bs_bs_banner
Research Paper
Journal of Pharmacy And Pharmacology
A comparative study on phenolic components and biological activity of some Senecio species in Turkey Sevil Albayraka, Ahmet Aksoyb, Lutfiye Yurtsevenc and Abit Yas¸arc a Science Faculty, Department of Biology and cGraduate School Natural Applied Science, Erciyes University, Kayseri, and bScience Faculty, Department of Biology, Akdeniz University, Antalya, Turkey
Keywords antimicrobial activity; antioxidant activity; phenolic; Senecio Correspondence Sevil Albayrak, Science Faculty, Department of Biology, Erciyes University, Kayseri 38039, Turkey. E-mail: [email protected] Received February 19, 2014 Accepted May 15, 2014 doi: 10.1111/jphp.12288
Abstract Objective The phenolic components and biological activity of nine Senecio species growing in Turkey were investigated. Methods Senecio species were extracted with methanol. The content of total phenols was determined using Folin–Ciocalteu method, while individual phenolic acids and flavonoids were detected using HPLC analysis. Also, to determine the antioxidant capacity, phosphomolybdenum assay and 1,1-diphenyl picrylhydrazyl (DPPH) radical-scavenging activity assay were used. Antimicrobial activity of extract was determined using agar diffusion and broth microdilution method. Key findings The total phenolic contents of the extracts were found to be highest in Senecio cilicius and Senecio mollis extracts (117.45 and 113.40 mg equivalent to gallic acid/g, respectively). S. salsuginea showed the strongest free radicalscavenging activity with IC50 (the concentration providing 50% inhibition) = 26.23 μg/ml and S. mollis showed the highest antioxidant capacity in the phosphomolybdenum method (434.48 mg equivalent to ascorbic acid/g). The extracts exerted promising antibacterial activity against most of the test bacteria (minimal inhibitory concentration = 6.25–12.5 mg/ml), but no activity was observed against Candida albicans. Conclusion The results demonstrated that nine Senecio species possess high antioxidant and antimicrobial activity in accordance with the high amount of phenolic contents in the extracts that might be natural agencies used in many areas such as food, pharmacy and alternative medicine.
Introduction Free radicals, with unpaired electrons, are produced in normal or pathological cell metabolism and reactive oxygen species (ROS) react easily with the free radicals to convert them into radicals. ROS are highly reactive molecules that include the superoxide anion radicals, hydroxyl radicals and hydrogen peroxide and peroxyl radicals.[1] Oxidative stress, a common feature of metabolic syndrome, is defined as an imbalance between the production and inactivation of ROS in biological systems. Overproduction of ROS results in cellular injury, including lipid peroxidation, protein oxidation and DNA damage.[2] The oxidative damage created by free radical generation is a critical aetiological factor implicated in several chronic human diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis and neurodegenerative diseases and also in the aging process.[3] Antioxidant activity is
defined as an inhibition of the oxidation of lipids, proteins, DNA or other molecules that occurs by blocking the propagation step in oxidative chain reactions.[4] The commonly used synthetic antioxidants such as butylhydroxyanisole and butylhydroxytoluene have potential health risks and toxicity. Therefore, these need to be replaced with natural antioxidants.[5] Phenolics are broadly distributed in the plant kingdom and are the most abundant secondary metabolites of plants. Plant polyphenols have drawn increasing attention because of their potent antioxidant properties and their marked effects in the prevention of various oxidative stress associated diseases such as cancer.[6] The antioxidant ability of phenolic components occurs mainly through a redox mechanism and allows the components to act as reducing
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
1
Bioactivity of Senecio
Sevil Albayrak et al.
agents, hydrogen donors, singlet oxygen quenchers and metal chelators.[4] Many studies have been carried out on some plants, which resulted in development of natural antioxidant formulations for food and other applications. However, scientific information on antioxidant properties of various plants, particularly those that are less widely used in medicine is still scarce. So, it is an interesting and useful task to find new sources for natural antioxidant and functional food.[7] In the last few years, the identification and development of phenolic compounds or extracts from different plants has become a major area of health and medical-related research.[6] On the other hand, several antimicrobials have been developed over the years to control microorganisms. However, the development of antimicrobial resistance and the relatively narrow spectrum of the antimicrobials have had limited success and the microbial contamination of food still poses an important public health and economic challenge.[8] Because of the resistance and the problems of emerging infectious disease have made it inevitable to search for new antimicrobials from nature especially plants.[5,7] Senecio is the largest genus of the tribe Senecioneae (Asteraceae) and more than 1500 species have been reported with a worldwide distribution.[9–11] It is represented by 39 species in Anatolia.[12] This genus is rich in pyrrolizidine alkoloids and furoeremophilanes, which are the most characteristic secondary metabolite, but sesquiterpenes, chalcones and flavonoids have also been reported.[10,11,13–15] Senecio species are used as food and in folk medicine in the Mediterranean area in the treatment of wounds and as anti-emetic, anti-inflammatory and vasodilator preparations.[11,13] It also is used in the treatment of various diseases in many part of the world. Senecio graveolens is used in Argentina as emmenagogue, digestive and cough suppressant. Senecio latifolius leaves were used by Zulu as an emetic and in the treatment of chest complaints.[15] Senecio cannabifolius is used as a traditional remedy for treating virus influenza, enteritis and pneumonia in China.[14] Senecio scandens is used in traditional and folk medicine for its anti-inflammatory, antipyretic and detoxification effects in China. Senecio vulagaris played a certain role as emmenagogue and in case of functional amenorrhoea in Europe.[16] Antibacterial and antifungal activity of compounds and extracts from Senecio species have been investigated by many researchers.[10,11,14,15,17–19] But, there are only three studies on antibacterial activity of Senecio species belonging to Turkish flora in the literature.[12,20,21] In our previous study, the phenolic contents and bioactivity of six Senecio species growing in the Black Sea region of Turkey were reported. This study is a part of the bioactivity examination of many Senecio species growing in the remaining six of seven region of Turkey. Accordingly, 2
this study was undertaken to test the phenolic components, antioxidant and antimicrobial activity of extracts from nine Senecio species belonging to Turkish flora.
Materials and Methods Plant materials Collection information of the nine plant species, which are individually numbered, is listed below: 1. S. cilicius Boiss., between Erzincan and Kelkit, Akdag˘ village, 2300 m, Erzincan, Turkey, July 2006 (Voucher No: AAksoy 2040), endemic. 2. S. inops subsp. karamanicus Hamzaog˘lu & Budak between Karaman and Bas¸yayla Tas¸kent, 1810 m, Karaman, Turkey, Jun 2006 (Voucher No: AAksoy 2310), endemic.[22] 3. S. mollis Willd. Taslıdere, 1330 m, Sivas, Turkey, July 2006 (Voucher No: AAksoy 2030). 4. S. olympicus Boiss., above of Uludag˘ Kırkpınar valley, 2110 m, Bursa, Turkey, July 2005 (Voucher No: AAksoy 2043), endemic. 5. S. othonnae Bieb., Fl. Taur.-Caucas, 1705 m, Sivas, Turkey, July 2006 (Voucher No: AAksoy 2050). 6. S. salsuginea H. Duman & Vural, straight Salt lake from Eskil, 910 m, Aksaray, Turkey, August 2006 (Voucher No: AAksoy 2077), endemic.[23] 7. S. sandrasicus Davis in Notes Roy. Bot. Gard. Edinburgh, Sandras mountain, 1545 m, Mug˘la Köyceg˘iz, Turkey, July 2006 (Voucher No: AAksoy 2031), endemic. 8. S. tauricolus Matthews in Notes R. B. G. Edinburgh, Yahyalı, mine road, 1850 m, Kayseri, Turkey, Jun 2006 (Voucher No: AAksoy 2075), endemic. 9. S. viscosus L., around to Yoncaklı village, 2280 m, Bayburt, Turkey, July 2005 (Voucher No: AAksoy 2080). Voucher specimens were identified by Dr Ahmet Aksoy and have been deposited at the Herbarium of the Department of Biology, Erciyes University, Kayseri, Turkey.
