In-Vitro Assessment of The Antidiabetic and Anti-Inflammatory Potential of The Artemisia absinthium, Artemisia vulgaris and Trigonella foenum-graecum Extracts Processed by Membrane Technologies

1. Introduction

Known and used for centuries, medicinal plants remain potential sources for finding new remedies useful in treating or ameliorating various diseases including diabetes and inflammatory diseases.

Diabetes is a metabolic disorder that generates severe dysfunctions such as neuropathy, vasculopathy, dyslipidemia, retinopathy, and cardiovascular disease [1]. The incidence of this condition is constantly increasing, from 400 million people in 2016 it is estimated that it will reach 642 million people with type II diabetes in 2040 [2]. Antidiabetic drugs such as acarbose, miglitol, and voglibose reduce the degradation and absorption of sugars by inhibiting the digestive enzymes - α-amylase and α-glucosidase, involved in these processes. However, these drugs also have side effects and that is why alternatives are sought from natural sources [3].

The drugs used to treat inflammation and pain are nonsteroidal drugs that generally have numerous side effects such as ulcers and hemorrhages [4,5]. Their long-term use even leads to cardiovascular, metabolic, endocrine and ophthalmological damage [5].

Medicinal plants have been used in folk medicine to treat many inflammatory diseases and remain important sources of new anti-inflammatory and antioxidant agents [6,7]. Inhibition of enzymes involved in the inflammatory process such as lipoxygenase and hyaluronidase is the basis for finding new treatments for allergies and inflammatory diseases [8,9]. Oxidative stress generated by reactive oxygen species (ROS) has been associated with pathological inflammatory processes and DNA distortions followed by carcinogenesis and tumors [10].

Lipoxygenase (LOX) is involved in inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, allergic rhinitis, atherosclerosis, and certain types of cancer. During LOX activity, peroxy radicals are formed in the reaction medium that can function as sources of free radicals. Thus, antioxidants that have free radical scavenging activity can also act as LOX inhibitors [11].

Hyaluronidase acts on hyaluronic acid which is an important part of the extracellular matrix and is involved in many physiological processes such as embryogenesis, wound healing, and cell migration [7,12]. Studies have led to the discovery in plants of hyaluronidase inhibitors (tannins, cucumbers, flavonoids) that can be used as anti-inflammatory and anti-allergic agents, as antitumor agents, in the treatment of bacterial infections and adjuvant in the treatment of arthritis [13,14].

Membrane technologies - micro- and ultrafiltration - are cheap and effective alternatives to traditional technologies for concentrating biologically active compounds. These processes feature low operating and maintenance costs, easy operation at moderate temperatures and pressure, high permittivity, and selective separations. [15,16]. These technologies can be successfully used for the purification and concentration of plant extracts while maintaining their functional and nutritional properties [17,18].

Artemisia vulgaris and Artemisia absinthium are two plants of the Asteraceae family (Compositae) used in traditional medicine due to their numerous therapeutic properties.

Artemisia vulgaris (known as mugwort) is a shrub of temperate zones in Europe, Asia, North Africa, and North America [19]. The plant has been used in folk medicine for the treatment of gastrointestinal and gynecological diseases, alleviation of hypertension and disorders of the nervous system [20,21] as well as for culinary purposes. Studies have revealed various pharmacological properties of A. vulgaris, such as anti-inflammatory, antioxidant, antitumor, and immunomodulatory activities [22,23].

Artemisia absinthium L. - also known as wormwood - has been known for centuries and used in folk medicine for gastrointestinal and urinary disorders, fever, and helminthiases [24,25]. A. absinthium has been shown to be effective as an antiparasitic and a digestive [25], as well as for reducing symptoms of leukemia, sclerosis, diabetes, malaria, and even some types of cancer in recent years [26].

Trigonella foenum-graecum (also known as fenugreek) is an annual herb from the Fabaceae family known for its medicinal and culinary properties since ancient times. Fenugreek is grown and used as a spice in many countries from Asia, Europe, and Africa and is considered to have a high nutraceutical value [27]. In folk medicine, this plant is used especially for immunity stimulation and for digestive and reproductive disorders [28]. Seed powder lowers blood sugar, and improves symptoms in patients with type 2 diabetes [29]. The seeds and leaves of this herb are rich in flavonoids, alkaloids, and saponins which confer medicinal properties [30]. While fenugreek seeds of fenugreek have been widely investigated for the treatment of inflammation, cancer, and diabetes, little is known about their leaves.

