Folin-Ciocalteu reagent, Gallic acid, 2,2-diphenyl-2-picryl-hydrazyl-hydrate (DPPH), 2,2‘-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), acarbose, alpha-glucosidase from Saccharomyces cerevisiae, ρ-nitrophenyl-α-D-glucopyranoside (ρNPG) were obtained from Sigma-Aldrich (Darmstadt, Germany). All reagents were of analytical grade.

All plant materials were gathered in the period July-August 2020, from protected area “Kailaka”, near the town of Pleven, Middle Danube plain, Bulgaria. The studied plant substances were as follows: leaves of Sambucus ebulus and Prunus mahaleb, flowering stems of Cichorium intybus and Satureja kitaibelii. The plant community of the Balkan endemic species Satureja kitaibelii falls under the habitat type 6240* Sub-pannonic steppes according to the Habitat Directive 92/43/EEC (1992). The bedrock in the locality is limestone and the altitude is 200 m a.s.l. The taxonomic identification of the species follows Delipavlov and Cheshmedzhiev (2003). For Satureja kitaibelii additional references of Ball et al. (1972), Velchev (1989) and Ciocârlan (2009) are considered. All plant substances were air-dried at room temperature. The yield ratios of fresh to dry herbal substances for the four species are as follows: Sambucus ebulus 3.9:1, Prunus mahaleb 2.9:1, Cichorium intybus 2.6:1, and Satureja kitaibelii 2.3:1.

The air-dried and grounded plant materials were extracted in triplicate with distillated water (1:10 w/v) in an ultrasonic bath (35 kHz) with gradual increase in temperature (T1 = 39 °C; T2 = 48 °C; T3 = 53 °C). The plant extracts were concentrated in a rotary vacuum evaporator, dried in vacuum drying oven and stored under freezing conditions. The yield of the extracts was calculated using the formula:

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Total phenolic content (TPC) was determined by the method of Singleton and Rossi (1965) with some modifications. To 0.2 mL of suitably pre-diluted extract 1.8 mL of dH₂O and 0.2 mL of Folin-Ciocalteu reagent were sequentially added. After five minutes 2 mL of 7% Na₂CO₃ were added and then the mixture was diluted up to 5 mL with dH₂O. The reaction continued in dark for 90 minutes. The absorbance was measured at 750 nm against dH₂O. The standard calibration curve was plotted using gallic acid (1–200 µg/mL). The total phenolic content was expressed as gallic acid equivalents per gram of dry extract (mg GA/g DE).

The radical scavenging ability was determined according to the method of Mensor et al. (2001). Samples with different concentrations were obtained from the initial extracts. To 2.5 mL of every sample 1 mL of 0.3 mM alcoholic DPPH solution was added. The absorbance was measured at 518 nm against a blank sample with ethanol after a stay of the samples for 30 minutes in dark. Solutions of various concentrations of rutin were used as a positive controls. Antiradical activity was expressed as IC₅₀ – the concentration of the extracts that causes 50% inhibition of the radical formation. The IC₅₀ is calculated by a linear graph equation that expresses the relationship between percentages of inhibitory activity and extract/standard concentration.

The method of Alara et al. (2019) was used with minor modifications. The ABTS radical was generated by mixing ABTS (7.0 mM in dH₂O) and potassium persulfate (2.45 mM in dH₂O) in equal amounts. The reaction continued for 16 h at room temperature in dark. Prior to analysis, 2.0 mL of the generated radical (ABTS^•) was diluted in ethanol (1:30; v/v) until obtaining a final absorbance of 0.7 ÷ 0.8 at 734 nm. After this preparation, 1710 μL ABTS solution was mixed with 90 μL of the sample extracts with various concentrations sequentially. The mixtures were kept in dark for 7 minutes and then the absorbencies were measured at 734 nm against a blank sample with ethanol. Solutions of various concentrations of rutin were used as positive controls. Antiradical activity was expressed as IC₅₀ values.

The method of Dong et al. (2012) was employed with slight modification. The reaction mixture of 100 µL 0.1 M phosphate buffer (pH 6.8), 20 µL enzyme solution and 40 µL of extracts or acarbose at different concentration was incubated in each well at 37 °C for 5 min. After incubation 40 µL 0.75 mM ρNPG solution was added to initiate the enzyme reaction. The released p-nitrophenol was monitored at 405 nm each 5 min for a total time of 20 min by a multimode microplate reader Mithras LB 943 MF (Berthold Technologies, Germany) in 96 micro well against a blank sample without enzyme. One unit of enzyme activity was defined as the amount of enzyme that released 1 mM of p-nitrophenol per minute under the assay conditions. The control was the enzyme reaction without inhibitors and the positive control was acarbose. The results of alpha-glucosidase inhibitory activity were expressed as percentages of inhibitory activity and relative enzyme activity. The IC₅₀ value was calculated in manner described above.

