Quantitative Anatomical Characteristics of the Leaf Blades of the Several Species of Crataegus L.

Introduction. The Crataegus L. (Hawthorn) is a common herb in numerous Pharmacopoeias. The State Pharmacopoeia of the Russian Federation provides hawthorn fruits and flowers for medical utilization. With that, the literature data confirms the medical utility of hawthorn leaves since the “leaves” and the “flowers with leaves” have pharmacopoeial status worldwide. Therefore, those are considered as prospective forms of Crataegus raw material for Russian pharmaceutical production. However, most species remain poorly pharmacognostically investigated regarding the quantitative microscopic characteristics (the sizes of stomatal apparatus (SA) and epidermal leaf blade (LB) trichomes), which could be substantial for establishing the authenticity of the raw material. Aim. Examine epidermal anatomy of Crataegus spp. Leaf blades (LBs) and perform a comparative study of several quantitative diagnostic features of LBs of hawthorn plants from the sect. Sanguineae and the sect. Crataegus , growing in diverse regions of the Russian Federation. Materials and methods. Samples of hawthorn leaves ( C. sanguinea , C. maximowiczii , C. dahurica , C. rhipidophylla , C. monogyna and C. pallasii ) were collected in natural habitats in Western Siberia (Kemerovo) and in arboretums of Botanical Gardens (Moscow, Stavropol). Measurements of anatomical structures were carried out using a light microscope accompanied by an ocular micrometre. Results and discussion. The LB surface phenotypic diversity within hawthorn species and sections was studied. The LBs were described in terms of meterages (longitude and width) of SA, meterages and shape of sedentary multicellular leaf teeth glands. The peculiarities of pubescence and the sizes of simple unicellular non-glandular trichomes were also observed. Conclusion. The results of quantitative anatomical examination provided the characteristic features determining these elements at the species and section levels. Thus, it may facilitate authentication and quality control of whole or ground Crataegus medicinal raw material.

The genus Crataegus L. estimates approximately 250 species, more than a half of which grow in North America, while the rest grow in the Old World [21]. The genus Crataegus is characterized by a complicated taxonomic structure [21,22]. Several studies displayed that majority of species were united into subgenera and sections based on the morphological features of leaves and fruits. According to literature, five subgenera were distinguished: Crataegus, Sanguineae Ufimov subgen. nov., Americanae El-Gazzar, Mespilus (L.) Ufimov & T. A. Dickinson, Brevispinae (Beadle) Ufimov & T. A. Dickinson, which mostly coincide with geographical zones [23,24]. Each subgenus included several sections [21,22,24,25]. Despite great diversity, only a few species are registered in pharmacopoeial monographs. For example, ten hawthorn species and two hybrids are approved for harvesting medicinal plant raw materials in the current State Pharmacopoeia of the Russian Federation the XIVth edition (RSPh XIV) [20]. At the same time, approximately 40 species of wild hawthorn were found in the eastern regions of forest, forest-steppe and steppe zones of the European and Asian parts of the Russian Federation [26].
The majority of hawthorn species included in the current RSPh XIV belong to the sect. Crataegus of the subgen. Crataegus [20]. C. monogyna Jacq. (also included in EPh and USP) [11,22], C. rhipidophylla Gand., C. pallasii Griseb. and other European species and their hybrids are the ordinary representatives of the sect. Crataegus [21]. Their growth area covers European countries, the middle zone of the European part of Russia, the Caucasus, Krasnodar and Stavropol Territories and the Crimea [21,25].
The species from sect. Sanguineae Zabel ex C. K. Schneider of the subgen. Sanguineae are widespread in the Asian part of the Russian Federation. However, only four species are listed in the RSPh XIV. C. sanguinea Pall remains the most extensive representative of the sect. Sanguineae [20]. It grows in Siberia, in the east of the European zone of Russia, in Zavolzhye, in the Middle and the South Urals and Altai Territory. C. dahurica Koechne ex Schneid. and C. maximowiczii C.K. Schneid. grow in Siberia, Amur and Primorye equally belong to the sect. Sanguineae [27][28][29][30].
Anatomical studies of medicinal products of Crataegus spp., especially leaves, are limited, furthermore quantitative microscopic characteristics are usually ungiven. Anatomical diagnostic features of Bashkortostan C. sanguinea leaves were studied by S. V. Trofimova [31]. The morphology and anatomy of C. sanguinea and C. monogyna petioles were described by N. A. Volkova [32]. T. A. Rezanova and S. A. Bakshutov had presented the results of morphological and anatomical investigation of leaves of 20 Crataegus spp. collected in Belgorod region during the beginning of fruiting, including three species from the sect. Sanguineae and three species from the sect. Crataegus [33]. V. Sharipova had investigated the leaf structure of C. korolkowii (sect. Sanguineae), collected in two different environmental locations [34]. The description of anatomic-morphological examination of leaves, flowers and fruits of the endemic plant C. almaatensis Pojark growing Almaty region of the Republic of Kazakhstan was given by E. N. Bekbolatova et al. [35].
Quantitative characterization of anatomical features is substantial for establishing the authenticity of the MRM. The sizes and density of microscopic diagnostic features commonly are unnormalized in microscopy atlases. Therefore, proceeded type of MRM could not be accurately characterized [36]. In some cases, it is necessary to normalize sizes or determine the density of anatomical features [37], considering the plasticity of diagnostic features in different environmental conditions [34,[38][39][40][41][42].
In this connection, the studies of anatomical features of medicinal products of various representatives of the genus Crataegus L. have become relevant. We set up a task to carry out the comparative study of some quantitative epidermal features of leaf blades (SA, glands and epidermal non-glandular trichomes) of hawthorn plants from the sect. Sanguineae and the sect. Crataegus, growing in diverse regions of the Russian Federation. Accompanying our previous study of flavonoid content in "Crataegi folia cum flores" further results can be used to refine the authenticity and quality criteria of the promising medicinal raw material [43].