The present paper analyzed the antidiabetic and anti-inflammatory activity of the plants: Artemisia vulgaris, A. absinthium, and Trigonella foenum-graecum by testing the ability to inhibit α-amylase, α-glucosidase activity, respectively, hyaluronidase (HYA) and lipoxygenase (LOX) activity.

2. Results and Discussion

2.1. Phytochemical Analysis and Antioxidant Capacity

The content in total polyphenols and flavones of Artemisia absinthium, Artemisia vulgaris, and Trigonella foenum-graecum extracts as well as the antioxidant activity of the extracts were analyzed by spectrophotometric and chromatographic methods presented in the previous chapter and the results are presented in the Table 1, Table 2 and Table 3.

The Artemisia vulgaris extracts have been shown to be richer in polyphenolic compounds with almost double the values of Artemisia absinthium. In concentrated extracts, a significant increase in the amount of active ingredients is obtained, especially in Artemisia vulgaris concentrate from 7877.5 ± 260.96 μg CA/mL to 11440.21 ± 49.96 μg CA/mL). The amount of flavones was also higher for Artemisia vulgaris extracts compared to Artemisia absinthium, obtaining even a doubling of the flavones amount by ultrafiltration: from 505.18 ± 30.12 μg QE / mL initially to 1020.12 ± 45.12μg QE / mL in concentrate, thus noting the efficiency of the ultrafiltration process. The analysis of the Trigonella foenum-graecum extract by HPLC–MS technique revealed the presence of polyphenolic compounds and showed a high amount of genistin.

The results regarding the antioxidant activity of the obtained and processed Artemisia extracts are presented in Table 1. It is unanimously accepted that the antioxidant activity must be verified by several alternative methods because only one method is not significant [31,32]. By all 3 methods used similar results were obtained, the extracts of T. foenum-graecum showed the highest antioxidant activity followed by A. vulgaris and A. absinthium, the antiradical capacity of the concentrated extracts was higher than microfiltrate. Data from the literature showed that T. foenum-graecum seeds extracts were a good antioxidant agent and this was attributed to the total phenolic compounds content [22,32].

There is a direct correlation between the amount of polyphenols and flavones and antioxidant activity, which is consistent with other studies [33,34]. The high content of polyphenols could explain the high antioxidant activity of Artemisia extracts [35]. Flavonoids also have antioxidant properties as shown by other studies on other Artemisia species [36]. In the case of Trigonella foenum-graecum extracts the HPLC analysis revealed the presence of a significant amount of genistin, which could explain the high antioxidant capacity.

The antioxidant activity is mainly attributed to phenolic compounds, as they are able to protect living systems from oxidative damage caused by free radicals [37]. Scientific studies have shown that through their antiradical capacity, antioxidants can prevent severe diseases such as cardiovascular diseases, and can also have anti-inflammatory and anti-carcinogenic effects [38].

Previous studies have shown that Artemisia vulgaris has increased antiradical activity compared to other species of the genus Artemisia [39] and that leaf extracts can be used as an effective antioxidant [40]. Regarding Artemisia absinthium, some studies have shown moderate antiradical activity [41] others have shown a high antiradical activity correlated with a large amount of polyphenols and flavonoids [42].

The quantitative analysis determined an increased amount of chlorogenic acid in the two species of Artemisia, significantly higher in Artemisia vulgaris. In Artemisia vulgaris, a higher amount of luteolin and isoquercitrin was also determined. The results are comparable with other data in the literature [39].

Rutin was determined in large quantity in both plants. Rutin has a high antioxidant capacity and has been reported to function for the treatment of several diseases associated with metabolic syndrome, including diabetes and as a treatment for neurodegenerative diseases associated with oxidative stress [43]. Numerous scientific reports claim that regular consumption of flavonoids or polyphenols decreases the risk of cardiovascular disease, diabetes, and cancer [44]. Rosmarinic acid has been identified in greater amount in Artemisia absinthium. In most extracts, the concentration of active principles is observed, which shows the efficiency of the ultrafiltration process.

2.2. α-Amylase and α-Glucosidase Inhibition Activity

Several pharmacological approaches are used to control diabetes. One of these refers to the control of postprandial hyperglycemia by inhibiting the absorption of glucose in the intestine [45]. This is achieved by using oral agents that interfere with glucose absorption, respectively inhibitors of pancreatic a-glucosidase and a-amylase.

The plants used in this study showed high inhibitory activity on α-amylase T. foenum-graecum concentrated extract, especially (IC₅₀ = 3.22±0.3 μg/mL), higher than the acarbose used as a standard, followed by A. vulgaris concentrated extract (IC₅₀ = 8.57±2.31 μg/mL). The inhibitory activity of the concentrated extracts is significantly higher than that of the microfiltrates, thereby highlighting the efficiency of the ultrafiltration process.