Statistical analyses were performed using Microsoft Excel 2010. All analyzes were carried out in triplicate and the results were expressed as a mean ±SD.

Aqueous extracts of medicinal plants are mixtures of multiple components, which may contain both primary and secondary metabolites in various concentrations. The large group of phenolic compounds are secondary plants metabolites, which possess many health benefits, such as anti-inflammatory, antimicrobial, antioxidant, antidiabetic, etc. (Cory et al. 2018).

The extraction yields, total polyphenolic content and antiradical capacity of the extracts against DPPH and ABTS expressed as IC₅₀ values are presented in Table 1. Тhe yield of the extracts, using ultrasound-assisted extraction with water varies from 45 to 50% in aqueous extract of Cichorium intybis (CIW). According to study published by Gupta et al. (2012) water based extraction with ultrasound leads to a 20% increase in extract yield. In addition to the high yield, the efficiency of the extraction, is directly correlating with the extract biological functions. The ultrasound assisted extraction is conducted in lower temperature than conventional methods such as infusion and decoction. That contributes directly to the greater extend for the preservation of thermally unstable components. Additionally, this method is appropriate for increase in polyphenolic yields in plant extracts according to (Dai and Mumper 2010).

Folin-Ciocalteu method is based on oxidation-reduction reactions between the reagents used and phenolic compound. The electron transfer measures the reducing capacity of components in plant extracts (Noreen et al. 2017). The extract that exhibited the highest total phenolic content was SKW (160.10 mg GAE/g DE), followed by extracts of SEW, CIW and PMW. TPC concentration of Satureja kitaibelii in our research correlates with the results of Gopčević et al. (2019) for the Serbian population of the same species. They reported 158.85 ± 15.02 mg GA/g in extract of stem, leaves and flowers The TPC obtained by them using ultrasound assisted extraction method were higher than the conventional methods of extraction which in above-mentioned case was bimaceration.

In the present study total phenolic content of dwarf elder’s extract was 108.59 ± 3.11 mg GAE/ g DE. In previous studies, the estimated concentration of polyphenolic compounds varies from 43.47 mg GAE/g to 116.3 mg GAE/g extract (Meriç et al. 2014; Topuzović et al. 2016; Cvetanović et al. 2017). Cvetanović et al. (2017) reported 116.3 mg chlorogenic acid equivalent in subcritical aqueous extract of Sambucus ebulus leaves with Serbian origin. Topuzović et al. (2016) reported 43.47 mg GAE per gram aqueous extract of leaves obtained by 24 h infusions. In conclusion, numerous environmental factors as climatic and atmospheric conditions in combination with technological parameters such as extraction method, temperature, extraction time, type and polarity of the solvent used, lead to significant differences in the concentration of total polyphenols in Sambucus ebulus (Cvetanović 2020).

The investigated aqueous extract of Cichorium intybus had a total phenolic content of 86.45 ± 1.54 mg GAE/ g DE. Abbas et al. (2015) showed a similar result of 85 mg GAE/ g DE in hydro-alcoholic extract. According to Taghizadeh et al. (2015) total phenolic content in Prunus mahaleb strongly depends on genotypes and ranging from 7.25 to 23.13 mg GAE/g DE. The result obtained in our study is 15.29 mg GAE/g DE and is in the middle of the given range.

To evaluate in vitro antioxidant effects of plant extracts, two methods against organic radicals, were used. These tests are the most accepted models for screening the free radical scavenging activity of any plant extracts. The samples exhibited a concentration-dependent radical inhibitory activity in both analyses. The DPPH assay is based on single electron transfer (SET) colorimetric reaction. There is high positive correlation between concentration of phenolic content and DPPH inhibitory activity because of similar mechanism of the methods (Moharram and Youssef 2014). The lower concentration of extracts shows more potential antiradical activity and measurement of the reducing ability of components in plant extracts. The lowest IC₅₀ value was obtained by SKW (0.087 mg/mL) where is the highest concentration of total phenols (160.10 ± 6.47 mg GAE/g DE). This trend is maintained in the analysis of all investigated plant extracts. The identified IC₅₀ concentration of Satureja kitaibelii extract in our study correspond to those reported by Gopčević et al. (2019) – 71.20 ± 9.55 μg/mL.

