Rhaponticum acaule (L) DC essential oil: chemical composition, in vitro antioxidant and enzyme inhibition properties

2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), trichloroacetic acid (TCA), potassium ferricyanide [K₃Fe(CN)₆], ferric chloride (FeCl₃), Folin–Ciocalteu reagent (FC reagent), metallic magnesium, hydrochloric acid (HCl), sulphuric acid (H₂SO₄), acetic anhydride, sodium hydroxide (NaOH), aluminum chloride (AlCl₃)_, Aspergillus niger α-glucosidase, Buttermilk grade I xanthine oxidase, p-nitrophenyl-α-D-glucopyranoside (pNPG), acarbose, p-nitrophenyl phosphate (pNPP) and xanthine were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used for the analyses were also obtained from Sigma-Aldrich. Turkey pancreatic lipase (TPL) was purified as described by Sayari and colleagues (Sayari et al. 2000). THL was a generous gift from Hoffmann la Roche (Basel, Switzerland.

The plants were collected at the flowering stage in January 2015 from the area of M’Saken (Sousse-Tunisia). The plant material was identified by Pr. Fethia Harzallah Skhiri (High Institute of Biotechnology of Monastir, Tunisia). A voucher specimen (N°.Ra15) has been deposited in the Herbarium of the Laboratory of Bioressources: Biologie Integrative and Valorization, High Institute of Biotechnology of Monastir, University of Monastir, Tunisia.

An amount of 100 g of R. acaule flowers freshly collected were cut in small pieces and submitted to hydrodistillation for 5–6 h with 500 ml of boiling distilled water using a Clevenger-type apparatus [17] in accordance with the European Pharmacopeia. The distilled essential oil was dried over anhydrous sodium sulfate, transferred to sealed dark vials and stored at 4 °C until use. The yield (0.018%) was calculated based on the fresh weight of the sample.

Analytical GC–MS: GC/EIMS analyses were carried out with a Varian CP-3800 gas-chromatograph equipped with a HP-5 capillary column (30 m × 0.25 mm; coating thickness 0.25 μm) and a Varian Saturn 2000 ion trap mass detector. The analytical conditions were: injector and transfer line temperatures 220 and 240 °C respectively; oven temperature programmed from 60 °C to 240 °C at 3 °C/min; carrier gas helium at 1 mL/min; injection of 0.2 μL (10% hexane solution); split ratio 1:30. Identification of the compounds was based on comparison of the retention times with those of authentic samples, comparing their linear retention indices relative to the series of n-hydrocarbons, and on computer matching against commercial (NIST 2014 and ADAMS) and home-made library mass spectra built up from pure substances and components of known essential oils and MS literature data [18‐21].

A spectrophotometric α-glucosidase assay was performed as previously described by Tao et al. [26] with slight modifications as detailed by Rengasamy et al. [27]. The mixture of α-glucosidase reaction contained 2.5 mM p-nitrophenyl-α-D-glucopyranoside (pNPG), 250 μL of essential oil in DMSO and 0.3 U/mL of α-glucosidase in phosphate buffer, pH 6.9. Pure control having 100% enzyme activity was conducted by replacing the essential oil with DMSO. Blank for pure control having 0% enzyme activity was conducted with DMSO and by replacing the enzyme with buffer. In positive controls, acarbose, which is clinically used as an α-glucosidase inhibitor, replaced the essential oil. Absorbance of the released p-nitrophenol (pNP) was measured (405 nm) and was considered proportional to the activity of the enzyme. Each sample was carried out in triplicate. Inhibition percentage by essential oil and acarbose were calculated using the following equation:

The IC₅₀, which is the necessary concentration of the sample to inhibit 50% of the enzyme, was determined.

For the kinetics study, the reaction mixture was performed as described above, except that the substrate concentration increased from 0.625 to 10 mM, and in the presence of different concentrations of essential oil (7.5, 15 and 30 μg/mL). The reaction was started by the addition of enzyme, and monitored at 405 nm, at 5 min intervals during 30 min. The initial reaction rates were determined using calibration curves constructed by varying p-nitrophenol concentrations. The results were used to construct Lineweaver–Burk plots to determine the type of inhibition, Michaelis-Menten constant (K_m) and maximum velocity (V_max) values. The values of inhibition constant (Ki) were determined from the secondary plots constructed using slopes or y-intercepts of Lineweaver–Burk plots. (Ki) expresses the equilibrium constant for the binding of RaEO to α-glucosidase.

