from Viscum album L. subspecies growing in Turkey are pre- sented in this paper. Introduction. In Turkey, Viscum L. (Loranthaceae) is represented by one.
Pharmaceutical Biology 2001, Vol. 39, No. 5, pp. 381–383
1388-0209/01/3905-381$16.00 © Swets & Zeitlinger
Evaluation of Antimycobacterial Activity of Viscum album Subspecies D. Deliorman1, F. Ergun1, B. S¸ener1 and P. Palittapongarnpim2 Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Ankara, Turkey and 2Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand
1
Abstract Continuing our investigations to search for antimycobacterial drugs from Turkish plants, the results of samples prepared from Viscum album L. subspecies growing in Turkey are presented in this paper.
Keywords: Antimycobacterial activity, Loranthaceae, subspecies, Viscum album L.
Materials and methods Introduction
Plants
In Turkey, Viscum L. (Loranthaceae) is represented by one species (V. album L.) and three subspecies, namely, ssp. album, ssp. abietis (Wiesb.) Abromeit and ssp. austriacum (Wiesb.) Vollmann, and known as “Ökse Otu” in Turkish (Davis, 1982; Ergun et al., 1997). The plant is semi-parasitic, growing on various host plants, such as trees and shrubs (Davis, 1982). V. album herbs are used for the treatment of various diseases. Their immunostimulatory, antitumor, hypotensive and anti-inflammatory effects have been recorded in the literature (Yes¸ilada et al., 1998). Tuberculosis is still a health problem that requires multidrug therapy. Isoniazid, rifampin, rifabutin and pyrazinamide have been used in the treatment of tuberculosis. Several factors, including immune disorders and drug malabsorption, have been resulted in drug-resistant tuberculosis. Single drug-resistant tuberculosis is relatively easy to treat, compared to multidrug-resistant tuberculosis. Therefore, the search for anti-mycobacterial drugs is an important component of new drug discovery programs. During our evaluation of Turkish plants, activity against Mycobacterium tuberculosis strain H37Ra using the microplate Alamar blue assay (MABA) has been determined. We currently report the results obtained with extracts prepared from Viscum album L. subspecies.
The host plants, localities, collection time, and herbarium numbers of V. album L. subspecies were as follows: ssp. album from Armeniaca vulgaris Lam. (apricot); Ankara, Baglum, June 1993 (AEF 18953); ssp. abietis (Wiesb.) Abromeit from Abies bornmülleriana Mattf. (fir), Bolu, Gölcük, April 1993 (AEF 18947); ssp. austriacum (Wiesb.) Vollmann from Pinus nigra Arn. ssp. pallasiana (pine), Ankara, Kızılcahamam, April (AEF 18939). Voucher specimens are deposited in the herbarium of Ankara University, Faculty of Pharmacy. Preparation of samples Plant materials were dried under shade and powdered. Each sample (1 kg) was extracted with 80% ethanol (25 l) at room temperature several times, based on tlc profiles. Combined ethanol extracts were evaporated to dryness in vacuo and dissolved in distilled water. The aqueous extract was fractionated by successive solvent extraction with petroleum ether (40–60 °C), diethylether, ethyl acetate and n-butanol (for each solvent 5 ¥ 1 liter). Each fraction was then evaporated to dryness in vacuo. The yields of the extracts and fractions were as follows: ssp. album; 432 g (EtOH) (43.20%), 6.1 g (pet. ether) (0.61%), 3.2 g (ether) (0.32%), 13.5 g (EtOAc)
Accepted: January 30, 2001 Address correspondence to: Prof. Dr. Fatma Ergun. Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, 06330, Ankara, Turkey. Fax: +90-312-2235018.
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D. Deliorman et al. Table 1. Antituberculosis effects of extracts of Viscum album subspecies against Mycobacterium tuberculosis H37Ra.
