Abstract
Context: Viscum triflorum DC. (Viscaceae) is a hemiparasitic plant used in traditional medicine on Réunion Island as a remedy to treat hypertension.
Objective: The in vitro angiotensin-converting enzyme (ACE) inhibitory activity of extracts of V. triflorum and the corresponding host plant species were examined to evaluate the use as a remedy against hypertension, and to investigate whether the host plants have an influence on the activity.
Materials and methods: Aqueous, ethanol and acetone extracts of 24 leaf samples of V. triflorum and the corresponding host plants, representing 10 plant species, were prepared. The ACE inhibitory activities of the extracts were measured by HPLC using dansyltriglycine as substrate.
Results and conclusion: Water extracts of Viscum samples from only two of the 10 host plants, namely Acacia heterophylla Willd. (Fabaceae) and Sophora denudata Bory (Fabaceae), showed significant inhibitory activity, ≥50% inhibition in a concentration of 0.33 mg crude plant extract in 1 mL test solution. From the two mentioned host plant species activity was only detected in the water extract from one of the six samples of A. heterophylla. Three host species showed pronounced activity without any detection of activity in the samples of V. triflorum. The results support the traditional use provided that V. triflorum is collected from A. heterophylla or S. denudata.
Introduction
The genus Viscum L. comprises about 150 species, all evergreen hemiparasitic plants rooted in the tissue of the host plants (CitationHeide-Jørgensen, 2008). The best known species is the European Viscum album L. (Viscaceae). This species is used for several medical purposes and the main areas of therapeutic applications are against hypertension, arteriosclerosis, cancer, and arthrosis (CitationWagner et al., 1986).
It has been previously demonstrated that V. album from different host plant species have different biological activities. In vitro studies of V. album have shown a host plant-dependent anti-inflammatory effect (CitationYesilada et al., 1998), antioxidant activity (CitationVicaş et al., 2008); and three preparations produced from V. album from different host trees exhibited different effects on human leukemia cells (CitationHülsen et al., 1986).
Several species of Viscum and other species belonging to the Viscaceae and Loranthaceae families are used for medical purposes such as a remedy to treat hypertension. Viscum triflorum DC. (Viscaceae) is used for several purposes in traditional medicine on Réunion Island and infusions are among other things used as an antihypertensive (CitationLavergne and Véra, 1989; CitationLavergne, 1990). In Argentine folk medicine, Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae) is used as a substitute for the European mistletoe and infusions of L. cuneifolia leaf and stem are used to treat high blood pressure (CitationFernández et al., 1998). The Japanese mistletoe, Taxillus yadoriki Danser (Loranthaceae) has been prescribed as a hypotensive or antidiabetic folk medicine in Japan (CitationFukunaga et al., 1989).
The use of varieties of V. album as a hypotensive remedy has been supported in in vivo studies. Crude aqueous leaf extract of V. album produced significant decrease in blood pressure in renal artery-occluded hypertensive rats, salt-induced hypertensive rats, and normotensive rats (CitationOfem et al., 2007) and aqueous leaf extract of Korean mistletoe, V. album L. var. coloratum Ohwi, was effective in the treatment of spontaneously hypertensive rats (CitationKim, 2006).
In a previous study, we have shown that different samples of the hemiparasitic epiphytes Cassytha filiformis L. (Lauraceae) and V. triflorum showed different ability to inhibit the angiotensin-converting enzyme (ACE) that plays an important role in the regulation of blood pressure and diuresis (CitationAdsersen and Adsersen, 1997). We had no opportunity to examine the corresponding host plants.
V. triflorum is an epiphytic branch parasite with Acacia heterophylla Willd. (Fabaceae) and Dombeya species (Sterculiaceae) as the most common host trees, it is indigenous to Réunion Island. The objective of the present study was to investigate the in vitro ACE inhibitory activity of samples of V. triflorum and their corresponding host plants to address the following questions:
Does the activity of V. triflorum depend on the host plant species on which it grows?
Does activity of the host imply activity in V. triflorum?
Is the traditional use of V. triflorum as a remedy against hypertension justified?
Materials and methods
Plant material
Leaf samples were collected at Maido, Forêt de Bebour and Plaine de Chicots, Réunion Island, in February 1997. Samples of V. triflorum were collected from 24 host plants representing 10 different plant species, all endemic to Réunion Island, and air-dried immediately after the collection. Voucher specimens of V. triflorum and the host plants are deposited at the Faculty of Pharmaceutical Sciences, University of Copenhagen. Identification was done by Thierry Pailler, Laboratoire de Biologie Végétale, l’Université de la Réunion. The material was collected according to the general collection authorization to this laboratory. The plant material was analyzed at the Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen in 1997.
