D. officinale, and D. moniliforme. The microscopic results show that different types of Dendrobium exhibit different states that can be distinguished under normal light normal and fluorescence microscopy. This comparative study of morphology and microscopy contributes to the development of identification and quality evaluation of Dendrobium.     

Fluorescence discrimination in a reflecting environment

A method to discriminate fluorescence from reflected or scattered light in epifluorescence microscopy is described. The method is based on the fact that the intensity of fluorescence measured with a long pass (LP) filter placed along the emerging beam (i.e. as a barrier filter) is higher than that measured with the same filter placed ahead of the objective (i.e. along the incident beam). On the other hand, when specific precautions are taken, the intensity of reflected or scattered light is identical to within a small difference. Within the margins of error of this small difference, it can be concluded that when the light intensity I measured with an LP filter along the emerging beam is higher than the intensity I' measured with the same filter along the incident beam, a fluorescent emission is present and the fraction of fluorescent light in the emerging beam is at least (I — I')/I.     

Morphological,microscopic, and physicochemical studies of Diospyros montana

Adulteration is the root cause of producing not only a chemically and pharmacologically inferior but also in some instances hazardous or poisonous drug. Despite availability of several techniques, microscopy and physicochemical analyses are the most practical approaches for crude drug authentication. Hence, the present study aimed to evaluate morphological, microscopic, and physicochemical properties of root, bark, leaf, and fruit of Diospyros montana Morphological properties were determined by sensory organs, whereas microscopic features of cross‐sections and powders were determined by light and scanning electron microscopy. The proximate and fluorescence analyses were performed using the standard guidelines. The physical examination of fresh, shade‐dried, and powdered material showed no significant change in color. The identifying cellular structures included cuboidal cork, pitted tracheids, scalariform, reticulate and spiral xylary vessels, and rosettes, raphide, and cuboidal calcium oxalate crystals. The stomatal number, stomatal index, vein‐islet and vein‐termination number, and palisade ratio in the leaf were found to be 293.91 ± 32.68 mm^?2, 64.18 ± 3.42%, 22.00 ± 3.81 mm^?2 and 38.40 ± 5.81 mm^?2, and 3.85 ± 0.60, respectively. Total ash, acid insoluble ash, water soluble ash and sulfated ash of leaf (9.00 ± 0.50%, 1.67 ± 0.23%, 2.00 ± 0.22% and 14.50 ± 0.99%, respectively), foaming index of bark and root (111.11 ± 2.11), and swelling index of fruit (19.00 ± 3.45) were higher than the other parts. The powder of different parts showed characteristic colors in the daylight and UV light upon treatment with various regents. The plant was found to be rich in saponins, fibers, and flavonoids. The results of the present study may serve as identifiers of different parts of Diospyros montana.     

Comparative morphology of the leaf epidermis in Lobelia (Lobelioideae) from China

The leaf epidermal morphology of 38 samples, representing 22 Lobelia species from China, was studied using light microscopy and scanning electron microscopy. The stomata and other epidermal features were largely consistent within species, and therefore represented good characters for taxonomic purposes. Diagnostic characters of the leaf epidermis were used to differentiate species of Lobelia. The species pairs L. clavata and L. pyramidalis, L. alsinoides and L. terminalis, and L. davidii and L. erectiuscula, were differentiated using leaf epidermal characters. Results at the micro‐structural level partly supported the current subdivisions of Lobelia. Previous taxonomic revisions of Lobelia in China were evaluated based on species epidermal characteristics. The results supported the recognition of Pratia merged with Lobelia, and of L. chevalieri as a distinct species. Lobelia brevisepala should not be included in L. montana. The status of Lobelia in the evolutionary history of Campanulaceae was assessed. The Lobelia species investigated retained many primitive leaf epidermal features.     

A prism combination for near isotropic fluorescence excitation by total internal reflection

Total internal reflection fluorescence (TIRF) microscopy is finding increasing application for selectively detecting molecules at or near a glass–water surface. As with all fluorescence methods, the efficiency of excitation of a fluorophore is potentially sensitive to the polarization state of the source. In TIRF, s‐polarized excitation produces an evanescent field that is perpendicular to the incident plane (y direction), whereas p‐polarized light generates a more complex pattern but one dominated by a field that is vertical to the surface (z direction). Thus, fluorophores whose absorption dipoles are fixed in the x direction are not favourably aligned for excitation. Here we describe a beam‐splitting prism arrangement that allows excitation by two orthogonal beams, thus giving isotropic excitation in the x–y plane with s‐polarized light. With linearly polarized light at the magic angle, near isotropic excitation in three dimensions should be achieved. This prism design should find application in polarized fluorescence microscopy to investigate the rotational motions of macromolecules or to minimize flickering of fluorescence emission arising from molecular rotations in single molecule studies.     

Multimodal and non‐linear optical microscopy applications in reproductive biology

A plethora of optical techniques is currently available to obtain non‐destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four‐dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567–582, 2016. © 2016 Wiley Periodicals, 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.     

Pollen morphology and its taxonomic potential in some selected taxa of Caesalpiniaceae observed under light microscopy and scanning electron microscopy

Pollen micromorphology is not only used to check the functional and structural evolution in plants but also to solve the taxonomic problem related to the classification of plants. Therefore, keeping in view the significance of pollen traits, selected taxa of the subfamily Caesalpiniaceae was collected from different geographical regions of Pakistan. The species were then analyzed under both light microscopy and scanning electron microscopy techniques to investigate the importance of micromorphological characters of pollen in the identification and classification of species. Great variation was recorded in equatorial shape, surface ornamentation, tectum, polar diameter, equatorial diameter, and exine thickness. However, little variation was observed in pollen type, polar shape, and fertility of pollen. The equatorial shape of five types was observed: prolate, prolate-spheroidal, spheroidal-subprolate, subspheroidal-prolate, and subspheroidal. Four types of surface ornamentation, psilate, granulate, clavate, and perforate, were recorded. Tectum of five types, intactate, reticulate regulate, medium reticulate, tactate, and striate, was observed. Sexine was thicker than nexine in all studied species. The largest polar diameter was observed in Caesalpinia pulcherrima 64.1 μm while the smallest in Parkinsonia aculeata 26.1 μm. The largest equatorial diameter was found in C. pulcherrima of 70.25 μm whereas the smallest in P. aculeata 27.57 μm. All the pollens analyzed were tricolporate. All studied species have a fertility ratio of more than 90%. A taxonomic key was developed to show the variation in pollen features and delimit species for the correct identification. In conclusion, the pollen traits were found useful to define species boundaries at various taxonomic ranks and will strengthen the taxonomy of this subfamily. Besides, this study also explored the palynological traits and their implication in the taxonomy of the subfamily Caesalpiniaceae.