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Micromorphological investigation on the leaves of the rock samphire(Crithmum maritimum L.): Occurrence of hesperidin and diosmin crystalsL. Cornaraa; C. D'Arrigob; F. Piolib; B. Borghesic; C. Bottinod; E. Patroneb; M. G. Mariottia

a DIPTERIS, Polo Botanico “Hanbury”, Università di Genova, Genova, Italy b ISMAC, CNR, Genova,Italy c DIPROVE, Università di Milano, Italy d IENI, CNR, Genova, Italy

To cite this Article Cornara, L. , D'Arrigo, C. , Pioli, F. , Borghesi, B. , Bottino, C. , Patrone, E. and Mariotti, M. G.(2009)'Micromorphological investigation on the leaves of the rock samphire (Crithmum maritimum L.): Occurrence ofhesperidin and diosmin crystals', Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology,143: 2, 283 — 292To link to this Article: DOI: 10.1080/11263500902722527URL: http://dx.doi.org/10.1080/11263500902722527

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Plant Biosystems

,

Vol. 143, No. 2, July 2009, pp. 283–292

ISSN 1126-3504 print/ISSN 1724-5575 online © 2009 Società Botanica ItalianaDOI: 10.1080/11263500902722527

Micromorphological investigation on the leaves of the rock samphire (

Crithmum maritimum

L.): Occurrence of hesperidin and diosmin crystals

L. CORNARA

1

, C. D’ARRIGO

2

, F. PIOLI

2

, B. BORGHESI

3

, C. BOTTINO

4

, E. PATRONE

2

,

& M. G. MARIOTTI

1

1

DIPTERIS, Polo Botanico “Hanbury”, Università di Genova, Genova, Italy,

2

ISMAC, CNR, Genova, Italy,

3

DIPROVE, Università di Milano, Italy and

4

IENI, CNR, Genova, Italy

Taylor and Francis

Abstract

A micromorphological study was carried out on

Crithmum maritimum

L. (rock samphire), a halophyte belonging to theApiaceae and used in folk medicine. The study led to the finding of relevant amounts of two closely related flavonoids,namely diosmin and hesperidin, which attract increasing interest for their biological properties. Anatomical investigationsof leaf tissue showed the presence of needle-shaped crystals forming plumose clusters, mainly located close to, or within,vascular bundles. Scanning electron microscopy (SEM) analysis provided a more detailed morphological characterizationof crystals, while the absence of mineral elements in their composition was assessed by SEM-EDX. High performanceliquid chromatography (HPLC) analysis of leaf tissue was conducted to detect hesperidin and diosmin and to quantify theiramounts. SEM analysis of two Rutaceae plants rich in hesperidin and diosmin, i.e.,

Citrus limon

and

Barosma betulina

,revealed the same plumose crystals found in

C. maritimum

, while in these plants the two flavonoids were also quantified byHPLC. The data clearly indicate the occurrence of hesperidin and diosmin crystals in

C. maritimum

leaves, thus providinga scientific basis for an exploitation of this plant in saline agriculture, as a crop or source of bioactive phytocompounds.

Keywords:

Leaf anatomy, flavone and flavanone glycosides, organic crystals, SEM-EDX,

Crithmum

Introduction

The benefit of herbal drugs has been highly regardedsince ancient times (Grabley & Thiericke 1999), andeven today many plants are investigated for thedevelopment of new phytocompounds. Rocksamphire,

Crithmum maritimum

L. (Apiaceae), is ahalophytic, succulent, highly branched perennialherb of up to 30–60 cm in height. This aromaticplant grows wild in rock crevices, rocky shores, andshingle beaches along the Mediterranean and BlackSea coasts, as well as along the Atlantic coast ofPortugal, and of south and south-west England,Wales, and southern Ireland. The plant also occursalong the coasts of other countries (e.g. Canada) asa naturalized species.