Preparation of the herb extracts Collected plant materials were dried at room temperature. Aerial parts of plants were ground to fine powder. Ground herbs (10 g) were extracted in a Soxhlet extractor (Nüve, Ankara, Turkey) with 100 ml methanol (50°C for 6 h). The extract was concentrated by using rotary evaporator (Rotavapor, Buchi, Switzerland; T < 40°C) under vacuum to get crude extracts. Dried extracts were stored at 4°C until use.
HPLC analysis of phenolic compounds in the extracts The extracts were dissolved in methanol at a concentration of 10 mg/ml. A high-performance liquid chromatograph (Shimadzu, Duisburg, Germany) was equipped with HPLC
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Sevil Albayrak et al.
pumps (LC-10ADvp; Shimadzu, Ontario, Canada) and a diode array detector (DAD) detector (278 nm; Shimadzu, Ontario, Canada). Eclipse XDB-C18 (5 μm) column (250 × 4, 60 mm) (Agilent 1200 Series; Agilent, Ontario, Canada) was used. The flow rate was 0.8 ml/min and the injection volume 20 μl. The analyses of the phenolic compounds were carried out at 30°C using two linear gradients of methanol. Caffeic acid, chlorogenic acid, cinnamic acid, eriodictyol, naringenin, p-coumaric acid, quercetin, and rutin were used as standard. Identification and quantitative analysis were done by comparison with standards.[24]
Determination of total phenolics The total phenolic amount in the extracts has been assessed with the Folin–Ciocalteau total phenols photometric assay.[25] Forty microlitre of methanol extract (1 mg/ml) were taken in test tubes. Also, 2.4 ml of distilled water, 200 μl of Folin–Ciocalteu reagent, 600 μl sodium carbonate (20%) and 760 μl of distilled water were added respectively. The tubes were mixed and allowed to stand at the dark for 2 h. The absorbance was measured at 765 nm against a blank, which contained 40 μl of methanol in place of extract. The total phenolic content was expressed as gallic acid equivalents (GAE) in mg/g of methanol extract.
Evaluation of total antioxidant capacity by phosphomolybdenum method The antioxidant power of the extracts has been assessed with the phosphomolybdenum reduction assay.[26] A 0.4 ml of methanol extract (1 mg/ml) was mixed with 4 ml of the reagent solution (0.6-m sulfuric acid, 28-mm sodium phosphate and 4-mm ammonium molybdate). For the blank, 0.4-ml methanol was mixed with 4 ml of the reagent. The samples were incubated in water bath at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance of the green phosphomolybdenum complex was measured at 695 nm. The antioxidant capacity of the extracts was expressed as the ascorbic acid equivalents (mg AAE/g of methanol extract).
Free radical-scavenging activity The 1,1-diphenyl picrylhydrazyl (DPPH) assay has been adopted for the evaluation of radical-scavenging capacity of the studied plant extracts.[25] The 50 μl of the extract dilution at the concentration range of 0.1–2 mg/ml was mixed with 450 μl of Tris-HCl buffer, pH = 7.4 and 1 ml of the methanol DPPH solution (0.1 mm). The absorbance has been measured at 517 nm after standing at room temperature for 30 min. The control contained 50 μl of methanol in place of extract. The butylated hydroxytoluene MeOH solution was used as a positive control. The decrease in
Bioactivity of Senecio
optical density of DPPH on addition of test samples in relation to the control was used to calculate the free radical-scavenging activity, as percentage inhibition (I %) of DPPH radical.
Percentage inhibition (I %) = (( A C − A S ) A C ) × 100 Where AC = absorbance of the control after 30 min; AS = absorbance of the test sample after 30 min. Extract concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage against extract concentration. The assay was carried out in triplicate.
Antimicrobial activity The 15 microorganisms that contain 13 bacteria and 1 yeast were used as test organisms: Aeromonas hydrophila ATCC 7965, Bacillus cereus FMC 19, Bacillus subtilis ATCC 6630, Escherichia coli ATCC 25922, Klebsiella pneumoniae FMC 5, Listeria monocytogenes 1/2B, Morganella morganii, Mycobacterium smegmatis RUT, Proteus mirabilis BC 3624, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium NRRLE 4463, Staphylococcus aureus ATCC 25923, Yersinia enterocolitica ATCC 1501 and Candida albicans ATCC 1223. These microorganisms were supplied by the Department of Food Engineering, Erciyes University, Kayseri, Turkey. Test yeast namely C. albicans and Y. enterocolitica were grown in malt extract and nutrient broths at 25°C for 18 h, respectively. The other microorganisms were grown in nutrient broth at 35°C for 18 h. All test microorganisms in nutrient broth or malt extract broth were enumerated by using the serial dilution method. Their final cell concentrations were 106–107 cfu/ml. The agar diffusion method was used to detect antimicrobial activity.[27] And 250 μl of each microorganism was added into a flask containing 25-ml sterile nutrient agar or malt extract agar at 45°C and poured into Petri dishes (9-cm diameter). Then, the agars were allowed to solidify at 4°C for 1 h. The holes were made in the agar using sterile cork borers (Ø = 6 mm). The extracts (50 μl) were prepared at 10% concentrations in absolute methanol and were applied to the holes using a pipettor, and absolute methanol without herb extract was used as a control. Y. enterocolitica and C. albicans were incubated at 25°C for 14–24 h in the inverted position. The other microorganisms were incubated at 35°C for 18–24 h. At the end of the period, inhibition zones, which formed on the medium, were measured in millimetres (mm). Tetracycline (10 mg/ml) (Sigma T3258-56; Sigma-Aldrich, St Louis, MO, USA) standard antibiotics was used as positive control. All the tests were performed in duplicate, and the results were presented as average.
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
3
Bioactivity of Senecio
Sevil Albayrak et al.
Results and Discussion
Determination of minimum inhibitory concentration The minimal inhibitory concentration (MIC) values were also studied for the microorganisms, which were determined as sensitive to the extracts in the agar- well diffusion assay. The inocula of the bacterial strains were prepared from 18-h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity (Biosan, Riga, Latvia). First of all, the extracts were dissolved in dimethylsulfoxide. Then they were diluted to the highest concentration (50 mg/ml) to be tested. Next, serial twofold dilutions were made of the extracts in a concentration range of 0.75– 50 mg/ml in 10-ml sterile test tubes containing nutrient broth. The MIC values of the extracts against bacterial strains were determined based on a micro-well dilution method.[28] In brief, 96-well plates were prepared by dispensing 95 μl of nutrient broth and 5 μl of the inocula into each well. Then, 100 μl of the extracts stock solutions was added into the first wells. Afterward, 100 μl of their serial dilutions was transferred into six consecutive wells. The last well, containing 195 μl of nutrient broth without compound and 5 μl of the inocula on each strip, was used as a negative control. The final volume in each well was 200 μl. The plate was covered with a sterile plate sealer. The contents of each well were mixed on a plate shaker at 300 rpm for 20 s and then incubated at 37°C (25°C for yeasts) for 24 h. The MIC was defined as the lowest concentration of the sample that prevented visible growth. Each assay in this experiment was repeated twice.
Statistical analyses Data from the experiments were subjected for the analysis of variance (ANOVA) using SPSS[29] for Windows. Percentage data were transformed using arcsine √x before ANOVA. Means were separated at the 5% significance level by the least significant difference test.
