Implication of scanning electron microscopy and light microscopy for morphology of some selected seed drugs: As a tool for authentication

Ramiza Munir; Basheer Khan; Ghulam Dastagir; Noor Ul Uza

doi:10.4103/jmau.jmau_104_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 11 | Issue : 1 | Page : 60-67

Implication of scanning electron microscopy and light microscopy for morphology of some selected seed drugs: As a tool for authentication

Ramiza Munir, Basheer Khan, Ghulam Dastagir, Noor Ul Uza
Department of Botany, University of Peshawar, Peshawar, Pakistan

Date of Submission	08-Nov-2022
Date of Decision	25-Dec-2022
Date of Acceptance	31-Dec-2022
Date of Web Publication	23-Feb-2023

Correspondence Address:
Ms. Noor Ul Uza
PHD Scholar at Department of Botany, University of Peshawar, Peshawar
Pakistan

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmau.jmau_104_22

Abstract

Background: The goal of our current study is to use Scanning electron microscopy (SEM) and light microscopy (LM) to evaluate the ultra-micromorphological properties of 14 seed drugs to correctly identify and validate them. There was no previous research on SEM-based evaluation of the selected seeds. These included: Solanum nigrum L., Physalis peruviana L., Cestrum diurrnum L., Withania somnifera L. Dunal, Achyranthus aspera L., Celosia argentia L., Chenopodium murale L., Cyperus alternifolius L., Cyperus rotundus L., Schoenoplectus litoralis (Schrad.) Palla, Oxalis corniculata L., Catharanthus roseus L., Canna indica L., and Parthenium hysterophorus L. belonging to 7 families (Solanaceae, Amaranthaceae, Cyperaceae, Oxalidaceae, Apocynaceae, Cannaceae, and Asteraceae). Methods: Quantitative characters (length, width, and weight of seeds) as well as qualitative characters (seed shape, color, texture, and surface level of seeds) were analyzed. Results: Seeds length ranged from 0.6 μm (C. indica) to 2.4 μm (A. aspera) while, the seeds width and weight ranged from 0.6 μm (C. indica) to 1.8 μm (W. somnifera) and 0.03 g (C. indica) to 3.7 g (C. argentia), respectively. The SEM revealed many types of surface texture. Five types of surface levels (raised, regular, smooth, rough, and ill-defined pattern) were observed in seeds. The variation was found to be quite significant for the taxonomic demarcation at generic and specific levels. Conclusions: SEM could be a valuable approach for hidden morphological features of seed drugs, which could aid further exploration, appropriate identification, seed taxonomy, and authenticity. SEM and LM also play an important role in drug discovery and development.

Keywords: Light microscopy, qualitative characters, quantitative characters, seed drugs, scanning electron microscopy

How to cite this article:
Munir R, Khan B, Dastagir G, Ul Uza N. Implication of scanning electron microscopy and light microscopy for morphology of some selected seed drugs: As a tool for authentication. J Microsc Ultrastruct 2023;11:60-7

How to cite this URL:
Munir R, Khan B, Dastagir G, Ul Uza N. Implication of scanning electron microscopy and light microscopy for morphology of some selected seed drugs: As a tool for authentication. J Microsc Ultrastruct [serial online] 2023 [cited 2023 Mar 20];11:60-7. Available from: https://www.jmau.org/text.asp?2023/11/1/60/370250

Introduction

Plant categorization is one of the most important topics for plant systematists and taxonomists. Researchers are currently attempting to lay a foundation for the classification. Over the last few years, plants have been classed and reclassified as needed. Because morphological features are seasonal and do not present throughout the year, many plants are classified based on their outward morphology.^[1],[2] As a result, the depiction of micro- and macro-morphological properties of seeds is extremely valuable and can be used to identify and classify medicinal plants at various levels.^[3],[4] The morphological characters of seeds are used to trace the evolutionary and phylogenetic relationship among various taxa.^[5],[6] These characteristics can also be used by agriculturists and foresters to distinguish the weeds seeds.^[7] Seed micromorphology is used as a reliable approach for assessing phylogenetic relationship.^[8],[9] The seed features used to make a key to species are shape, seed ornamentation, anticlinal boundaries, and epidermal cell shape.^[10],[30]

