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.     

Significance of leaf morphoanatomical markers for the authentication of adultered drugs sold in herbal markets of district Lahore,Pakistan

A number of herbal plants sold in herbal markets of Lahore are under adulteration threat which can pose harmful health effects to end‐user. This adulteration problem of medicinal plants can be resolved by the implication of some valuable taxonomic parameter such as leaf epidermal anatomical characteristics. Hence, this research was aimed to provide viable anatomical markers in order to resolve this adulteration issue persisted in some common marketed medicinal plants of district Lahore, that is, Cinnamomum verum Presl., Cinnamomum tamala (Buuch.‐Ham.) T.Nees&Eberm., Gymnema sylvestre (Retz.) R.Br.ex Sm., Sphaeranthus indicus Linn., Artemisia maritima Linn., Achillea millifolim L., Adhatoda vasica Nees, Butea monosperma (Lam.) Taub, and Morus nigra L. Overall multiple anatomical variations (epidermal cell shape, their length and width, type of stomata, length and width of guard cells along with presence or absence of trichomes) had been reported in the study that could be worthwhile for the correct identification of medicinal plants. Irregular shapes of epidermal cells were observed in Cinnamomum verum and Achillea millifolium while pentagonal and polygonal cells were found in their adultaerants, that is, Canella winterana and Adhatoda vasica, respectively. Types of stomata were also strikingly varied among genuine plant and its adulterant, for example, anisocytic stomata were observed in Artemisia maritima while in its adulterant (Artemisia absinthium) anomocytic stomata were found. Similarly, paracytic stomata were observed in Butea monosperma, whereas its adulterant plant (Averrhoa carambola) characteristically possessed anisocytic stomata. Hence, anatomical characteristics were proved to be a valuable taxonomic tool in resolving the adulteration issue of medicinal plants.     

Light and scanning electron microscopy-based foliar morpho-anatomical comparison of selected family Rosaceae members distributed in District Lahore,Punjab, Pakistan

In the present study morpho-anatomical characterization of selected Rosaceae members distributed in District Lahore was performed. Light and scanning electron microscopy was used for systematic characterization of the selected 19 species. Distinguished morpho-anatomical features such as size and shape of epidermal cells, size and type of stomatal cells, size and shape of trichomes, oil droplets, and silica bodies were contrasted. Results reported remarkable variations which could be taxonomically useful in identification of these members. Polygonal epidermal cells were observed in Eriyobotraya japonica, Potentilla bifurca, Potentilla supina, and Prunus amygdalus. However, Prunus cerasus possessed irregular-shaped epidermal cells that can be distinguished from hexagonal epidermal cells of Prunus persica. Similarly, stomatal type varied among some members. Paracytic or perisocytic stomata were observed in E. japonica whereas P. bifurca observed paracytic and anisocytic stomata. Lengths of guard cells were also of variable sizes. The average length of guard cells ranged from 53 (52–54) μm to 74 (73–75) μm in abaxial view. Potentilla supina had biggest, while Rosmarinus officinalis had tiny guard cells. Trichomes were tubular, stellate, cylindrical, ribbon-like, glandular, and nonglandular. Silica bodies in the present investigation were bilobed, rounded, and oval-shaped. It is inferred that diverse anatomical features proved to be valuable taxonomic tools that could be fruitfully helpful in identification of plants at specific as well as generic level.     

Micro‐morphological,environmental and phytochemical investigation of herbal medicines by using LM and SEM: A quality control tool

The use of quality control tool for authentication of Jadwar (Delphinium denudatum Wall. ex Hook.f. & Thomson), a folk herbal drug used for the treatment of different ailments, was studied. People face problems of adulteration for this drug at global, regional, national and local levels. Two different plant species are commercially marketed in the Indo‐Pak Subcontinent under the same trade name of Jadwar. One is D. denudatum Wall. ex Hook.f. & Thomson and the other is Aconitum heterophyllum Wall. ex Royle. To focus on this problem, a marketable available drug sample of Jadwar was authenticated by using basic microscopy tools (LM) and advanced chemo‐taxonomic markers. Authentication, quality and standardization of this drug was achieved using morphology, organoleptography, UV and IR analyses, scanning electron microscopy of pollen and anatomical investigations. The techniques used for authentication marked the clear difference between the studied plants. Microscopic studies, chemotaxonomic investigation and other techniques used in this project provided the basis for the authentication of this species.     

