Journal of Dental Hygiene Science Vol. 3, No. 2, pp. 67~ 70 (2003) Screening for Biological Activity of Crude Extracts from Medicinal plants Jung-Sook Kwag†1, Hyun-Ju Oh, Hyun-Ok Lee2, Nigel B. Perry3 and Seung-Hwa Baek Department of Herbal Resources, Professional Graduate School of Oriental Medicine and Institute of Basic Natural Sciences, Wonkwang University, Iksan 570-749, Korea 1Department of Dental Hygiene, Mokpo Science College, Mokpo 530-390, Korea 2Department of Dental Hygiene, Wonkwang Health Science College, Iksan 570-750, Korea 3Plant Extracts Research Unit, New Zealand Institute for Crop & Food Research Ltd., Department of Chemistry, Otago University, Box 56, Dunedin, New Zealand The biological effects of the crude extracts from medicinal plants, Brachyglottis monroi and Trichocolea hatcheri were investigated. The crude ethanol extract inhibited the growth of the Gram positive bacterium Bacillus subtilis (ATCC 19659, 1 mm inhibition zone at 150 µg/disc) and the dermatophyte Trichophyton mentagrophytes (ATCC 28185, 6 µ µ µ mm inhibition zone at 150 g/disc), and was toxic to P388 murine leukaemia cells (IC50 23.96 g/mL at 75 g/disc). B. monroi ethanol extract showed stronger antiviral activity than that of T. hatcheri against Herpes simplex Type I virus (ATCC VR 733) and Polio Type I virus (Pfizer vaccine strain) (50% activity, @ 5 mg/ml at 150 µg/disc). The crude ethanol extract of T. hatcheri showed stronger antimicrobial activity than that of B. monroi. However, this extract was inactive against P388 murine leukaemia cells. Brachyglottis monroi, Trichocolea hatcheri, Bacillus subtilis, Trichophyton mentagrophytes, P388, Cytotoxicity I. INTRODUCTION MATERIALS AND METHODS There is world-wide interest in bioactive natural products 1. General experimental procedures from plant as new agents for controlling human and agricultural All solvents were distilled before use and were removed by diseases and pests. Research groups in many countries are rotary evaporation at temperatures up to 35oC. Octadecyl engaged in the screening of plant extract. Liverworts are functionalized silica gel (C 18, Aldrich) was used for reversed- closely related to mosses, and the two groups together form a phase flash chromatography, and Davisil, 35-70 µm, 150 Å large and important division of the plant kingdom, technically was used for Si gel flash chromatography. Preparative silica known as the Bryophytes1). Liverworts of the genus Trichocolea gel TLC was carried out using Merck DC-plastikfolien (family Trichocoleaceae) are a treasury of isoprenyl phenyl Kieselgel 60 F254, visualized with an UV lamp then by dipping in ethers. Trichocolea hatcheri Hodgs, which grows throughout a vanillin solution (1% vanillin, 1% H2SO4 in EtOH) and heating. New Zealand, is distinguished from T. mollissima by its smaller size, dark green color, and prostrate habit2). 2. Plant material A chemical abstracts search of Trichocolea revealed that Trichocolea hatcheri (T. hatcheri) was collected from a only T. tomentella3-5), T. lanata3), T. mollisima3,6), T. hatcheri7) steep earth bank in the Morrisons Creek area, Dunedin, New and T. pluma8) have been studied for their chemical constituents. Zealand, in Fabruary 1996 [University of Otago Herbarium Brachyglottis monroi (Hook. f) B. Nordenstam (Asteraceae (OTA) specimen no. 048094]. compositae), previously Senecio monroi, is a shrub endemic Brachyglottis monroi (B. monroi) was collected from the to New Zealand9,10). B. monroi has been widely used in Maori Dunedin Botanical Garden, Dunedin, New Zealand, in June traditional medicine for treatment of sores and wounds11). 1998. This was identified by Dr. Glenny, Landcare Research, In this study, the antiviral, antimicrobial activities and and a voucher specimen, OTA 980309-63, has been kept in cytotoxicity of crude ethanol extracts from medicinal plants the Otago University herbarium. were examined. 3. Preparation of the extract Air-dried medicinal plants were marcate in redistilled † Corresponding author ethanol in a Waring Blender, and then filtered. The residual Tel: 061-270-2721 Fax: 061-270-2721 marc was re-extracted in the same way with more ethanol and E-mail: [email protected] chloroform. The combined filtrates were evaporated under Screening for Biological Activity of Crude Extracts from Medicinal plants reduced pressure to give a dark green gum, which was stored RESULTS AND DISCUSSION at 4oC until tested. 