The morphometric of seeds micromorphology solve various problems related to taxonomic-identification, refinement as well as validation of actual sources of seeds from their adulterants in several plant taxa.^[11] Seed characters' study is useful for comparative analysis at each level of the taxonomic hierarchy. Typically, taxonomic account of seed micromorphology includes two stages: the description of characters associated to seed surface and the description of seed kinds.^[12],[35] These studies particularly the seed coat patterns (microscopy) have been used as an important tool for solving various taxonomic problems such as tracing the evolutionary relationship, solving classificatory problems, or the delimitation of genera and species.^{[13],[14],[15]}

According to Singh et al., seeds are a well-known source of biodiesel, recognizing them is a critical step that can be achieved by examining their morphology. Seeds can be used as a great source of biodiesel production to help overcome energy problems because they offer several advantages.^[16] Such studies assist in seed categorization and authentication in biodiesel manufacturing in the future. The innovative form of microscopic studies is scanning electron microscopy (SEM). It is valuable for describing the micromorphology of the samples and can distinguish closely related taxonomic characters up to incredible level.^[17] It is used for the standardization of plant products from their adulterants and plays a crucial role in the quality control of crude drugs. Among such plant products, the most common are seed drugs.^[18],[19] SEM comprises using high magnification for the quantitative study of several elements of seed ultrastructure (seed surface patterns, wall levels, and cell configurations) that are difficult to examine using light microscopy (LM).^[20],[21]

There has, however, been no research toward detecting minute seed medicines based on morphological characteristics. It is crucial to distinguish tiny seed medicines appropriately. On the other hand, the seeds are difficult to spot with the naked eye. As a result, microscopic studies to aid in the morphological identification of these seeds have been conducted. Overall, the data suggest that studying seed morphology at a microscopic level is a simple and cost-effective way of seed identification. It was demonstrated that morphological and micromorphological features can be used to distinguish between genuine herbal remedies and adulterants using microscopic imaging techniques.

This study will pave the way for the finding of morphological traits at both the macro and micro scales, as well as the evaluation of systematic links between related species. Furthermore, by integrating micromorphological traits, this investigation will serve as a point of view, supporting analysts in the accurate recognition and identification of seeds.

Methods

Collection of Seeds

Fresh seeds of 14 plant spp. belonging to 7 families Solanaceae (4 spp.), Cyperaceae (3 spp.), Amaranthaceae (3 spp.), Oxalidaceae (1 sp.), Apocynaceae (1 sp.), Cannaceae (1 sp.), and Asteraceae (1 sp.) were collected from Azakhel Botanical Garden and from Peshawar University Campus and were allowed to dry for 3–4 weeks.

Light microscopy

For LM, seeds were examined under the light microscope, and microphotographs were taken by polarized camera (DCM35 350K pixel USB 2.0) installed at Botany Department, University of Peshawar. Photographs were taken from different angles by rotating seeds.

Scanning electron microscopy

For SEM, seeds were mounted directly onto stubs, using single-sided adhesive tape, and coated with gold-palladium using a (JEOL JFC 1100 E) ion sputtering device and observed under the Scanning Electron Microscope (Model JEOL JSM-5910) installed at Central Resource Laboratory, Department of Physics, University of Peshawar. SEM photos were taken using Polaroid P/N 665 film in the middle area of the ventral face for each sample. Micrometry was done with the help of micrometer.

The terms used for describing the seed surface patterns have been adopted according to the works of Stearn and Koul et al.^[22],[23]

Results

The seed morphological characters for the studied taxa of the families Solanaceae, Cyperaceae, Amaranthaceae, Oxalidaceae, Apocynaceae, Cannaceae, and Asteraceae are summarized in [Table 1]. Quantitative and qualitative parameters such as seed color, seed shape, seed texture, seed surface level, 100-seed weight (g), seed length (μm), and seed width (μm) are recorded in [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6],[Figure 7],[Figure 8],[Figure 9],[Figure 10],[Figure 11],[Figure 12],[Figure 13],[Figure 14],[Figure 15],[Figure 16].