Microscopic and phytochemical techniques as a tool for authentication of herbal drug chiraita: Swertia cordata (G. Don) C.B. Clarke

Swertia cordata (G. Don) C.B. Clarke is one of the potential medicinal plants extensively used in eastern traditional medicine such as Unani, Ayurveda, Siddha, and in traditional Tibetan and Chinese medicine. S. paniculata is the common adulterant of S. cordata at herbal shops and markets but S. paniculata is also used in number of herbal formulations. The present study was conducted to use microscopic, pharmacognostic, and phytochemical techniques as a tool for the authentication of herbal drug chiraita (S. cordata). In herbal markets, mixing, adulteration, and use of spurious materials as substitute have become a major concern for herbal practitioners, local user, and industry for reasons of safety and efficacy. Therefore, authentication of medicinal plants is of utmost importance at each level of drug research. In the present study, anatomical features of two species showed a great diversity, as irregular epidermal cells and nonglandular, unicellular trichomes were found in S. cordata while in S. paniculata epidermal cells were hexagonal in shape and trichomes were A‐shaped. Antioxidant activity of two species showed a great variation where IC₅₀ value recorded for S. cordata was 208 μg/mL, while for S. paniculata IC₅₀ was 624 μg/mL. The study can serve as an important source of information to achieve the authenticity and to evaluate the quality and purity of the plant material in accordance to WHO guidelines. As this species is greatly exploited, so conservation is highly recommended.     

Scanning Electron Microscopy (SEM) and Light Microscopy (LM)‐based Palyno‐morphological views of Solanaceae in Western Himalaya

In this study, plants belonging to family Solanaceae growing in Western Himalaya region have been observed palynologically under Light Microscope and Scanning electron microscope. Present investigation comprises of 10 genera and 23 species, namely, Atropa acuminata, Capsicum decoraticus, Capsicum frutescens, Cestrum aurantiacum, Cestrum diurnum, Cestrum nocturnum, Datura alba, Datura innoxia, Datura stramonium, Hyoscymus niger, Lycopersicon esculentum, Nicotiana rustica, Nicotiana tabacum, Petunia alba, Petunia hybrida, Solanum erianthum, Solanum melongena, Solanum miniatum, Solanum pseudocapsicum, Solanum surratense, Solanum tuberosum, Withania coagulans, Withania somnifera. Solanaceae is a eurypalynous family. Grains are usually Tricolporate and Tetracolporate, radially symmetrical, isopolar, prolate‐spheroidal to oblate‐spheroidal to oblate‐spheroidal to subprolate to per prolate or suboblate to oblate, size range: 8.55–72 μm, amb circular, semi‐angular or subangular, aperture drop‐type, labrum common‐type, exine usually 2 μm thick, nexine 1–1.5 μm thick. Tectum usually psilate, sexine reticulate, granulate or striato‐reticulate, with obscure pattern, sexine 1–2 μm thick, nexine 1–1.5 μm thick, and intine 0.5–1 μm thick. Most striking variation has been found in the shape class, aperture‐type, and tectal surface. Based on these characters, taxonomic keys have been made for correct identification of members in Solanaceae. However, the grains of this family are usually tricolporate and have direct relationship with certain members of the family Scrophulariaceae. Palyno‐morphological characters of family Solanaceae have been studied for the first time in Western Himalayan region of Pakistan. These palyno‐morphological characters are significant for identification of the members of family Solanaceae.     

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.     

Micromorphological evaluation of Daphne mucronata Royle and Myrtus communis L. using scanning electron microscopic techniques

The present study was undertaken to establish micromorphological standards of Daphne mucronata Royle and Myrtus communis (L.). Daphne mucronata Royle is a xerophytic perennial shrub while, Myrtus communis is an evergreen shrub or small tree. Micromorphological investigation is one of the cheapest methods. It will be helpful in the accurate identification of these plants from other species. Results of macroscopic evaluation showed various organoleptic features. Anatomy of the selected plant parts was carried out. It showed a typical dicot structure as it revealed the presence of epidermal cells, crystals, trichomes, cuticle, mesophyll tissues, xylem, phloem, paracytic, and dicytic stomata. Anatomical study facilitates the detection of adulteration with inferior commercial varieties, exhausted drugs, cheaper natural substances, and chemicals. Stomatal number, types, and size of stomata, and palisade ratio of Myrtus communis were observed. These observations will be helpful in the preparation of taxonomic key for the identification of selected species. Powder drug study [scanning electron microscopy (SEM)] showed characteristics histological differentiation. The methods cited here can be measured as characteristics to identify and validate these drugs.     

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.     