The leaves of

C. maritimum

are used in folk medi-cine as appetizer, tonic, carminative, diuretic, andvermifuge. Moreover, due to significant amounts of

vitamin C contained in the leaves (Franke 1982),these have been traditionally eaten by sailors as aprotection from scurvy. The diuretic action of

C.maritimum

and its use in renal therapy has beenreported in Pliny the Elder’s

Natural Historia

and inDioscorides’

De Materia Medica

, and long after-wards, this use was also referred to in Plenck’s

IconesPlantarum Medicinalium

(De Santo et al. 2002). Theplant is also used as a condiment and pickle, or asalad ingredient, and according to folk veterinarymedicine in Italy, it is fed to rabbits for preventivepurposes (Viegi et al. 2003).

Recent studies have also reported antimicrobial,antibacterial, antioxidant, and insecticidal activitiesof the essential oil extracted from this plant (Flaminiet al. 1999; Ruberto et al. 2000; Senatore et al.2000; Tsoukatoua et al. 2001). The plant alsocontains mineral elements, with highest levels ofsodium, as well as iodine compounds, pectin, lipids,

Correspondence: M. G. Mariotti, DIPTERIS, Polo Botanico “Hanbury”, Università di Genova, Corso Dogali 1M, I-16136 Genova, Italy. Tel: +39 010 2099376. Fax: +39 010 2099377. Email: m.mariotti@unige.it

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sulpholipids, and polyphenols (Gastaldo 1987;Guil-Guerrero et al. 1998; Guil-Guerrero &Rodríguez-García 1999; Male

[scaron]

et al. 2003).Moreover, the biological properties of rock samphirehave been recognized by the cosmetic (e.g.Block & Wagner 2005, International Patent No.WO2005023212) and the agro-foodstuffs industry(e.g. Tramier & Helmlinger 1991, French PatentNo. FR2650941).

In the present paper, we report novel findingsbased on the micromorphological analysis of

C.maritimum

leaves, in particular the abundance ofneedle-shaped crystals forming plumose aggregates.Investigations of the leaf tissue performed usingscanning electron microscopy (SEM), energy disper-sive X-ray (SEM-EDX), and high performanceliquid chromatography (HPLC) indicate that thesecrystalline structures are formed by hesperidin anddiosmin, two flavonoids of remarkable biologicalinterest. These data have been validated by carryingout similar analyses on two Rutaceae species, i.e.,

Citrus limon

(L.) Burm f. and

Barosma betulina

Bartl.& Wendl. f., which are known for their high contentof the two flavonoids (Tyler et al. 1988; Kanes et al.1993).

Materials and methods

Plant material

Plants of

C. maritimum

were sampled on maritimerocks near Framura along the Ligurian coast (WGS-84 coordinates: Lat. North 44

°

11

′

55

″

, Long. East9

°

33

′

38

″

) from September to November 2005.Specimens of the collected material are kept in theHerbarium (GE) at the University of Genova,Italy. Herbal preparations of dehydrated lemon peel(

C. limon

) and of dried leaves of buchu (

B. betulina

)were obtained from A. Minardi & Figli s.r.l. (Bagna-cavallo, RA, Italy). Fresh peels of green lemon fruitswere from plants growing in the Botanical Garden ofthe University of Genova.

SEM and SEM-EDX

Small pieces of leaves were fixed in FAA (forma-lin:acetic acid:60% ethanol, 5:5:90, v:v:v) for 24hours. Specimens were dehydrated in a gradedethanol series, critical point dried with liquid CO

2

,sputtered with gold, and viewed with a LEO 1450 VPScanning Electron Microscope at an accelerationvoltage of 20 kV. Chemical composition and spectrawere obtained using an OXFORD Inca 300 EnergyDispersive Spectrometer. Microanalysis wasperformed at 20 kV, the working distance was 15 mm,and the chamber vacuum was 6.66

×

10

−

4

Pa. Imageswere obtained with secondary electrons (SEI).