Table 1
The methanol extracts obtained from nine Senecio species growing in Turkey were evaluated for their phenolic contents, antioxidant and antimicrobial activity. The total phenolics of the methanol extracts were measured using Folin–Ciocalteu’s assay and results are presented in Table 1. The results show that all extracts tested in this study contain significantly high total phenolic contents ranging from 11.63 ± 2.1 mg GAE/g extract in Senecio viscosus to 117.45 ± 1.8 mg GAE/g extract in S. cilicius. Also, the yield of crude extract from S. viscosus was the least (16.94%), whereas S. cilicius had the highest yield of 35.00% (Table 1). In our previous study, it had been reported that the total phenolic contents of six Senecio species growing in the Black Sea region of Turkey (S. pandurifolius, S. trapezuntinus, S. integrifolius subsp. aucheri, S. hypochionaeus var. argaeus, S. hypochionaeus var. ilkasiensis and S. lorentii) varied from 19.54 to 81.78 mg GAE/g dry extract.[21] In comparison with our previous study, the phenolic contents of S. cilicius (117.45 mg GAE/g), S. mollis (113.40 mg GAE/g), S. othonnae (94.46 mg GAE/g), S. salsuginea (95.82 mg GAE/g), S. sandrasicus (87.70 mg GAE/g) were higher than these species, whereas S. viscosus (11.63 mg GAE/g) had the lower phenolic content than all other tested species.[21] The phenolic compounds of Senecio species tested were determined by the HPLC method. The amount of each compound is presented in Table 2 as μg/g dry residue. Chlorogenic acid, caffeic acid, p-coumaric acid, rutin, eriodictyol, cinnamik acid, quercetin and naringenin were identified by comparison with the retention times and ultraviolet spectra of authentic standards analyzed under identical analytical conditions, while the quantitative data were calculated from their respective calibration curves. The major component present in the extracts was identified as chlorogenic acid (118526.9 μg/g) followed by rutin
The yields, total phenolic contents, total antioxidant activity and 50% inhibition values of Senecio methanolic extracts
Senecio taxa
Yield (%)
Total Phenolic Content (mg GAE/ g extract)
Total Antioxidant Activity (mg AAE/ g extract)
1,1-Diphenyl picrylhydrazyl IC50 (μg /ml)
S. cilicius S. inops subsp. karamanicus S. mollis S. olympicus S. othonnae S. salsuginea S. sandrasicus S. tauricolus S. viscosus
35.00 ± 2.5 31.97 ± 2.1 27.93 ± 1.5 21.93 ± 1.3 34.21 ± 1.4 26.43 ± 12.0 29.56 ± 3.2 22.47 ± 0.7 16.94 ± 0.0
117.45 ± 1.8a 81.62 ± 1.2d 113.40 ± 0.0a 46.12 ± 3.3e 94.46 ± 1.2b 95.82 ± 0.6b 87.70 ± 1.5c 27.86 ± 0.6f 11.63 ± 2.1g
404.12 ± 0.9b 300.73 ± 1.2d 434.48 ± 0.3a 274.87 ± 0.8g 280.97 ± 0.5f 346.66 ± 0.7c 248.31 ± 0.9h 233.13 ± 0.8i 288.14 ± 0.1e
30.01f 43.15d 26.47h 46.81c 27.98g 26.23i 33.30e 73.74b 150.32a
In each column, means of three independent experiments (± standard deviation) with different superscript letters are significantly different (P < 0.05). Total phenolic activity expressed as gallic acid equivalent (GAE), total antioxidant activity expressed as ascorbic acid equivalent (AAE).
4
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Sevil Albayrak et al.
Bioactivity of Senecio
(93690.0 μg/g) and quercetin (2459.0 μg/g). The least abundant compound present in the extracts was cinnamic acid (57.5 μg/g). Chlorogenic acid, caffeic acid and p-coumaric acid are identified in the all tested species. While the highest amount compound was found in the S. cilius, the least amount compound was found in the S. viscosus. Chromatogram of standard compounds is shown in Figure 1. Senecio contain pyrrolizidine alkaloids (PA), which are principally cytotoxins and are incriminated as a common cause of poisoning especially in livestock following consumption of these plants or their derived products.[30] A large variety of sesquiterpenoids, triterpenoids, diterpenoids, pyrrolizidines and shikimic acid had been previously characterized from Senecio species.[31] Phenolic/ aromatic acids, flavonoid glycosides and PA from S. scandens and S. vulgaris were previously identified by
Table 2
using ultraperformance liquid chromatography-DAD/ electrospray ionization-mass spectrometry. In this previous study, jacaranone derivatives were only detected in S. scandens and jacaranone ethyl ester was detected as the predominant constituent. In the fingerprint of the n-butanol extracts, quercetin and kaempferol glycosides derivatives were detected in these species.[16] Phenolics are able to act as antioxidants in a number of ways. Phenolic hydroxyl groups are good hydrogen donors: hydrogen-donating antioxidants can react with reactive oxygen and reactive nitrogen species in a termination reaction, which breaks the cycle of generation of new radicals. The antioxidant capacity of phenolic compounds is also attributed to their ability to chelate metal ions involved in the production of free radicals.[32] The total antioxidant capacities of the methanol extracts obtained from nine Senecio are presented in Table 1. The
The quantity of some phenolic compounds determined in methanolic extracts by HPLC Compounds (μg/g)
Senecio taxa
1
2
3
4
5
6
7
8
Total
S. cilicius S. inops subsp. karamanicus S. mollis S. olympicus S. othonnae S. salsuginea S. sandrasicus S. tauricolus S. viscosus Total
15406.5 9732.8 16413.0 18033.6 16766.1 8586.3 16261.1 10070.4 7257.1 118526.9
94.1 63.9 252.8 123.2 89.6 152.6 280.2 172.9 187.4 1416.7
159.1 79.2 278.6 58.4 178.1 344.2 110.0 77.0 54.2 1338.8
29091.9 16576.0 — — 24627.0 11480.6 11914.5 — — 93690.0
— 102.3 — — 87.5 199.7 — — 261.0 650.5
— 27.8 — 23.9 — — — — 5.8 57.5
74.4 441.2 965.8 110.9 199.1 594.3 73.3 — — 2459.0
— — 456.7 — — — — 158.1 31.2 646.0
44826.0 27023.2 18366.9 18350.0 41947.4 21357.7 28639.1 10478.4 7796.7 218785.4
Compounds: 1, chlorogenic acid; 2, caffeic acid; 3, p-coumaric acid; 4, rutin; 5, eriodictyol; 6, cinnamik asit; 7, quercetin; 8, naringenin. —, Not detected
mAU 278 nm, 4 nm (1.00) 225 200 175 150 125 100
1 2
6
3
75 50
4
8 7
5
25 0 –25 –50 10
20
30
40
50
60
70
80
min
Figure 1 Chromatogram of standard compounds. 1, Chlorogenic acid; 2, caffeic acid; 3, p-coumaric acid; 4, rutin; 5, eriodictyol; 6, cinnamic acid; 7, quercetin; 8, naringenin. © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
5
Bioactivity of Senecio
Sevil Albayrak et al.
120
%Inhibition
100 80 60 40 20 0 8.3 S. cilicius S. olympicus S. tauricolus Butylated hydroxytoluene
Figure 2
16.6
33.3 Concentrations S. inops subsp. karamanicus S. othonnae S. salsuginea
100
S. mollis S. sandrasicus S. viscosus
% 1,1-Diphenyl picrylhydrazyl inhibition values of Senecio taxa at different concentration (μg/ml).
