Application of microscopy to Digitalis thapsi x Digitalis purpurea ...

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Oleandrin: A cardiac glycosides with potent cytotoxicity. Pharmacognosy Review. 2013; 7:131-139. [5] Real Jardin Botânico, Digitalis in: Flora iberica – Plantas ...
Microscopy: advances in scientific research and education (A. Méndez-Vilas, Ed.) __________________________________________________________________

Application of microscopy to Digitalis thapsi x Digitalis purpurea natural hybrid identification R. Serrano, S. Frazão, J. Silva, E. T. Gomes and O. Silva iMED.UL, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal The dried leaf of Digitalis purpurea L. (foxglove leaf) was introduced as botanical drug into the London Pharmacopoeia of 1650 and remains actually in all Occidental Pharmacopoeias (digitalis purpureae folium). During last year’s, some specimens of Digitalis L. genus collected in the Northeast region of Portugal were identified by us as natural hybrids Digitalis thapsi L. x Digitalis purpurea L.. Traditionally, hybridization in plants has been generally detected using morphological characters and the leaf is one of the most common organs used in this kind of studies. The aim of this chapter is to present a complete description of the microscopic characteristics, founded by us in the leaf of the collected samples, that allows the identification of the natural hybridization based on the application of light microscopy and scanning electron microscopy. Quantification and statistical data analysis of the major micromorphologic D. thapsi x D. purpurea leaf distinctive characters were also presented. This work emphasizes the importance and actuality of the use of microscopic techniques as a tool of identification of pharmaceutical botanical raw materials. Keywords: Botanical identification; Digitalis purpurea; Digitalis thapsi; Medicinal plants; Microscopy; Natural hybrids

1. Introduction Digitalis purpurea L. leaf (foxglove leaf) is a medicinal plant used to extract cardiac glycosides like digitoxin, taken as a medication to treat cardiac insufficiency. The dried leaf of D. purpurea were introduced as botanical drug into the London Pharmacopoeia of 1650 and remains actually in all Occidental Pharmacopoeias like European Pharmacopoeia 8th edition (digitalis purpureae folium) [1]. The Iberian endemism Digitalis thapsi L. is also characterized by the cardiac glycosides content [2,3]. Current trend shows the cytotoxic activity of some compounds from this chemical class and its consequent possible application as antitumor agents [4]. Both species (D. purpurea and D. thapsi) were present in the Iberian Peninsula [5]. During last year’s, some specimens of Digitalis L. genus collected in the Northeast region of Portugal were identified by us as natural hybrids [2,3]. Traditionally, hybridization in plants has been generally detected using morphological characters and the leaf is one of the most common organs used in this kind of studies. As referred, the leaf is the only part of the plant used as medicine concerning Digitalis genus. The aim of this chapter is to present a complete description of all specific microscopic characteristics founded by us in the leaf of the collected samples that allows the identification of the natural hybridization (D. thapsi x D. purpurea) and its consequent distinction from the parents, based on the application of light microscopy (LM) and scanning electron microscopy (SEM). Quantification and statistical data analysis of the major micromorphologic D. thapsi x D. purpurea leaf distinctive characters were also presented. 1.1 Biogeography of Portuguese Digitalis species Formally known to belong to the family of Scrophulariaceae Juss., Digitalis L. is nowadays classified as a genus of the Plantaginaceae family [6]. According to Flora European two species of Digitalis L. were described in Portugal namely Digitalis thapsi L. (perennial) plant endemic of the Iberian Peninsula and Digitalis purpurea L. (biennial) represented by three subspecies: Digitalis purpurea L. ssp. purpurea var. purpurea, Digitalis purpurea ssp. heywoodii P. Silva & M. Silva, and Digitalis purpurea L. ssp. mariana (Boiss.) Rivas Goday [7]. Concerning the geographical distribution, D. purpurea ssp. purpurea var. purpurea and D. thapsi are widespread in Portugal and D. purpurea ssp. mariana, considered a rare species, have similar distribution, while D. purpurea ssp. heywoodii is an endemic species distributed only around Reguengos de Monsaraz [8,9]. Thought, “Flora Iberica Plantas Vasculares de la Península Ibérica e Islas Baleares” [5] in the taxonomic revision of the genus refer seven Iberian species, three of these cited as present in Portugal, namely D. purpurea, D. thapsi and D. mariana Boiss. subsp. heywoodii (P. Silva & M. Silva) Hinz. (exclusively detected at Alto Alentejo region), D. purpurea subsp. purpurea (with a wide distribution) and D. purpurea subsp. amandiana (Samp.) Hinz distributed in Baixo Alentejo, Douro Litoral and Trás-os-Montes. Besides, one notes that D. purpurea subsp. purpurea, show a great polymorphism and introgression with D. thapsi. Furthermore, the hybrids D. lutea x D. purpurea subsp. purpurea and D. purpurea subsp. purpurea x D. thapsi are described in Spain. In Portugal a hybrid between D. purpurea and D. thapsi named “Coutinhii Samp. (D. purpurea x D. thapsi) was been described [10] and some voucher specimens of this were deposited at “Instituto de Botânica Dr. Gonçalo Sampaio”