Light microscopy

Handmade sections were made using a razor bladeon fresh leaves. In addition, leaf samples were fixedand dehydrated as described above, and embeddedin JB4 resin (Polyscience Inc., Warrington, PA,USA) in BEEM capsules (Brinn & Pickett 1979).Tissue blocks were sectioned at intervals of 5–10

µ

musing a Reichert Om U2 ultramicrotome equippedwith a glass knife and then stained with ToluidineBlue O (TBO) at pH 4.4, as a metachromatic stain(Feder & O’Brien 1968), and with the Yasue (1969)method for Ca

2+

detection.For fluorescence microscopy, fresh leaves of

C.maritimum

were transversely hand cut, obtainingsections of about 20

µ

m, and then treated with FluorolYellow 088 for lipophilic substances, particularlyterpenes (Brundrett et al. 1991). All sections wereobserved with a Leica DM2000 microscope,equipped with DFC320 camera, and images weredigitized with IM1000 and Qwin software. For fluo-rescence observations a HBO 50 W mercury vapourlamp and a filter block H

3

: BP 420–490 were used.Polarized light was used to detect birefringent crystals.

HPLC characterization of diosmin and hesperidin

A HPLC method for qualitative and quantitativedetermination of diosmin and hesperidin in

C.maritimum

leaves was developed. The identificationof flavonoid glycosides was made by comparing theirretention times and UV spectra with those of stan-dards. The experimental data were compared withthe HPLC analysis of two different kinds of

C. limon

peel, where hesperidin was the major flavonoid, andwith

B. betulina

having similar levels of diosmin andhesperidin.

Diosmin (3

′

,5,7-trihydroxy-4

′

-methoxyflavone 7-rutinoside), hesperidin (3

′

,5,7-trihydroxy-4

′

-methox-yflavanone 7-rhamnoglycoside), rhoifolin (apigenin7-O-neohesperidoside), HPLC-grade methanol,dimethyl sulphoxide (DMSO), and water weresupplied from Sigma-Aldrich (Milan, Italy).

Material from herbal preparations was weighed,broken into small pieces, and powdered. Fresh mate-rial from both rock samphire and lemon peel waspreviously dried at 40

°

C for 96 hours in a stove, thenweighed, broken into small pieces, and powdered.The analyses were performed at room temperatureusing a Beckman System Gold HPLC, equippedwith two programmable pumps, Solvent Module 126and Scanning detector Module 167. The column wasa LiChrosorb RP-18 (10

µ

m) 25 cm long, and aPerkin Elmer HS5 C-18 (5

µ

m), 15 cm long. Themobile phase consisted of eluent A [water/TFA(99.95/0.05, v/v)] and eluent B [methanol/TFA(99.95/0.05, v/v)] with a linear gradient from 13% to

s

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Hesperidin and diosmin in

Crithmum

285

100% of B, at a flow rate of 0.5 ml/minute; thecolumn effluent was monitored at 345 nm.

Stock solutions of diosmin, hesperidin, and theexternal standard rhoifolin were separately preparedby dissolving appropriate amounts in methanol/DMSO (1:1) to achieve concentrations of 1 mg/mlfor each compound. The use of 50% DMSO wasbased on the finding that diosmin and hesperidin aremore soluble in DMSO than in methanol. Calibra-tion standard samples, containing diosmin andhesperidin each at 10, 25, 50, 75, 100, and 150

µ

g/ml, and rhoifolin as external standard at 25

µ

g/ml,were freshly prepared from stock solutions byappropriate dilutions with methanol/DMSO (1:1).Calibrations curves were obtained by plotting theconcentration of each flavonoid against the corre-sponding peak area.

Diosmin and hesperidin were extracted from theanalysed samples by sonicating 500 mg of powderedmaterial, at room temperature for 15 minutes, with4

×

20 ml of methanol/DMSO (1:1). The combinedextract was filtered through 0.45

µ

m PTFE filters,and the volume was adjusted to 20 ml using metha-nol/DMSO (1:1). One ml of this solution wasdiluted to 5 ml with methanol/DMSO (1:1), and anappropriate volume (usually 20

µ

l) was injected intothe HPLC system for analysis.

The extraction of diosmin and hesperidin from allthe examined samples left no detectable amounts ofthe two flavonoids, as confirmed by further extrac-tion and HPLC analysis.

