Corresponding author: Rumyana Vitanska-Simeonova ( rvitanska@gmail.com ) Academic editor: Plamen Peikov
© 2021 Gazela Nassar-Eddin, Dimitrina Zheleva-Dimitrova, Nikolay Danchev, Rumyana Vitanska-Simeonova.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Nassar-Eddin G, Zheleva-Dimitrova D, Danchev N, Vitanska-Simeonova R (2021) Antioxidant and enzyme-inhibiting activity of lyophilized extract from Clinopodium vulgare L. (Lamiaceae). Pharmacia 68(1): 259-263. https://doi.org/10.3897/pharmacia.68.e61911
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Clinopodium vulgare L. (Lamiaceae) was used in the traditional Bulgarian medicine for treatment of wounds, diabetes and gastric ulcers. The aim of the present study was to evaluate the antioxidant capacity of the extract (CVE) and fractions from C. vulgare (CV) using DPPH, ABTS and FRAP methods. Enzyme inhibitory activity against acetylcholinesterase, α-glucosidase and α-amylase was also investigated. Rosmarinic acid was used as a positive control. The fraction CV3 demonstrated the highest radical scavenging activity with IC50 values of 0.02 mg/ml (DPPH) and 0.0002 mg/ml (ABTS), as well as the strongest ferric reducing potential (FRAP) of 0.89 mM TE/mg dw. The crude aqueous-methanol extract of C. vulgare also showed high activity with IC50 values of 0.05 mg/ml (DPPH), 0.04 mg/ml (ABTS) and 0.89 mM TE/mg dw (FRAP). Moreover, CV3 demonstrated moderate α-glucosidase and α-amylase inhibitory potential.
ABTS, α-glucosidase, α-amylase, Clinopodium vulgare, DPPH, FRAP
Clinopodium vulgare L. (Lamiaceae) is a perennial herbaceous plant widespread in Bulgaria. Aerial parts are used in the Bulgarian folk medicine for treatment of diabetes, gastric ulcers and cancer. The genus Clinopodium L. consist of flowering plants, widely distributed in southern and southeastern Europe, North America, Latin America and Asia (
Plants have been used for many years in the traditional medicine to treat various diseases and conditions. Over the last decades it was established that the oxidative stress is involved in the initial development of many diseases including Alzheimer’s disease, (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS) (
Based on accurate masses, MS/MS and comparison with standards, a variety of flavonoids, caffeic acid oligomers and saponins were tentatively elucidated in CVE. Rosmarinic acid (RA) was the major compound. Prevoius UHPLC-HRMS analysis revealed CVE as a new rich source of water soluble caffeic acid oligomers (
In the present study we provided evidence for the antioxidant and enzyme-inhibiting effects of the investigated lyophilized extract (CVE) and fractions from C. vulgare (CV).
C. vulgare aerial parts were collected in July 2017 from region of German village near Sofia, Bulgaria (voucher specimen SO 107606). Air-dried powdered aerial parts (50 g) were triplicate extracted with water (500 ml) by ultrasound assisted extraction (15 min each time). A lyophilized C. vulgare extract (CVE) (5 g) was used for further phytochemical and pharmacological assays.
1 g of the obtained crude CVE was dissolved in 5 ml of distilled water and applied to a low-bar liquid chromatographic system (Lobar, RP18, Merck). Elution was performed with 100 ml of distilled water solution and increasing concentrations of methanol (0 → 70%). Fractions of 30 ml were collected. The composition of the fractions was monitored by HPLC-UV, on an RP 18 reversed phase column and water-methanol gradient elution. Fractions of similar composition were combined. The procedure was repeated three times. 3 combinations with similar composition were selected for the present work – CV1, CV2, CV3, as well as the raw CVE. Rosmarinic acid was found to be the main compound in CVE (
2,2’-Diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azinobis-(3ethylbenzothiazine-6-sulfonic acid) (ABTS), 6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tripyridyl-s-triazine (TPTZ), FeCl3·6H2O, sodium acetate, potassium persulphate, acetylcholinesterase (AChE) type VI-S, from electric eel 349 U/mg solid, 411 U/mg protein, acetylthiocholine iodide (AChI) were purchased from Sigma-Aldrich. All the others chemicals including the solvents were of analytical grade.