results are reported as mg ascorbic acid equivalents/g extract. S. mollis has the higher total antioxidant activity (434.48 ± 0.3 mg AAE/g extract) than the other eight species, which showed total antioxidant activity in the order: S. cilicius (404.12 ± 0.9), S. salsuginea (346.66 ± 0.7), S. inops subsp. karamanicus (300.73 ± 1.2), S. viscosus (288.14 ± 0.1), S. othonnae (280.97 ± 0.5), S. olympicus (274.87 ± 0.8), S. sandrasicus (248.31 ± 0.9) and S. tauricolius (233.13 ± 0.8) (Table 1). In our previous study, it had been reported that the total antioxidant capacities of six Senecio species tested varied from 103.16 and 165.21 mg AAE/g extract.[21] These values were very lower than that of nine Senecio species tested in this study. The radical-scavenging activity of the methanol extracts of Senecio samples were estimated using DPPH assay. The extracts exhibited the scavenging activity of various degrees and were dose dependent in all species (Figure 2). The IC50 values are reported in Table 1. The methanol extracts of nine Senecio species showed high free radicalscavenging activity in order: S. salsuginea > S. mollis > S. othonnae > S. cilicius > S. sandrasicus > S. inops subsp. karamanicus > S. olympicus > S. tauricolius > S. viscosus with IC50 values of 26.23, 26.47, 27.98, 30.01, 33.30, 43.15, 46.81, 73.74 and 150.32 μg/ml, respectively (Table 1). It had been previously determined that IC50 values of S. pandurifolius, S. trapezuntinus, S. integrifolius subsp. aucheri, S. hypochionaeus var. argaeus, S. hypochionaeus var. ilkasiensis and S. lorentii were 23.32; 35.30; 56.38; 32.89; 15.99 and 37.29 μg/ml, respectively.[21] The high potency in inhibiting lipid peroxidation, strong superoxide and hydroxyl radical-scavenging activity of S. scandens aqueous extract was showed by Liu and Ng.[33] Conforti et al.[34] showed that the methanolic extract and its ethyl acetate 6
66.6
fraction of S. gibbosus subsp. gibbosus aerial parts a significant antioxidant effect in DPPH assay (IC50 = 0.022 and 0.010 mg/ml for methanolic extract and ethyl acetate fraction, respectively). In another study of the same researches, it had been reported that the ethyl acetate extract of Senecio inaequidens possesses a great antioxidant activity compared with S. vulgaris (61.60 and 44.57% of inhibition, respectively, at 0.312 mg/ml).[13] Antioxidant activity of the methanol extract and its fractions obtained from S. herzogui were reported previously by the DPPH technique (IC50 = 1.4–49.4 μg/ml).[35] Linear correlation between the total antioxidant capacity and the total phenolic contents of the nine Senecio methanol extracts (r2 = 0.68) was observed. Also, negative correlation between the total phenolic contents and free radical-scavenging activity (r2 = −0.85) was observed. But there is no correlation (r2 = −0.355) between total antioxidant capacity and free radical-scavenging activity of the extracts (Figure 3). These data are in accordance with results of Kumaran and Karunakaran,[36] who have shown that the content of phenolics in the extracts correlates with the antioxidant activity. High correlation between radicalscavenging activity and total phenolic content of the extracts obtained from various natural sources has also observed in other studies.[4,37] Thus, significant antioxidant activity can be attributed to the presence of high phenolic compounds in the extracts. On the other hand, Yu et al. have shown that there is no correlation between the content of these main antioxidant compounds and the radicalscavenging capacity.[38] The results obtained by us do not support these claims. The antimicrobial activity of Senecio methanol extracts were assayed in vitro by agar diffusion and a broth
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Sevil Albayrak et al.
Bioactivity of Senecio
500
400
Antioxid
300
200 0
20
40
60
80
100
120
40
60
80
100
120
Phenolic 160 140 120 100 80 60
IC50
40 20 0
20
Phenolic 160 140 120 100 80 60
IC50
40 20 200 Antioxid
Figure 3
300
The results of correlation analyses.
400
500
microdilution method against eight Gram (−), five Gram (+) bacteria and C. albicans. The extracts showed significant antibacterial activity against tested bacteria but no activity was observed against the C. albicans. The inhibitory activity of the extracts in Gram (+) bacteria was significantly higher than in Gram (−) bacteria (Table 3). All Senecio taxa, except the S. salsuginea, tested in this study showed antibacterial activity against at least one of eight Gram (−) test bacteria, with inhibition zones ranging 7–12 mm. S. mollis was only effective against P. mirabilis, while S. tauricolius was only effective against A. hydrophila among the tested Gram (−) bacteria. None of the tested Senecio taxa had antibacterial activity against E. coli, S. typhimurium and S. aureus. Among the tested Senecio taxa, only S. inops subsp. karamanicus had inhibitory activity against M. morganii. The most active plant against bacteria was S. viscosus, whereas the least active plant was S. salsuginea compared with other tested Senecio taxa. MIC values of the extracts in comparison with tetracycline are presented in Table 4. The lowest MIC for A. hydrophila was observed with S. viscosus (6.25 mg/ml). The lowest MIC of S. olympicus, S. sandrasicus and S. viscosus was 6.25 mg/ml for K. pneumoniae. MIC of S. inops subsp. karamanicus for M. morganii was 12.5 mg/ ml. P. aeruginosa inhibited at 6.25 mg/ml by both S. olympicus and S. viscosus extracts. The lowest MIC for P. mirabilis was 6.25 mg/ml. The MIC values for Y. enterecolitica were found to be 12.5 mg/ml (S. inops subsp. karamanicus) and 6.25 mg/ml (S. olympicus). The MIC value of S. inops subsp. karamanicus, S. olympicus and S. tauricolius for B. cereus was 12.5 mg/ml. B. subtilis, L. monocytogenes and M. smegmatis were inhibited by extracts at 6.25 mg/ml. These data are in accordance with results of our previous study, which have determined that no activity was observed against the C. albicans except S. hypochionaeus var. ilkasiensis (6.0 mm) among the six Senecio species tested.[21] Similarly, it had been reported that none of six Senecio species tested except S. lorentii was shown inhibitory effect against E. coli. On the contrary, all of the six Senecio species tested showed inhibitory effect against S. aureus and M. morganii (except S. trapezuntinus).[21] El-Shazly et al.[10] reported that the oil obtained from S. aegyptius var. discoideus showed moderate effect against Gram (+) bacteria, while, it had weak activity against Gram (−) bacteria, in contrary of our results, it had significant level of antifungal activity against C. albicans. The antifungal and antibacterial active compound as β-sitosterol from Senecio lyratus methanol extract was isolated and identified by Kiprono et al.[18] Loizzo et al.[11] who reported that S. vulgaris methanol extract showed antimicrobial activity against the B. subtilis and S. aureus (MIC = 0.5 and 0.125 mg/ml, respectively) while that of S. inaequidens showed no
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
7
Bioactivity of Senecio
Table 3
Antibacterial activity of Senecio taxa
Gram (−) A. hydrophila E. coli K. pneumoniae M. morganii P. mirabilis P. aeruginosa S. typhimurium Y. enterocolitica Gram (+) B. cereus B. subtilis L. monocytogenes M. smegmatis S. aureus a
Sevil Albayrak et al.
S. cilicius
S. inops subsp. karamanicus
S. mollis
S. olympicus
S. othonnae
S. salsuginea
S. sandrasicus
S. tauricolus
S. viscosus
Tetracycline (10 mg/ml)
7.0a — 7.0 — 7.0 — — —
10.0 — 7.0 7.0 — — — 9.0
— — — — 8.0 — — —
11.0 — 10.0 — 7.0 10.0 — 7.0
7.0 — 7.0 — 7.0 — — —
— — — — — — — —
— — 7.0 — 9.0 — — —
7.0 — — — — — — —
12.0 — 11.0 — — 11.0 — —
25.0 26.0 25.0 18.0 21.0 23.0 18.0 29.0
— 10.0 10.0 7.0 —
12.0 11.0 9.0 10.0 —
— 10.0 10.0 9.0 —
12.0 11.0 11.0 11.0 —
— 10.0 9.0 9.0 —
— 8.0 9.0 8.0 —
— 11.0 8.0 8.0 —
8.0 — — 10.0 —
— 16.0 16.0 12.0 —
33.0 30.0 27.0 17.0 22.0
Tetracycline (μg/ml)
Inhibition zones include diameter of hole (6 mm). Sample amount 50 μl. —, Not active.