The T. foenum-graecum showed also a high inhibitory activity on a-glucosidase (IC₅₀ = 11.14 ± 0.9 μg/mL) and the concentrated extract of A. absinthium showed moderate inhibitory activity on a-glucosidase (IC₅₀ = 31.90 ± 1.89 μg/mL). Concentrated extracts showed higher inhibitory activity than microfiltrate. The results are presented in Table 3.

Other plants of the genus Artemisia such as Artemisia campestris, and Artemisia herba-alba Asso, are used in traditional medicine to treat diabetes in countries such as Algeria, Morocco, Pakistan, Mexico. The hypoglycemic action of Artemisia absinthium extracts by inhibiting α-glucosidase has been confirmed by other studies [46].

In vitro studies have reported the inhibitory activity of α-amylase activity by a 70% ethanolic extract of Artemisia herba-alba. This resulted in an 11% reduction in α-amylase activity, which may be a mechanism by which this extract can reduce blood glucose [47]. Studies performed on another species of Artemisia - Artemisia indica have shown the antihyperglycemic and antihyperlipidemic effect of some methanolic and chloroform extracts comparable to that of glibenclamide [48]. Antidiabetic activity has also been shown in other species of the genus Artemisia such as A. amygdalina [49], A. pallens [50].

The inhibitory effect of Trigonella foenum-graecum extracts on α-amylase and α-glucosidase has been scarcely described in the literature. The results obtained are in agreement with previous reports which proved the inhibitory effect of T. foenum-graecum on amylase activity [51].

2.3. Lipoxygenase (LOX) and Hyaluronidase (HYA) Inhibition Activity

Synthetic drugs - non-steroidal anti-inflammatory drugs, and anti-rheumatic drugs, reduce the inflammatory process but can also have a number of side effects. Herbal therapies could be beneficial because they contain several medically important chemical constituents, are readily available, the costs are low and the side effects negligible [52].

In the inflammatory process and in pathological conditions in general, enzymes such as lipoxygenase (LOX), cyclooxygenase (COX), and hyaluronidase (HYA) enzymes are activated leading to the synthesis of prostaglandins involved in the inflammatory process and allergic diseases [11].

Studies have shown that plant-derived bioactive compounds such as phenolic acids and flavonoids with antioxidant activity, also possess anti-hyaluronidase activities, showing positive correlations between the amount of these compounds and anti-hyaluronidase activity [7].

The extracts tested in this study showed accentuated anti-inflammatory activity by inhibiting LOX: T. foenum-graecum and A. absinthium concentrate extracts showed similar inhibitory activities, IC₅₀ = 19.69 μg / mL, respectively IC₅₀ = 19.71 μg / mL, even higher than the polyphenolic compound used as the reference standard (eg. rutin - IC₅₀ = 22.34 μg/mL) (Table 4). The Artemisia vulgaris and T. foenum-graecum concentrated extracts showed moderate HYA inhibition activity: IC₅₀ = 17.18 μg / mL IC₅₀ = 17.57 μg / mL compared to ibuprofen, the reference standard used - IC₅₀ = 5.73 μg/mL.

The anti-inflammatory activity of Artemisia vulgaris extracts has been attributed to the presence of flavonoids [53]. Previous studies have shown that Artemisia vulgaris extracts have an anti-inflammatory action by inhibiting cyclooxygenase [54].

In vitro studies have shown the anti-inflammatory activity of Artemisia campestris extracts by inhibiting lipoxygenase [55]. This species is rich in bioactive compounds such as phenolic acids, flavonoids, and terpenoids, many of the plant's properties such as antioxidant, antidiabetic and anti-inflammatory are attributed to these compounds.

Another species of Artemisia - Artemisia nilagirica, commonly found in India, has anti-inflammatory potential, as well as antioxidant, antimicrobial, antifungal, and anticancer, properties associated with the presence of phytochemicals such as tannins, flavonoids, alkaloids, saponins, coumarins, steroids and phenols [56,57]. Studies have shown anti-inflammatory properties in other species of the genus Artemisia: Artemisia maritima L [58] Artemisia sieversiana Ehrh. [59].

3. Materials and Methods

3.1. Chemicals

All reagents used for analysis were of analytical purity and were purchased from Sigma Chemical Company (Sigma Aldrich, Germany), Roth (Carl Roth GmbH, Germany), and Fluka (Switzerland).

