Bioactivity of secoiridoid glycosides from Centaurium erythraea. (Short Communication).
As part of our on-going search for bioactive compounds from Scottish plants, two secoiridoid glycosides, swertiamarin and sweroside, have been isolated from the aerial parts of Centaurium erythraea Rafn (Family: Gentianaceae) by reversed-phase preparative HPLC coupled with a photo-diode-array detector. The structures of these compounds were elucidated unambiguously by UV, FARMS and extensive 1D and 2D NMR spectroscopic analyses and also by comparing experimental data with literature data. Antibacterial, free radical scavenging activities and general toxicity of these glycosides have been assessed. Both compounds inhibited the growth of Bacillus cereus, Bacillus subtilis, Citrobacter freundii and Escherichia coli. While swertiamarin was also active against Proteus mirabilis and Serratia marcescens, sweroside inhibited the growth of Staphylococcus epidermidis. Swertiamarin and sweroside exhibited significant general toxicity in brine shrimp lethality bioassay and the [LD.sub.50] values were 8.0 [micro]g/ml and 34 [micro]g/ml, respectively, whereas that of the positive control podophyllotoxin, a well known cytotoxic lignan, was 2.79 [micro]g/ml. Chemotaxonomic implications of these compounds in the family Gentianaceae have also been discussed briefly.
Key words: Centaurium erythraea, Gentianaceae, sweroside, swertiamarin, antibacterial activity, brine shrimp toxicity, DPPH assay
Centaurium erythraea Rafn (Family: Gentianaceae), commonly known as "feverfoullie", "gentian" or "centaury" is a glabrous annual from Scotland and also found in other parts of Europe, from Sweden southwards, Mediterranean regions, Azores, and naturalised in America (Flora Celtica database, 2001; Flora Europaea database 2002; GRIN database, 2002). Historically, centaury was one of the traditional panaceas used for practically everything except diarrhoea (HerbMed database, 2002; Grieve, 2002; Beith, 1995). Previous phytochemical investigations on C. erythraea yielded a variety of plant secondary metabolites including alkaloids, coumarins, phenolic acids, secoiridoids, triter penes and xanthone derivatives (Dictionary of Natural Products, 1999; ISI database, 2002; Phytochemical and Ethnobotanical Database, 2002). Secoiridoid glucosides, swertiamarin and sweroside, are present in various traditional medicine preparations and are reported to have hepatoprotective activity (Pauli et al., 1995; Liu et al., 1994; Zho u, 1991). Choleretic, hepatoprotective, pancreatic and sialogogue activities of swertiamarin have also been reported (Phytochemical and Ethnobotanical Database, 2002). As part of our continuing search for biologically active compounds from Scottish plants (Cooper et al., 2002; Ferguson et al., 2002; Kumarasamy et al., 2002a, b, c, d; Ribeiro et al., 2002; Sarker et al., 2001; Tomlinson et al., 2002), we now report on the prep-HPLC-PDA assisted isolation of bioactive secoiridoid glycosides, swertiamarin and sweroside, from the aerial parts of C. erythraea, and antibacterial, free radical scavenging activities and general toxicity of these compounds.
UV spectra were obtained in MeOH. NMR spectra were recorded in [CD.sub.3]OD using a Varian Unity INOVA 400 MHz NMR Spectrometer. High-resolution FAB mass spectra were acquired on a JEOL SX102 mass spectrometer using glycerol as matrix. HPLC separation was performed in a Dionex prep-HPLC System coupled with Gynkotek GINA50 autosampler and Dionex UVD340S Photo-Diode-Array detector. A Luna [C.sub.18] preparative HPLC column (10 [micro]m particle size, 250 mm x 21.2 mm) was used. Sep-Pak Vac 35 cc (10 g) [C.sub.18] cartridge (Waters) was used for pre-HPLC cleaning of the MeOH extract.
The aerial parts of C. erythraea Rafn (cat. no. 402392) were purachased from B & T World Seeds Sarl, Paguignan, 34210 Olonzac, France. A voucher specimen (PH300020) has been deposited in the herbarium of Plant and Soil Science Department, University of Aberdeen, Scotland (ABD).
Extraction and isolation
Ground aerial parts (140 g) of C. erythraea were Soxhlet-extracted, successively, with n-hexane, dichloromethane and MeOH (1.1 1 each). The MeOH extract was run through Sep-Pak [C.sub.18] (10 g) cartridge eluting with 100% MeOH to clean it up from any non-polar material. Preparative-HPLC (eluted with a linear gradient of water: MeCN from 80:20 to 00:100 over 30 min, followed by 100% MeCN for 10 min, 20 ml/mm, monitored by photo-diode-array detector) of the Sep-Pakcleaned MeOH extract yielded swertiamarin (1) and sweroside (2).
Swertiamarin (1): Brown gum (90 mg; yield = 0.064%; Rt = 11.0 mm), UV [[lambda].sub.max] nm = 246. [H.sup.1] and [C.sup.13] NMR (Boros and Stermitz, 1991). FABMS (positive ion mode) m/z 375 [[M+H].sup.+].