Table 4
Minimal inhibitory concentration values (mg/ml) of Senecio taxa
Gram (−) A. hydrophila K. pneumoniae M. morganii P. mirabilis P. aeruginosa Y. enterocolitica Gram (+) B. cereus B. subtilis L. monocytogenes M. smegmatis
S. cilicius
S. inops subsp. karamanicus
S. mollis
S. olympicus
S. othonnae
S. salsuginea
S. sandrasicus
S. tauricolus
S. viscosus
12.5 12.5 — 12.5 — —
12.5 12.5 12.5 — — 12.5
— — — 12.5 — —
12.5 6.25 — 12.5 6.25 6.25
12.5 12.5 — 6.25 — —
— — — — — —
— 6.25 — 6.25 — —
12.5 — — — — —
6.25 6.25 — — 6.25 —
Research Paper
Journal of Pharmacy And Pharmacology
A comparative study on phenolic components and biological activity of some Senecio species in Turkey Sevil Albayraka, Ahmet Aksoyb, Lutfiye Yurtsevenc and Abit Yas¸arc a Science Faculty, Department of Biology and cGraduate School Natural Applied Science, Erciyes University, Kayseri, and bScience Faculty, Department of Biology, Akdeniz University, Antalya, Turkey
Keywords antimicrobial activity; antioxidant activity; phenolic; Senecio Correspondence Sevil Albayrak, Science Faculty, Department of Biology, Erciyes University, Kayseri 38039, Turkey. E-mail: [email protected] Received February 19, 2014 Accepted May 15, 2014 doi: 10.1111/jphp.12288
Abstract Objective The phenolic components and biological activity of nine Senecio species growing in Turkey were investigated. Methods Senecio species were extracted with methanol. The content of total phenols was determined using Folin–Ciocalteu method, while individual phenolic acids and flavonoids were detected using HPLC analysis. Also, to determine the antioxidant capacity, phosphomolybdenum assay and 1,1-diphenyl picrylhydrazyl (DPPH) radical-scavenging activity assay were used. Antimicrobial activity of extract was determined using agar diffusion and broth microdilution method. Key findings The total phenolic contents of the extracts were found to be highest in Senecio cilicius and Senecio mollis extracts (117.45 and 113.40 mg equivalent to gallic acid/g, respectively). S. salsuginea showed the strongest free radicalscavenging activity with IC50 (the concentration providing 50% inhibition) = 26.23 μg/ml and S. mollis showed the highest antioxidant capacity in the phosphomolybdenum method (434.48 mg equivalent to ascorbic acid/g). The extracts exerted promising antibacterial activity against most of the test bacteria (minimal inhibitory concentration = 6.25–12.5 mg/ml), but no activity was observed against Candida albicans. Conclusion The results demonstrated that nine Senecio species possess high antioxidant and antimicrobial activity in accordance with the high amount of phenolic contents in the extracts that might be natural agencies used in many areas such as food, pharmacy and alternative medicine.
Introduction Free radicals, with unpaired electrons, are produced in normal or pathological cell metabolism and reactive oxygen species (ROS) react easily with the free radicals to convert them into radicals. ROS are highly reactive molecules that include the superoxide anion radicals, hydroxyl radicals and hydrogen peroxide and peroxyl radicals.[1] Oxidative stress, a common feature of metabolic syndrome, is defined as an imbalance between the production and inactivation of ROS in biological systems. Overproduction of ROS results in cellular injury, including lipid peroxidation, protein oxidation and DNA damage.[2] The oxidative damage created by free radical generation is a critical aetiological factor implicated in several chronic human diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis and neurodegenerative diseases and also in the aging process.[3] Antioxidant activity is
defined as an inhibition of the oxidation of lipids, proteins, DNA or other molecules that occurs by blocking the propagation step in oxidative chain reactions.[4] The commonly used synthetic antioxidants such as butylhydroxyanisole and butylhydroxytoluene have potential health risks and toxicity. Therefore, these need to be replaced with natural antioxidants.[5] Phenolics are broadly distributed in the plant kingdom and are the most abundant secondary metabolites of plants. Plant polyphenols have drawn increasing attention because of their potent antioxidant properties and their marked effects in the prevention of various oxidative stress associated diseases such as cancer.[6] The antioxidant ability of phenolic components occurs mainly through a redox mechanism and allows the components to act as reducing
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
1
Bioactivity of Senecio
Sevil Albayrak et al.
agents, hydrogen donors, singlet oxygen quenchers and metal chelators.[4] Many studies have been carried out on some plants, which resulted in development of natural antioxidant formulations for food and other applications. However, scientific information on antioxidant properties of various plants, particularly those that are less widely used in medicine is still scarce. So, it is an interesting and useful task to find new sources for natural antioxidant and functional food.[7] In the last few years, the identification and development of phenolic compounds or extracts from different plants has become a major area of health and medical-related research.[6] On the other hand, several antimicrobials have been developed over the years to control microorganisms. However, the development of antimicrobial resistance and the relatively narrow spectrum of the antimicrobials have had limited success and the microbial contamination of food still poses an important public health and economic challenge.[8] Because of the resistance and the problems of emerging infectious disease have made it inevitable to search for new antimicrobials from nature especially plants.[5,7] Senecio is the largest genus of the tribe Senecioneae (Asteraceae) and more than 1500 species have been reported with a worldwide distribution.[9–11] It is represented by 39 species in Anatolia.[12] This genus is rich in pyrrolizidine alkoloids and furoeremophilanes, which are the most characteristic secondary metabolite, but sesquiterpenes, chalcones and flavonoids have also been reported.[10,11,13–15] Senecio species are used as food and in folk medicine in the Mediterranean area in the treatment of wounds and as anti-emetic, anti-inflammatory and vasodilator preparations.[11,13] It also is used in the treatment of various diseases in many part of the world. Senecio graveolens is used in Argentina as emmenagogue, digestive and cough suppressant. Senecio latifolius leaves were used by Zulu as an emetic and in the treatment of chest complaints.[15] Senecio cannabifolius is used as a traditional remedy for treating virus influenza, enteritis and pneumonia in China.[14] Senecio scandens is used in traditional and folk medicine for its anti-inflammatory, antipyretic and detoxification effects in China. Senecio vulagaris played a certain role as emmenagogue and in case of functional amenorrhoea in Europe.[16] Antibacterial and antifungal activity of compounds and extracts from Senecio species have been investigated by many researchers.[10,11,14,15,17–19] But, there are only three studies on antibacterial activity of Senecio species belonging to Turkish flora in the literature.[12,20,21] In our previous study, the phenolic contents and bioactivity of six Senecio species growing in the Black Sea region of Turkey were reported. This study is a part of the bioactivity examination of many Senecio species growing in the remaining six of seven region of Turkey. Accordingly, 2
this study was undertaken to test the phenolic components, antioxidant and antimicrobial activity of extracts from nine Senecio species belonging to Turkish flora.
Materials and Methods Plant materials Collection information of the nine plant species, which are individually numbered, is listed below: 1. S. cilicius Boiss., between Erzincan and Kelkit, Akdag˘ village, 2300 m, Erzincan, Turkey, July 2006 (Voucher No: AAksoy 2040), endemic. 2. S. inops subsp. karamanicus Hamzaog˘lu & Budak between Karaman and Bas¸yayla Tas¸kent, 1810 m, Karaman, Turkey, Jun 2006 (Voucher No: AAksoy 2310), endemic.[22] 3. S. mollis Willd. Taslıdere, 1330 m, Sivas, Turkey, July 2006 (Voucher No: AAksoy 2030). 4. S. olympicus Boiss., above of Uludag˘ Kırkpınar valley, 2110 m, Bursa, Turkey, July 2005 (Voucher No: AAksoy 2043), endemic. 5. S. othonnae Bieb., Fl. Taur.-Caucas, 1705 m, Sivas, Turkey, July 2006 (Voucher No: AAksoy 2050). 6. S. salsuginea H. Duman & Vural, straight Salt lake from Eskil, 910 m, Aksaray, Turkey, August 2006 (Voucher No: AAksoy 2077), endemic.[23] 7. S. sandrasicus Davis in Notes Roy. Bot. Gard. Edinburgh, Sandras mountain, 1545 m, Mug˘la Köyceg˘iz, Turkey, July 2006 (Voucher No: AAksoy 2031), endemic. 8. S. tauricolus Matthews in Notes R. B. G. Edinburgh, Yahyalı, mine road, 1850 m, Kayseri, Turkey, Jun 2006 (Voucher No: AAksoy 2075), endemic. 9. S. viscosus L., around to Yoncaklı village, 2280 m, Bayburt, Turkey, July 2005 (Voucher No: AAksoy 2080). Voucher specimens were identified by Dr Ahmet Aksoy and have been deposited at the Herbarium of the Department of Biology, Erciyes University, Kayseri, Turkey.
