In this study, antioxidant, cytotoxic, larvicidal, antimicrobial and anthelmintic effects and phenolic contents of ethanol, methanol and acetone extracts of leaf and tuber parts of Cyclamen alpinum were investigated. DPPH, ABTS, β-carotene assays were carried out in antioxidant activity and total phenolic and flavonoid contents were tested in determination assay. 9 phenolic contents were determined by HPLC. Artemia salina was used in the cytotoxic effect. Larvicidal effect was investigated against Culex pipiens. Disc diffusion method was used in antimicrobial effect. The tuber part was found to be more toxic than the leaf part in the anthelmintic activity assay. The highest value obtained from the antioxidant activity experiment was observed with value of 86.73 ± 0.16 (%) in DPPH assay. The lowest LC50 value in larvicidal effect was determined 0.151 mg/mL after 72 hours. Consequently, there is need for further studies on the Cyclamen alpinum, which has the ability to fight against diseases such as cancer, cardiovascular diseases, etc.
Free radicals are substances in which atoms in normal or pathological cells contain one or more unpaired electrons and they are very unstable [
Antioxidants are compounds that protect molecules by inhibiting the initiation or propagation of oxidant chain reactions caused by free radicals. Antioxidants are divided into natural and synthetic [
The genus Cyclamen, which possesses geofit plant species, belongs to the family of Primulaceae [
Although this study is not the first study of C. alpinum species, it has been carried out to fill the gaps in the literature and to expand the studies on this species. In a study by Arslan et al. [
The aim of this study was to investigate the phenolic compounds, antioxidant, cytotoxic, larvicidal, antimicrobial and anthelmintic activity of various solvent extracts of C. alpinum tubers and leaves. The findings obtained from this study can increase the overall value of the medical potential of the plant.
Leaves and tubers of C. alpinum were collected from their habitats in Denizli, Turkey, in March-April 2015, identified from the book of Flora of Turkey [
Dried samples were pulverized with blender then 10 g samples were transferred to each 250 mL’s erlenmeyers and ethanol, methanol or acetone were poured 100 mL into erlenmeyers. Erlenmeyers were put in a shaker water bath (Memmert WNB 22) at 49˚C for 6 h. Then the mixture of extraction was filtered with Whatmann No: 1 filter paper. This process was repeated twice time. The solvents were separated by rotary evaporator (Ika RV 10) at 48˚C - 49˚C. The water inside extracts were frozen at −80˚C and drawn out with freze-dryer (Labconco Freezone 6) at −54˚C. Extracts were stored in −20˚C [
2.2-diphenyl-1-picrylhydrazyl (DPPH) free radical was used for determined the scavenging activity according to method of Wu et al. [
where Ab is the absorbance value of the control (methanol) and As is the absorbance value of sample involving extract.
The antioxidant activity of extracts was measured with β-carotene-linoleic acid model system according to method of Amin et al. [
where A0s is the initial absorbance value of sample, A0c is the initial value of control, A2s is the absorbance value of sample at 2 h and A2c is the absorbance value of control at 2 h.
2.2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical cation was used for determined the scavenging activity according to method of Re et al. [
where Ab is the absorbance value of the control (methanol) and As is the absorbance value of sample involving extract.