In general, there are four types of enzyme inhibition: competitive, non-competitive, uncompetitive and mixed. For mixed-type inhibition, the Lineweaver-Burk equation in a double reciprocal form can be expressed as follows:

Secondary plots can be constructed fromor

Xanthine oxidase activity was measured spectrophotometrically at 295 nm by continuously measuring uric acid formation, according to the protocol of Kong et al. [28]. The reaction mixture is composed of 250 μL of test solution, 300 μL of 70 mM phosphate buffer (pH 7.5) and 300 μL of substrate solution (150 μM xanthine in the same buffer). After preincubation at 25 °C during 15 min, the reaction was triggered by the addition of 150 μL enzyme solution (0.1 units/mL in 70 mM phosphate buffer (pH 7.5) freshly prepared before use. Pure control having 100% enzyme activity was conducted by replacing the essential oil with DMSO. Blank for pure control having 0% enzyme activity was conducted with DMSO and by replacing the enzyme with buffer. Allopurinol was used as a positive control. The RaEO was tested for xanthine oxidase inhibitory activity at various concentrations. Each sample was carried out in triplicate. The inhibitory activity was determined by IC₅₀, which was obtained from percent inhibition calculated by the following equation:

Percent inhibition (%) = (1 − (ΔOD_sample/ΔOD_control)) × 100.

The IC₅₀, which is the concentration of the sample required to inhibit 50% of the enzyme was determined for each sample.

For the kinetics study, the reaction mixture was as described above, except that the substrate concentration increased from 37.5 to 300 μM, and in the presence of different concentrations of RaEO (2.5, 3.75 and 5 μg/mL). The reaction was initiated by the addition of enzyme, and monitored at 295 nm and at 5 min intervals during 30 min. The obtained results were used to construct Lineweaver–Burk plots to determine the inhibition mode, Michaelis-Menten constant (K_m) and maximum velocity (V_max) values. The inhibition constant (Ki) value was calculated from the secondary plot constructed using Y-intercepts of Lineweaver–Burk plots. (Ki) expresses the equilibrium constant for the binding of RaEO to xanthine oxidase.

For a general analysis of non-competitive inhibition, the Lineweaver-Burk equation can be written in double-reciprocal form:

Secondary plots can be constructed from

The secondary replot of Y-intercept vs. [I] is linearly fitted assuming a single inhibition site or a sing class of inhibition sites.

The means and standard deviation (SD) of data were calculated from independent experiments. The IC₅₀ (α-glucosidase, xanthine oxidase and pancreatic lipase inhibition) and EC₅₀ (ABTS, reducing power and phosphomolybdenum methods) values were calculated by linear regression analysis and the limits of their confidence intervals were carried out under the normality assumption. Data analysis was carried out using an unpaired Student’s t-test. GraphPad Software (USA) was used to fit sigmoid curves models and data were analyzed using a statistical analysis computer software (Graphpad Instat v.3.0a for MacIntosh, San Diego, CA, USA). For all statistical tests, P-values that were less than 0.05 were considered to be significant.

The total yield of the volatile fraction obtained from aerial part of R. acaule was 0.018% (w/w). The obtained essential oil was yellow with a pleasant odour. The chemical composition of the essential oil obtained from the aerial part was investigated using both GC and GC/MS techniques. The percentages and the retention indices of the identified oil components were listed in Table 1 in the order of their elution on the HP-5MS column. As shown in this table, 26 components were identified which represent 97.4% of the total oil. The major constituents were germacrene D (49.2 ± 1.1%), methyl eugenol (8.3 ± 0.28%), (E)-β-ionone (6.2 ± 0.18%), β-caryophyllene (5.7 ± 0.17%), (E,E)-α-farnesene (4.2 ± 0.1%), bicyclogermacrene (4.1 ± 0.12%) and (Z)-α-bisabolene (3.7 ± 0.08%). Table 1 showed also that the essential oil was characterized by the dominance of sesquiterpene hydrocarbons (74.2%).