Preparation of the herb extracts Collected plant materials were dried at room temperature. Aerial parts of plants were ground to fine powder. Ground herbs (10 g) were extracted in a Soxhlet extractor (Nüve, Ankara, Turkey) with 100 ml methanol (50°C for 6 h). The extract was concentrated by using rotary evaporator (Rotavapor, Buchi, Switzerland; T < 40°C) under vacuum to get crude extracts. Dried extracts were stored at 4°C until use.
HPLC analysis of phenolic compounds in the extracts The extracts were dissolved in methanol at a concentration of 10 mg/ml. A high-performance liquid chromatograph (Shimadzu, Duisburg, Germany) was equipped with HPLC
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Sevil Albayrak et al.
pumps (LC-10ADvp; Shimadzu, Ontario, Canada) and a diode array detector (DAD) detector (278 nm; Shimadzu, Ontario, Canada). Eclipse XDB-C18 (5 μm) column (250 × 4, 60 mm) (Agilent 1200 Series; Agilent, Ontario, Canada) was used. The flow rate was 0.8 ml/min and the injection volume 20 μl. The analyses of the phenolic compounds were carried out at 30°C using two linear gradients of methanol. Caffeic acid, chlorogenic acid, cinnamic acid, eriodictyol, naringenin, p-coumaric acid, quercetin, and rutin were used as standard. Identification and quantitative analysis were done by comparison with standards.[24]
Determination of total phenolics The total phenolic amount in the extracts has been assessed with the Folin–Ciocalteau total phenols photometric assay.[25] Forty microlitre of methanol extract (1 mg/ml) were taken in test tubes. Also, 2.4 ml of distilled water, 200 μl of Folin–Ciocalteu reagent, 600 μl sodium carbonate (20%) and 760 μl of distilled water were added respectively. The tubes were mixed and allowed to stand at the dark for 2 h. The absorbance was measured at 765 nm against a blank, which contained 40 μl of methanol in place of extract. The total phenolic content was expressed as gallic acid equivalents (GAE) in mg/g of methanol extract.
Evaluation of total antioxidant capacity by phosphomolybdenum method The antioxidant power of the extracts has been assessed with the phosphomolybdenum reduction assay.[26] A 0.4 ml of methanol extract (1 mg/ml) was mixed with 4 ml of the reagent solution (0.6-m sulfuric acid, 28-mm sodium phosphate and 4-mm ammonium molybdate). For the blank, 0.4-ml methanol was mixed with 4 ml of the reagent. The samples were incubated in water bath at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance of the green phosphomolybdenum complex was measured at 695 nm. The antioxidant capacity of the extracts was expressed as the ascorbic acid equivalents (mg AAE/g of methanol extract).
Free radical-scavenging activity The 1,1-diphenyl picrylhydrazyl (DPPH) assay has been adopted for the evaluation of radical-scavenging capacity of the studied plant extracts.[25] The 50 μl of the extract dilution at the concentration range of 0.1–2 mg/ml was mixed with 450 μl of Tris-HCl buffer, pH = 7.4 and 1 ml of the methanol DPPH solution (0.1 mm). The absorbance has been measured at 517 nm after standing at room temperature for 30 min. The control contained 50 μl of methanol in place of extract. The butylated hydroxytoluene MeOH solution was used as a positive control. The decrease in
Bioactivity of Senecio
optical density of DPPH on addition of test samples in relation to the control was used to calculate the free radical-scavenging activity, as percentage inhibition (I %) of DPPH radical.
Percentage inhibition (I %) = (( A C − A S ) A C ) × 100 Where AC = absorbance of the control after 30 min; AS = absorbance of the test sample after 30 min. Extract concentration providing 50% inhibition (IC50) was calculated from the graph of inhibition percentage against extract concentration. The assay was carried out in triplicate.
Antimicrobial activity The 15 microorganisms that contain 13 bacteria and 1 yeast were used as test organisms: Aeromonas hydrophila ATCC 7965, Bacillus cereus FMC 19, Bacillus subtilis ATCC 6630, Escherichia coli ATCC 25922, Klebsiella pneumoniae FMC 5, Listeria monocytogenes 1/2B, Morganella morganii, Mycobacterium smegmatis RUT, Proteus mirabilis BC 3624, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium NRRLE 4463, Staphylococcus aureus ATCC 25923, Yersinia enterocolitica ATCC 1501 and Candida albicans ATCC 1223. These microorganisms were supplied by the Department of Food Engineering, Erciyes University, Kayseri, Turkey. Test yeast namely C. albicans and Y. enterocolitica were grown in malt extract and nutrient broths at 25°C for 18 h, respectively. The other microorganisms were grown in nutrient broth at 35°C for 18 h. All test microorganisms in nutrient broth or malt extract broth were enumerated by using the serial dilution method. Their final cell concentrations were 106–107 cfu/ml. The agar diffusion method was used to detect antimicrobial activity.[27] And 250 μl of each microorganism was added into a flask containing 25-ml sterile nutrient agar or malt extract agar at 45°C and poured into Petri dishes (9-cm diameter). Then, the agars were allowed to solidify at 4°C for 1 h. The holes were made in the agar using sterile cork borers (Ø = 6 mm). The extracts (50 μl) were prepared at 10% concentrations in absolute methanol and were applied to the holes using a pipettor, and absolute methanol without herb extract was used as a control. Y. enterocolitica and C. albicans were incubated at 25°C for 14–24 h in the inverted position. The other microorganisms were incubated at 35°C for 18–24 h. At the end of the period, inhibition zones, which formed on the medium, were measured in millimetres (mm). Tetracycline (10 mg/ml) (Sigma T3258-56; Sigma-Aldrich, St Louis, MO, USA) standard antibiotics was used as positive control. All the tests were performed in duplicate, and the results were presented as average.
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
3
Bioactivity of Senecio
Sevil Albayrak et al.
Results and Discussion
Determination of minimum inhibitory concentration The minimal inhibitory concentration (MIC) values were also studied for the microorganisms, which were determined as sensitive to the extracts in the agar- well diffusion assay. The inocula of the bacterial strains were prepared from 18-h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity (Biosan, Riga, Latvia). First of all, the extracts were dissolved in dimethylsulfoxide. Then they were diluted to the highest concentration (50 mg/ml) to be tested. Next, serial twofold dilutions were made of the extracts in a concentration range of 0.75– 50 mg/ml in 10-ml sterile test tubes containing nutrient broth. The MIC values of the extracts against bacterial strains were determined based on a micro-well dilution method.[28] In brief, 96-well plates were prepared by dispensing 95 μl of nutrient broth and 5 μl of the inocula into each well. Then, 100 μl of the extracts stock solutions was added into the first wells. Afterward, 100 μl of their serial dilutions was transferred into six consecutive wells. The last well, containing 195 μl of nutrient broth without compound and 5 μl of the inocula on each strip, was used as a negative control. The final volume in each well was 200 μl. The plate was covered with a sterile plate sealer. The contents of each well were mixed on a plate shaker at 300 rpm for 20 s and then incubated at 37°C (25°C for yeasts) for 24 h. The MIC was defined as the lowest concentration of the sample that prevented visible growth. Each assay in this experiment was repeated twice.
Statistical analyses Data from the experiments were subjected for the analysis of variance (ANOVA) using SPSS[29] for Windows. Percentage data were transformed using arcsine √x before ANOVA. Means were separated at the 5% significance level by the least significant difference test.