Plasmodium species aware based quantification of malaria parasitemia in light microscopy thin blood smear

Malaria is a serious worldwide disease, caused by a bite of a female Anopheles mosquito. The parasite transferred into complex life round in which it is grown and reproduces into the human body. The detection and recognition of Plasmodium species are possible and efficient through a process called staining (Giemsa). The staining process slightly colorizes the red blood cells (RBCs) but highlights Plasmodium parasites, white blood cells and artifacts. Giemsa stains nuclei, chromatin in blue tone and RBCs in pink color. It has been reported in numerous studies that manual microscopy is not a trustworthy screening technique when performed by nonexperts. Malaria parasites host in RBCs when it enters the bloodstream. This paper presents segmentation of Plasmodium parasite from the thin blood smear points on region growing and dynamic convolution based filtering algorithm. After segmentation, malaria parasite classified into four Plasmodium species: Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, and Plasmodium malaria. The random forest and K‐nearest neighbor are used for classification base on local binary pattern and hue saturation value features. The sensitivity for malaria parasitemia (MP) is 96.75% on training and testing of the proposed approach while specificity is 94.59%. Beside these, the comparisons of the two features are added to the proposed work for classification having sensitivity is 83.60% while having specificity is 94.90% through random forest classifier based on local binary pattern feature.     

Pollen morphological investigations of family Cactaceae and its taxonomic implication by light microscopy and scanning electron microscopy

The family Cactaceae is the diversified group of angiosperm plants whose pollen statistics has been used for taxonomic identification. In this article, we present the pollen morphology of eight species belong to seven taxonomically complex genera of Cactaceae including Astrophytum, Cylindropuntia, Echinocereus, Echinopsis, Mammillaria, Opuntia, and Thelocactus using light and scanning electron microscopy. The pollen grains were acetolyzed, measured, described, and electron photomicrographs were taken. Cactaceae can be characterized by presenting different palynomorphological features including pollen type, sculpturing, polar and equatorial diameter, aperture orientation, exine thickness, P/E ratio, and echini features. Four types of pollen shapes, that is, prolate spheroidal (three species), subprolate (two species), prolate (two species), and oblate spheroidal in Echinocereus reichenbachii were observed. The polar and equatorial diameter observed maximum in O. ficus indica 116.95 and 112.27 μm while minimum in M. compressa 38.42 and 21.05 μm. Pollen of two types, tricolpate in members of subfamily Cactioideae and pantoporate in the Opuntioideae were examined. The fertility percentage has been observed maximum in Opuntia macrocentra (83.84%) and minimum in Opuntia ficus‐indica (57.89%). Exine sculpturing showing great variations such as granulate, reticulate, granulate perforate and micro‐echinate foveolate ornamentation was examined only in Echinopsis eyriesii. A key to species, based on pollen micromorphological attributes, has been constructed for correct identification of complex cactus species.     

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Microscopic investigation of palyno‐morphological features of melliferous flora of Lakki Marwat district,Khyber Pakhtunkhwa,Pakistan

The present study was carried out to investigate the polliniferous bee flora of Lakki Marwat district, Khyber Pakhtunkhwa, Pakistan. A total of 18 species, belonging to 13 different families were identified for the foraging activities of honeybees, namely, Cenchrus biflorus, Cestrum nocturnum, Citrus limon, Combretum indicum, Datura innoxia, Duranta erecta, Hamelia patens, Helianthus annuus, Ipomoea cairica, Luffa aegyptiaca, Nerium oleander, Ocimum basilicum, Parthenium hysterophorus, Pennisetum typhoides, Prosopis cineraria, Prosopis juliflora, Saccharum spontaneum, and Ziziphus jujuba. The identified melliferous flora was collected for the morpho‐palynological investigation. Pollen were acetolyzed, measured, and described qualitatively using light microscopy. The pollen types varied from psilate to echinate and colpi from tricolpate to hexacolpate. Quantitative characteristics including polar diameter, equatorial diameter, polar to equatorial ratio, colpi length, colpi width, spine length, spine width, and exine thickness of the pollen were determined and analyzed statistically using software IBM SPSS Statistics 20. The results of the present study highlighted the significance of pollen morphology of the bee flora of the area which may lead to the identification of the potential and useful botanical sources for beekeepers and to check the honey quality marketed from the study area.     

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.     