Total phenolic contents were determined with Folin-Ciocalteu method according to Slinkard and Singleton [
Total flavonoid contents were determined used method by Arvouet-Grand et al. [
The Brine Shrimp cytotoxicity activity with Artemia salina of the extracts was determined according to method of Krishnaraju et al. [
Analysis of phenolic compounds in High Performance Liquid Chromatography (HPLC) with standards was performed with modified according to method of Caponio et al. [
Larvicidal activity to mosquito (Cx. pipiens) larvae of the extracts was investigated according to method of Çetin et al. [
The antimicrobial activity of the extracts with disc diffusion method was determined according to method of Collins et al. [
The anthelmintic activity of theextracts were determined with modifications according to method of Ajaiyeoba et al. [
In all experiments, three replicates of each concentration were run at the same time. The standart errors of mean of experiments were analyzed and all experiments except the anthelmintic activity test were compared with the ANOVA-Duncan test by using SPSS Version 23.0 [
Free radicals can result in various diseases such as cancer, cataract, aging, etc. Therefore, antioxidants that can quench free radicals may be implicated in the prevention of these diseases [
DPPH radical scavenging activity test is widely used to test for compounds acting as hydrogen donor for the free radical scavenging [
In leaf part, there is statistical difference between all concentrations at ethanol and methanol solvents (p < 0.005), no statistical difference between 0.8 mg/mL and 1.0 mg/mL at acetone solvent in terms of concentration (p > 0.005). There is no statistical difference between ethanol and methanol at concentration of 0.4 mg/mL in terms of solvent (p > 0.005). In tuber part, there is statistical difference between all concentrations of whole solvents in terms of concentration (p < 0.005). There is statistical difference between overall concentrations of all solvents in terms of concentration (p < 0.005). In standard of BHA, there is statistical difference between all concentrations of all solvents in terms of concentration (p < 0.005). There is no statistical difference between ethanol and acetone at concentration of 0.4 mg/mL and statistical difference between all solvents of 0.6 mg/mL, 0.8 mg/mL 1.0 mg/mL in terms of solvent (p > 0.005), no statistical difference between whole solvents at concentration of 0.2 mg/mL (p > 0.005) (
As the plant concentrations used in the study increased, the amount of DPPH free radicals increased. It was observed that the tuber part was more active than the leaf part and showed that it could act as a free radical scavenger when 1.0 mg/mL was taken into consideration. The reason showing different antioxidant activity of the same plants extracts can be polarities of the solvents [
The ABTS radical cation scavenging activity is generally one of the methods used to measure the hydrogen donating and chain breaking capacity of plant extracts [
Leaf Part | Tuber Part | BHA | |||||||
---|---|---|---|---|---|---|---|---|---|
Concentrations | Ethanol | Methanol | Acetone | Ethanol | Methanol | Acetone | Ethanol | Methanol | Acetone |
0.2 mg/mL | 22.07 ± 0.30 ax, Ay | 26.19 ± 0.14 b, A | 33.01 ± 0.33 c, A | 13.11 ± 0.32 ax, Ay | 23.43 ± 0.40 b, A | 20.98 ± 0.27 c, A | 50.20 ± 0.66 ax, Ay | 58.46 ± 0.44 b, A | 55.70 ± 0.66 c, A |
0.4 mg/mL | 37.41 ± 0.32 a, B | 28.33 ± 0.16 a, B | 61.66 ± 0.34 b, B | 24.37 ± 0.24 a, B | 27.07 ± 0.25 b, B | 48.40 ± 0.16 c, B | 68.47 ± 0.86 ab, B | 69.47 ± 0.50 b, B | 66.47 ± 0.55 a, B |
0.6 mg/mL | 48.17 ± 0.53 a, C | 31.90 ± 0.30 b, C | 82.97 ± 0.18 c, C | 31.10 ± 0.34 a, C | 31.07 ± 0.14 b, C | 73.43 ± 0.71 c, C | 76.22 ± 0.63 a, C | 79.23 ± 0.87 b, C | 78.23 ± 0.43 ab, C |
0.8 mg/mL | 58.76 ± 0.67 a, D | 33.81 ± 0.33 b, D | 84.85 ± 0.62 c, D | 35.23 ± 0.37 a, D | 34.57 ± 0.29 b, D | 83.79 ± 0.21 c, D | 88.74 ± 0.75 a, D | 90.74 ± 0.38 a, D | 89.99 ± 0.63 a, D |
1.0 mg/mL | 68.84 ± 0.43 a, E | 37.93 ± 0.08 b, E | 85.82 ± 0.40 c, D | 37.68 ± 0.40 a, E | 37.19 ± 0.79 b, E | 86.73 ± 0.16 c, E | 96.50 ± 0.50 a, E | 97.50 ± 0.50 a, E | 96.75 ± 0.50 a, E |
x: If lower cases are same in a line, there is no statistical difference in Duncan’s multiple range test (p ≤ 0.05). y: If lower cases are same in a column, there is no statistical difference in Duncan’s multiple range test (p ≤ 0.05).
