Free radical scavenging, α-glucosidase inhibitory and lipase inhibitory activities of eighteen Sudanese medicinal plants

Background Lifestyle-related diseases such as diabetes are steadily increasing worldwide. In Sudan, there are a variety of plant species used traditionally for the treatment of diabetes, obesity and other symptoms which need to be validated through scientific studies for their claimed traditional uses. Therefore, in the current study, the free radical scavenging activity, α-glucosidase inhibitory and pancreatic lipase inhibitory activities of 70% ethanol and water extracts of eighteen Sudanese medicinal plants were investigated using various in vitro assays. Moreover, the cytotoxicity and genotoxicity were assessed for the bioactive plant extracts. Methods Eighteen plants were selected on the basis of their traditional uses and extracted with 70% ethanol and water to obtain thirty-six extracts. The obtained extracts were screened using different in vitro bioassays namely, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, α-glucosidase inhibitory and pancreatic lipase inhibitory assays. Furthermore, the active plant extracts were investigated for their cytotoxicity and genotoxicity on HeLa cell line using HCS DNA Damage Assay. Results Both 70% ethanol and water extracts of Acacia nilotica, Ziziphus spina-christi, Abrus precatorius, and Geigeria alata along with the 70% ethanol extract of Martynia annua showed potent free radical scavenging activity. Regarding the α-glucosidase inhibition assay, both extracts of Acacia nilotica, Ziziphus spina-christi, Geigeria alata, and Cyperus rotundus showed potent activity. In general, 70% ethanol extracts were more potent compared to water extracts with exception of Cordia sinensis and Cymbopogon proximus, for which water extracts also showed potent enzyme inhibitory activity. Similarly, water extracts of Acacia nilotica and Ziziphus spina-christi showed potent inhibitory activity against pancreatic lipase enzyme. Some of the extracts also showed significant genotoxicity and cytotoxicity at the concentration range used for bioactivities. Conclusion The extracts of Acacia nilotica, Ziziphus spina-christi, Geigeria alata, Martynia annua and Abrus precatorius exhibited an appreciable range of activity on antioxidant and enzyme inhibitory assays.