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University of Oporto. Nowadays, it’s not considered in the Portuguese and European Floras nor in the literature related to spontaneous hybridization. Besides the cited species, recent ICN documents (ICN, Plano Sectorial da Rede Natura 2000) on Natura 2000 Network concerning rocky habitats named Western Mediterranean and thermophilous screens in Portugal, include D. purpurea subsp. carpetana, exclusively in “Serra Estrelense Sector”, among other perennial plant taxa as bioindicators. D. thapsi, is distributed in Portugal mainly from Alentejo to Trás-os-Montes along the border regions between Portugal and Spain [8, 9], and according “Flora Iberica” inexistent at Algarve, but present in Beira Litoral, and perhaps in Douro Litoral and Minho. 1.2 Constituents and medicinal uses of Digitalis species The chemistry of Digitalis has engaged the attention of many researchers since 1820 and allowed the discovery of the chemical group of cardenolides, responsible for the founded cardiotonic activity of this plant genus. The primary glycosides, purpurea glycoside A and purpurea glycoside B, are the principal active constituents of the fresh leaf of D. purpurea, and digitoxin and gitoxin the main active components of the dried drug [11]. D. purpurea leaf remains the main industrial source of purpurea glycosides A, B and digitoxin for clinical proposes. D. purpurea leaf also contain flavonoids like apigenin, crysoeriol, digicitrin, hispidulin, luteolin and nepetin [12]. Concerning the cardiac glycosides, other Digitalis species are alternative industrial sources of these active constituents, like D. lanata Ehrh. This species still the raw material for digoxin and lanatoside C obtention. Actually, these compounds and some other semi-synthetic derivatives are the main compounds therapeutically used to treat heart diseases [11]. D. thapsi show a different biosynthetic profile on secondary metabolites from that of the main species used by the pharmaceutical industry as materials for extraction of the cardiotonic drugs. Though D. thapsi is not nowadays a raw material for the extractive industry [9,13]. This species is able to produce simultaneously purpurea glycosides and lanatosides and is richer in lipophilic surface flavonoids [12,14] than other Digitalis species giving to the leaf a greenyellowed color. For this reason, local Portuguese populations names the species by the vernacular name of “abeloura amarelada” to distinguish from D. purpurea known in Portugal, among other names, by “abeloura”. Flavonoids, act as solar filters and are important plant metabolites for their antioxidant and scavenging activities. Recent pharmacological studies points out the cardiotonic glycosides interest as antitumor agents, this is illustrated by several papers and some US Patents claiming their use in the treatment of the cell-proliferative diseases including cancer [15,18] 1.3 Micromorphologic studies on Digitalis species The morphological and histological characterization of the leaf was undertook for Digitalis of Spain (D. lutea, D. parviflora, D. obscura, D. ambigua, D. purpurea, D. dubia, D. nevadensis, D. thapsi, D. mariana and D. tomentosa) and based on leaf surface observation, the most useful characters for identification proposals were epidermal cells shape and type of trichomes (non-glandular or glandular trichomes) and stomata index (SI) [19]. Interspecific hybrids produced experimentally were also characterized according the same microscopic characters [20]. In Portugal some morphoanatomical studies have been performed on D. thapsi leaf [9] also by our team [21,22]. Our studies also included a natural hybrids morphoanatomical characterization [2,3,23]. Morphological and anatomical studies on fruits and seeds in the context of the family Scrophulariaceae in all the genera present in the southwest of Spain were also published [24,25].