Leaf Part | Tuber Part | BHA | |||||||
---|---|---|---|---|---|---|---|---|---|
Concentrations | Ethanol | Methanol | Acetone | Ethanol | Methanol | Acetone | Ethanol | Methanol | Acetone |
10 mg/mL | 35.90 ± 0.29 ax, Ay | 39.56 ± 0.24 b, A | 38.59 ± 0.55 b, A | 30.55 ± 0.36 ax, Ay | 35.54 ± 0.32 b, A | 32.16 ± 0.20 c, A | 62.40 ± 0.22 ax, Ay | 69.20 ± 0.39 b, A | 68.17 ± 0.22 c, A |
20 mg/mL | 55.84 ± 0.15 a, B | 68.20 ± 0.40 b, B | 65.21 ± 0.21 c, B | 52.57 ± 0.38 a, B | 58.50 ± 0.33 b, B | 55.84 ± 0.15 c, B | 85.10 ± 0.33 a, B | 90.00 ± 0.29 b, B | 88.40 ± 0.38 c, B |
40 mg/mL | 63.11 ± 0.20 a, C | 72.86 ± 0.22 b, C | 76.21 ± 0.32 c, C | 61.86 ± 0.81 a, C | 65.52 ± 0.37 b, C | 63.11 ± 0.20 c, C | 97.10 ± 0.33 a, C | 95.85 ± 0.23 b, C | 96.50 ± 0.32 b, C |
x: If lower cases are same in a line, there is no statistical difference in Duncan’s multiple range test (p ≤ 0.05). y: If lower cases are same in a column, there is no statistical difference in Duncan’s multiple range test (p ≤ 0.05).
(p < 0.005). There is no statistical difference between methanol and acetone at concentration of 10 mg/mL (p > 0.005) and but there is statistical difference between ethanol and methanol, ethanol and acetone at concentration of 10 mg/mL in terms of solvent (p < 0.005). Additionally, there is statistical difference between all solvents at the other concentrations (20 mg/mL, 40 mg/mL) in terms of solvent (p < 0.005). In tuber part, there is statistical difference between all concentrations with overall solvents in terms of concentration (p < 0.005). There is statistical difference between all solvents at whole concentrations in terms of solvent (p < 0.005). In standard of BHA, there is statistical difference between in all concentrations of all solvents in terms of concentration (p < 0.005).
The result of DPPH scavenging activity was found to be higher than that of ABTS cation scavenging activity. Wang et al. [
In the β-carotene assay, the speed of β-carotene color change in the presence of antioxidants in the linoleic acid medium decreases and the characteristic yellow color is preserved. In this assay, antioxidant activity can be mentioned when the color is preserved [
Antioxidant activity occurs due to the natural polyphenolic compounds present in plant extracts [
Leaf Part | Tuber Part | BHA | ||||||
---|---|---|---|---|---|---|---|---|
Ethanol | Methanol | Acetone | Ethanol | Methanol | Acetone | Ethanol | Methanol | Acetone |
62.40 ± 0.28 ax | 72.27 ± 0.37 b | 83.35 ± 0.29 c | 63.44 ± 0.58 ax | 66.08 ± 0.16 b | 69.74 ± 0.16 c | 92.50 ± 0.23 ax | 94.05 ± 0.18 b | 94.70 ± 0.18 c |
x: If upper cases are same in a line, there is no statistical difference in Duncan’s multiple range test (p ≤ 0.05).
Total Phenolic Contents (mg GAE/g) | |||||
---|---|---|---|---|---|
Leaf Part | Tuber Part | ||||
Ethanol | Methanol | Acetone | Ethanol | Methanol | Acetone |
8.45 ± 0.25 ax | 8.60 ± 0.23 a | 8.95 ± 0.17 a | 7.42 ± 0.22 ax | 8.05 ± 0.11 a | 8.10 ± 0.28 a |
Total Flavonoid Contents (mg QE/g) | |||||
---|---|---|---|---|---|
Leaf Part | Tuber Part | ||||
Ethanol | Methanol | Acetone | Ethanol | Methanol | Acetone |
47.25 ± 0.50 ax | 55.33 ± 0.45 b | 92.63 ± 0.45 c | 3.85 ± 0.20 ax | 3.98 ± 0.19 a | 14.32 ± 0.36 b |
x: If upper cases are same in a line, there is no statistical difference in Duncan’s multiple range test (p ≤ 0.05).
