Pharmacognostical Comparison of Three Species of Himatanthus

MATERIALS AND METHODS

Plant material: Botanical specimens were collected (2003) in the Adolpho Ducke Reserve, Amazonas, Brazil, by Silo Soares. Vouchers are deposited in the INPA Herbarium under accession numbers 200263 (H. bracteatus), 180485 (H. stenophyllus) and 209858 (H. sucuuba).

Morphological methods: Fresh specimens used for macroscopic descriptions were studied with a hand lenses or through the naked eye inspection. Anatomical studies were made on histological transverse sections from the lower mid-third portion of the leaf lamina and from the bark pieces. The leaf blades were sectioned with a Ranvier microtome and then diaphanized by the sodium hypochlorite and stained by the double coloration method (Dop and Guatie, 1928), using Iodine Green and Congo Red. Slides were mounted in glycerin, examined and photographed with a scale on an Olympus BX40 microscope equipped with microphotographic system.

Chromatographic analysis: The ground leaves and barks of each Himatanthus species were extracted by infusion and decoction, respectively (2:10, plant: H₂O, w/v). After 30 min, the mixtures were filtered and lyophilized. The latex of each species was centrifuged for 5 min (3000 rpm) and the aqueous fraction was lyophilized. From aqueous extract of latex of H. sucuuba, the iridoids, plumieride and isoplumieride, could be isolated in pure form by medium-pressure liquid chromatography and HPLC analyses and identified by NMR spectroscopy. The solutions of the extracts and the iridoids (10 and 3 mg mL^-1 (plumieride) and 1 mg mL^-1 (isoplumieride), respectively) were prepared using methanol as solvent. Five microliter (5 μL) of the extracts and iridoids were loaded as 4 mm streak on the plate at 4 mm distance between two streaks, using Linomat IV, a Camag semi-automatic spotter.

The analysis were carried out on precoated silica gel 60 F₂₅₄ (Merck) HPTLC plate (0.2 mm of layer thickness and 10 x 10 cm size) using CHCl₃:MeOH (8:2) as developing system. Plate was dried and the spots were visualized by spraying with detection reagent (vanillin-H₂SO₄), followed by heating at 110°C.

RESULTS AND DISCUSSION

The leaves from three species of Himatanthus from the Ducke Reserve showed the typical morphological characteristics of both this genus and the Apocynaceae´s family, as cited by Barros (1988) and Larrosa and Duarte (2005a). Being confirmed by microscopic analysis, it showed that the mesophyll was dorsiventral (Fig. 1a, c , e), the midvein had bicollateral vascular bundles and non-articulated laticifer ducts that sometimes accompanied the vascular bundles. The mesophyll varied from 1-2 layers of palisade parenchyma, with predominance of the 2-layered type (Fig. 1a, c, e). Solereder (1908), Newcombe and Patel (1966) and Fjell (1983) described idioblasts with crystals in the leaves of various species of Apocynaceae and Larrosa and Duarte (2005a) noted their occurrence in H. sucuuba, however crystals with a specific, well-defined origin were not observed in the mesophyll and midvein of any of the species of Himatanthus being studied. The existence of isolated groups of phloem in the medullar parenchyma, observed by Barros (1988) in H. lancifolius, was noted in the three species.

As to the macroscopic aspects, the leaves of the three species were coriaceous in texture. The shape of the laminas varies, being usually spatulate or elliptic in H. sucuuba (Fig. 2a); oblong-elliptic in H. bracteatus (Fig. 2b) and often larger in size in this latter species than in the other two species. H. stenophyllus showed lanceolate laminas (Fig. 2c), yet the three species had attenuate leaf bases. The leaf apices were more acute or rounded in H. sucuuba (never obtuse) (Fig. 2a) and cuspidate in H. bracteatus (Fig. 2b) and in H. stenophyllus (Fig. 2c). Important differences were observed in leaf architecture: in H. sucuuba, the secondary veins present the midvein at an acute angle, arched and unite to form a submarginal vein 2-3 mm from the margin; in H. bracteatus the secondary, veins present the midvein at a less acute angle than in H. sucuuba and are straight or only slightly arched; in H. stenophyllus, the secondary veins present the midvein nearly at right angles and unite into a submarginal vein close to the margin (Ribeiro et al., 1999). The study of the petiole showed that this is canaliculate in H. sucuuba and H. stenophyllus, plane or somewhat plane on the upper surface in this latter species. H. bracteatus has a cylindrical petiole which is flattish on the upper surface and rounded-convex on the lower surface.

The barks of Himatanthus stenophyllus (Fig. 1f) and H. sucuuba (Fig. 1b) exhibited a large quantity of prismatic calcium oxalate crystals that had already been observed in this latter species (Costa et al., 2002; Larrosa and Duarte 2005b). Larrosa and Duarte (2005b) describe calcium oxalate druses in the stem cortex of H. sucuuba, however this crystalline form was not detected in the bark of this species from the ducke reserve specimens. The cortex of the three species had starch grains as reserve substance as noted for H. sucuuba bark.