Sweroside (2): Brown gum (87 mg; yield = 0.062%; Rt = 12.0 mm), UV [[lambda].sub.max] am = 245. [H.sup.1] and [C.sup.13] NMR (Boros and Stermitz, 1991). FABMS (positive ion mode) m/z 359 [[M+H].sup.+].
2,2-Diphenyl-1-picrylhydrazyl (DPPH), molecular formula [C.sub.18][H.sub.12][N.sub.5][O.sub.6], was obtained from Fluka Chemie AG, Bucks. Quercetin was obtained from Avocado Research Chemicals Ltd. Shore road, Heysham, Lancs. The method used by Takao et al. (1994) was adopted with suitable modifications to our particular circumstance (Kumarasamy et al., 2002a). DPPH (8 mg) was dissolved in MeOH (100 ml) to obtain a concentration of 80 [micro]g/ml. Compounds 1 and 2 were dissolved in MeOH to obtain a concentration of 0.5 mg/ml.
Antibacterial activity of 1 and 2 was assessed against 14 species of Gram-positive and Gram-negative bacteria (Table 1) using the 96-well micro-plate-based broth dilution method, also known as the checkerboard assay (Lorian, 1991; Richards and Xing, 1993; Kumarasamy et al., 2002a). Isosensitised nutrient broth was obtained from Oxoid, Basingstoke, Hampshire, England. Microtitre plates were from Sero-wel, Bibby sterilin, Stone, STaffs, UK. Eppendorf pipettes were purchased from Netheter-hinz-Gmbh, 2231, Hamburg, Germany. Bacterial suspension (20 [micro]l) in double strength nutrient broth at a concentration of 5 x [10.sup.5] CFU/ml was used. Test compounds [1, 2] were dissolved in DMSO to obtain the stock concentration of 1 mg/ml. Serial dilution was done in microwell plates. Ciprofloxacin, a well known broad-spectrum antibiotic, was used as positive control. The minimum inhibitory concentration (MIC) was determined for each compound and compared with that of the positive control, ciprofloxacin, a broad-spectr um antibiotic (Table 1).
Brine shrimp lethality assay
The method of Meyer et al. (1982) was adopted to study the general toxicity of the compound. Waterlife[R] brand brine shrimp (Artemia sauna) eggs were purchased from The Pet Shop, Kittybrewster Shopping Complex, Aberdeen, UK. The eggs were hatched in a conical flask containing 300 ml artificial seawater made by dissolving a commercial marine salt preparation in deionised and distilled water. The flasks were well aerated with the aid of an air pump, and kept in a water bath at 29-30[degrees]C. A bright light was left on. The nauplii hatched within 48 h. The compounds 1 and 2 were dissolved in DMSO to obtain a concentration of 1 mg/ml. These were serially diluted ten-times. Solution of each concentration (1 ml) was transferred into clean sterile universal vials with a pipette and aerated seawater (20 ml) was added. About 10 nauplii were transferred into each vial with a pipette. A check count was performed. The number alive after 24 h was noted. The mortality endpoint of this bioassay was defined as the absence of controlled forward motion during 30 sec of observation. The experiment was carried out in triplicate and the average values were noted. The controls used were DMSO, normal saline, and podophyllotoxin (3 [micr o]g/ml). Abbots formula was used to correct the values, i.e., P = Pi - C/l - C), where P denotes the observed non-zero mortality rate and C represents the mortality rate of the MeOH control.
* Results and discussion
These scoiridoid glycosides, 1 and 2, were active against a number of bacterial species and also showed significant toxicity in brine shrimp lethality assay. However, none of 1 and 2 showed any antioxidant activity in DPPH assay. Both swertiamarin (1) and sweroside (2) exhibited antibacterial activity against Bacillus cereus, B. subtilis, Citrobacter freundii and Escherichia coli (Table 1). While 1 inhibited the growth of Proteus mirabilis and Serratia marcescens, 2 was active against Staphylococcus epidermedis. However, none of these compounds, at test concentrations, showed any inhibitory activity against methicillin resistant Staphylococcus aureus NCTC11940 and [beta]-lactamase producing Escherichia coli NCTC11954. Swertiamarin (1) was most effective against C. freundii (MIC = 5 x [l0.sup.-3] mg/ml).
