1118 ISSN 0326-2383

KEY WORDS: Ecological anatomy, Ginkgo biloba, Maidenhair, Medicinal plant, Pharmacognosy, Plant anatomy.

* Author to whom correspondence should be addressed. E-mail: marciard@ufpr.br

Latin American Journal of Pharmacy(formerly Acta Farmacéutica Bonaerense)

Lat. Am. J. Pharm. 32 (8): 1118-23 (2013)

Regular Article

Received: July 8, 2013Accepted: July 17, 2013

Comparative Microscopic Charactersof Ginkgo biloba L. from South America and Asia

Márcia R. DUARTE 1*, Danielle C. SOUZA 2 & Rosana E. COSTA 2

1 Laboratório de Farmacognosia, Departamento de Farmácia, Universidade Federal do Paraná,Av. Pref. Lothário Meissner, 632, CEP 80210-170, Curitiba, PR, Brazil

2 Curso de Farmácia, Setor de Ciências da Saúde, Universidade Federal do Paraná, Brazil

SUMMARY. Ginkgo biloba, the single species of Ginkgoales, is a deciduous tree displaying bilobe dichoto-mously veined leaf. It is native to China and cultivated around the world because of its therapeutic rele-vance. Leaf phytopharmaceutical products are mainly used for treating cognitive deficits and vascular de-mentia. This work has compared the leaf and stem microscopic characters of different specimens fromSouth America (Curitiba, Brazil) and Asia (Beijing, China and Hiroshima and Tokyo, Japan), aiming toinvestigate some anatomical variations according to environmental factors. All the specimens from SouthAmerica and Asia have shown similar characters, except for the amount of phenolic compounds. Thesebioactive metabolites are visually alike in all the Asian samples and higher comparatively to the SouthAmerican specimens. These results are useful for the quality control of the raw material and standardiza-tion of leaf extracts for phytotherapy.

INTRODUCTIONGinkgo biloba L. (Ginkgoaceae), commonly

named maidenhair or ginkgo, is a living fossilbelonging to Gymnospermae 1 and the only ex-isting representative of the order Ginkgoales 2. Itis a tree that has straight trunk and ample crownand shows slow growth, although it can reachvery high height 3. It is dioecious (either male orfemale plant), deciduous 4 and long living (ca.1,000 years) 5. The leaves are alternate and sim-ple, displaying a unique feature: bilobe or fla-belliform shape with dichotomous venation 6

(Figs. 1A-C).Ginkgo is native to China 7 and cultivated in

different latitudes around the world due to itstherapeutic and economic importance. Phy-topharmaceutical preparations of its leaves havegreat popularity for reducing symptoms associ-ated with cognitive deficits, vascular dementia,early stage of Alzheimer’s disease, ischemicheart disorder and peripheral claudication 8-12.Several secondary metabolites such as ter-

penoids, polyphenols and allyl phenols havebeen isolated from the species, although themain bioactive constituents are terpene trilac-tones and flavonoid glycosides 13,14, which areconsidered responsible for vasoactive and freeradical-scavenging properties and the modula-tion of cholinergic function 15,16.

Despite the myriad of investigations dealingwith morphological, chemical and pharmacolog-ical analyses as well as plant fossil records, fewones have carried out comparative ecologicalapproaches which can evaluate the environmen-tal influence on vegetal development, such ason cultivated American and Asian plants. Basedon this, the present work has compared the mi-croscopic characters of the leaf and stem of dif-ferent specimens of G. biloba from South Ameri-ca (Brazil) and Asia (China and Japan), in orderto provide some foundation for understandinganatomical variations according to environmen-tal factors, e.g. climate and pollutants. Thesefindings are useful for the micromorphological

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quality control of the raw material as the firststep for the production of standardized extracts.

MATERIALS AND METHODSApical vegetative branches were studied

from Ginkgo biloba L. (Ginkgoaceae) grown inSouth America and Asia in autumn. In SouthAmerica, the plant material was collected fromspecimens in the city of Curitiba, Brazil, in May2007. Asian samples were obtained from China(Beijing) and Japan (Hiroshima and Tokyo), inOctober 2010. All the plant sites were sunny,open and exposed to natural and urban roaddust and motor vehicle exhaust. General geo-graphic data and environmental factors of eachlocality are shown in Table 1. Rainfall levels,temperature monitoring and atmospheric partic-

Figure 1. Ginkgo biloba L.: A. Apical branches, B. De-tail of a vegetative branch, C. Bilobe leaf; bl: leafblade, pet: petiole. Bar = 2 cm.

ulate matter concentrations were presented inlong term estimation for ginkgo being a longliving species, despite annual leaf renewals. Mu-seum curators identified the species by crossingthe reference voucher MBM 287187 from theherbarium permanent collection at the MuseuBotânico Municipal de Curitiba.