Background
Diabetes mellitus (DM) is a serious chronic metabolic disorder, with life-threatening complications. The condition is defined by a state of chronically elevated blood glucose levels (hyperglycemia) with disturbances of carbohydrate, fat and protein metabolism, and mainly caused by deficiency or diminished effectiveness of endogenous insulin [1]. DM is an important cause of mortality and morbidity globally and has considerable health-system costs consequences. According to the World Health Organization (WHO), the total number of people with DM worldwide has substantially increased between 1980 and 2014, rising from 108 million to current numbers of 422 million, which are around four times higher [2]. This rise in the burden of the disease is greatly observed in the middle-and low-income countries compared to high-income countries [3]. In 2017, the International Diabetes Federation (IDF) estimated that 9.6% adults aged 20-79 years are living with DM in the Middle East and Northern Africa region, about 49.1% of them are still undiagnosed and 83.8% are living in low or middle-income countries, such as Sudan [4].
In Sudan, non-communicable diseases (NCD), including diabetes mellitus have become the principal cause of morbidity and mortality, with an overall prevalence of 10.9% among adults, according to the IDF [4,5] . Furthermore, DM accounts for about 6% of total deaths from non-communicable diseases in Sudan [6]. In spite of that, diabetes care in Sudan is still very poor to achieve proper glycemic goals and a good quality of life for diabetic patients [7]. According to a recent study conducted in Khartoum state, Sudan, the main factors of the insufficient diabetes services at primary health care centers are the lack of the follow up system for diabetic patients, the insufficient investigations, the lack of diet therapy guidelines and poor patient knowledge and behavior towards the disease management [8]. Moreover, treatment with the current available antidiabetic drugs is still not satisfactory to rectify a normal pattern of glycemic control, and unable to prevent the long-term diabetic complications as well [9]. Therefore, the need for effective, safe and affordable alternative treatment options has become crucial. As an alternative source of medicine, medicinal plants and herbal remedies have always gained a growing public interest across the globe. In Sudan, the use of medicinal plants was always inherent in Sudanese traditional medicine. People in many areas of the country, especially in rural areas, depend mainly on herbal medicine to cure their health problems as they believe in their safety, efficacy, besides to their availability and affordability for a huge sector of the population [10]. One important property of medicinal plants associated with DM treatment is antioxidant activity. There are several scientific reports and clinical evidence that the oxidative stress condition associated with the generation of reactive oxygen species (ROS) increases in both type 1 and type 2 diabetes [11]. It is believed to play an important role in the development of DM vascular complications through endothelial dysfunction and inflammation [12]. In addition, it has been suggested that the cause of the vascular complications in diabetic patients can be due to the insufficient response of the cellular antioxidants against the oxidative stress induced by hyperglycemia [13]. In this context, medicinal plants with potential antioxidant activity may be of great benefit to diminish oxidative stress in diabetic patients and profoundly contribute to reducing the severity and complications of the disease. On the other hand, obesity is a worldwide health problem, which is strongly associated with a number of chronic diseases such as DM [14]. Inhibition of nutrients digestion and absorption is an important target for the treatment of obesity and related diseases like DM. Pancreatic lipase and α-glucosidase enzymes inhibitors can lower blood glucose levels by reducing the intestinal absorption of fats and carbohydrates, respectively [15,16].
Fortunately, the flora of Sudan has an immense diversity of indigenous plant species, owing to the fact that they extend over six agro-ecological zones, including desert and semi-desert in the northern part, high and low rainfall savannah to the south, mountain region and flood zone. Several floristic studies revealed that a total of 3137 species of flowering plants representing 170 families exist in Sudan [17]. More than 136 species of them are used commonly as medicinal plants in Khartoum State, from which, 90 plant species indigenous to Sudan [18]. Previous studies have reported a considerable range of biological activities of Sudanese medicinal plants, such as antibacterial [19,20], anthelmintic [21], antiparasitic, antiplasmodial [22], anticancer [23], antidiabetic [24], and many other activities. However, many other Sudanese medicinal plants did not receive any test for their efficacy to validate their claimed traditional uses. In a different study, we screened six medicinal plants from Sudan for their antioxidant and enzyme inhibitory activities using different extraction solvents, and found that the water and aqueous ethanol extracts were suitable solvents for extraction of phenolic compounds [25]. In this context, in the present study, thirty-six extracts derived from 18 plant species belonging to 14 families collected from North Kurdufan and Khartoum states, Sudan (Table 1) were investigated for their potential antioxidant, α-glucosidase inhibitory and pancreatic lipase inhibitory activities. The adverse effects and toxicity due to the presence of genotoxic and cytotoxic agents in medicinal plants is important from the point of view of their therapeutic application [26]. Therefore, ten active extracts in antioxidant and enzyme inhibitory assays were further investigated for their potential genotoxicity and cytotoxicity on HeLa cells using HCS DNA Damage assay.

Plant materials
Eighteen plant species (  Table 1.

Preparation of plant extract
About 20 g of each plant sample was ground and extracted with two different solvents (70% aqueous ethanol and water) by the use of ultra-sonication assisted extraction at 50°C for two hours. Both extracts were then filtered. 70% Ethanolic extracts were concentrated and evaporated under reduced pressure using rotatory evaporator. Whereas, water extracts were freeze-dried.

DPPH free radical scavenging activity
The free radical scavenging activity was measured according to the modified method of Shimamura et al. [27], One hundred μL of sample extract (in 50% (v/v) ethanol) with varying concentrations were mixed with 50 μL of 200 mM MES buffer (pH 6.0) and 50 μL of 800 μM DPPH in 99.5% ethanol solution. After that the assay plate was kept in dark at room temperature for 20 min, then the absorbance was measured at 520 nm. Trolox was used as a positive control and the percentage radical scavenging capacity (SC%) of samples was obtained from the following equation: where AC is the absorbance of the control, which contained all reagents except test sample, A0 is absorbance of the blank which contained the sample and all reagents except DPPH and AS is the absorbance of the test samples. From these data, the SC 50 value was calculated, which is defined as the concentration of the extract required for 50% reduction of the DPPH radical absorbance. All samples were assayed in triplicate.
α-Glucosidase inhibitory activity α-Glucosidase inhibitory activity was measured according to the method by Yang et al. [28] with slight modifications. Briefly, 10 μL of the sample solution with varying concentrations was mixed with 60 μL of phosphate buffer (200 mM pH 6.8) and 10 μL of α-glucosidase (1 U/ml) in phosphate buffer. After incubation at 37°C for 5 min, 20 μL of PNGP (4 mM) was added to start enzyme reaction. The absorbance of the reaction solutions was measured right after the addition of substrate for 12 min at 405 nm. Acarbose was used as the positive control. The percentage inhibition of α-glucosidase activity was calculated via the following equation: % inhibition = [1-(AS at 12 min − AS at 0 min)/ (AC at 12 min − AC at 0 min)] × 100, where AS is the absorbance readings of samples, AC is the absorbance readings of the control which contain all solvents except the sample. The concentration of the tested samples giving 50% inhibition of the enzyme activity (IC 50 ) was estimated from the plots of the concentration vs. the inhibitory activity, all samples were assayed in triplicate.