According to literature, few reports exist for the inhibition activity of α-glucosidase by essential oils. The low IC₅₀ values obtained in this assay indicated a high inhibition activity. From our results, RaEO shows interesting antidiabetic activity with IC₅₀ value of 6.7 ± 0.10 μg/mL which is 42-fold lower than that of the commercial inhibitor acarbose (280 ± 10.01 μg/mL).

To investigate the type of enzyme inhibition and to determine the inhibition constant (Ki), the kinetics was performed according to the procedure detailed in the Materials and Methods section. The Lineweaver–Burk plots analysis indicated that the lines coincided on a point on the left side of the Y-axis and intercepted both the Y- and the X-axes at different points. What can be also observed are a decrease in apparent V_max value and an increase in the value of apparent K_m, indicating that the nature of inhibition is of mixed-type (Fig.2).

Moreover, from Fig. 2, it can be noted that the intersecting lines on the graph converge at the left side of the Y-axis and above the X-axis, indicating that the value of α (a constant that defines the degree to which inhibitor binding affects the affinity of the enzyme for substrate) is greater than 1 [32]. This confirms that the inhibitor prefers binding to the free enzyme rather than to the enzyme substrate complex. Therefore, the mode of inhibition caused by the essential oil is a mixed-type one, but it seems that it has strong competitive components. The inhibition constant (Ki) value derived from the secondary plot (Fig. 2) was 37.5 μg/mL.

The RaEO was tested for xanthine oxidase inhibitory activity at various concentrations and thereafter the IC₅₀ value was deduced. It can be observed that this essential oil has a very interesting IC₅₀ value (2.20 ± 0.10 μg/mL), which is comparable to that of the reference compound, allopurinol (2.6 ± 0.16 μg/mL).

Under non-inhibitory conditions, kinetic parameters for xanthine oxidase were K_m = 0.003 μM and V_max = 1.052 ΔOD/min. Assays with the essential oil induced a modification on V_max (from 0.26 to 0.66 ΔOD/min), whereas the K_m value was not changed (Fig. 3). As illustrated in Fig. 3, the slope and the vertical axis intercept increase with increasing essential oil concentration (2.5, 3.75 and 5 μg/mL). This result indicates that the essential oil components affected the velocity of the reaction catalyzed by xanthine oxidase, without affecting the Michaelis constant (K_m). From these results, we can conclude that this inhibition is a non-competitive one, indicating that the components of the essential oil bind to a site other than the active one of the enzyme, without competing with the substrate.

The Ki value derived from the secondary plot (Fig. 3) was 10 μg/mL, indicating that this essential oil tended to bind more easily to the xanthine oxidase. Indeed, smaller value of inhibition constant indicates stronger inhibition, which explains that the inhibitor-enzyme binding affinity exceeds the binding affinity of the enzyme-substrate.

In this work, TPL was used to measure the anti-lipase activity of RaEO. This enzyme was totally purified and its relation structure–function is previously determined [33]. These authors showed that the biochemical properties and structures of TPL are similar to those of mammals. For this reason, TPL was used instead of the mammalian one. The anti-lipase activity of the essential oil was conducted using two methods, which differ in the order of the inhibitor addition.

According to the literature, the comparison of the chemical composition of our essential oil (RaEO) with that of Rhaponticum carthamoides roots studied by Skała et al. [34], revealed important similarity. In fact, these authors reported sesquiterpene hydrocarbons as the main class of chemicals in this essential oil (54.7%). Furthermore, Havlik et al. [35] noted that sesquiterpene lactones exist in all parts of Rhaponticum carthamoides while the sesquiterpene hydrocarbons were identified only in essential oil. On the other hand, Benyelles et al. [36] reported that the essential oil extracted from the roots of R. acaule was characterized by a large amount of alcohol derivatives (69.2%). Finally it is worth to note that for the essential oil of the aerial part of the same species (R. acaule), Boussaada et al. [16] described a composition richer in diterpenoids (23.7%), aromatic compounds (23.6%) and oxygenated sesquiterpenes (21.3%). However, these authors collected the plant material in the completely different habitat of Tunisia. Indeed, the differences between the chemical compositions of the essential oil obtained from the same species may be attributable to environmental factors (geographical, climatic, and seasonal), development stage and genetic variability [37‐40]. Additionally, chemotypes and individual variability may appear within the same plant species, resulting in the differences in chemical compositions of the raw materials [41].