2. Material and Methods 2.1 Plant material Different samples of the leaf of Digitalis spp. were collected in the Northeast region of Portugal. Voucher specimens were preserved at “João de Carvalho e Vasconcelos” Herbarium of the “Instituto Superior de Agronomia", Lisbon, Portugal. Methodology used includes microscopic analysis of about 30 specimens of two different populations of Digitalis, one from “Barragem da Marateca” witch is only present D. thapsi and other from Seia where D. thapsi and D. purpurea co-occur leading to specimens with different morphological appearance, corresponding to natural hybrids. Plant material was dried in the dark under controlled conditions of humidity (75 ± 5%) and temperature (21 ± 1ºC). 2.2 Microscopic analysis 2.2.1 Light microscopy The dried plant material was previously hydrated in water. For the anatomical analysis, each leaf sample was sectioned freehand. Lamina transverse sections (midrib and distal part of the blade) and tangential longitudinal sections (leaf

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surface) were cleared and mounted in a 60% chloral hydrate aqueous solution, according to European Pharmacopoeia (EDQM, 2010). Microscopic analysis of the prepared leaf sections was conducted on an Olympus CX40 upright microscope, coupled with an Olympus ColorView IIIu camera. Image analysis was performed with Cell D 2006 Olympus Software. 2.2.2 Scanning electron microscopy The dried plant material was sectioned, dehydrated at 35 ºC for 24 h and directly mounted on stubs using double-side adhesive tape. The samples were sputtered with a thin layer of gold in a JEOL JSM-1200 Fine Coater and observed in a JEOL JSM-T220 scanning electron microscope at 15 kV, with a digital image acquisition integrated system. 2.3 Statistical data analysis Statistical values have been calculated using the Microsoft Excel 2010 software. Stomatal index (SI) was determined by the following formula: SI ൌ

ୗൈଵ଴଴ ୗା୉

, where (S) represents the number of stomata in a given area of the leaf and (E), the

number of epidermal cells in the same area of the leaf (EDQM, 2010).

3. Results and Discussion At naked eye, all specimens are similar to D. thapsi, except for samples of D. purpurea. The statistical analysis of the principal microscopic results obtained are summarized in Tables 1-2 and illustrated in Figures 1-7. Light microscopy (LM) analysis of transversal sections of the leaf of D. thapsi x D. purpurea and D. thapsi (Fig. 1) shows in all the species a pronounced convex midrib on the lower surface and a zone of collenchyma underlies both epidermises in the midrib region. The crescent shaped midrib bundle is enclosed in an endodermis with one or two cells thick. The pericycle is parenchymatous above and collenchymatous below. The xylem is toward the upper epidermis and the phloem is toward the lower epidermis. Sclerenchymatous fibres are absent.

a)

b)

Fig. 1 LM micrographs of leaf transverse section. Midrib structure: D. thapsi x D. purpurea a); D. thapsi b). Scale bar = 200 µm a); 500 µm b)

By LM an asymmetric organization with a typical bifacial structure was observed in the leaf transverses sections (Fig. 2). The mesophyll has palisade parenchyma constituted by 1-3 cell layers and a thin spongy parenchyma.

a)

b)

Fig. 2 LM micrographs of leaf transverse section. Mesophyll asymmetric with palisade parenchyma and spongy parenchyma: D. thapsi x D. purpurea a); D. thapsi b). Scale bar = 50 µm

LM (Figures 3-4) and Scanning electron microscopy (SEM) (Figures 5-6) of the leaf surface of D. thapsi x D. purpurea and D. thapsi showed the presence of sinuous epidermal cells in upper and lower epidermis. Also observed, the anomocytic stomata surrounded by a ring of four to five subsidiary cells were more frequent in the lower epidermis.

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Stomata and non-glandular trichomes and glandular trichomes were found on both surfaces but were more frequent on the lower epidermis. Glandular trichomes of six general types occur: short with a unicellular stalk and a bicellular head (Fig. 7 A) or short with a unicellular stalk and unicellular head (Fig. 7 B), or long with a uniseriate stalk (2-5 cells) and unicellular head (Figures 7 C-F). The non-glandular trichomes were rare in D. thapsi, multicellular, uniseriate, with three to five cells (Figures 7 G-H), with smooth walls and narrow lumen, and often with one or more of the cells collapsed inward. They were frequently broken off, leaving a characteristic scar. Calcium oxalate crystals were absent in the leaf of all specimens.

b)

a)

Fig. 3 LM micrographs of leaf surface view showing anomocytic stomata on upper epidermis: D. thapsi x D. purpurea a); D. thapsi b). Scale bar = 50 µm

b)

a)