Table 1
The methanol extracts obtained from nine Senecio species growing in Turkey were evaluated for their phenolic contents, antioxidant and antimicrobial activity. The total phenolics of the methanol extracts were measured using Folin–Ciocalteu’s assay and results are presented in Table 1. The results show that all extracts tested in this study contain significantly high total phenolic contents ranging from 11.63 ± 2.1 mg GAE/g extract in Senecio viscosus to 117.45 ± 1.8 mg GAE/g extract in S. cilicius. Also, the yield of crude extract from S. viscosus was the least (16.94%), whereas S. cilicius had the highest yield of 35.00% (Table 1). In our previous study, it had been reported that the total phenolic contents of six Senecio species growing in the Black Sea region of Turkey (S. pandurifolius, S. trapezuntinus, S. integrifolius subsp. aucheri, S. hypochionaeus var. argaeus, S. hypochionaeus var. ilkasiensis and S. lorentii) varied from 19.54 to 81.78 mg GAE/g dry extract.[21] In comparison with our previous study, the phenolic contents of S. cilicius (117.45 mg GAE/g), S. mollis (113.40 mg GAE/g), S. othonnae (94.46 mg GAE/g), S. salsuginea (95.82 mg GAE/g), S. sandrasicus (87.70 mg GAE/g) were higher than these species, whereas S. viscosus (11.63 mg GAE/g) had the lower phenolic content than all other tested species.[21] The phenolic compounds of Senecio species tested were determined by the HPLC method. The amount of each compound is presented in Table 2 as μg/g dry residue. Chlorogenic acid, caffeic acid, p-coumaric acid, rutin, eriodictyol, cinnamik acid, quercetin and naringenin were identified by comparison with the retention times and ultraviolet spectra of authentic standards analyzed under identical analytical conditions, while the quantitative data were calculated from their respective calibration curves. The major component present in the extracts was identified as chlorogenic acid (118526.9 μg/g) followed by rutin
The yields, total phenolic contents, total antioxidant activity and 50% inhibition values of Senecio methanolic extracts
Senecio taxa
Yield (%)
Total Phenolic Content (mg GAE/ g extract)
Total Antioxidant Activity (mg AAE/ g extract)
1,1-Diphenyl picrylhydrazyl IC50 (μg /ml)
S. cilicius S. inops subsp. karamanicus S. mollis S. olympicus S. othonnae S. salsuginea S. sandrasicus S. tauricolus S. viscosus
35.00 ± 2.5 31.97 ± 2.1 27.93 ± 1.5 21.93 ± 1.3 34.21 ± 1.4 26.43 ± 12.0 29.56 ± 3.2 22.47 ± 0.7 16.94 ± 0.0
117.45 ± 1.8a 81.62 ± 1.2d 113.40 ± 0.0a 46.12 ± 3.3e 94.46 ± 1.2b 95.82 ± 0.6b 87.70 ± 1.5c 27.86 ± 0.6f 11.63 ± 2.1g
404.12 ± 0.9b 300.73 ± 1.2d 434.48 ± 0.3a 274.87 ± 0.8g 280.97 ± 0.5f 346.66 ± 0.7c 248.31 ± 0.9h 233.13 ± 0.8i 288.14 ± 0.1e
30.01f 43.15d 26.47h 46.81c 27.98g 26.23i 33.30e 73.74b 150.32a
In each column, means of three independent experiments (± standard deviation) with different superscript letters are significantly different (P < 0.05). Total phenolic activity expressed as gallic acid equivalent (GAE), total antioxidant activity expressed as ascorbic acid equivalent (AAE).
4
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Sevil Albayrak et al.
Bioactivity of Senecio
(93690.0 μg/g) and quercetin (2459.0 μg/g). The least abundant compound present in the extracts was cinnamic acid (57.5 μg/g). Chlorogenic acid, caffeic acid and p-coumaric acid are identified in the all tested species. While the highest amount compound was found in the S. cilius, the least amount compound was found in the S. viscosus. Chromatogram of standard compounds is shown in Figure 1. Senecio contain pyrrolizidine alkaloids (PA), which are principally cytotoxins and are incriminated as a common cause of poisoning especially in livestock following consumption of these plants or their derived products.[30] A large variety of sesquiterpenoids, triterpenoids, diterpenoids, pyrrolizidines and shikimic acid had been previously characterized from Senecio species.[31] Phenolic/ aromatic acids, flavonoid glycosides and PA from S. scandens and S. vulgaris were previously identified by
Table 2
using ultraperformance liquid chromatography-DAD/ electrospray ionization-mass spectrometry. In this previous study, jacaranone derivatives were only detected in S. scandens and jacaranone ethyl ester was detected as the predominant constituent. In the fingerprint of the n-butanol extracts, quercetin and kaempferol glycosides derivatives were detected in these species.[16] Phenolics are able to act as antioxidants in a number of ways. Phenolic hydroxyl groups are good hydrogen donors: hydrogen-donating antioxidants can react with reactive oxygen and reactive nitrogen species in a termination reaction, which breaks the cycle of generation of new radicals. The antioxidant capacity of phenolic compounds is also attributed to their ability to chelate metal ions involved in the production of free radicals.[32] The total antioxidant capacities of the methanol extracts obtained from nine Senecio are presented in Table 1. The
The quantity of some phenolic compounds determined in methanolic extracts by HPLC Compounds (μg/g)
Senecio taxa
1
2
3
4
5
6
7
8
Total
S. cilicius S. inops subsp. karamanicus S. mollis S. olympicus S. othonnae S. salsuginea S. sandrasicus S. tauricolus S. viscosus Total
15406.5 9732.8 16413.0 18033.6 16766.1 8586.3 16261.1 10070.4 7257.1 118526.9
94.1 63.9 252.8 123.2 89.6 152.6 280.2 172.9 187.4 1416.7
159.1 79.2 278.6 58.4 178.1 344.2 110.0 77.0 54.2 1338.8
29091.9 16576.0 — — 24627.0 11480.6 11914.5 — — 93690.0
— 102.3 — — 87.5 199.7 — — 261.0 650.5
— 27.8 — 23.9 — — — — 5.8 57.5
74.4 441.2 965.8 110.9 199.1 594.3 73.3 — — 2459.0
— — 456.7 — — — — 158.1 31.2 646.0
44826.0 27023.2 18366.9 18350.0 41947.4 21357.7 28639.1 10478.4 7796.7 218785.4
Compounds: 1, chlorogenic acid; 2, caffeic acid; 3, p-coumaric acid; 4, rutin; 5, eriodictyol; 6, cinnamik asit; 7, quercetin; 8, naringenin. —, Not detected
mAU 278 nm, 4 nm (1.00) 225 200 175 150 125 100
1 2
6
3
75 50
4
8 7
5
25 0 –25 –50 10
20
30
40
50
60
70
80
min
Figure 1 Chromatogram of standard compounds. 1, Chlorogenic acid; 2, caffeic acid; 3, p-coumaric acid; 4, rutin; 5, eriodictyol; 6, cinnamic acid; 7, quercetin; 8, naringenin. © 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
5
Bioactivity of Senecio
Sevil Albayrak et al.
120
%Inhibition
100 80 60 40 20 0 8.3 S. cilicius S. olympicus S. tauricolus Butylated hydroxytoluene
Figure 2
16.6
33.3 Concentrations S. inops subsp. karamanicus S. othonnae S. salsuginea
100
S. mollis S. sandrasicus S. viscosus
% 1,1-Diphenyl picrylhydrazyl inhibition values of Senecio taxa at different concentration (μg/ml).