Fig. 1:	H. sucuuba: (a) dorsiventral mesophyll with vascular bundles, (b) cortical and phloem regions with sclereids and prismatic calcium oxalate crystals; H. bracteatus: (c) dorsiventral mesophyll, (d) cortical region with laticifers and sclereids; H. stenophyllus: (e) dorsiventral mesophyll with vascular bundles and (f) cortical region with sclereids and prismatic calcium oxalate crystals. pp: Palisade parenchyma, vb: Vascular bundles, ae: Adaxial epiderm, oc: Prismatic calcium oxalate crystals, ld: Laticifer ducts, st: Sclerenchymatous tissue, c: Cork

Although the vascular system of Himatanthus is typically bicolateral, as typical characteristic observed in the Apocynaceae, this could not be verified in the bark fragment due to its thinness (the usual thickness of the commercial bark), which does not show internal phloem. Non-articulated laticifers were present in the cortical (Fig. 1b, d, f) and phloematic regions of the barks, a universal character of this family and abundant sclerenchymatous tissue was found in the cortical and phloematic parenchyma of the bark of the three species (Fig. 1b, d, f) (Larrosa and Duarte, 2005b).

The bark fragments showed a plane surface in H. sucuuba (Fig. 2d) and a curved surface in H. bracteatus and in H. stenophyllus (sometimes in scroll-like shape in this latter species) (Fig. 2e, f). The three species showed a lighter-colored internal surface. The surface of transversal fracture was granulose in the external portion and fibrous in the internal portion in H. sucuuba, shiny in H. bracteatus and slightly granulose in H. stenophyllus.

Therefore, the morphological analysis of the adult leaves added to the barks of Himatanthus sucuuba, Himatanthus bracteatus and Himatanthus stenophyllus provided the basis for differentiating the species. The contour of the leaf lamina, apex and the petiole aspects and the veination patterns were important features for the leaves. With regard to the barks, the inner, outer and transversal fracture surfaces must be conjointly analyzed. By the microscopic analysis of barks collected twice, the main difference among the species was the lack of the prismatic calcium oxalate crystals in the H. bracteatus.

Fig. 2:	H. sucuuba: (a) leaf abaxial face, (b) leaf adaxial face, (c) leaf abaxial face (d) bark inner surface, H. bracteatus: (e) bark inner surface, H. stenophyllus: and (f) bark outer and inner surface

Fig. 3:	HPTLC fingerprint of H. bracteatus leaves, bark and latex extracts (1, 2, 5), H. stenophyllus leaves, bark and latex extracts (3, 4, 6), H. sucuuba latex, leaves and bark extracts (7, 8, 9). P: Plumieride, I: Isoplumieride. [CHCl3:MeOH (8:2)]

HPTLC chromatographic profile: Preliminary samples evaluations were made with silica gel and RP-18 TLC for the purpose of choosing the phase and solvents system, before the utilization of HPTLC. For the silica gel TLC, there were applied the solvents systems CH₂Cl₂:AcOEt (7:3), CHCl₃: AcOEt :MeOH (7:2:1), CHCl₃:MeOH (8:2) and for the RP-18 TLC, H₂O with 0,05% TFA: MeOH (6:4) and H₂O with HOAc 0,1%: CH₃CN (7:3). The best chromatographic condition used to develop the HPTLC fingerprint was silica gel 60 F₂₅₄ (Merck) plate (0.2 mm of layer thickness and 10x10 cm size) using CHCl₃:MeOH (8:2) (Fig. 3). The chromatographic analysis of the extracts of the plants exhibited considerable differences in the profile. In relation to the revelation of the iridoids, the chromatogram showed in a first moment a characteristic green color and after 2 min under 110°C heat, the color of these substances has been intensified, resulting in brown or green-brown zones. In the chromatographic conditions mentioned, the R_f values found for these substances were 0.35 (plumieride) and 0.26 (isoplumieride). The spots present in the extracts at Rfs above showed quenching like that of the both iridoids. All samples loaded on the plate had plumieride presence. Under analysis conditions, this substance appeared in larger quantity in samples 1, 5, 7 and 8 and in smaller proportions in samples 2, 4 and 6 (Fig. 3). The isoplumieride was found in smaller proportions in all samples.

CONCLUSION

The analysis of the leaves, bark and latex of the three Himatanthus species studied by HPTLC presented different profiles for the samples and correlation with two important iridoids of this genus through simple and rapid procedure. Additionally, the morphological analyses of the leaves and bark provided data for differentiation among the species by means of the characteristics botanical.

ACKNOWLEDGMENTS

The researchers would like to thank CAPES (RENOR Project), CNPq/PADCT Project 620072/2004-0 ACT, FAPEAM and FIOCRUZ (Accord number 01/1998) for the financial support and June Justa by the revision of this study.