Matures leaves, from the fourth node on, andyoung stems, 5-15 cm from the shoot, werefixed in FAA 70 17 and stored in 70% ethanol(v/v) 18. For light microscopy (LM), semiperma-nent slides were made by freehand sectioning intransverse and longitudinal planes, and stainingwith astra blue and basic fuchsine 19. For perma-nent slides, leaf and stem fragments were dehy-drated in a graded ethanol series, embedded insynthetic resin, sectioned using a rotary micro-tome and stained with toluidine blue 20.

With reference to elementary microchemicaltests, in order to detect visually phenolic com-pounds focusing on flavonoids and tannins in-distinctly, freehand sections of the fixed materialunderwent reaction with ferric chloride 17, andto reveal the chemical nature of the crystalsplant sections were treated with sulfuric acid 21.

Scanning electron microscopy (SEM) wasperformed using small leaf fragments dehydrat-ed in ascending ethanol series and CO2 criticalpoint drying. Then, the material was coatedwith gold and examined in high vacuum 22.

RESULTS All the specimens from South America (Cu-

ritiba, Brazil) and Asia (Beijing, China and Hi-roshima and Tokyo, Japan) have shared similaranatomical characters, as it follows: the leaf, inface view of both surfaces, has striate cuticleand polygonal (sub-rectangular) shaped epider-mal cells with wavy and moderate thick anticli-nal walls (Figs. 2A-D, 4A-D). The cells of theadaxial side are comparatively larger than theones of the abaxial surface. Stomata are restrict-ed to the abaxial side and randomly distributedon the blade (Figs. 2A, 2C, 4A, and 4B). Theguard-cells are kidney-shaped and surroundedby approximately five or six cells (Figs. 2C and4B). Fine epicuticular wax, deposited in smalldepressions of the epidermal surface, is onlyseen in specimens from Hiroshima (Figs. 4C and4D).

In cross-section, the cuticle is moderate thickand the epidermis is uniseriate and shows con-vex external periclinal walls with more promi-nent curvature on the abaxial side (Figs. 2E, 3A,3B, 4E-H). The guard-cells are sunken due to

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projecting surrounding cells that partially coverthe stomatal aperture (Figs. 3B, 4F, and 4H).The mesophyll has dorsiventral organizationand comprehends one to three rows of plicate

Figure 2. Ginkgo biloba L., leaf, Brazilian specimens:A, B. Face view of the adaxial side of the epidermis inLM and SEM, C, D. Face view of the abaxial side of theepidermis in LM and SEM, E. Transverse section of theintervein region; ep: epidermis, LM: light microscopy,phc: phenolic compounds, plp: plicate parenchyma,sc: secretory cavity, SEM: scanning electron mi-croscopy, sp: spongy parenchyma, st: stomatum, vb:vascular bundle. Bar = 50 µm (E), 20 µm (A, C).

Figure 3. Ginkgo biloba L., leaf in transverse section,Brazilian specimens: A, C. Detail of the plicateparenchyma, phenolic compounds and druse, B.Stomatum inserted in a small depression, D. Secretorycavity in the mesophyll, E. Collateral vascular bundleand transfusion tissue; ct: cuticle, dr: druse, ep: epi-dermis, ph: phloem, phc: phenolic compounds, plp:plicate parenchyma, sc: secretory cavity, st: stoma-tum, tr: transfusion tissue, vb: vascular bundle, xy:xylem. Bar = 20 µm.

Elevation Time ofAverage

Mean and extreme ClimateAir pollution

City Coordinates(m) collection

rainfall*temperatures* (°C) type

(PM10** -(mm) µg/m3)

Curitiba, 25°26’S 930 May 1,560 18 (-5 and 35) subtropical/ 27Brazil 49°14’W 2007 humid

Beijing, 39°54’N 36 October 640 13 (-17 and 42) temperate/ 106China 116°31’E 2010 continental moonson

Hiroshima, 34°23’N 11 October 1,550 16 (-7 and 38) temperate/ 31Japan 132°27’E 2010 maritime

Tokyo, 35°41’N 5 October 1,530 16 (-5 and 37) temperate/ 43Japan 139°49’E 2010 humid

Table 1. Geographic data and environmental conditions of South American and Asian cities where specimens ofGinkgo biloba were collected 23,24. *In long term. **Concentrations of particle matter smaller than 10 µg.