Preparation of crude extracts
Of the dried powdered plant material, 1 g was extracted with 10 mL water, ethanol (96%), or acetone separately for half an hour in an ultrasonic bath. The extracts were filtered and concentrated in vacuo except for the water extracts, which were freeze-dried. Test solutions were made by dissolving 1 mg dry extract in 1 mL HEPES assay buffer, or buffer with 10% ethanol or acetone, corresponding to a final concentration of 0.33 mg crude plant extract in 1 mL test volume.
ACE inhibitory activity assay
In vitro ACE inhibitory activity was measured as described by CitationElbl and Wagner (1991) and later modified by CitationHansen et al. (1995). Dansyl l-glutamic acid, dansylglycine, and ACE from rabbit lung (EC 3.4.15.1; 3 U/mg protein) were obtained from Sigma. Dansyl triglycine was synthesized in our laboratory as described by CitationHansen et al. (1995). Captopril was used as a positive standard; the IC50 value was 12.0 ± 2.6 nM.
Instrument
HPLC determination of ACE inhibitory activity was performed using a Shimadzu LC 10 AS pump, a Shimadzu SIL 10 A injector, a Shimadzu SPD 10 A UV spectrophotometric detector, a Shimadzu SCL 10 A system controller, and a Shimadzu C-R 6A chromatopac integrator.
Results
Samples were considered active if ACE inhibition was 50% or more in one of the three extracts in a concentration of 0.33 mg crude extract in 1 mL test solution. Results are presented in .
Table 1. Angiotensin-converting enzyme (ACE) inhibitory activity of studied plant species.
Viscum samples from two of the 10 host plant species showed pronounced ACE inhibitory activity in the water extracts. All six samples of V. triflorum with A. heterophylla as host plant showed activity with inhibition in the range 64–87%. Only one of the corresponding host plants showed activity with 52% inhibition in the water extract. V. triflorum with Sophora denudata Bory (Fabaceae) as host was active in the water extract with 86% inhibition; there was no inhibition in the extracts from the host.
Pronounced ACE inhibitory activity was detected in the host species Monimia rotundifolia Thou. (Monimiaceae), Dombeya ciliata Cordem. (Sterculiaceae), and Dombeya filcunea Baill. (Sterculiaceae) without any detection of activity in the corresponding samples of V. triflorum. The two samples of M. rotundifolia showed activity in the water and ethanol extract and one of the samples was active in the acetone extract as well. Of the 11 Dombeya samples, representing four species, two species showed activity and only in the ethanol extract namely one of the three samples of D. filcunea and both samples of D. ciliata.
Discussion
Our bioassay analyses show that the ACE inhibitory activity of V. triflorum is host plant-dependent but apparently there is no connection between ACE inhibitory activity in the host and the corresponding sample of V. triflorum, only one of the six samples of A. heterophylla showed activity as well.
All samples of V. trifolium from A. heterophylla and the sample from S. denudata showed pronounced ACE inhibitory activity in the water extract. Both host plant species belongs to the plant family Fabaceae (subfamily Mimosoideae and Faboideae, respectively).
In V. album, several subspecies have been recognized. The nominal subspecies V. album ssp. album parasitizes angiosperm species, whereas V. album ssp. abietis Abrom. is found on Abies (fir) and V. album ssp. austriacum Vollm. is found on Pinus (pine). Apart from this, it has not been possible to demonstrate host specificity, but there is some evidence that V. album plants parasitizing different host species can be separated by their DNA (CitationZuber and Widmer, 2000). This has not been studied in V. triflorum. If a similar pattern can be detected here the results above may be due to differentiation in the parasite, rather than a direct induction from the host plant.
The activity levels were only consistently high in V. triflorum from the Fabaceous trees A. heterophylla and S. denudata. The results justify the use of V. triflorum as an antihypertensive remedy provided that it has been collected from the two mentioned species.
Many Viscum spp. and other Loranthaceae are in focus as drugs or producers of chemicals of pharmacological interest. Our results show that it is important in such studies to take into account the identity of the host plants and the host–parasite relations.
Acknowledgement
We thank Dr. Dominique Strasberg and Thierry Pailler, Laboratoire de Biologie Végétale, l’Université de la Réunion for help with this project.
Declaration of interest
The authors report no declarations of interest.
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