Table 1: Morphological characteristics of some selected seed drugs

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Figure 1: (a) Solanum nigrum L. seed LM (b) SEM of S. nigrum L. (c) Seed apex of S. nigrum L. (d) Surface texture of S. nigrum L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 2: (a) Physalis peruviana L. seed LM. (b) SEM of P. peruviana L. (c) P. peruviana L. (d) Surface texture of P. peruviana L. LM: Light Microscopy, SEM: Scanning electron microscopy

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Figure 3: (a) Cestrum diurrnum L. seed LM, (b) SEM of C. diurrnum L. (c) Seed surface pattern of C. diurrnum L. (d) Surface texture of C. diurrnum L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 4: (a) Withania somnifera L. LM, (b) SEM of W. somnifera L. (c) Surface texture of W. somnifera L. (d) W. somnifera L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 5: (a) Achyranthus aspera L. LM (b) SEM A. aspera L. (c) Apex of A. aspera L. seed. (d) Surface texture of A. aspera L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 6: (a) Celosia argentia L. seed LM (b) SEM of C. argentia L. (c) Seed surface texture of C. argentia L. (d) C. argentia L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 7: (a) Chenopodium murale L. LM (b) SEM C. murale L. (c) Seed surface of C. murale L. (d) Seed surface texture of C. murale L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 8: (a) Cyperus alternifolius L. LM (b) SEM C. alternifolius L. (c) Seed surface of C. alternifolius L. (d) Seed surface of C. alternifolius L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 9: (a) Cyperus rotundus L. LM (b) SEM C. rotundus L. (c) Seed surface of C. rotundus L. (d) Seed surface of C. rotundus L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 10: (a) Schoenoplectus litoralis LM (b) SEM S. litoralis. (c) Seed surface of S. litoralis (d) Seed surface of S. litoralis. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 11: (a) Oxalis corniculata L. LM (b) SEM O. corniculata L. (c) Seed surface of O. corniculata L. (d) Seed surface of O. corniculata L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 12: (a) Canna indica L. LM (b) SE) C. indica L. (c) Surface texture of C. indica L. (d) Surface texture of C. indica L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 13: (a) Catharanthus roseus L. LM (b) SEM C. roseus L. seed (c) Seed surface of C. roseus L. (d) Surface texture of C. roseus L. seed. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 14: (a) Parthenium hysterophorus L. LM (b) SEM P. hysterophorus L. (c) Seed surface of P. hysterophorus L. (d) Seed surface of P. hysterophorus L. LM: Light Microscopy, SEM: Scanning Electron Microscopy

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Figure 15: (a) Micrometry of SEM of seeds (Size in μm). (b) Micrometry of SEM of seeds (Size in μm). SEM: Scanning Electron Microscopy

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Figure 16: Weight of selected seed drugs in grams

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Solanaceae

Solanum nigrum L.

The seeds of Solanum nigrum were off-white in color, ovate in shape, broad and rounded at the base, and tapering at the end; egg-shaped [Figure 1]a. The texture was rough hard consisted of reticulate “net-like” ribs, and the surface level was raised [Figure 1]b, [Figure 1]c, [Figure 1]d. Edges of the seeds were irregular in some cases. The length was 1.68 μm, and the width was 1.2 μm [Figure 15]a. The weight of 100 seeds was 0.05 g [Figure 16].

Physalis peruviana L.

The seeds were white in color, elliptical, longer than broad with blunt or rounded ends, ribs of surface level were raised [Figure 2a]. The seeds were ruminate in texture, i.e., penetrated by irregular channels giving an eroded appearance and running in different directions [Figure 2]b, [Figure 2]c, [Figure 2]d. The weight of seeds was 0.38 g [Figure 16], length was 1.61 μm, and width was 1.30 μm [Figure 15]a.

Cestrum diurrnum L.

The seeds were white in color, variable in shape, but mostly obovate seeds were observed [Figure 3]a. Seeds had abrupt wavy margins with more or less wrinkled surface texture [Figure 3]b and [Figure 2]c and undulate margins with ill-defined surface level [Figure 3]d. The weight of 100 seeds was 0.22 g [Figure 16], length was 1.63 μm, and width was 1.45 μm [Figure 15]a.

Withania somnifera L.