Results

Anatomical and histochemical analyses

Similarly to most halophytic, succulent plants, theleaves of

C. maritimum

are of a greenish-grey colour,due to a thick cuticle and abundant epicuticularwaxes covering the epidermal layers (Figure 1A).Many sunken stomata are distributed on both theupper and lower epidermis (Figure 1B, arrows). Themesophyll consists of a two- to three-cell layeredpalisade tissue, and of an abundant water storageparenchyma with vascular bundles (Figure 1B), andsecretory canals lined with specialized epithelial cells(Figure 1B, arrowheads; Figure 1C). The secretorycanals are of variable sizes (Figure 2A and B,arrows), the largest ones typically located nearthe vascular bundles, in particular at the leaf tips(Figure 2A, arrow), while the smallest ones tend tobe located just under the epidermal layers. Theproduction and release of essential oil into the lumenof the ducts were revealed by using Fluorol Yellowstaining (Figure 2C–D). In addition, this dye high-lighted the thickness of the cuticular layer overlayingthe epidermal cells (Figure 2C, arrow).

Figure 1. SEM micrographs of

C. maritimum

leaf. (A) Stomata sunken in the epidermal layer, covered by epicuticular waxes. (B) Transverse section of a leaf, showing the mesophyll, the main vascular bundle and two secretory ducts (arrowheads). Both the upper and the lower epidermisshow sunken stomata (arrows). (C) A secretory duct, lined with specialized epithelial cells, and located within the leaf parenchyma. (A–C) bar = 0.1 mm.

Within the mesophyll, plumose crystal aggregateswere also visible within vascular bundles, and inparenchyma cells located around vascular bundlesand secretory ducts (Figure 2E–G). These crystalsappeared birefringent under polarized light (Figure2F) and did not react using the Yasue (1969) method

Figure 1. SEM micrographs of C. maritimum leaf. (A) Stomatasunken in the epidermal layer, covered by epicuticular waxes. (B)Transverse section of a leaf, showing the mesophyll, the mainvascular bundle and two secretory ducts (arrowheads). Both theupper and the lower epidermis show sunken stomata (arrows).(C) A secretory duct, lined with specialized epithelial cells, andlocated within the leaf parenchyma. (A–C) bar = 0.1 mm.

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Figure 2. Light (A–B; E–H) and fluorescent (C–D) micrographs of C. maritimum leaves. (A–B; E–F) TBO pH 4.4; (C–D) Fluorol yellow.(A–B) Secretory ducts of different sizes are visible within the mesophyll. (C–D) Fluorol yellow staining of the essential oil, and of the thickcuticular layer (C, arrow). (E) Fair amounts of crystalline aggregates with the characteristic plumose shape, and their location close to thevascular bundles are shown. (F) The crystals are birefringent under polarized light. (G) Hand-cut section of a fresh leaf showing acicularcrystal needles near the phloem in the vascular bundle and in the parenchyma surrounding it (arrows). (H) Hand-cut section of a leafshowing a crystal needle aggregate within an epidermal cell (arrow). (A–C) bar = 15 µm; (D) bar = 4 µm; (E–G) bar = 6 µm; and (H)bar = 18 µm. (The colour version of this figure is available online.).

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Hesperidin and diosmin in

Crithmum

287

for Ca

2+

detection. The crystals were also visible infresh leaf sections and appeared mainly located nearthe phloem in the vascular bundles (Figure 2G) andwithin the epidermal layer (Figure 2H).

Figure 2. Light (A–B; E–H) and fluorescent (C–D) micrographs of

C. maritimum

leaves. (A–B; E–F) TBO pH 4.4; (C–D) Fluorol yellow. (A–B) Secretory ducts of different sizes are visible within the mesophyll. (C–D) Fluorol yellow staining of the essential oil, and of the thickcuticular layer (C, arrow). (E) Fair amounts of crystalline aggregates with the characteristic plumose shape, and their location close to the vascular bundles are shown. (F) The crystals are birefringent under polarized light. (G) Hand-cut section of a fresh leaf showing acicular crystalneedles near the phloem in the vascular bundle and in the parenchyma surrounding it (arrows). (H) Hand-cut section of a leaf showing a crystal needle aggregate within an epidermal cell (arrow). (A–C) bar = 15

µ

m; (D) bar = 4

µ

m; (E–G) bar = 6

µ

m; and (H) bar = 18

µ

m. (The colourversion of this figure is available online.).