Lipase inhibitory activity
Porcine pancreatic lipase inhibitory activity was measured according to the modified method of Bitou et al. [29], the assay was performed using 96-well microplate. Briefly, the reaction mixture was prepared with 50 μL of porcine pancreatic lipase (

Cell culture
The HeLa cell line was cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). The cells were maintained at 37°C in a CO 2 incubator with 90% humidity and 5% CO 2 . Cells were subcultured regularly using trypsin/EDTA.
Cell genotoxicity/cytotoxicity assay using the HCS DNA damage kit HeLa cells were seeded at a density of 7500 cells/100 μL /well on 96-well plate. After 24 h incubation, 100 μL of the plant extracts with different concentrations was added to cells along with the control drug (Valinomycin) and incubated for 24 h. Treated cells were then stained by adding 50 μL of Image-iT® Dead Green™ viability stain and incubated for 30 min. Then the medium was removed and cells were fixed, permeabilized and blocked by incubation with 4% paraformaldehyde, Triton® X-100 in PBS and bovine serum albumin (BSA) in PBS solutions, respectively. Cells were then incubated with 50 μL of pH2AX mouse monoclonal primary antibody solution for 1 h at room temperature. After washing in

Statistical analysis
For quantitative analysis, the result represents the mean ± SD (n = 3) and the data were analyzed using the Scheffe's test by a Statview software as indicated in each figure legend. The level of significance was set at p < 0.05.

Extraction yield
The extraction yield (%) of the 18 Sudanese medicinal plants using 70% ethanol and water as solvents is shown in Table 2.

Free radical scavenging activity
In the present study, DPPH radical scavenging assay was used to evaluate the in vitro antioxidant activity for all plant extracts. Results are presented as SC 50 , the lower SC 50 values indicate the stronger antioxidant activity (Table 3). Trolox, a water-soluble derivative of vitamin E was used as positive control, with an SC 50

α-Glucosidase inhibitory activity
In this study, we aimed to explore α-glucosidase inhibitors of plant origin. Thus, we investigated the inhibitory effect of thirty-six ethanolic and water extracts on α-glucosidase enzyme obtained from Saccharomyces cerevisiae.  Acarbose was used as a positive control shows the results of the in vitro assay of α-glucosidase inhibitory activity for all extracts. Acarbose was used as a positive control in this assay (IC 50 = 240.00 ± 0.03 μg/ml). It was noted that 70% ethanolic extracts had higher inhibitory activity as compared to their corresponding water extracts. Our results demonstrated that the highest potential α-glucosidase inhibitory activity was shown by the ethanolic and water extracts of A. nilotica and Z. spina-christi with IC 50 values ranging between 1.0-5.0 μg/ml, which were significantly higher than that of the positive control, Acarbose. In addition, the ethanolic extract of M.

Lipase inhibitory activity
In the current study, pancreatic lipase inhibitory activity was evaluated for all extracts, using Cetilistat as a positive control, with the IC 50 value of 4.66 ± 0.05 μg/ml (  (Fig. 1a). As shown in Fig. 1b, the 70% ethanol extracts of A. nilotica, Z. spina-christi, and M. annua showed genotoxicity at concentrations > 20 μg/ml. In addition, a significant level of genotoxicity was observed at concentrations > 20 μg/ml for the water extracts of Z. spina-christi and 2 μg/ml for the water extracts of A. precatorius. Whereas, water extract of A. nilotica showed genotoxicity at concentrations > 20 μg/ml (Fig. 1c). precatorius (2, 20, 200 μg/ml) exhibited a significant level of cytotoxicity as well (Fig. 2).