The seeds were similar to Physalis and Cestrum in some respects. White in color, obovate in shape [Figure 4]a, and ruminate in texture with raised surface level [Figure 4]b, [Figure 2]c, [Figure 2]d. The weight estimated was 0.14 g [Figure 16], length was 2.13 μm, and width was 1.81 μm. These seeds were much wider as compared to others [Figure 15]a.

Amaranthaceae

Achyranthus aspera L.

The seeds were black in color and papillate in shape [Figure 5]a. The surface level was regular almost but rough at the tip [Figure 5]b and [Figure 2]c. The texture was characterized by lineolate margins, i.e., marked with fine broken lines [Figure 5]d. Seeds were the longest among collection about 2.40 μm in length and 2.27 μm in width [Figure 15]a. The weight of 100 seeds was 0.10 g [Figure 16].

Celosia argentia L.

The seeds were shiny black in color, rounded, and circular in shape [Figure 6a]. The surface was fine and smooth while, the texture of seed was areolate [Figure 6]b, [Figure 2]c, [Figure 2]d. The seeds weighted up to 3.70 31 g [Figure 16]. The length was 1.18 μm, and the width was 1.06 μm [Figure 15]a.

Chenopodium murale L.

The seeds showed similarities to Achyranthus and Celosia in black color and smooth surface level. The shape was such as Celosia rounded or globular [Figure 7]a. The texture was pusticulate, i.e., with small broad slight elevations not so high or abundant as on a colliculate surface and not having as abrupt elevations as a minutely tuberculate surface; covered with small pustule or blister-like elevations [Figure 7]b, [Figure 2]c, [Figure 2]d. The weight estimated was 1.50 g [Figure 16], length was 1.13 μm, and width was 1.04 μm [Figure 15]a.

Cyperaceae

Cyperus alternifolius L.

The seed was off-white in color and cymbiform in shape [Figure 8]a. The texture was favulariate, i.e., with the surface finely ribbed, the ribs separated by zigzag furrows. The surface was rough [Figure 8]b, [Figure 2]c, [Figure 2]d. The weight of seeds was 0.55 g [Figure 16], length was 1.46 μm, and width was 0.43 μm [Figure 15]b.

Cyperus rotundus L.

The seeds were brown in color and obovate in shape [Figure 9]a. The texture was slightly reticulate with fine margins and with a regular surface level [Figure 9]b, [Figure 9]c, [Figure 9]d. The weight estimated was 0.92 g [Figure 16], length was 2.02 μm followed by width of 0.04 μm [Figure 15]b.

Schoenoplectus litoralis (Schrad.) Palla L.

Seeds were white in color and tear-shaped [Figure 10]a, areolate in texture with regular surface level as in Cyperus rotundus [Figure 10]b, [Figure 9]c, [Figure 9]d. The weight was 0.56 g [Figure 16], length was 1.49 μm, and width was 0.96 μm [Figure 15]b.

Oxalidaceae

Oxalis corniculata L.

The color of seeds was blood-red during collection but dark-red in dried condition. The shape was mucronate, and the edges or margins of seed were dentate [Figure 11]a. The seeds had reticulate texture “net-like” with a raised network of narrow and sharply angled lines frequently presenting a geometric appearance, each area or depression outlined by the reticulum being an interspace with raised surface level [Figure 11]b, [Figure 11]c, [Figure 11]d. The weight of seeds estimated was 0.12 g [Figure 16], the length was 1.73 μm, and the width was 1.13 μm [Figure 15]b.

Cannaceae

Canna indica L.

The seeds were minute, white in color initially but black in dried condition, globose in shape [Figure 12]a, undulate in texture, and surface level in highly ill-defined [Figure 12]b, [Figure 12]c, [Figure 12]d, respectively. The seeds had a minimum weight of 0.03 g [Figure 16], the smallest length and width of 0.66 μm equally [Figure 15]b.

Apocynaceae

Catharanthus roseus L.

Seeds were dark black in color and oblong in shape, i.e., elliptical and two to four times longer than wide, with approximately parallel sides [Figure 13]a. Verrucate in texture, i.e., with irregular projections or knobs and the surface level was raised [Figure 13]b, [Figure 13]c, [Figure 13]d. The seed was 2.3 μm in length, 1.18 μm in width [Figure 15]b, and 1.42 g in weight [Figure 16].