SEM observations yielded detailed views of theneedle-shaped crystals (Figure 3A–B), showing thatthey frequently end with a small hook at their tips(Figure 3C, arrow). In these observations, crystals

Figure 3. SEM micrographs of C. maritimum leaf (A–C); C. limon peel (D); and B. betulina leaf (E–F). In the C. maritimum leaf, crystalaggregates are visible within the vessels of the vascular bundles (A) and in neighbouring cells of the parenchyma (B). Crystals are formedby aggregates of numerous needle-shaped crystals showing a small hook at their tips (C, arrow). Similar crystals are visible within theparenchyma cells of C. limon peel (D, arrows). In the B. betulina leaf, many crystals, either isolated or clusters, are visible in the mesophyllbeneath the epidermis (E). Spherical aggregates of crystals can be found within epidermal cells (F). (A–B) bar = 3 µm; (C–E) bar = 10µm; and (F) bar = 2 µm.

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L. Cornara et al.

were also found within the vessels of vascularbundles (Figure 3A), as well as in parenchyma cells(Figure 3B). Crystals, quite similar to those found in

C. maritimum

leaves, were also observed in samplesfrom herbal preparations and fresh specimens oflemon peels (Figure 3D, arrows), as well as insamples from herbal preparations of buchu leaves(Figure 3E and F).

Figure 3. SEM micrographs of

C. maritimum

leaf (A–C);

C. limon

peel (D); and

B. betulina

leaf (E–F). In the

C. maritimum

leaf, crystal aggregates are visible within the vessels of the vascular bundles (A) and in neighbouring cells of the parenchyma (B). Crystals are formed byaggregates of numerous needle-shaped crystals showing a small hook at their tips (C, arrow). Similar crystals are visible within the parenchyma cells of

C. limon

peel (D, arrows). In the

B. betulina

leaf, many crystals, either isolated or clusters, are visible in the mesophyll beneath theepidermis (E). Spherical aggregates of crystals can be found within epidermal cells (F). (A–B) bar = 3

µ

m; (C–E) bar = 10

µ

m; and (F) bar = 2

µ

m.

SEM-EDX analysis

The only elements detected by SEM-EDX in

C.maritimum

crystals were carbon and oxygen (Figure4A). Conversely, no trace of calcium and silicon,commonly found in plant crystals, was found. Asimilar analysis performed on the crystals present inlemon peels (Figure 4B), and in buchu leaves(Figure 4C), yielded the same result. In contrast,EDX-analysis of typical calcium oxalate druses, scat-tered among the mesophyll of buchu leaves, showeda high Ca

2+

signal (Figure 4D).

Figure 4. SEM-EDX analysis of crystals found in

C. maritimum

leaf (A);

C. limon

peel (from the Botanical Garden) (B); and

B. betulina

leaf (C–D). The elemental composition of crystals only shows the presence of carbon and oxygen (A–C), whereas the same analysis of a typicaldruse of calcium oxalate, located within the leaf parenchyma of

B. betulina

, shows a high peak of calcium.

HPLC analysis

A typical chromatogram of a mixture of standarddiosmin (tR, 30.9 minutes) and hesperidin (tR, 27.4minutes) is shown in Figure 5. For the simultaneousdetermination of diosmin and hesperidin, twocalibration graphs were obtained over the ranges of10–150

µ

g/ml. The regression analysis of experimen-tal data points showed a linear relationship, withexcellent correlation coefficient (R > 0.999), and thelinear regression equations were:

where Y is the peak area and X is the concentration.

Figure 5. HPLC chromatogram of a mixture at 10

µ

g/ml of hesperidin and diosmin standards.