3.2. The Obtaining of Extracts

The plants were first dried, finely ground with a GRINDOMIX 200GM mill, then hydroalcoholic extracts Artemisia vulgaris and Artemisia absinthium were obtained in 50% EtOH (10% mass). Extracts were obtained by ultrasound-assisted solvent extraction (UAE) the room temperature for 1 hour, followed by filtration.

The extracts were processed by membrane methods: micro- and ultrafiltration through a KMS Laboratory Cell CF-1 laboratory facility purchased from Koch Membrane (Germany).

Microfiltration was performed by microfiltration membranes with the pore size of 0.45 µm and ultrafiltration by regenerated cellulose membranes with a cut-off of 3,000 Da; the concentration was achieved in the ratio 1: 3 and the pressure used was 6 bar.

3.3. The Bioactive Compounds Determination

Determination of the total polyphenols content was realized using Folin Ciocalteu's technique with slight modification [60]. Spectrophotometric measurements were realized at 760 nm. The experiments were performed in triplicates and the polyphenols concentration was calculated using an etalon curve of chlorogenic acid (CA).

Determination of the total flavonoid content was done using the aluminum chloride colorimetric assay [61]. The flavonoid content was calculated using the rutin calibration curve and was expressed in μg rutin equivalent (RE)/mL of extract.

3.3.1. HPLC Analysis

The chromatographic analysis was performed using a complete HPLC SHIMADZU system, a C18 Nucleosil 3.5, 4.6 × 50mm, Zorbax column. The system was coupled to an MS detector, LCMS-2010 detector (liquid chromatograph mass spectrometer), equipped with an ESI interface. The samples were filtrated before injection using Syringe Driven Filter Unit 0.2 µm (Macherey-Nagel).

All the other reagents, (acetonitrile, formic acid) were analytical pure or of chromatographic grade and were used after filtration. The ultra-pure water was obtained using a system for purification of water, Elix 3 (Millipore).

All standards used: chlorogenic acid, gallic acid, ellagic acid, caffeic acid, rosmarinic acid, coumaric acid, rutin, luteolin, quercetin, quercetin 3-β-D-glucoside, apigenin, umbelliferone, kaempferol, and genistin stock solutions, 1mg/mL, were prepared in ethanol Stock samples were stored at dark and -4^°C between the experiments.

The used HPLC method for the analysis of polyphenolic compounds was previously published by Alecu, et al. [62].

3.4. Antioxidant Assays

The antioxidant activity was measured using 3 methods, ascorbic acid was used as the control in all methods.

• The method was based on decreasing the maximum absorbance of ABTS to 731 nm in the presence of the antioxidant [63]; antioxidant activity was expressed in TEAC equivalents (Trolox Equivalent Antioxidant Capacity) using the formula

$${\mathrm{TEAC}}_{\mathrm{sample}}=C_{Trolox}\cdot f\cdot \frac{A_{sample}-A_{blank}}{A_{Trolox}-A_{blank}}$$

(1)

where: A _blank = control absorbance; A _Trolox = Trolox absorbance.A _sample = sample absorbance; f =dilution factor; C_Trolox = Trolox concentration

• DPPH radical scavenging activity

The scavenging activity on the DPPH radical of samples was determined by measuring the decrease in the DPPH maximum absorbency at 517 nm after 10 min [64] and was calculated:

radical scavenging activity (%) = [(A_B – A_A)/A_B] x 100

(2)

where: A_B = control absorbance and A_A = sample absorbance.

• Reducing Power Activity (Iron (III) to iron (II) reduction)

Reducing power was determined according to a previously described procedure [65]. The absorbance was measured spectrophotometrically at 700 nm and calculation was done using the formula:

Reducing power (%) = [(A_A – A_B)/A_A] x 100

(3)

where: A_A = sample absorbance, A_B = control absorbance

3.5. Enzyme Inhibitory Activity Assay

3.5.1. Testing the Antidiabetic Capacity of the Extracts

• α Amylase inhibition assay

The α-amylase inhibition assay was realized by the Ranilla method slight modification [66]. So, 100 μL of extract were mixed with 250 μL α - amylase from hog pancreas (EC 3.2.1.1) (0.5 mg/mL in 0.02 M sodium phosphate buffer, pH 6.9 with 0.006 M NaCl) and were incubated at 37⁰ C, 20 min; then 250 μL of the starch solution (1% in sodium phosphate buffer) were added, and the mixture was re-incubated for 30 min at 370; afterward, 500 μL dinitrosalycilic acid (DNS) was added and the mixture was been boiling for 5 min. Finally, 5 mL of distilled water was added to the reaction mixture. The absorbance measurement was realized at 540 mm using a UV-visible spectrophotometer (Jasco-V630) with acarbose as the positive control. The calculation of the results was realized using the formula:

$$\% \mathrm{Amylase} \mathrm{inhibition} = \frac{\Delta A_{control}-\Delta A_{sample}}{\Delta A_{control}}\times 100$$

(4)

The IC₅₀ values were calculated by the linear regression analysis. Significant statistical differences were considered p<0.05.