all solvents terms of concentration (p < 0.005). Polyphenols play an active role in preventing important diseases like cancer risk and in protecting human health [
The brine shrimp lethality assay is a rapid, inexpensive and simple biological assay of plant extracts, generally showing positive correlation with cytotoxic and antitumor properties [
The percentage of mortality in which the ethanolic extracts of tuber and leaf
Leaf Part 24 h Later | Tuber Part 24 h Later | |
---|---|---|
Control (dH2O) | 0 Ay | 0 Ay |
0.1 mg/mL | 20 ± 3.33 B | 20 ± 3.33 B |
0.25 mg/mL | 30 ± 5.77 B | 50 ± 5.77 C |
0.5 mg/mL | 70 ± 11.55 C | 70 ± 5.77 D |
1 mg/mL | 90 ± 3.33 D | 90 ± 5.77 E |
LC50 (min) (mg/mL) | 0.167 | 0.121 |
LC50 (mg/mL) | 0.306 | 0.257 |
LC50 (max) (mg/mL) | 0.518 | 0.434 |
LC90 (mg/mL) | 1.171 | 1.073 |
x2 | 0.61 | 0.01 |
y: If lower cases are same in a column, there is no statistical difference in Duncan’s multiple range test (p ≤ 0.05).
parts of the C. alpinum plant affect the 2nd and 3rd larval stages of Cx. pipiens are shown in
Leaf Part 24 h later | Leaf Part 48 h later | Leaf Part 72 h later | Tuber Part 24 h later | Tuber Part 48 h later | Tuber Part 72 h later | |
---|---|---|---|---|---|---|
Control (dH2O) | 0 ax, Ay | 0 a, A | 0 a, A | 0 ax, Ay | 0 a, A | 0 a, A |
0.1 mg/mL | 0 a, A | 0 a, A | 0 a, A | 0 a, A | 0 a, A | 10 ± 3.33 a, B |
0.25 mg/mL | 0 a, A | 0 a, A | 10 ± 0.33 a, A | 0 a, A | 70 ± 5.77 b, B | 100 ± 0.00 c, C |
0.5 mg/mL | 0 a, A | 10 ± 0.33 b, A | 40 ± 0.57 c, B | 0 a, A | 80 ± 3.33 b, C | 100 ± 3.33 c, C |
1 mg/mL | 70 ± 0.88 a, B | 80 ± 1.20 a, B | 90 ± 1.33 a, C | 10 ± 3.33 a, A | 100 ± 3.33 b, D | 100 ± 0.00 b, C |
LC50 (min) (mg/mL) | 0.636 | 0.577 | 0.382 | 0.176 | 0.164 | 0.108 |
LC50 (mg/mL) | 0.924 | 0.760 | 0.534 | 1.383 | 0.244 | 0.151 |
LC50 (max) (mg/mL) | 1.343 | 1.021 | 0.769 | 1.827 | 0.341 | 0.195 |
LC90 (mg/mL) | 1.119 | 1.152 | 1.059 | 1.915 | 0.502 | 0.277 |
x2 | 8.28 | 28.30 | 6.93 | 3.35 | 0.94 | 0.59 |
x: If lower cases are same in a line, there is no statistical difference in Duncan’s multiple range test (p ≤ 0.05). y: If lower cases are same in a column, there is no statistical difference in Duncan’s multiple range test (p ≤ 0.05).
tract cells. In this study, the same effect may be observed due to saponins in the content of C. alpinum plant [
Phenolic compounds and quantities determined by HPLC from C. alpinum plant are given in
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
---|---|---|---|---|---|---|---|---|---|
Quantity (μg/g) | 10.01 | 33.65 | 125.33 | 2.73 | 121.35 | 0.13 | 5.78 | 3.25 | 2.25 |
Retention Time (min) | 7.8 | 12.2 | 18.1 | 19.9 | 22.1 | 23 | 30.3 | 35.7 | 71.1 |
1: Gallic acid, 2: 3.4 dihydroxy benzoic acid, 3: 4 hydroxy benzoic acid, 4: Chlorogenic acid, 5: Vanillic acid, 6: Caffeic acid, 7: p-coumaric acid, 8: Ferulic acid, 9: Cinnamic acid.