Typical chromatograms of extracts from

C. marit-imum

leaves,

B. betulina

leaves, and

C. limon

peels(from herbal preparations and from the BotanicalGarden of the University of Genova) are shown inFigure 6A–D. Peaks due to the two flavonoids werewell resolved from each other and from other co-extracted materials. The concentrations of diosminand hesperidin found in the extracts are shown inTable I.

Figure 6. HPLC chromatograms of

C. maritimum

leaves (A);

B. betulina

leaves (B);

C.

limon

peel herbal preparation (C); and

C. limon

from the Botanical Garden (D). Note: h = hesperidin and d = diosmin. (For operating conditions see Materials and Methods.)

Hesperidin was the major flavonoid glycosidefound in the two

Citrus

peels tested: 1.96% (herbalpreparation) and 3.49% (Botanical Garden).Diosmin and hesperidin were in equal concentrationsin

C. maritimum leaves (about 0.6% w/w), and inalmost equal concentrations, though at higheramounts, in B. betulina leaves (diosmin 2.64%,hesperidin 2.84%).

Discussion

Diosmin and hesperidin have similar structures andbiological activities and are widely used to improvethe resistance of blood vessels (Michiels et al. 1991;Stantus et al. 1991; Hitzenberger 1997), and formedical and clinical applications such as diuretic,anti-hypertensive, and hypolipidemic activities(Manforte et al. 1995; Galati et al. 1996). In addition,these flavonoids have been proposed as cancer-preventing agents, e.g., on colon and urinary bladdercancer (Tanaka et al. 1997; Yang et al. 1997).

In plant anatomical studies, hesperidin anddiosmin have been described as needle-shaped orplumose crystals, and due to their similar appear-ance they have been often confused with each other.Diosmin has been mentioned by pharmacognosistsas yellow dendritic, plumose, or spheric crystals inbuchu leaves (Solereder 1899). Solereder (1908)and Metcalfe and Chalk (1950) refer to plumosecrystals of a substance thought to be hesperidin innine genera of Apiaceae (Aethusa, Bupleurum,Chaerophyllum, Conium, Coriandrum, Cuminum,Oenanthe, Sium, and Trinia). Spherical aggregationsof yellowish acicular crystals were also noted at Kewin the epidermis and vessels of the petiole of C.maritimum (Metcalfe & Chalk 1989), but noevidence of this fact has been provided until now.

Based on these literature data, the morphologicalobservations of the present study provide a strongindication that the crystals found in C. maritimumleaves are formed by hesperidin and diosmin.Furthermore, the elemental composition of thecrystals, obtained by SEM-EDX analysis, was verysimilar to those obtained for the crystals of lemon peeland buchu leaves. In agreement with these data, ourHPLC analyses showed the occurrence of diosminand hesperidin in the leaves of C. maritimum, and thisfinding was confirmed by the detection of the sameflavonoids in lemon peel and buchu leaves. OurHPLC data also indicate that the extraction methodplays an important role in the detection and quanti-fication of these flavonoids. The high recovery of ourmethod derives from the use of 50% DMSO, whereasin a study by El-Shafae and El-Domiaty (2001),made on buchu leaves, the use of 10% DMSO yieldedlower amounts of diosmin and hesperidin (0.82%and 0.12%, respectively). This hypothesis is consis-tent with the reports of Saleh et al. (1983), who usingalcoholic extraction on C. maritimum were able tofind luteolin, quercetin, and kaempferol, whereasneither diosmin nor hesperidin were detected.

In the aerial parts of C. maritimum, the content inflavonoids, tannins, and polyphenols show highvariability as a function of growth site and stage(Male[scaron] et al. 2003). Therefore, the high amounts ofcrystals detected within the leaves could be related

Y X dios

Y X hesperidin

= += +

2 07247 1 59877

0 25529 0 19273

. . ( min)

. . ( )

s

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Hesperidin and diosmin in Crithmum 289

Figure 4. SEM-EDX analysis of crystals found in C. maritimum leaf (A); C. limon peel (from the Botanical Garden) (B); and B. betulinaleaf (C–D). The elemental composition of crystals only shows the presence of carbon and oxygen (A–C), whereas the same analysis of atypical druse of calcium oxalate, located within the leaf parenchyma of B. betulina, shows a high peak of calcium.