Parthenium hysterophorus L.

The seeds were black in color and ovoid in shape, i.e., egg-shaped [Figure 14]a. A large number of species had this type of seeds which were otherwise difficult to describe. The seeds were scalar form in texture, i.e., ladder like; having transverse bars or markings like the rounds of a ladder [Figure 14]b, [Figure 14]c, [Figure 14]d. The surface level was regular with length of 2.57 μm, width of 1.25 μm [Figure 15]b, and weight of 0.20 g [Figure 16].

Discussion

According to Barthlott, the species bearing fruits/seeds with similar surface ornamentations may have common genetic-phylogenetic relations.^[24] In the present study, seed color, shape, texture, surface level, weight, and length were observed. Differences were noticed in seeds of Solanaceae in color (white/off-white), shape (ovate, elliptical, and obovate), and texture (ruminate, reticulate, and undulate) which resembles with the work of Zhang and Liu.^[26] S. nigrum is reticulate in texture, according to the findings of Gunn and Gaffney, which resembles the present work.^[27] All present seeds in Amaranthaceae have a smooth surface and black in color but quite different in shape and texture. Some species of Chenopodiaceae were observed by Karcz et al. previously showed resemblance in smooth surface level with respect to present work.^[28] C. rotundus seeds are obovate in shape while oblong according to Shalabi and Gazer, and similar (reticulate) in texture.^[29] C. alternifolius (off-white) weighed 0.55 g while the length estimated was 1.46 mm which falls in the range according to the work of Shalabi and Gazer. The seeds of Oxalis corniculata were blood red, mucronate in shape, and reticulate in texture which is not supported by the previous findings of Obone, where orange-brown and elliptic seeds have been observed.^[31] The seeds of C. roseus were black in color, oblong in shape and verrucate in texture in the present study, which mismatch the finding of Roy et al. where brown color, ovoid shape, and reticulate texture were observed.^[32] The seeds of C. indica were ovoid, smooth, and black, according to the findings of Graven et al., while the present study showed a globose shape and undulate texture.^[25],[33] The seeds of Parthenium hysterophorus were black, ovoid in shape, and scalariform in texture. Various other workers have also investigated the seed diversity of medicinal plants and stressed that it is a reliable method for evaluating phenetic relationships.

According to Meo and Hostels, SEM is widely used to observe the surface topology of a wide range of plants species. It has a key role in the validation of entire botanicals and as well in powder state. Safety and quality of the important medicinal plants may be accomplished through accurate identification.^[34] Comparison, based on current work and published literature showed that taxonomic investigation of oil-yielding seeded plants is of the prodigious extent to be used as a significant feature to distinguish the genuine drugs from their adulterant.^{[36],[37],[42]} Seed surface study through SEM is used in resolving problems of plant systematics and phylogenetic relationships.^{[38],[39],[40]} Despite the fact that medicinal plants play an important role in the pharmaceutical sector and health care, there has been little research on seed identification. Seed morphology is important in theoretical botany and can be utilized for seed identification, quarantine, forensics, seed dispersal and soil seed bank research, and macrofossil identification in archaeobotanical studies.^{[41],[43],[44]}

Conclusions and Recommendations

Macro and micromorphological characteristics of 14 seed drugs of different families were investigated using scanning electron microscope and light microscope. Key to species was based on seed color, shape, texture, surface level, weight, length, and width. This is a preliminary study to determine the applicability of the tested characters, and further researches covering all taxa of the species are needed for determining the entire variations and better understanding systematics of taxa. Seed micromorphology confirmed that the developmental variation in seed characters is taxonomically useful because it gives us a better understanding of sculpture development. It is recommended that this study will be helpful in identifying seeds of the same family and supportive in classification, authentication, and standardization of medicinal plants. The biodiesel sector might easily be operated by morphological and logistical data analysis for building new technology procedures. Academicians, researchers, drug testing laboratories, health professionals and regulatory authorities, plant-based pharmaceutical enterprises, crude drug sellers, and those involved in the identification and standardization of crude pharmaceuticals will all benefit from these findings.

Acknowledgments

The authors are thankful to Centralized Research Laboratory, Department of Physics, University of Peshawar, for providing SEM facilities.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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