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.     

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.     

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.     

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.     

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.     

Systematic significance of pollen morphology and foliar epidermal anatomy of medicinal plants using SEM and LM techniques

Alternative medicinal source is a hot issue within medicine community now a days due to various side effects of allotropic drugs; most importantly the multidrug resistance phenomena. Medicinal plants provide an attractive and effective alternative to modern synthetic drugs as they contain different types of bioactive compounds, having great potential in treatment of various diseases. The present study deals with identification of micromorphological characteristics of 10 ethnomedicinally important plants of Northern Pakistan, that is, Aster himalaicus C. B. Clarke, Artemisia maritima (Berg.) Wellkomm, Isodon rugosus (Wall. ex Benth.), Myricaria squamosal Desv, Pedicularis groenlandica Retz. Polygonum aviculare L, Salvia himmelbauer E. Peter, Swertia kingie Hook. f., Tanacetum dolichophyllum (Kitam) Kitam, Verbascum epixanthinum Boiss. & Heldr. as potential source for pharmaceutical industry. The characters studied were shape and size of pollen, polar diameter and equatorial diameter, epidermal cell shape and size, stomata width and length, trichome shapes, and type. Most of the species had tricolporate pollen type but hexacolporate pollen was also observed in Isodon rugosus, Polygonum aviculare, Salvia himmelbauer. Different types of exine sculpturing observed were foveolate, scabrate, lophate, regular, steriate, and alveolate. Minimum equatorial diameter was noted in Tanacetum dolichophyllum (12.4 μm) and maximum in Myricaria squamosal (20.9 μm). Shape of epidermis cell ranged from rectangular and irregular, polygonal and pentagonal. Different types of stomata like anisocytic, actinocytic, paracytic, and diacytic were noticed. Variations in microscopic characters observed in the current study have great potential to be used as identification tools by the taxonomists for further studies.     

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.     

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.     

Taxonomic study of Triblemma by using light and scanning electron microscopy

Triblemma Ching is a genus proposed by Ching Ren‐Chang in 1978, and it is composed of two species, Triblemma lancea (Thunb.) Ching and Triblemma zeylanica (Hook.) Ching. There has been much debate on the systematic position of Triblemma, and this genus has always been included in Diplazium. Here, we have described new features of tracheary elements, epidermis, spore, scale, and rachis of Triblemma revealed using light and scanning electron microscopy and proposed that Triblemma is closely related to Athyriopsis, which conflicts with the traditional viewpoint.     

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.     

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.     

Nondestructive estimation of growth year in ginseng cultivars using the means of mathematical modeling on the basis of allometry

Growth‐year authentication has extraordinary significance for plant growth, structure and development research, and has a wide range of applications in value assessment of economic crops. Panax ginseng is the most commonly used medicinal plant in Asian countries. The fix number of growth‐year is an important quality evaluation which is difficult to be obtained accurately in current technical conditions. Preliminary authentication theory for growth‐year has been described in previous studies using a short‐lived perennial medicinal plant (Paeonia lactiflora pall.) as the research material. In this research, we focused on the growth‐year estimation in ginseng cultivars, and attempt to explore the age estimation method for vascular plants according to mathematical simulation of the root structure development. Micro data was obtained from 204 individuals of 3 different kinds of ginseng cultivars, which have a series of gradient age and a clear growth record. Outer diameter of the vascular cambium (b) and the radius of cross section (r) were measured with ordinary stereo microscope. We further designed and established two different kinds of authentication model based on the taproot structure development for growth year authentication (P and M = K*X₁^a1X₂^a2). Moreover, the models were applied to identify the growth year of ginseng without damage using Micro‐CT or DEI reconstruction. A potential method, have been recently described, the age of ginseng can be analyzed by telomere length and telomerase activity. However, we found that there are different results indicated in other species. We concluded that microscopic methods perceived currently were provided a more effective means for growth‐year authentication. Microsc. Res. Tech. 79:98–105, 2016. © 2016 The Authors Microscopy Research and Technique Published by Wiley Periodicals, Inc.     

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.     

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.     

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.     