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290 L. Cornara et al.

to the growth stage of the examined plants, i.e., theend of the flowering period and beginning of fruitripening.

The location of diosmin and hesperidin in theleaves of C. maritimum is in agreement with literaturedata. For example, in Hyssopus officinalis L. diosminwas found to occur at highest levels in aerial parts,mainly in sepals and leaves, and the amounts ofthese flavonoids gradually increased with age (Marinet al. 1998). As for flavonoid translocation, Castilloet al. (1992) have shown the presence of substantialconcentrations of two flavonoids, neohesperidin andnaringin, in the vascular fluids of Citrus aurantiumL., and have hypothesized that both compounds canbe transported from the leaves to other plant organs.In accord with this view, a study on Rosmarinusofficinalis L. reported a relevant amount of hesperi-din and diosmin in the vascular system (Del Bañoet al. 2004), while flavonoid translocation has beenFigure 5. HPLC chromatogram of a mixture at 10 µg/ml of

hesperidin and diosmin standards.

Figure 6. HPLC chromatograms of C. maritimum leaves (A); B. betulina leaves (B); C. limon peel herbal preparation (C); and C. limonfrom the Botanical Garden (D). Note: h = hesperidin and d = diosmin. (For operating conditions see Materials and methods.)

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Hesperidin and diosmin in Crithmum 291

suggested to occur through the phloem in a study byMacleod and Pridham (1966) based on the aphid-head technique. Hence, literature data seem tosuggest that flavonoids are synthesized in the leavesor aerial parts, and transported to other organsthrough the phloem. Our observations on freshleaves are in agreement with this hypothesis sincecrystals were found near the phloematic tissue ofvascular bundles. The SEM analysis revealed thatcrystals were also visible within the xylem vessels ofvascular bundles, but such a location could alsodepend on SEM procedures, taking into account theclose proximity of phloem elements and xylemvessels.

Taken together, the data obtained in this studyindicate that C. maritimum is able to synthesize andaccumulate hesperidin and diosmin in its leaves. Thisfinding provides a scientific basis to the traditionaluses of this plant (e.g. in relation to its anti-scurvyproperties) since bioflavonoids are known to exertvitamin C like effects. In addition, the use of the plantas a diuretic, well known since ancient times(Dioscorides, I sec. A.D.), can be associated with thediuretic properties recently reported for these twoflavonoids (Galati et al. 1996). The occurrence in C.maritimum leaves of these flavonoids, as well asthe presence of essential fatty acids and carotenes(Guil-Guerrero & Rodríguez-García 1999), and of anessential oil with antioxidant and antibacterial prop-erties (Ruberto et al. 2000; Senatore et al. 2000),highlight the need to revaluate C. maritimum as aninteresting source for the production of pharmaceu-ticals and nutraceuticals. In addition, this is an ediblespecies showing important ecological features, beinga perennial halophyte able to grow on alkaline andsaline soils, and to tolerate maritime exposure. Forthis reason, this plant could represent a new potentialcrop in saline environments, where only few plantspecies can survive.

Acknowledgements

We thank Dr Cecilia Balbi (“La Massocca” CulturalAssociation, Framura, SP, Italy) for her encourage-ment and helpful comments.

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s Z Z c s c c

Table I. Diosmin and hesperidin (% w/w) in C. maritimum leaves,C. limon peel, and B. betulina leaves.

Compound Diosmin Hesperidin

Crithmum maritimum 0.60 (±0.02%) 0.63 (±0.02%)Citrus limon (herbal preparation) 0.22 (±0.03%) 1.96 (±0.01%)Citrus limon (botanical gardens) 0.34 (±0.03%) 3.49 (±0.01%)Barosma betulina (herbal

preparation)2.64 (±0.09%) 2.84 (±0.06%)

Note: Results are the average of five determinations and SD%values are given in parenthesis.

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