DPPH method for determination of radical scavenging activity
DPPH assay was done according to the method of (
ABTS radical scavenging activity
One mL of a 7 mM solution of 2, 2’-azino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was mixed with 1 ml of a 2.4 mM K2S2O8 solution. This mixture was left in the dark for approximately 12 hours, after which 60 ml of MeOH were added to a few ml of it to obtain an absorption of about 0.7 at 734 nm. Then, 0.3 ml of this solution was added to 0.3 ml of the solution of CVE or CV fraction. The absorbance of the mixture was measured after 7 minutes at 734 nm. Mixture of ABTS and methanol served as a control. Decreased absorption due to the discoloration of the ABTS solution indicates higher radical scavenging activity. At pronounced radical scavenging activity, the IC50 concentration at which the % ABTS radical scavenging activity decreased by 50% was also calculated (
Ferric reducing/antioxidant power (FRAP method)
Total antioxidant activity – FRAP, was conducted by the method of (
Determination of the antioxidant activity in a linoleic acid system
Inhibition of lipid peroxidation of the CVE and CV fractions was performed by so-called ammonium-thiocyanate method (
AChE inhibition assay
The acetylcholinesterase inhibitory activity of the CVE or of selected fractions of Clinopodium vulgare and rosemary RA acid was determined by the method of (Ellman 1961). Briefly in 96-well plate, 10 µl solution of the CVE or the fractions, 15 µl solution of AchE in phosphate buffer (0.03 U/ ml) and 200 μl buffer (pH 8) were mixed. After 30 minutes incubation 15 μl solution of DTNB (0.3 mM) and 15 μl solution of acetylthiocholine iodide (1.8 mM) were aded. The mixture was incubated for another 15 minutes at room temperature and the absorbance at 403 nm was measured. Absorption was recorded on a Microplate Reader Biochrom EZ 800. Galantamine was used as a positive control.
Determination of α-glucosidase inhibitory activity
Alfa-glucosidase inhibitory activity was determined by the method described by (
Determination of α-amylase inhibitory activity
Alfa-amylase inhibitory activity was determined by the Caraway-Somogyi method with minor modifications (
The reference standard rosemary acid (RA) and butylhydroxytoluene (BHT) were used as positive controls.
Statistical analysis
For all the experiments all the assays were carried out in triplicate. Results were expressed as a mean ± SD (n = 3). Values of p < 0.05 were considered statistically significant. The differences between the groups were analyzed using one-way analysis of variance (ANOVA).
Various antioxidant activity tests were performed to determine the antioxidant profile of the crude plant extract CVE and 3 different fractions. Tests based on different mechanisms were used in the research. The results for DPPH, ABTS and FRAP activity are presented in Table
DPPH, ABTS и FRAP activity of the fractions and raw extract of Clinopodium vulgare.
Sample | DPPH | ABTS | FRAP |
---|---|---|---|
IC50 mg/ml | IC50 mg/ml | mM TE/mg dw | |
CV1 | 0.19 | 0.03 | 0.69 |
CV2 | 0.1 | 0.05 | 0.44 |
CV3 | 0.02 | 0.002 | 0.89 |
CVE | 0.05 | 0.04 | 0.85 |
RA | 0.0004 | 0.007 | 1.9 |
Inhibition of lipid peroxidation of the combined fraction CV3 and the crude CVE are presented in Figure
In in vitro assays for acetylcholinesterase (AchE) inhibitory activity, the extract, fractions and pure RA did not show inhibitory activity at the concentrations tested. Therefore, the test was performed with higher concentration (10 mg/ml) for CV3, CVE and rosemary acid, but the results also showed a lack of acetylcholinesterase inhibitory activity.
In our previous in vivo experiments (
On the other hand, all samples tested showed significant α-glucosidase inhibitory activity. Fraction CV3 again showed the highest activity, higher than rosmarinic acid and comparable to that of acarbose. This is also established after the calculations of the IC50 of the studied fractions. CV3 and CVE exhibited the most pronounced statistically significant inhibitory activity (Table
Sample | IC50 mg/ml |
---|---|
CV3 | 0.03 |
CVE | 0.05 |
RA | 0.13 |
Acarbose | 0.027 |
In addition, it is likely that the other components in the composition of the crude extract and CV3 have such activity.
Data on α-amylase inhibitory activity are presented in Table
Sample | Concentration (mg/ml) | Inhibitory activity (%) |
---|---|---|
CV1 | 1.88 | 12.52 |
CV3 | 1.56 | 18.56 |
CVE | 1.12 | 16.61 |
RA | 1.10 | 17.32 |
Acarbose | 1.29 | 84.78 |
The most active fraction CV3 was analyzed by UHPLC-HRMS. Based on the retention times, MS and MS/MS accurate masses, fragmentation patterns and comparison with reference standards, the main compounds in CV3 were identified as clinopodic acid K, followed by salvianolic acid A and salvianolic acid L (Fig.
In conclusion, the studied extract and fractions from C. vulgare containing a variety of phenylpropanoids possess a moderate antioxidant and enzyme inhibitory potential that could be explored in further in vivo experiments
The study was carried out with the financial support by the Medical University-Sofia (Council of Medical Science, Project № 8232/2018, Contract № D-147/2019).