In vivo second‐harmonic generation and ex vivo coherent anti‐stokes raman scattering microscopy to study the effect of obesity to fibroblast cell function using an Yb‐fiber laser‐based CARS extension unit

Nonlinear microscopy techniques are being increasingly used to perform in vivo studies in dermatology. These methods enable us to investigate the morphology and monitor the physiological process in the skin by the use of femtosecond lasers operating in the red, near‐infrared spectral range (680–1,300 nm). In this work we used two different techniques that require no labeling: second harmonic generation (SHG) for collagen detection and coherent anti‐Stokes Raman scattering (CARS) to assess lipid distribution in genetically obese murine skin. Obesity is one of the most serious public health problems due to its high and increasing prevalence and the associated risk of type 2 diabetes and cardiovascular diseases. Other than these diseases, nearly half of patients with diabetes mellitus suffer from dermatological complications such as delayed wound healing, foot ulcers and several other skin changes. In our experiment we investigated and followed the effects of obesity on dermal collagen alterations and adipocyte enlargement using a technique not reported in the literature so far. Our results indicate that the in vivo SHG and ex vivo CARS imaging technique might be an important tool for diagnosis of diabetes‐related skin disorders in the near future. Microsc. Res. Tech. 78:823–830, 2015. © 2015 Wiley Periodicals, Inc.     

Comparative light and scanning electron microscopy in authentication of adulterated traded medicinal plants

The medicinal plants are utilized globally considering the cheap and chemical free source, but their correct identification and authentication is prerequisite for safety and efficacy of plant‐based medicines. The present study encompassed traded medicinal plants (16) with high therapeutic value from diverse families like Brassicaceae, Berberidaceae, Malvaceae, Salicaceae, Myrtaceae, Papilionaceae, Ascelpiadaceae, Colchicaceae, Violaceae, and Vitaceae for detailed microscopic study of characters that is, morphology, pollen shape and sizes, P/E ratio, pore length and width, spine length, colpi dimensions, and exine sculpture pattern. The plants showed noteworthy differences in microscopy of Wattakaka volubilis having pollinia, translator and corpusculum like structures while pores were visible in Colchicum luteum, Alcea rosea, and Hibiscus syriacus. The spines were observed in Centipeda minima, A. rosea, and H. syriacus being dimorphic spines in A. rosea and monomorphic in H. syriacus. The exine sculpturing pattern was reticulate in mostly studied plants however distinctive exine pattern was noted in Berberis aristata and Berberis lyceum. The highest polar diameter, equatorial diameter and exine thickness among studied plants were observed in H. syriacus (161 μm), C. luteum (50 μm) and Vitis jacquemontii (1.10), respectively. Thus, microscopy of medicinal plants in addition to other taxonomic evidence offers a supportive skill in authentication, consequently utilization by local consumers and pharmaceutical industries.     

Comparison of single‐particle analysis and electron tomography approaches: an overview

Three‐dimensional structure of a wide range of biological specimens can be computed from images collected by transmission electron microscopy. This information integrated with structural data obtained with other techniques (e.g., X‐ray crystallography) helps structural biologists to understand the function of macromolecular complexes and organelles within cells. In this paper, we compare two three‐dimensional transmission electron microscopy techniques that are becoming more and more related (at the image acquisition level as well as the image processing one): electron tomography and single‐particle analysis. The first one is currently used to elucidate the three‐dimensional structure of cellular components or smaller entire cells, whereas the second one has been traditionally applied to structural studies of macromolecules and macromolecular complexes. Also, we discuss possibilities for their integration with other structural biology techniques for an integrative study of living matter from proteins to whole cells.     

Vascular imaging with contrast agent in hard and soft tissues using microcomputed‐tomography

Vascularization is essential for many tissues and is a main requisite for various tissue‐engineering strategies. Different techniques are used for highlighting vasculature, in vivo and ex vivo, in 2‐D or 3‐D including histological staining, immunohistochemistry, radiography, angiography, microscopy, computed tomography (CT) or micro‐CT, both stand‐alone and synchrotron system. Vascularization can be studied with or without a contrast agent. This paper presents the results obtained with the latest Skyscan micro‐CT (Skyscan 1272, Bruker, Belgium) following barium sulphate injection replacing the bloodstream in comparison with results obtained with a Skyscan In Vivo 1076. Different hard and soft tissues were perfused with contrast agent and were harvested. Samples were analysed using both forms of micro‐CT, and improved results were shown using this new micro‐CT. This study highlights the vasculature using micro‐CT methods. The results obtained with the Skyscan 1272 are clearly defined compared to results obtained with Skyscan 1076. In particular, this instrument highlights the high number of small vessels, which were not seen before at lower resolution. This new micro‐CT opens broader possibilities in detection and characterization of the 3‐D vascular tree to assess vascular tissue engineering strategies.