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parenchyma and multiseriate spongy parenchy-ma (Figs. 2E, 3A, 3C, and 4E-H). This representsabout 60% of the chlorenchyma height. Thereare collateral vascular bundles (Figs. 2E, 3E, 4E,4G, and 4H), transfusion tissue (Fig. 3E) and se-cretory cavities lined by two or three layers ofepithelial cells (Figs. 2E, 3D, and 4F). Largedruses of calcium oxalate are scattered in themesophyll (Figs. 3C, 4E, 4G, and 4H).

Figure 4. Ginkgo biloba L., leaf, Asian specimens: A.Face view of the adaxial side of the epidermis in LM(Beijing), B. Face view of the abaxial side of the epi-dermis in LM (Hiroshima), C, D. Adaxial and abaxialsurfaces of the epidermis in SEM (Hiroshima), E, G,H. Detail of epidermis, druse and minor vascular bun-dle (E: Tokyo, G: Beijing, H: Hiroshima), F. A secre-tory cavity in the mesophyll (Beijing); ct, cuticle, dr:druse, ep: epidermis, LM: light microscopy, phc: phe-nolic compounds, plp: plicate parenchyma, sc: secre-tory cavity, SEM: scanning electron microscopy, sp:spongy parenchyma, st: stomatum, vb: vascular bun-dle. Bar = 20 µm.

Figure 5. Ginkgo biloba L., leaf and stem in transversesection, Brazilian specimens: A. Medium region of thepetiole, B. Caulinar organization, C. Detail of the epi-dermis and external cortex of the stem, showinggelatinous fibres and a secretory cavity; ep: epidermis,gf: gelatinous fibre, ph: phloem, phc: phenolic com-pounds, pi: pith, sc: secretoy cavity, vb: vascular bun-dle, xy: xylem. Bar = 200 µm (A), 100 µm (B, C).

Phenolic compounds are present in thechlorenchyma, as well as in the cells encirclingthe vascular bundles and in the epithelium ofthe secretory cavities (Figs. 2E, 3C, 3E, and 4E-H). These metabolite concentrations are visuallysimilar in all the Asian samples and higher com-paratively to the South American specimens.

The petiole, in cross-section, has plano-con-vex contour in the proximal and medium re-gions (Figs. 5A and 6A) to concave-convexshape in the distal one (Fig. 6B). There are unis-eriate epidermis, two collateral vascular bundlessituated side by side, secretory cavities analo-gous to the ones previously described, groupsof fibres with lignified walls and cells eithercontaining druses of calcium oxalate or phenoliccompounds (Figs. 5A, 6A-C).

The stem, in incipient secondary develop-ment, has circular cross-section and presentsone-layered epidermis coated with thick cuticle(Figs. 5C and 6E). It follows a few strata of col-lenchyma and some groups of gelatinous fibres(Figs. 5B, 5C, 6D, and 6E). The phellogen hasperipheral origin, in the first layers of the cortex.It forms suber externally which consists of largevacuolated cells (Fig. 6E). It is also encounteredmultiseriate cortical parenchyma, secretory cavi-ties with the same characteristics of the leaf, lig-nified and gelatinous fibres (Figs. 5B, 5C, and6D-G). In the vascular cylinder, the phloem andxylem are traversed by narrow rays and the

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xylem shows homogeneous pattern, consistingof elements (tracheids, fibres and parenchymacells) with small diameter (Figs. 5B and 6D).Gelatinous fibres, cells with amyloplasts and se-cretory cavities are present in the pith. Cellsbearing phenolic compounds are found in thecortex and pith (Figs. 5B, 5C, 6D and 6E) anddistributed alike in all the specimens analysedfrom South America and Asia.