1. Biological screening of the crude extract of T. 4. Screening for antiviral activity hatcheri The extract was applied (15 L of a 5 mg/mL solution) to a Trichocolea hatcheri Hodgs (family Trichocoleaceae) grows small filter-paper disc, dried, and assayed for antiviral activity throughout New Zealand. Foliage plant collected from a steep using Schroeder et al.’s 12) methods. The results were earth bank in the Morrisons Creek area in Dunedin. observed cell death (cytotoxicity), inhibition of virus replication, An extract of T. hatcheri was prepared by grinding dried no effect (i.e., all of the cells show viral infection), or a plant material and extracting separately with ethanol then combination of all three. The results were noted as the chloroform. The two extracts were combined, as their 1H- approximate size of the circular zone, radiating from the NMR spectra were similar. A crude extract was cytotoxic to extract sample, from 1+ to 4+ representing 25% through to P388 murine leukemia µ whole well sized zones. The notation used is inhibition/antiviral (IC50 > 12,500 g/mL) and BSC monkey kidney cells (50% activity. The type of antiviral effect, indicated by a number of well at 150 µg/mL). Table 1 shows the mediocre antiviral after the size of the zone, was also considered important and activity against Herpes simplex Type I virus (ATCC VR 733) may give some indication as to the mode of cytotoxic action. and Polio Type I virus (Pfizer vaccine strain) (50% activity, @ 5 mg/mL at 150 µg/disc). The crude extract inhibited the 5. Screening for antibacterial and anti-yeast activities growth of the Gram-positive bacteria and fungus of the extract Activity against the following bacterial strains and yeast prepared from New Zealand medicinal plant. As indicated in was tested: multi-resistant B. subtilis (ATCC 19659), and Table 1, this crude extract inhibited the growth of the Gram- Candida albicans (ATCC 2091). Extracts were dissolved and positive bacterium Bacillus subtilis ATCC 19659 (1 mm diluted in an appropriate solvent (usually ethanol : water) to a inhibition zone at 150 µg/disc) and the dermatophyte concentration of 5 mg/mL. Test plates are prepared from Trichophyton mentagrophytes ATCC 28185 (6 mm inhibition Müller Hinton agar containing extract to give a final zone at 150 µg/disc). No activity was observed against the concentration of 100 µg extract/mL agar. Activity growing fungus Candida albicans at 150 µg/disc. This extract showed cultures of the test strains were diluted in saline so as to weaker antimicrobial activity than chloramphenicol and nystatin deliver 104 colony forming units onto the test, control (solvent), (Table 1). However, this crude extract showed stronger and blank (agar only) plates with a multipoint inoculator. antimicrobial activity than the crude extract of B. monroi and Inoculated plates were incubated overnight at 37oC. Growth this extract was inactive against P388 murine leukaemia cells. on the blank and control plates were checked and, if satisfactory, growth on the test plates was scored for each test Biological assays of the crude extract from T. hatcheri − strain as follows: ( ) inhibition, no reduction in growth Cytotoxicity compared with the control, (+) inhibition, no growth. Herpes simplex Extract BSCa Polio virus b virusb 6. Screening for antifungal activity Activity against the following fungal strain was tested: ++ ++ ++ Trichophyton mentagraphytes [(ATCC 28185) local strain]. Antimicrobial activityc Fungal spore suspensions of the test organisms were applied B. subtilis C. albicans T. mentagrophytes to dextrose agar plates. Aliquots of the extract solutions were Extract SM 1 - HM 2 applied to filter paper discs, at 15 µg extract/disc, and dried at 37oC for two hours. These discs were applied to the agar Chloramphenicol SM 12 0 0 plates, two per plate, and incubated at 28oC. Nystatin 0 SM 11 HM 8 P388 cytotoxicity 7. Screening for cytotoxic activity against animal cell d This is a measure of the ability of a sample to inhibit the Mitomycin C 59.7 multiplication of murine leukaemia cells. The sample was Extract > 12,500e dissolved in a suitable solvent, usually ethanol, at 5 mg/mL, and a% of well showing cytotoxic effects. @ 5 mg/mL, 150 µg/disc; ++: 50% 15 µL of this solution was placed in the first well of a multiwell activity. bCytotoxicity in antiviral assays. @ 5 mg/mL, 150 µg/disc; Zone of plate. Seven two-fold dilutions were made across the plate. After cytotoxic activity: ++: 50% activity. addition of the cell solution, the concentration range in the test cWidth of zone of inhibition in mm; 150 µg/disc; -: no reduction in growth, 0: not determined. Chloramphenicol; 30 mcg/disc, Nystatin; 100 unit/ wells was 25,000 down to 195 ng/mL. After incubation for three disk. SM; Sharp margin, HM; hazy margin, numbers refer to zone of days, the plates were read using an ELISA palte reader at 540 inhibition (mm) d µ nm wavelength. Automated reading of the plates was possible Toxicity of sample to P388 murine leukaemia cells in ng/ml at 0.075 g/ disc. P388; Concentration of the sample required to inhibit cell growth to with the addition of a MTT tetrazolium salt (yellow color). 50% of a solvent control. e Healthy cells reduce this salt to MTT formazan (purple color). Toxicity of sample to P388 murine leukaemia cells in ng/mL at 75 µg/disc Journal of Dental Hygiene Science Vol. 3, No. 2, pp. 67~70 (2003) 2. Biological screening of the crude extract of B. In conclusion, these crude extracts inhibited the growth of monroi the Gram positive bacterium Bacillus subtilis ATCC 19659 (1 Brachyglottis monroi (Hook.