ABTS method was reported to use both mechanisms – hydrogen atom transfer (HAT) and SET (Prior et al. 2005). Between 2.33 (Sambucus ebulus) and 4.6 times (Prunus mahaleb) higher concentrations of extracts are required to obtain 50% inhibition of the ABTS radical in comparison with DPPH. Positive control of rutin (quercetin-3-rhamnosyl glucoside) which is a natural flavone derivative, was used in the antiradicals’ analysis. The scavenging effect of samples decreased in the following order: rutin > SKW > SEW > CIW > PWM. The minimum IC₅₀ concentration was obtained by SKW (0.33 mg /mL) which is 1.7 times higher than the positive control.

Many experimental studies exhibited exacerbated relationship between oxidative stress and diabetes by measuring markers of oxidative stress (Yang et al. 2011; Ullah et al. 2016). The high antiradical activity demonstrated in this experimental research is the reason for evaluating in vitro antidiabetic potential of the plants. Alpha-glucosidase inhibitors play a key role in early phase of the treatment of type 2 diabetes. The decrease in the activity of the α-glucosidase leads lower postprandial blood glucose. Acarbose is used as an oral antihyperglycemic agent (Laar 2008). The most prominent gastrointestinal side effects such as diarrhea and flatulence occurring often in patients population treated with acarbose during the course of therapy, make the substances from natural origins a suitable alternative due to expected lack of side effects (Kumar and Sudha 2012).

The α-glucosidase inhibition correlates with the increasing the concentration of tested plant extracts. Their inhibitory activity was compared as a percentage as shown in Table 2. The two different concentrations of each extract tested were observed side by side. The lowest concentration of positive control acarbose of 1 mg/mL demonstrated 5.47% inhibitory activity. After increase the concentrated 7.5 times (7.5 mg/mL) the inhibition raised up to 53.39%. The two concentrations of SKW 0.695 mg/mL and 5.56 mg/mL showed inhibition rates of 28.22% and 92.02%, respectively, which clearly exceeded the inhibitory activities of acarbose. To the best of our knowledge this is the first report of α-glucosidase inhibitory activity of Satureja kitaibelii which shows that the plant has a good potential as a blood glucose regulating agent. At lower tested concentrations, the aqueous extract of chicory showed an inhibition of 11.91% which increased to 48.01% with a tenfold increase in concentration. Both values obtained for the leafy flowering stems of Cichorium intybus are comparable to the performance of acarbose. Dalar and Konczak (2014) reported a pronounced inhibitory activity of the leaf extract against α-glucosidase (IC50:4.25 ± 0.08 mg/ml). Anti-diabetic activity of the whole plant and the fruit (cypsella) were proved through in vivo analysis (Pushparaj et al. 2007; Draz et al. 2010; Chandra et al. 2018). Some other studies research directly the anti-diabetic and related activities of the roots (Kanj et al. 2019), leaves (Mathusamy et al. 2008; Ahmed 2009; Brieudes et al. 2016) or aerial parts (Azay-Milhau et al. 2013) of C. intybus. Our original results are the first evidence for α-glucosidase inhibitory activity of the upper parts of the flowering stems of C. intybus. The properties of the stems of this plant are very poorly studied according to the literature and data on their antidiabetic potential is practically lacking. Probably this is due to their dryness, hollowed pith and significant participation of transport and support tissues, which can lead to the assumption that they do not possess biological activity. Our results show that the chemical composition of the flowering upper part of the stems of needs further research in order to be explained the significant α-glucosidase inhibitory activity which could not be attributed only to the heads in different flowering stage and the few small leaves on this part of the stem. Moreover, the most abundant and easy to collect part of C. intybus as a perennial herbaceous plant is the flowering top part of the stem. On the other hand, SEW and PMW showed only moderate activities with 16.60% and 17.38% inhibition rate. Samples with the lower tested concentrations showed higher inhibition potential described in percentages compared to the positive control – acarbose.

The baseline of the enzyme activity is shown as 100% for the non-inhibited enzyme reaction (Fig. 1A, B). The relative enzyme activity of α-glucosidase decreases within the range from 7% to 64% in dose-dependent manner by adding increasing concentrations of SKW from 0.7 to 5.6 mg/mL (Fig. 1A). The most active amongst the tested plants’ extracts including positive control is SKW. This is also confirmed by comparing IC₅₀ concentrations (Table 3). Therefore, 1.18 mg/mL from SKW extract was needed to achieve 50% inhibition of enzyme, which is approximately 6 times lower than acarbose (6.86 mg/mL).

Figure 1. 

Relative enzyme activity of α-glucosidase by adding different concentration of (A) SKW and (B) acarbose IC₅₀ values and relative enzyme activity were calculated for the samples which exhibited higher than 50% inhibition of α-glucosidase.

After the studies accomplished, it could be concluded that only SKW could be applied as potential alternative of synthetic inhibitors against the digestive action of α-glucosidase.