DISCUSSIONIn general, the leaf anatomical characters de-

scribed in this work are comparable to the find-ings by other authors 6,25 who have studied the

Figure 6. Ginkgo biloba L., leaf and stem in trans-verse section, Asian specimens: A. Medium region ofthe petiole (Beijing), B. Distal region of the petiole,showing concave-convex shape (Tokyo), C. Detail ofthe previous figure, indicating a secretory cavity, D,G. Stem (Hiroshima), displaying secretory cavity,druses and gelatinous fibres, E, F. Stem (Tokyo), pre-senting phellogen origin and suber, gelatinous andlignified fibres; dr: druse, ep: epidermis, fb: lignifiedfibre, gf: gelatinous fibre, ph: phloem, phc: phenoliccompounds, phl: phellogen, sc: secretory cavity, su:suber, vb: vascular bundle, xy: xylem. Bar = 200 µm(A, B), 100 µm (D), 50 µm (C), 20 µm (E-G).

same species: striate cuticle covering a uniseri-ate epidermis with wavy anticlinal and convexpericlinal walls, sunken stomata exclusively onthe abaxial epidermal surface (hypostomaticleaf), internal secretory structures and vascularbundles of the collateral type. However, somedifferences can be mentioned, as Neinhuis &Barthlott 26 have stressed that the ginkgo leaf ishomogeneously covered with a dense layer ofwax tubules and only small amount of alter-ations can be detected until late summer andautumn. This differs from the present investiga-tion for epicuticular wax having been only no-ticed in specimens from Hiroshima, although allplant materials were collected in middle au-tumn. Despite the common stomatum featuresdisplayed by all specimens investigated in thiswork, Denk & Velitzelos 7 have stated that thereis a high variability in the degree of the papilos-ity of surrounding cells of stomata in G. biloba.They are prominent in mature sun leaves andlacking in shade leaves suggesting an environ-mental influence on this trait. This variation wasnot found in this work possibly because all theanalysed ginkgo trees were exposed to sun. Ac-cording to the British Pharmacopoeia 27, theginkgo leaf is amphistomatic and for Denk &Velitzelos 7 stomata also on the adaxial side(amphistomatic leaf) and trichomes on the leafbase may occasionally occur in ginkgo, al-though these characteristics were not seen inany of the specimens studied herein.

Diverging from the present results, Stan &Simeanu 6 have reported homogeneous meso-phyll, secretory ducts and absence of calciumoxalate crystals in the leaf of G. biloba. With ref-erence to these secretory structures, McCargo etal. 25 have cited the occurrence of ducts in theginkgo leaf and have classified them asschizogenous. There are some contradictory as-sumptions on this matter, since some researcheshave shown that the species has secretory cavi-ties 2,28,29, as corroborated in this study. Besides,their origin is not well defined. In Ameele’sopinion 28, their formation is lysigenous, but onthe authority of Wang et al. 29 and Peng et al. 2

they follow the schizo-lysigenous pattern, beingmature cavities round in shape and surroundedby flat secretory cells and peripheral sheathcells.

The caulinar description presented in this re-search is in conformity with Stan’s publication 3,except for the previously indicated controversy.This author has observed secretory ducts andhas not reported calcium oxalate crystals orgelatinous fibres in the stem of G. biloba.

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As pointed out by Lindorf 30, it is wellknown that climatic factors such as temperatureand water availability, as well as geographicvariables including latitude and altitude affectthe plant development. Apart from the amountof phenolic compounds, all the specimens haveshown quite similar features. Considering thelimited ecological perspective of this work,among the Asian samples, the Japanese ones(Hiroshima and Tokyo) have shared commonenvironmental factors, differing from those fromChina (Beijing) as the latter have grown underlower average rainfall and higher air pollutants.Even so, this seems not to influence the featuresanalysed in this survey. Phenolic compoundshave been visually lower in South Americanspecimens (Curitiba, Brazil), although there isno discrepancy with reference to rainfall, tem-perature and air pollution between Brazilianand Japanese samples.

In conclusion, the results described hereinindicate that ginkgo specimens exhibit quitesimilar characters independently from the geo-graphic origin and environmental factors, exceptfor the amount of phenolic compounds. Thesefindings confirm that the standardization of leafginkgo extracts is needed to achieve a therapeu-tic product for phytotherapy because phenoliccompounds are related to the concentration offlavonoids, one of the plant bioactive groups ofmetabolites.

Acknowledgments. Museu Botânico Municipal deCuritiba for the taxonomic identification and Centrode Microscopia Eletrônica - UFPR for the electron mi-crographs.

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