Fig. 4 LM micrographs of leaf surface view showing anomocytic stomata on lower epidermis: D. thapsi x D. purpurea a); D. thapsi b). Scale bar = 50 µm

a)

b)

Fig. 5 SEM micrographs of D. thapsi x D. purpurea leaf surface view showing stomata detail a) and abundant trichomes b). Scale bar = 5 µm; 50 µm b)

a)

b)

Fig. 6 SEM micrographs of D. thapsi leaf surface view showing stomata detail a) and abundant trichomes b). Scale bar = 10 µm a); 50 µm b)

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Fig. 7 LM micrographs of details of the different glandular trichomes types (A, B, C, D, E, F) and covering (non-glandular) trichomes (G, H) founded in D. thapsi x D. purpurea and D. thapsi. Scale bar = 50 µm (A, B, C, D); Scale bar = 100 µm (E, G, H); Scale bar = 200 µm (F)

Concerning the “Barragem da Marateca” specimens, our microscopic analysis results clearly showed the presence of similar leaf characters to all analyzed samples, with the presence of typical glandular trichomes, scarce non-glandular trichomes and frequent anomocytic stomata. On “Seia” specimens we found some leaf distinctive quantitative characteristics associated with the thickness of the cuticle, the number of layers of palisade and spongy parenchyma, the midrib and trichomes, although it is also possible to draw analogies between these specimens (Tables 1 and 2). At microscopic level (Tables 1 and 2), the samples of natural of D. thapsi x D. purpurea hybrids from “Seia” have the cuticle of the epidermal cells and the type and form of glandular trichomes similar to those observed in D. thapsi from Barragem da Marateca, and the type and shape of trichomes and number of layers of spongy parenchyma cells leaf similar to D. purpurea. The palisade parenchyma of the D. thapsi x D. purpurea hybrid specimens showed a number of cell layers intermediate showed intermediate cell number relative to the D. purpurea and D. thapsi. In the main vein, D. thapsi x D. purpurea specimens also showed higher abundance of glandular trichomes erect and free, such as D. thapsi. Both (hybrid and D. thapsi) were characterized by the presence of stalk trichomes with 4-5 cells and unicellular head. The glandular trichomes of D. thapsi x D. purpurea are similar to D. thapsi while the nonglandular trichomes are similar to those observed in D. purpurea. D. thapsi and D. thapsi x D. purpurea hybrid present a grooved cuticle. Stomata index of D. thapsi and of D. thapsi x D. purpurea were similar. The results suggest and confirm the hybrid nature of the specimens collected at “Seia”. Having regard to the approaches undertaken in this investigation, we confirm that the natural hybrid observed resulted from the crossing D. thapsi x D. purpurea, taking into account the predominance of characters of D. thapsi observed. D. thapsi x D. purpurea specimens can be identified by the presence of a midrib (pronounced) with convex form on the lower surface of the leaf, a dorsiventral mesophyll with palisade parenchyma constituted by 2 cell layers and a thin spongy parenchyma, a upper epidermis with irregularly shaped cells, striated cuticle and few anomocytic stomata, a lower epidermis containing epidermal cells with a smooth cuticle and sinuous anticlinal walls and numerous anomocytic stomata with 4-6 subsidiary cells, glandular trichomes (abundant) – long with a uniseriate stalk (2-5 cells) and unicellular head, or short with a unicellular stalk and bicellular head, or short with a unicellular stalk and unicellular head and non-glandular trichomes (few) multicellular, uniseriate, 1-5 cells.

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Table 1

Comparative analysis of the leaf micromorphologial characters.

Parameter

D. purpurea

D. thapsi x D. purpurea D. thapsi

mean (standard deviation) Thickness (µm) upper cuticle lower cuticle total mesophyll (µm) *