• α-Glucosidase inhibition assay

The α-glucosidase inhibition assay was realized using the Queiroz et al. method slight modification [67]. Thus, 120 μL α-glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20) (0.5 U/mL) with 720 μL sodium phosphate buffer (0.1 M, pH 6.9) and 60 μL extract were mixed, then the mixture was incubated at 37^°C for 15 min. After the pre-incubation, 120 μL p-nitrophenyl-α-D-glucopyranoside (5mM/L) solution was added and the reaction mixture was incubated at 37⁰C for 15 min. The absorbance measurement was realized at 405 nm using a UV-visible spectrophotometer with acarbose as the positive control. The calculation of the results was realized using the formula:

$$\% \mathrm{Glucosidase} \mathrm{inhibition} = \frac{\Delta A_{control}-\Delta A_{sample}}{\Delta A_{control}}\times 100$$

(5)

The IC₅₀ values were calculated by the linear regression analysis. Significant statistical differences were considered p<0.05.

3.5.2. Testing the Anti-Inflammatory Capacity of the Extracts

• o Hyaluronidase inhibition assay

The hyaluronidase activity (Hyaluronidase EC 3.2.1.35 from bovine testicles – Sigma) manifested by the hydrolysis action of hyaluronic acid was tested by the modified Morgan-Elson method (1949) [68] which involves the measurement at 585 nm of the complex formed by enzymatic hydrolysis products by reaction with p-Dimethylaminobenzaldehyde.

Pipette 100 μl of enzyme solution (1 mg/ml) together with 50 μl of plant extract and preincubate for 30 minutes at 37°C to exert the action of inhibiting the centers of hyaluronidase activity. To the reaction mixture is added 100 μl of substrate solution (sodium salt of hyaluronic acid in the vitreous bovine humor - 2.5 mg /ml) is incubated for 60 minutes at 37 ° C - the time required to carry out the enzymatic reaction. The blank is prepared in the same way by replacing the vegetable extract with a buffer solution.

The reaction is stopped by maintaining for 3 minutes at 100°C and the reaction products are highlighted by staining with p-Dimethylaminobenzaldehyde and spectrophotometrically quantification at 585 nm of the pink complex; the calculation of the results was done according to the formula:

$$\% \mathrm{inhibition} = \frac{\Delta A_{control}-\Delta A_{sample}}{\Delta A_{control}}\times 100$$

(6)

The IC₅₀ values were calculated by the linear regression analysis. Significant statistical differences were considered p<0.05.

• o Lipoxygenase inhibition assay

Lipoxidase inhibition testing was performed according to a Sigma-Aldrich protocol [69]: 0.017% (v/v) linoleic acid substrate and a lipoxygenase solution (2200 units/mL) (EC 1.13.11.12); it was determined to increase the absorbance for approximately 5 minutes at 234 nm, using the maximum linear rate for both the sample and the blank, the calculation of the results being made according to the formula:

$$\% \mathrm{inhibition} = \frac{\Delta A_{control}-\Delta A_{sample}}{\Delta A_{control}}\times 100$$

(7)

The IC₅₀ values were calculated by the linear regression analysis. Significant statistical differences were considered p<0.05.

4. Conclusions

The phytochemical screening, the antidiabetic and anti-inflammatory potential of Artemisia vulgaris, Artemisia absinthium, and Trigonella foenum-graecum concentrated hydroalcoholic extracts by inhibiting α-amylase and α-glucosidase, respectively lipoxygenase and hyaluronidase were analyzed.

The T. foenum-graecum extracts showed a higher genistin content and Artemisia species showed a higher polyphenols and flavones content. The T. foenum-graecum extracts showed high antioxidant activity by all methods of analysis.

The T. foenum-graecum extracts showed a high inhibitory activity on all enzymes studied (α-amylase, α-glucosidase, hyaluronidase, and lipoxygenase). The Artemisia absinthium concentrated extract showed high inhibitory activity on LOX, while Artemisia vulgaris concentrated extract has a significant inhibitory effect on HYA. Thus, these herbs can be potential sources of phytocompounds useful in the management of type 2 diabetes and inflammatory diseases.

This section is not mandatory but can be added to the manuscript if the discussion is unusually long or complex.