results are reported as mg ascorbic acid equivalents/g extract. S. mollis has the higher total antioxidant activity (434.48 ± 0.3 mg AAE/g extract) than the other eight species, which showed total antioxidant activity in the order: S. cilicius (404.12 ± 0.9), S. salsuginea (346.66 ± 0.7), S. inops subsp. karamanicus (300.73 ± 1.2), S. viscosus (288.14 ± 0.1), S. othonnae (280.97 ± 0.5), S. olympicus (274.87 ± 0.8), S. sandrasicus (248.31 ± 0.9) and S. tauricolius (233.13 ± 0.8) (Table 1). In our previous study, it had been reported that the total antioxidant capacities of six Senecio species tested varied from 103.16 and 165.21 mg AAE/g extract.[21] These values were very lower than that of nine Senecio species tested in this study. The radical-scavenging activity of the methanol extracts of Senecio samples were estimated using DPPH assay. The extracts exhibited the scavenging activity of various degrees and were dose dependent in all species (Figure 2). The IC50 values are reported in Table 1. The methanol extracts of nine Senecio species showed high free radicalscavenging activity in order: S. salsuginea > S. mollis > S. othonnae > S. cilicius > S. sandrasicus > S. inops subsp. karamanicus > S. olympicus > S. tauricolius > S. viscosus with IC50 values of 26.23, 26.47, 27.98, 30.01, 33.30, 43.15, 46.81, 73.74 and 150.32 μg/ml, respectively (Table 1). It had been previously determined that IC50 values of S. pandurifolius, S. trapezuntinus, S. integrifolius subsp. aucheri, S. hypochionaeus var. argaeus, S. hypochionaeus var. ilkasiensis and S. lorentii were 23.32; 35.30; 56.38; 32.89; 15.99 and 37.29 μg/ml, respectively.[21] The high potency in inhibiting lipid peroxidation, strong superoxide and hydroxyl radical-scavenging activity of S. scandens aqueous extract was showed by Liu and Ng.[33] Conforti et al.[34] showed that the methanolic extract and its ethyl acetate 6
66.6
fraction of S. gibbosus subsp. gibbosus aerial parts a significant antioxidant effect in DPPH assay (IC50 = 0.022 and 0.010 mg/ml for methanolic extract and ethyl acetate fraction, respectively). In another study of the same researches, it had been reported that the ethyl acetate extract of Senecio inaequidens possesses a great antioxidant activity compared with S. vulgaris (61.60 and 44.57% of inhibition, respectively, at 0.312 mg/ml).[13] Antioxidant activity of the methanol extract and its fractions obtained from S. herzogui were reported previously by the DPPH technique (IC50 = 1.4–49.4 μg/ml).[35] Linear correlation between the total antioxidant capacity and the total phenolic contents of the nine Senecio methanol extracts (r2 = 0.68) was observed. Also, negative correlation between the total phenolic contents and free radical-scavenging activity (r2 = −0.85) was observed. But there is no correlation (r2 = −0.355) between total antioxidant capacity and free radical-scavenging activity of the extracts (Figure 3). These data are in accordance with results of Kumaran and Karunakaran,[36] who have shown that the content of phenolics in the extracts correlates with the antioxidant activity. High correlation between radicalscavenging activity and total phenolic content of the extracts obtained from various natural sources has also observed in other studies.[4,37] Thus, significant antioxidant activity can be attributed to the presence of high phenolic compounds in the extracts. On the other hand, Yu et al. have shown that there is no correlation between the content of these main antioxidant compounds and the radicalscavenging capacity.[38] The results obtained by us do not support these claims. The antimicrobial activity of Senecio methanol extracts were assayed in vitro by agar diffusion and a broth
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
Sevil Albayrak et al.
Bioactivity of Senecio
500
400
Antioxid
300
200 0
20
40
60
80
100
120
40
60
80
100
120
Phenolic 160 140 120 100 80 60
IC50
40 20 0
20
Phenolic 160 140 120 100 80 60
IC50
40 20 200 Antioxid
Figure 3
300
The results of correlation analyses.
400
500
microdilution method against eight Gram (−), five Gram (+) bacteria and C. albicans. The extracts showed significant antibacterial activity against tested bacteria but no activity was observed against the C. albicans. The inhibitory activity of the extracts in Gram (+) bacteria was significantly higher than in Gram (−) bacteria (Table 3). All Senecio taxa, except the S. salsuginea, tested in this study showed antibacterial activity against at least one of eight Gram (−) test bacteria, with inhibition zones ranging 7–12 mm. S. mollis was only effective against P. mirabilis, while S. tauricolius was only effective against A. hydrophila among the tested Gram (−) bacteria. None of the tested Senecio taxa had antibacterial activity against E. coli, S. typhimurium and S. aureus. Among the tested Senecio taxa, only S. inops subsp. karamanicus had inhibitory activity against M. morganii. The most active plant against bacteria was S. viscosus, whereas the least active plant was S. salsuginea compared with other tested Senecio taxa. MIC values of the extracts in comparison with tetracycline are presented in Table 4. The lowest MIC for A. hydrophila was observed with S. viscosus (6.25 mg/ml). The lowest MIC of S. olympicus, S. sandrasicus and S. viscosus was 6.25 mg/ml for K. pneumoniae. MIC of S. inops subsp. karamanicus for M. morganii was 12.5 mg/ ml. P. aeruginosa inhibited at 6.25 mg/ml by both S. olympicus and S. viscosus extracts. The lowest MIC for P. mirabilis was 6.25 mg/ml. The MIC values for Y. enterecolitica were found to be 12.5 mg/ml (S. inops subsp. karamanicus) and 6.25 mg/ml (S. olympicus). The MIC value of S. inops subsp. karamanicus, S. olympicus and S. tauricolius for B. cereus was 12.5 mg/ml. B. subtilis, L. monocytogenes and M. smegmatis were inhibited by extracts at 6.25 mg/ml. These data are in accordance with results of our previous study, which have determined that no activity was observed against the C. albicans except S. hypochionaeus var. ilkasiensis (6.0 mm) among the six Senecio species tested.[21] Similarly, it had been reported that none of six Senecio species tested except S. lorentii was shown inhibitory effect against E. coli. On the contrary, all of the six Senecio species tested showed inhibitory effect against S. aureus and M. morganii (except S. trapezuntinus).[21] El-Shazly et al.[10] reported that the oil obtained from S. aegyptius var. discoideus showed moderate effect against Gram (+) bacteria, while, it had weak activity against Gram (−) bacteria, in contrary of our results, it had significant level of antifungal activity against C. albicans. The antifungal and antibacterial active compound as β-sitosterol from Senecio lyratus methanol extract was isolated and identified by Kiprono et al.[18] Loizzo et al.[11] who reported that S. vulgaris methanol extract showed antimicrobial activity against the B. subtilis and S. aureus (MIC = 0.5 and 0.125 mg/ml, respectively) while that of S. inaequidens showed no
© 2014 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
7
Bioactivity of Senecio
Table 3
Antibacterial activity of Senecio taxa
Gram (−) A. hydrophila E. coli K. pneumoniae M. morganii P. mirabilis P. aeruginosa S. typhimurium Y. enterocolitica Gram (+) B. cereus B. subtilis L. monocytogenes M. smegmatis S. aureus a
Sevil Albayrak et al.
S. cilicius
S. inops subsp. karamanicus
S. mollis
S. olympicus
S. othonnae
S. salsuginea
S. sandrasicus
S. tauricolus
S. viscosus
Tetracycline (10 mg/ml)
7.0a — 7.0 — 7.0 — — —
10.0 — 7.0 7.0 — — — 9.0
— — — — 8.0 — — —
11.0 — 10.0 — 7.0 10.0 — 7.0
7.0 — 7.0 — 7.0 — — —
— — — — — — — —
— — 7.0 — 9.0 — — —
7.0 — — — — — — —
12.0 — 11.0 — — 11.0 — —
25.0 26.0 25.0 18.0 21.0 23.0 18.0 29.0
— 10.0 10.0 7.0 —
12.0 11.0 9.0 10.0 —
— 10.0 10.0 9.0 —
12.0 11.0 11.0 11.0 —
— 10.0 9.0 9.0 —
— 8.0 9.0 8.0 —
— 11.0 8.0 8.0 —
8.0 — — 10.0 —
— 16.0 16.0 12.0 —
33.0 30.0 27.0 17.0 22.0
Tetracycline (μg/ml)
Inhibition zones include diameter of hole (6 mm). Sample amount 50 μl. —, Not active.
Table 4
Minimal inhibitory concentration values (mg/ml) of Senecio taxa
Gram (−) A. hydrophila K. pneumoniae M. morganii P. mirabilis P. aeruginosa Y. enterocolitica Gram (+) B. cereus B. subtilis L. monocytogenes M. smegmatis
S. cilicius
S. inops subsp. karamanicus
S. mollis
S. olympicus
S. othonnae
S. salsuginea
S. sandrasicus
S. tauricolus
S. viscosus
12.5 12.5 — 12.5 — —
12.5 12.5 12.5 — — 12.5
— — — 12.5 — —
12.5 6.25 — 12.5 6.25 6.25
12.5 12.5 — 6.25 — —
— — — — — —
— 6.25 — 6.25 — —
12.5 — — — — —
6.25 6.25 — — 6.25 —