Discussion
The increasing reliance on traditional herbal medicines as an approach to therapy has been observed worldwide. Herbal remedies are prepared traditionally as macerates, potions or decoctions using water as a solvent and given either as a mixture or as a single medicinal plant [10]. However, the use of these herbal remedies and medicinal plants without scientific validation of their claimed activity can be harmful and sometimes fatal. Accordingly, we designed this study with the aim of discovering potential and safe antidiabetic agents from plant origin and to validate the traditional uses of these plants as antidiabetic medicines.
In the current study, different parts of eighteen Sudanese medicinal plants were selected according to their traditional uses ( Table 1), many of which are commonly used by Sudanese people to treat different ailments, including  For each extract, 70 represents 70% EtOH extract and W represents water extract. As a control experiment, HeLa cells were treated with 120 μM valinomycin for 24 h at 37°C/5% CO 2 . Imaging and analysis were performed using a BZ-X700. At 120 μM valinomycin, cells showed cytotoxic effects as demonstrated by the positive Image-iT® DEAD Green™ viability stain fluorescence diabetes mellitus. Two solvents were used for extracting plant samples, 70% ethanol and water, which plays an important role in isolating different phytochemical from the plant and consequently different activities will be obtained [25,30]. The use of solvents of different polarities has a direct association with the percentage yield of the extract. It was noted that the yield percentage of water extracted samples was relatively higher than that of 70% ethanol, which may be attributed to the higher solubility of carbohydrates and proteins in water than in ethanol [31]. Thereafter, the obtained extracts were screened for their potential antioxidant activity as well as, α-glucosidase inhibitory and pancreatic lipase inhibitory activities.
Oxidative stress occurs when production of oxidants or reactive oxygen species (ROS) exceeds local antioxidant capacity and it is thought to be associated with the pathogenesis of many diseases including diabetes mellitus [32]. Antioxidants are molecules that decelerate or quench free radical reactions and hence, delay or prevent cellular damage [33]. Medicinal plants are rich sources of secondary metabolites that act as natural antioxidant such as phenolic compounds (cinnamic acids, benzoic acids, flavonoids, proanthocyanidins, stilbenes, coumarins, lignans, and lignins), ascorbic acid and carotenoids [34,35]. According to our results, A. nilotica showed the most potent antioxidant activity among other tested plants. There are several phytochemical studies on the pods of A. nilotica, revealed the presence of various polyphenolic compounds, such as isoquercetin, rutin, leucocyanidin, different galloylated derivatives of catechin, which possess free radical scavenging properties [36,37]. Furthermore, the ethanolic extracts of Z. spina-christi and A. precatorius had relatively similar activity as the positive control, which is consistent with previous phytochemical studies that reported the presence of polyphenols, flavonoids, and tannins in both species and can be responsible for their potent antioxidant activities as well [38,39]. Similarly, the ethanolic extracts of M. annua and G. alata, had moderate free radical scavenging activities. Kenwat et al. (2014) investigated the phytoconstituents of fruit oil of M. annua and reported the presence of flavonoids, tannins and phenolic compounds that may be attributed to the plant antioxidant activity [40]. To the best of our knowledge, this is the first screening study on M. annua species from Sudan [41]. A recent study by Zheleva-Dimitrova et al.
(2016) investigated the phenolic acids contents of G. alata roots and leaves and identified chlorogenic acids that revealed antioxidant potentials [42]. Notably, the extracts obtained using 70% ethanol, illustrated stronger radical scavenging capacity than water extracts, which may be due to the ability of the organic/aqueous solvent to extract more phenolic compounds (a higher number of hydroxyl groups) that possess antioxidant activity, than extraction with water alone. Do et al. (2014) has reported a similar effect of extraction solvents on the radical scavenging activity, with stronger antioxidant activity in ethanolic/aqueous extracts compared to water extracts [43].