The brine shrimp lethality assay, which has been proven to be an effective and rapid assay method to screen compounds for potential cytotoxic activity (Meyers et al., 1982), was applied to determine the general toxicity of these secoiridoid glycosides (1, 2). Both compounds were found to be quite toxic in this assay. From Probit analysis (Finney, 1971), the [LD.sub.50] values of 1 and 2 were determined as 8.0 [micro]g/ml and 34 [micro]g/ml, respectively, whereas that of the positive control podopohyllotoxin, a well known cytotoxic lignan, was 2.79 [micro]g/ml. General toxicity of 1 was ca. 3 times less than podophyllotoxin, but ca. 4 times more than 2. The higher toxicity oft (compared to 2) may be due to the presence of the extra hydroxyl group in 1 (Figure 1). Significant correlations with brine shrimp toxicity have previously been shown for cytotoxicity and antitumour activity (Anderson et al., 1991). Therefore, from the low [LD.sub.50] values of these compounds, it can be speculated that these compounds m ight have some anticancer potential. On the basis of very high % yield of 1 and 2 and their bioactivities found in this study, it is reasonable to assume that these compounds contribute, at least partly, to some of the traditional medicinal properties of C. erythraea.
Within the genus Centaurium, apart from C. erythraea, swertiamarin (1) and sweroside (2) have previously been reported from C. turcicum (Nikolova Damyanova and handjieva, 1996) and C. littorale (Vandersluis and Labadie, 1981), respectively. However, compounds 1 and 2 have been isolated from various other species of the genera Gentiana and Swertia from the family Gentianaceae (Dictionary of Natural Products, 1999; ISI database, 2002). Among the swertiamarin-producing species, Centaurium erythraea, Gentiana lutea and Gentiana scabra have been noted to produce the highest amount of this compound (Phytochemical and Ethnobotanical Database, 2002). Iridoid/secoiridoid glucosides have previously been used as chemotaxonomic markers in the Loasoideae (Muller et al., 1999). Cooccurrence of the secoiridoid glycosides 1 and 2 among the species of the Gentianaceae might have some chemotaxonomic implications.
Table 1 Antibacterial activity of sertiamarin (1) and sweroside (2). Bacterial species MIC (mg/ml) Ciprofloxacin Bacillus cereus 9689 (a) 2.5 x [10.sup.-8] Bacillus subtilis 7801 (a) 2.5 x [10.sup.-6] Citrobacrer freundii 9750 (a) 2.5 x [10.sup.-7] Escherichia coli 8110 (a) 2.5 x [10.sup.-7] Escherichia coli 11954 (a) 2.5 x [10.sup.-6] Klebsiella aerogenes 9528 (a) 2.5 x [10.sup.-6] Proteus mirabilis 60 (a) 2.5 x [10.sup.-10] Pseudomonas aeruginosa 9689 (a) 2.5 x [10.sup.-8] Salmonella goldcoast 13175 (a) 2.5 x [10.sup.-6] Serratia marcescens 1377 (a) 2.5 x [10.sup.-6] Staphylococcus aureus 10788 (b) 2.5 x [10.sup.-8] Staphylococcus aureus (MRSA) 11940 (a) 2.5 x [10.sup.-5] Staphylococcus epidermedis 8558 (a) 2.5 x [10.sup.-8] Staphylococcus hominis 11320 (a) 2.5 x [10.sup.-7] Bacterial species MIC (mg/ml) 1 2 Bacillus cereus 9689 (a) 2 x [10.sup.-2] 2 x [10.sup.-2] Bacillus subtilis 7801 (a) 2 x [10.sup.-1] 2 x [10.sup.-1] Citrobacrer freundii 9750 (a) 5 x [10.sup.-3] 1 x [10.sup.-2] Escherichia coli 8110 (a) 2 x [10.sup.-1] 2 x [10.sup.-1] Escherichia coli 11954 (a) -- -- Klebsiella aerogenes 9528 (a) -- -- Proteus mirabilis 60 (a) 2 x [10.sup.-1] -- Pseudomonas aeruginosa 9689 (a) -- -- Salmonella goldcoast 13175 (a) -- -- Serratia marcescens 1377 (a) 2 x [10.sup.-2] -- Staphylococcus aureus 10788 (b) -- -- Staphylococcus aureus (MRSA) 11940 (a) -- -- Staphylococcus epidermedis 8558 (a) -- 2 x [10.sup.-1] Staphylococcus hominis 11320 (a) -- -- -- not inhibition of growth at test concentrations, (a) - NCTC, (b) - ATCC
We thank EPSRC National Mass Spectrometry Service Centre (Department of Chemistry, University of Wales, Swansea, Swansea, Wales, UK) for FABMS analysis.
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Y. Kumarasamy (1)
L. Nahar (2)
P.J. Cox (1)
M. Jaspars (3)
S.D. Sarker (1)
(1.) Phytopharmaceutical Research Laboratory, School of Pharmacy, The Robert Gordon University, Aberdeen, UK
(2.) Japp Laboratory, Department of Chemistry, University of Aberdeen, Old Aberdeen, Aberdeen, UK
(3.) Marine Natural Products Laboratory, Department of Chemistry, University of Aberdeen, Old Aberdeen, Aberdeen, UK
S. D. Sarker, Phytopharmaceutical Research Laboratory, School of Pharmacy, the Robert Gordon University, Schoolhill, Aberdeen AB10 1FR, UK Tel.: ++44-0-1224 262547; Fax: ++44-0-1224-262555; e-mail: S.Sarker@rgu.ac.uk