3,28 (0,33) a 2,79 (0,33) a 109,69 (7,86) a

7,80 (0,93) b 8,62 (0,96) b 105,16 (3,24) a

7,15 (1,70) b 8,74 (0,64) b 282,60 (60,30) b

palisade parenchyma

41,95 (5,50) a

41,24 (1,24) a

106,02 (45,85) b

Spongy parenchyma

67,75 (9,53) a

58,42 (2,93) a

110,73 (58,04) b

Cells number upper epidermis lower epidermis

799* 1101*

2984,60 (494,47) a 2414,50 (401,13) a

1691,30 (262,30) b 1245,30 (225, 50) b

upper epidermis stomata

30*

54,60 (33,51) a

49,30 (34,44) b

lower epidermis stomata

193*

195,80 (65,13) a

138,60 (57,23) b

upper epidermis cell

43,76 (7,58) a

25,87 (9,36) b

27,75 (1,62) b

lower epidermis cell

37,31 (5,42) a

31,37 (6,03) a

23,94 (4,65) b

upper epidermis stomata

20 (2,44) a

20,69 (2,79) a

20,20 (1,52) a

lower epidermis stomata

23,88 (6,35) a

19,49 (3,06) b

20,98 (1,06) c

upper epidermis cell

27,01 (8,37) a

16,35 (4,92) b

19,84 (5,39) b

lower epidermis cell

19.79 (6,23) a

17,54 (3,12) a

17,75 (3,96) a

upper epidermis stomata

15,68 (1,01) a

15,29 (1,68) a

15,69 (1,83) a

Lower epidermis stomata

15,33 (4,71) a

16,11 (3,46) b

15,11(1,02) a

Width / length ratio upper epidermis cells lower epidermis cells upper epidermis stomata lower epidermis stomata

1,62 (11,85) a 1,89 (12,39) a 1,28 (3,05) a 1,56 (6,05) a

1,58 (6,73) a 1,78 (9,77) a 1,35 (3,82) b 1,21 (2,39) b

1,40 (5,59) b 1,35 (4,38) b 1,28 (3,19) a 1,39 (4,16) c

upper epidermis

3,70* a

1,79 (1,29) b

3,81 (1,39) a

lower epidermis

12,20* a

7,50 (1,73) b

7,65 (2,82) b

Stomata ratio (upper/lower epidermis)

1:6,4*

1:3

1:2,5

Width (µm)

Length (µm)

Stomatal index

a, b, c - difference statistically significant between species (p < 0,05).; * Data of Martinez (1946) [19].

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Table 2

Principal distinctive microscopic trichomes features. D. purpurea

D. thapsi x D. purpurea

D. thapsi

Glandular trichomes

Stalk (µm)

Head L/W ratio

Stalk (µm)

Head L/W ratio

Stalk (µm)

Head L/W ratio

Stalk (1 cell) Head (1 cell)

20,46 (1,41) a

1,47 (0,05) a

19,34 (1,67) a

0,87 (0,05) b

18,92 (0,99) b

1,55 (0,11) a

Stalk (2 cells) Head (1 cell)

41,29 (0,78) a

0,86 (0,01) a

59,49 (2,06) b

0,79 (0,02) b

73,22 (1,10) c

1,02 (0,01) c

Stalk (3 cells) Head (1 cell)

145,77 (6,88) a

0,82 (0,08) a

308,71(19,84) b

1,05 (0,05) b

123,68 (1,89) c

1,05 (0,01) b

Stalk (4 cells) Head (1 cell)

416,17 (5,70) a

0,86 (0,01) a

486,15 (31,41) b

1,32 (0,42) b

539,23 (4,33) c 1,60 (0,02) c

910,66 (35,39) a

1,08 (1,32) a

224,72 (3,33) b 0,78 (0,99) b

30,66 (5,84) b

0,74 (0,04) b

6,86 (0,35) c

Stalk (5 cells) Head (1 cell) Stalk (6 cells) Head (1 cell)

41,91 (1,86) a

Non glandular 518,32 (96,84) a Trichomes

0,97 (0,01) a

1,01 (0,00) c

254,29 (1,53) b

L = length; W = width; a, b, c - difference statistically significant between species (p < 0,05).

4. Conclusions On the basis of the identified micromorphological characters, we confirm the occurrence of natural hybridization between D. thapsi and D. purpurea in the Northeast region of Portugal, and the usefulness of the LM and SEM analysis to identify and distinguish Digitalis species and hybrids. The identification of spontaneous hybrids could be of great interest for future plant breeding of these species of pharmacological interest. Molecular studies are also undertaken to prove hybrid nature of different samples. Advances in microscope technology and improvements in LM and SEM have increased the accuracy and capabilities of microscopy as a mean of herbal medicines identification. This work emphasizes the importance and actuality of the use of microscopic techniques as a tool of identification of pharmaceutical botanical raw materials. Acknowledgements We would like to thank Telmo Nunes from the Microscopy and Image Analysis Laboratory of the Centre for Environmental Biology, Faculty of Sciences, University of Lisbon, for technical assistance on SEM observations.

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