Plant material
The

Microscopy
Microscopic samples of leaf surface were studied. Stem leaves from the middle tier of main shoot were observed. It is known that the number of stomata per surface unit varies within different parts of а Методы анализа лекарственных средств Analytical Methods leaf. The sites approaching the average number of stomata are located in the middle third of LB: between ribs and а midrib [44]. Therefore, this site was taken for anatomical investigations and measurements. For C. sanguinea, C. maximowiczii, C. dahurica and C. rhipidophylla two specimens per species and for C. monogyna and C. pallasii one specimens per species were observed. At least 3 individual leaves were used. For each sample 3 measurements were made. Microscopic samples of LB were prepared according to the General Pharmacopoeial Monograph 1.5.3.0003.15 RSPh XIV "Technique of microscopic and microchemical studies of medicinal plant materials and herbal medicines" [45]. The samples were observed under the light Mikmed-1 microscope with AU-12 1.5x binocular (10x eyepiece, WF10x wide-angle eyepiece with a scale, 8x, 10x, 20x, 40x lenses) (Lomo, Russia) and the MSP-1 binocular (Lomo, Russia). Pictures were taken with Fugifilm DIGITAL CAMERA FinePix JX500 (Fugifilm, Japan) and edited in Picasa (ver. 3.9 141.259, Google, USA).
Anatomical diagnostic features were studied according to the requirements of General Pharmacopoeial Monograph 1.5.1.0003.15 "Folia" in RSPh XIV [45]. Measurements of anatomical structures were carried out using an ocular micrometre according to the method described in the General Pharmacopoeial Monograph 1.5.3.0003.15 "Technique of microscopic and microchemical studies of medicinal plant materials and herbal medicines" [45]. The linear size between two most distant points (the cell wall of the periostic epidermal cells), measured horizontally was considered as the length of the SA [46]. The linear size across the cell walls of the periosteum cells of the epidermis, measured horizontally was considered as the width of SA [47].

Statistical analysis
Statistical calculations were carried out using Excel (ver. 2016, Microsoft, USA) and RStudio for Windows (ver. Desktop 1.4.1103, RStudio PBC, USA) software package. The normality of all data was analyzed by Shapiro-Wilk's test. All normally distributed data were analyzed using ANOVA with post-hoc Tukey HSD Test for intergroup comparisons. In other cases, Nemenyi non-parametric allpairs comparison test for Kruskal-type ranked data was applied. Comparisons between two sections were performed using Mann-Whitney U-test. Data on figures are presented as medians and 25 th -, 75 th -percentiles. Data in tables are presented as means±SD. Student t-test with Bonferroni correction for multiple comparisons was applied for two-group comparison if the data were normally distributed; p ≤ 0.05 were considered to be significant.

RESULTS AND DISCUSSION
Leaves of observed species were hypostomatic. Stomata were elongated, rarely rounded, surrounded by 5-7 periostic cells (anomocytic type of SA). The striations of cuticle were folding around stomata (figure 1).
The stomatal length varied from 20.1 to 50.3 μm and width varied from 10.1 to 36.9 among investigated species. Significant differences regarding SA width among the sect. Sanguineae representatives were detected neither among distinct species nor within the species harvested in different environmental conditions. However, С. dahurica from Kemerovo had significantly greater SA longitude than other studied accessions and С. dahurica from Moscow as well. The C. rhipidophylla and C. pallasii (the sect. Crataegus) had the equivalent SAs meterages and pretended to have significantly wider and longer SAs than C. monogyna SA (table 2).
Comparing the data on both sections, we found that the sect. Crataegus representatives had significantly greater SA meterages (figure 2).
Multicellular marginal glandular trichomes were typical for Crataegus species LBs. Similar trichomes were previously described for several genera in fami-