Authentication of Polygonati Odorati Rhizoma and other two Chinese Materia Medica of the Liliaceae family by pharmacognosy technique with GC–MS analysis

Yuzhu (Polygonati Odorati Rhizoma), Kangdingyuzhu (Polygonati Prattii Rhizoma), and zhugenqiyuzhu (Disporopsis Fuscopictae Rhizoma) are of the same family, but of different genera. They have all often used in Chinese Materia Medica (CMM) as Polygonati Odorati Rhizoma in China market. Three species of CMM are confused. For better application, we need to identify these plants accurately. This study use pharmacognosy technique and GC‐MS analysis, three species of CMM were authenticated. In macroscopic characteristics, the fruit of Polygonati Odorati Rhizoma is blue‐black, while the other two are maroon and dark purple orderly. Nodes of Polygonati Odorati Rhizoma are upward and light uplift, about 1 cm spacing, while the other are not. As for microscopic characteristics, the cortex of Polygonati Odorati Rhizoma only occupies about 1/5 of the radius of the transverse section with inconspicuous endodermis, which is much smaller than others. The type of vascular bundles of Polygonati Odorati Rhizoma is closed collateral, but the other is amphivasal. Raphides of calcium oxalate are scattered, but Raphides of the other two are like brooms and neat rows. GC–MS analysis of essential oil could provide different characteristics to distinguish three species. Twenty‐three compounds were identified from essential oil of Polygonati Odorati Rhizoma and the main components were n‐hexadecanoic acid (49.45%), while n‐hexadecanoic acid of the other two are 23.92% and 9.45%. The content of n‐hexadecanoic is strongly different. This research was aimed to establish a method by pharmacognosy and GC–MS analysis to identify three CMM and for providing scientifical data to ensure accuracy of origin of three species. Microsc. Res. Tech. 78:119–127, 2015. © 2014 Wiley Periodicals, Inc.     

Morphology of the fertilizable mature egg in the Acanthopagrus latus,A. schlegeli and Sparus sarba (Teleostei: perciformes: sparidae)

Through appropriate scanning electron microscope (SEM) processing, the shape and the surface structures of just‐mature eggs are preserved integrally for observation and measuring to guarantee accurate comparison and prediction on the phylogenetic relationships among three species of teleost, i.e. Acanthopagrus latus, Acanthopagrus schlegeli and Sparus sarba of Sparidae. Eggs in all three species can be categorized as type III eggs. Specifically, a regular pattern is found in the arrangement of micropyle and its surrounding pore canals from the inspected surface structures of the mature eggs in three species. Other than these similarities in egg surface structures, several differences in micropyle structures, micropyle region and size of pore canals are also observed in eggs of respective species. Comprehension and accurate data are statistically computed and analyzed through hierarchical cluster analysis to provide solid species‐specific evidences and standardized empirical reference for accurate species identification and phylogeny determination.     

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.     

Structural characterization of four <i>Rhododendron</i> spp. chloroplast genomes and comparative analyses with other azaleas

Azalea is a general designation of Rhododendron in the Ericaceae family. Rhododendron not only has high ornamental value but also has application value in ecological protection, medicine, and scientific research. In this study, we used Illumina and PacBio sequencing to assemble and annotate the entire chloroplast genomes (cp genomes) of four Rhododendron species. The chloroplast genomes of R. concinnum, R. henanense subsp. lingbaoense, R. micranthum, and R. simsii were assembled into 207,236, 208,015, 207,233, and 206,912 bp, respectively. All chloroplast genomes contain eight rRNA genes, with either 88 or 89 protein-coding genes. The four cp genomes were compared and analyzed by bioinformatics, and the phylogenetic analysis based on chloroplast genomes of 26 species of Ericaceae, Actinidiaceae, and Primulaceae under Ericales was conducted. A comparison of the linear structure of cp genomes of four Rhododendron showed that there were substantial sequence similarities in coding regions, but high differences in non-coding regions. A phylogenetic analysis, based on chloroplast whole genome sequences, showed that all Rhododendron species are in the clade Ericaceae. This study provides valuable genetic information for the study of population genetics and evolutionary relationships in Rhododendron and other azalea species.     

The morphology of stigma of Asteraceae observed by scanning electron microscopy

The morphology of stigma has taxonomic values. To further explore the taxonomy of family Asteraceae, the morphological characteristics of stigma of 28 genera, 32 species, and two varieties in the family were observed using scanning electron microscopy. The results indicated that the stigma morphology of these Asteraceae plants could be divided into 10 types, of which eight are reported for the first time. The morphological characteristics of stigma support the close relationship between genera Aster and Erigeron and among genera Sonchus, Taraxacum, and Youngia. Our results enriched the stigma type diversity data and provided a morphological basis for the study of the phylogenetic evolution of Asteraceae.