Oral antidiabetic (hypoglycemic) drugs such as Acarbose act by competitive and reversible inhibition of intestinal α-glucosidases, which slows down carbohydrate digestion and thus leads to a reduction of postprandial blood glucose levels [44]. As mentioned above, the strongest α-glucosidase inhibitory activity was shown by A. nilotica extracts. On the basis of the previous reports, it is possible that the presence of polyphenols, flavonoids, and tannins are responsible for the observed antidiabetic activity. Our results are also consistent with the in vivo study on the therapeutic effect of A. nilotica pod on diabetic rats, Enayat et al. (2012) has reported a significant reduction in blood glucose levels in the A. nilotica treated group in comparison to diabetic control rats [45], which support the α-glucosidase inhibitory activity of A. nilotica as a mechanism of lowering blood glucose levels. In addition, the strong α-glucosidase inhibitory activity of the ethanolic extract of M. annua, and G. alata suggest a correlation with their antioxidant activities due to the presence of bioactive compounds. For the best of our knowledge, it is the first report of the α-glucosidase inhibitory activity of the fruit of M. annua.
Inhibitors of pancreatic lipase such as Cetilistat, act by diminishing the absorption of dietary fats. Many reported secondary metabolites in plants, have already shown profound inhibition of pancreatic lipase enzyme such as phenolics, glycosides, saponins, and terpenes [46]. Our results showed that the majority of the tested extracts demonstrated some extent of inhibitory activity against lipase enzyme, particularly the extracts of A. nilotica and Z. spina-christi. This strongly suggests that these plants can be a source of potential leads for pancreatic lipase inhibition in order to treat and/or prevent obesity and other lifestyle-related diseases.
Prominently, during the evaluation of the bioactivities of the crude extracts, it is crucially important to consider the cytotoxicity testing of the bioactive extracts to exclude the toxic compounds that can impediment their therapeutic indications [47]. In the current study, 10 bioactive extracts from 5 plants species namely; A. nilotica, Z. spina-christi, M. annua, A. precatorius, and G. alata were selected for further investigations of their genotoxicity and cytotoxicity on HeLa cell line. Regarding the genotoxicity effect in comparison with the antioxidant activity, the IC 50 values of A. nilotica and G. alata were non-genotoxic for both extracts along with the ethanolic extracts of A. precatorius and Z. spina-christi. Contrarily, the ethanolic extracts of M. annua and the water extracts of A. precatorius and Z. spina-christi were considered as genotoxic at their IC 50 concentrations. Furthermore, A. nilotica and Z. spina-christi extracts were considered to be non-cytotoxic at their IC 50 concentrations, in addition to the ethanolic extract of A. precatorius and the water extract of G. alata. In contrast, the ethanolic extracts G. alata and M. annua, along with the water extract of A. precatorius were found to be cytotoxic for HeLa cells at their activity concentrations.
On the other hand, in comparison with the IC 50 values of the α-glucosidase inhibitory and lipase inhibitory activities of the extracts, it was found that both extracts of A. nilotica along with the ethanolic extract of Z. spina-christi were found to be non-genotoxic and non-cytotoxic at their IC 50 values, which support the use of these bioactive crude extracts as safe anti-diabetic agents. Consequently, these findings recommend the use of these bioactive crude extracts as antioxidant, anti-diabetic, and anti-obesity agents under consideration to their toxicity levels. In addition, it necessitates further investigations for the bioactive extracts to identify the responsible compounds for the observed antioxidant, αglucosidase, and lipase inhibitory activities.

Conclusion
In the present study, eighteen of the Sudanese medicinal plants were screened for their potential antioxidant activity, α-glucosidase inhibitory and porcine pancreatic lipase inhibitory activities. Based upon the results, it could be concluded that Acacia nilotica, Ziziphus spina-christi, Geigeria alata, Martynia annua, Abrus precatorius, Cordia sinensis and Boswellia papyrifera exhibited an appreciable range of activity on the tested bioactivities, which highlights and supports their potential use as natural antidiabetic agents with strong antioxidant and anti-hyperlipidemic effects. Moreover, the genotoxicity and cytotoxicity assay findings suggest that although some of the tested bioactive extracts can be cytotoxic and/or genotoxic, however, their use within the safe concentration limits or as pure bioactive compounds can be of great benefit therapeutically. Our results will be a guide for the selection of plant species for further investigation in the discovery of new bioactive compounds from natural origin. Further supportive studies in chemical isolation and animal model experiments can be performed to confirm the in vitro antioxidant and enzymatic inhibition activities of the extracts.