Методы анализа лекарственных средств Analytical Methods
ly Rosaceae: Prunus L., Malus L., etc. [48]. Chin and co-authors had presented the classification of multicellular glands and had described their structural variants for several species from the genus Prunus [49]. According to that classification, we had observed leaf teeth glands located near vascular zones. The epidermis of the gland was formed by palisade cells. The hypodermal zone was intensely brown coloured ( figure 3).
The secretion of glandular trichomes of flowering plants naturally includes various classes of secondary metabolites. Terpenoids are the most studied [50]. Phenolic compounds, particularly flavonoids, are considered the most common components in glands secretion of woody plants [51,52]. The chemical composition of the leaf teeth glands exudation in hawthorns is scarce [53].
The sizes of leaf teeth glands in C. sanguinea, C. dahurica, and C. maximowiczii (sect. Sanguineae) from two regions: Moscow (central region of the Non-Chernozem Zone) and Kemerovo (south of Western Siberia) were studied comparatively. C. dahurica (Kemerovo) had the widest leaf teeth glands in comparison to C. maximowiczii from the same location and the species from Moscow. C. sanguinea (Kemerovo) could be significantly distinguished from Moscow specimens: C. dahurica and C. maximowiczii (p ≤ 0.05), C. sanguinea (p ≤ 0.1) (table 3).
The specimens from Moscow and C. maximowiczii from Kemerovo had equally wide leaf teeth glands. Regarding the longitude of leaf teeth glands: within the
Within the sect. Sanguineae, the length/width ratio of the leaf teeth gland varied among species. C. sanguinea had 0.47-0.55 length/width ratio, while C. maximowiczii samples demonstrated comparable results: 0.43-0.45. Thus, the glands were twice as wide as long. The length/width ratio of C. dahurica leaf teeth glands varied: the sample from Kemerovo -0.72 and the sample from Moscow -0.67. So, the shape of these glands was close to cubic (in the plane -square).
Among species within the sect. Crataegus and individual species collected in different regions (e.g., C. rhipidophylla from Moscow and Stavropol) significant differences in leaf teeth glands meterage were unfound. Unlike the sect. Sanguineae, hawthorns from the sect. Crataegus mainly had a spherical (in the plane -rounded) leaf teeth gland shape with 0.78 ratiо -C. monogyna. The length/width ratio of 0.65 was C. pallasii and 0.67 -C. rhipidophylla (Stavropol). The C. rhipidophylla from the arboretum of the MBG RAS (Moscow) is distinguished by more elongated glands -0.51 length/width ratio. The sizes of leaf teeth glands of specimens collected in the arboretum of the MBG RAS (Moscow) were comparable in both sections (table 3). With that, sect. Sanguineae plants had significantly wider leaf teeth glands (figure 4). Our data is consistent with the results obtained in the Belgorod region [33].
According to the Pharmacopoeias and WHO Monographs on medicinal plants, epidermal non-glandular trichomes on Crataegus leaf blade surface are considered as a diagnostic feature [2, 11-13, 16, 20]. Observed leaf blades were covered with simple unicellular trichomes of 117-660 μm long (table 4), excluding the C. rhipido-

CONCLUSIONS
The anatomical study of SA and trichomes on leaf epidermis of C. sanguinea, С. dahurica, C. maximowiczii, C. monogyna, C. rhipidophylla, C. pallasii LBs was performed. Microscopy has revealed characteristic features: hypostomatic leaf type, anomocytic SA, multicellular glands, simple unicellular hairs. Studied species had comparable sizes of LBs' SA within both sections. With that, plants from the sect. Crataegus from both regions demonstrated significantly greater sizes of SA in comparison with the sect. Sanguineae plants.
Both sections did not demonstrate significant differences in leaf teeth glands longitude among species collected within the same location. Comparing the sections concerning leaf teeth glands length the difference was also unfound. Furthermore, significantly wider leaf teeth glands differentiated C. dahurica and C. sanguinea (Kemerovo) from the other species of the sect. Sanguineae, collected in Moscow. The sections Sanguineae and Crataegus were also distinguished by the leaf teeth gland shape: cuboid and spherical, respectively. With that, in the sect. Sanguineae plants had significantly wider leaf teeth glands.
The studied species also had various features of pubescence of LB and the sizes of simple unicellular non-glandular trichomes. The species of both sections were pubescent with trichomes of equal length on both sides, except Siberian C. sanguinea, which had longer trichomes on the adaxial side. Within both sections, non-glandular trichomes assembled in clusters in the region of the midrib.
Identified distinctive features could be applicable for authentication of the MRM of hawthorn ("leaves" or "flowers with leaves") harvested in different geographical zones. Possible variations of studied parameters on species and sections should be taken into account during the quality control process of hawthorn MRM since it will help screen out the herbal adulterants.