Despite the many publications on the chemical composition and antimicrobial activity of essential oils for the Rhaponticum genus, little information is available about their antioxidant activity [42]. Therefore, this is the first report about the antioxidant activity of the essential oil of R. acaule. Overall, according to the three tests used in this study (ABTS radical scavenging activity, Reducing power and Phosphomolybdenum assay), the obtained antioxidant activity of the RaEO might be attributed to the presence of high percentages of mainly germacrene D (49.2 ± 1.1%), methyl eugenol (8.3 ± 0.28%), (E)-β-ionone (6.2 ± 0.18%) and β-caryophyllene (5.7 ± 0.17%) (Table 1). Similarly to our results, some works reported that several essential oils having low phenolic compounds amounts also exhibits interesting antioxidant capacity [43, 44]. However, it’s difficult to correlate the antioxidant activity of a total essential oil to one or few active compounds. Both minor and major compounds should make a significant contribution to the activity of the oil.

Among the available approaches, inhibition of α-glucosidase has appeared to be a potential therapeutic target for the treatment and prevention of type 2 diabetes mellitus (DM) [45]. For a long time plant food rich in polyphenols have been reported to cause effects similar to insulin in the utilization of glucose and act as good inhibitors of key enzymes like α-glucosidase associated with type 2 diabetes and lipid peroxidation in tissues [46]. On the other hand, according to literature, few reports exist for the inhibition activity of α-glucosidase by essential oils. In this study, our results revealed that the RaEO exhibited good antidiabetic activity which is 42-fold higher than that of acarbose used as commercial inhibitor. This interesting antidiabetic activity may be due to the large amount of sesquiterpene hydrocarbons, especially germacrene D, as hypothesized by Sarikurkcu et al. [47] using the essential oils of three Phlomis species.

It is well known that allopurinol, an inhibitor of xanthine oxidase which is structurally related to xanthine, binds to the active site of this enzyme, causing its inhibition. This drug has been clinically used for more than 40 years. Unfortunately, the appearance of several side effects including fever, allergic reactions, skin rashes, hepatitis, and nephropathy limit the clinical use of allopurinol. For this reason, the search for novel natural xanthine oxidase inhibitors would be beneficial to treat gout and other diseases.

In our case, the significant xanthine oxidase inhibition exercised by the RaEO may be explained by the high amount of sesquiterpene hydrocarbons contained in this essential oil. Our results are in good agreement with those obtained by Kaurinovic et al. [43] with the essential oil of Marrubium peregrinum L., which is rich in β-caryophyllene, bicyclogermacrene and germacrene D.

Using the method A (preincubation of lipase with inhibitor), the RaEO exhibited promising anti-lipase activity which is comparable with that of pure THL used as a standard inhibitor against pancreatic lipase. In the same way and according to the literature, the inhibition of pancreatic lipase by the essential oil of Salvia judaica (Lamiaceae) flowers was determined colorimetrically and compared with Orlistat used as a positive control by Afifi et al. [48]. The IC₅₀ value (0.108 mg/mL) obtained in this study was very close to that obtained with RaEO (0.2 mg/mL). In this work, similarly to our essential oil, the aroma profile of the Salvia judaica (Lamiaceae) flowers was found to be mainly composed by sesquiterpene hydrocarbons (71.8%).

To determine the type of inhibition exerted by the RaEO on the TPL, the method B (inhibition during lipolysis) was used. Our results revealed that the obtained lipase inhibition was irreversible. Similar results have been reported by Gargouri et al. [49] using pure THL as inhibitor for porcine pancreatic lipase. In this case, the lipase hydrolysis rate of olive oil decreased rapidly to reach 10%, with final THL concentration of 200 μM. Gargouri et al. [50] have described two types of inhibition: the reversible inactivation obtained with amphiphiles compounds, such as detergents and some proteins. This inhibition is due to the modification of the interface quality, which prevents the binding of lipase to the interface [51]; the irreversible inhibition was obtained in the presence of colipase and bile salt and cannot be suppressed by renewal or excess of interface. The irreversible inhibition was obtained with specific reagents, such as THL, C12:0-TNB and E600 [49].