Subspecies (Extracts) ssp. austriacum (n-BuOH) ssp. album (n-BuOH) ssp. abietis (n-BuOH) ssp. austriacum (EtOAc) ssp. album (EtOAc) ssp. abietis (EtOAc) ssp. album (Et2O) ssp. austriacum (Et2O) ssp. abietis (Et2O) ssp. austriacum (PE) ssp. album (PE) ssp. abietis (PE) ssp. austriacum (EtOH) ssp. album (EtOH) ssp. abietis (EtOH)
Sample labeled
Results against TB at 200 mg/ml
MIC (mg/ml)
DD-1 DD-2 DD-3 DD-4 DD-5 DD-6 DD-7 DD-8 DD-9 DD-10 DD-11 DD-12 DD-13 DD-14 DD-15
Inactive Inactive Inactive Inactive Inactive Inactive Active Active Active Active Inactive Active Inactive Inactive Inactive
– – – – – – 200 200 200 200 – 200 – – –
(1.35%), 65.3 g (n-BuOH) (6.53%), ssp. abietis; 450.7 g (EtOH) (45.07%), 0.4 g (pet. ether) (0.04%), 3.3 g (ether) (0.33%), 9.6 g (EtOAc) (0.96%), 37.5 g (n-BuOH) (3.75%), ssp. austriacum; 411.9 g (EtOH) (41.19%), 1.9 g (pet. ether) (0.19%), 3.8 g (ether) (0.38%), 12.5 g (EtOAc) (1.25%), 36.0 g (n-BuOH) (3.6%). The yields are based on dry plant weight. Equal amounts (about 25 mg) of each extract and fraction were weighed and evaluated for antituberculosis activity. Microbiological assay Testing for antituberculous activity was done using the microplate Alamar blue assay (MABA) (Collins & Franzblau, 1997). Suspensions of Mycobacterium tuberculosis H37Ra strains were prepared at a concentration of about 105 cells/ml, 100 ml of the bacterial suspension were added to each well of microtiter plate, together with the plant extracts in Middlebrook 7H9 medium to the final volume of 200 ml, and the final concentration of the plant extracts were 50, 100 and 200 mg/ml. After incubation for about 7 days, 20 ml of Alamar blue dye were added to the control well. If the dye turned pink, indicating bacterial growth, the dye was then added to all remaining wells in the plate. The results were read in the following day. If the extracts were active at 50 mg/ml, the minimum inhibitory concentration (MIC) of the extracts was determined. For standard tests, the MIC values of rifampin, isoniazid and kanamycin were determined each time. The acceptable MIC ranges of the drugs were 0.0047–0.0095, 0.05–0.1 and 2.5–5.0 mg/ml, respectively.
Results and discussion Extracts of Viscum album subspecies were evaluated for antituberculosis activity against Mycobacterium tuberculosis
H37Ra (Table 1). Bacterial growth is indicated by the change in the color of Alamar blue from blue, in oxidized form, to pink, in reduced form. The reduction reflects the consumption of oxygen, which is essential for the growth of the obligate-aerobic bacterium. Observation of color was performed visually. Samples exhibited varying degrees of inhibition in the in vitro primary screen conducted at 200 mg/ml. Active extracts were retested at lower concentration to determine the actual MIC values. The extracts coded DD-7, 8, 9, 10 and 12 were found to be active at 200 mg/ml. The extracts of DD-7-9 were prepared using ether, and DD-10, 12 were petroleum ether extracts. DD-8 and DD-10 were prepared from the subspecies of austriacum, DD-9 and DD-12 were obtained from the subspecies of abietis. These extracts were compared by tlc for their chemical composition and found have similar nonpolar compounds. Although, the petroleum ether extract (DD-11) of ssp. album was inactive, its ether extract (DD-7) was active. Therefore, it is difficult to state that nonpolar compounds can be responsible for antiTB activity. On the other hand, it is not yet known if the anti-TB activity depends on the host plants (Pinus, Abies, Armeniaca) resulted from these subspecies (Ergun & Deliorman, 1995). This is the first report on the antimycobacterial activity of Viscum album subspecies.
References Collins L, Franzblau SG (1997): Microplate Alamar blue assayversus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother 41: 1004–1009.
Evaluation of antimycobacterial activity of Viscum album subspecies Davis PH (1982): Flora of Turkey and the East Aegean Islands. Edinburgh, Edinburgh University Press. Ergun F, Deliorman D (1995): Studies on the active principles of the various Viscum album L. collected from different host plants J Fac Pharm Ankara 24: 127–139. Ergun F, Deliorman D, Özçelik B, Abbasog˘lu U (1997): Screen-
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ing of antifungal activities of various Viscum album L. samples. GUEDE, J Fac Pharm Gazi 14: 1–4. Yes¸ilada E, Deliorman D, Ergun F, Takaishi Y, Ono Y (1998): Effects of the Turkish subspecies of Viscum album on macrophage-derived cytokines. J Ethnopharmacol 61: 195–200.