end of the HPLC may vary according to the method of extraction of the plant and the conditions of the region it is growing [
The study of Ruiz-Ruiz [
Test microorganisms | Antibiotics and plant extracts | |||||||
---|---|---|---|---|---|---|---|---|
A | P | CTM | CTE | CTA | CLM | CLE | CLA | |
Escherichia coli ATCC 35218 | 21 | nt | 8 ± 2 | 11 ± 2 | - | 4 ± 1 | - | 8 ± 1 |
Staphylococcus aureus ATCC 25923 | nt | 34 | 6 ± 1 | 8 ± 2 | 6 ± 1 | - | 5 ± 0 | 8 ± 0 |
Staphylococcus aureus ATCC 12598 | nt | 6 | - | 7 ± 2 | - | 6 ± 0 | - | 5 ± 1 |
Pseudomonas aeruginosa NRLL B-23 | 4 | nt | 8 ± 2 | - | 7 ± 1 | - | 6 ± 0 | 9 ± 1 |
Bacillus subtilis ATCC 6633 | nt | 14 | - | 4 ± 0 | 8 ± 1 | - | 11 ± 2 | 5 ± 1 |
Bacillus cereus RSKK 863 | nt | 27 | 6 ± 1 | 9 ± 1 | - | 10 ± 2 | 5 ± 1 | - |
Candida albicans ATCC 10239 | 3 | nt | 9 ± 2 | 6 ± 1 | 12 ± 2 | - | 9 ± 2 | 6 ± 1 |
nt: not tested, (-): no inhibition, A: Ampicillin (10 µg), P: Penicillin (10 U), CLA: Cyclamen-Leaf-Acetone, CLE: Cyclamen-Leaf-Ethanol, CLM: Cyclamen-Leaf-Methanol, CTA: Cyclamen-Tuber-Acetone, CTE: Cyclamen-Tuber-Ethanol, CTM: Cyclamen-Tuber-Methanol.
action was Staphylococcus aureus (ATCC 25923) with 34 mm. The ethanol extract of the tuber part is partially active (11 ± 2 mm) against the Escherichia coli (ATCC 35218), acetone extract of the tuber part is partially active (12 ± 2 mm) against candida albicans (ATCC 10239), methanol extract of the leaf part is partially active (10 ± 2 mm) against Bacillus cereus (RSKK 863) and ethanol extract of the leaf part is partially active (11 ± 2 mm) against Bacillus subtilis (ATCC 6633). The results of all other extracts from C. alpinum plant were inactivated.
The percentage of mortality in which the ethanolic extracts of tuber and leaf parts of the C. alpinum plant against Tubifex tubifex are shown in
The results showed that processed C. alpinum extracts exhibited in varying degrees of phenolic content, antimictobial and antioxidant properties and obtained weaken oxidative stress by its antioxidant properties. C. alpinum consumption may play a role in preventing human diseases that interfere with free radicals such as cancer, cardiovascular disease and aging. However, further investigation is needed on phenols, flavonoids, active substance, antioxidant activity and different antioxidant mechanisms, larvicidal effects of mosquito and antimicrobial properties.
Leaf Part 2 min later | Leaf Part 4 min later | Leaf Part 6 min later | Tuber Part 2 min later | Tuber Part 4 min later | Tuber Part 6 min later | |
---|---|---|---|---|---|---|
Control (dH2O) | 0 | 0 | 0 | 0 | 0 | 0 |
1 mg/mL | 0 | 20 | 50 | 0 | 30 | 60 |
2.5 mg/mL | 0 | 40 | 60 | 0 | 50 | 80 |
5 mg/mL | 0 | 50 | 70 | 0 | 60 | 80 |
10 mg/mL | 20 | 70 | 90 | 20 | 60 | 90 |
20 mg/mL | 30 | 70 | 90 | 40 | 70 | 90 |
40 mg/mL | 40 | 80 | 100 | 50 | 80 | 100 |
LC50 (min) (mg/mL) | 25.58 | 3.85 | 0.41 | 28.99 | 2.28 | 0.23 |
LC50 (mg/mL) | 44.07 | 4.99 | 1.32 | 36.47 | 3.44 | 0.52 |
LC50 (max) (mg/mL) | 215.73 | 6.32 | 2.33 | 49.52 | 4.75 | 0.86 |
LC90 (mg/mL) | 214.46 | 87.91 | 13.92 | 212.37 | 126.26 | 1.035 |
x2 | 12.07 | 0.75 | 0.75 | 4.3 | 1.6 | 0.9 |
This project supported by Scientific Projects Administration Unit (BAP) of Pamukkale University, Turkey (BAP Project No: 2015FBE002).
Murat Turan did the assays, collected test results, analysed the statistical data and wrote the article. Ramazan Mammadov, designed the study, interpreted the results of data and helped with references.
Turan, M. and Mammadov, R. (2018) Antioxidant, Antimicrobial, Cytotoxic, Larvicidal and Anthelmintic Activities and Phenolic Contents of Cyclamen alpinum. Pharmacology & Pharmacy, 9, 100-116. https://doi.org/10.4236/pp.2018.94008