Stipa tenacissima L.: A New Promising Source of Bioactive Compounds with Antioxidant and Anticancer Potentials

Background: Stipa tenacissima L. (S. tenacissima), called Esparto grass, is a cultivated species used for industrial purposes, including textile production. This species has never been studied for its medical potential before, nor has it been used in traditional medicines. It is thus fitting that the present study aimed to investigate the pharmacological potential of S. tenacissima. To achieve this goal, this work was conducted to study the chemical composition, antioxidant properties, and antiproliferative effects of S. tenacissima against cancerous cell lines, including the human colorectal adenocarcinoma cell line (HT-29) and human breast adenocarcinoma cell line (MDA-MB-231). Fractionation and characterization of S. tenacissima extract showed the presence of promising bioactive fractions. The fractions obtained from S. tenacissima extract exhibited interesting antioxidant properties, with IC50 values ranging from 1.26 to 1.85 mg/mL. All fractions, such as F1, F2, F3, and F4, induced an important antiproliferative effect on the cancer cell lines MDA-MB-231, scoring IC50 values ranging from 63.58 ± 3.14 to 99.880 ± 0.061 µg/mL. These fractions (F1, F2, F3, and F4) also exhibited a potent antiproliferative effect versus HT-29 cell lines, with IC50 values ranging from 71.50 ± 4.97 to 87.500 ± 1.799 µg/mL. Therefore, S. tenacissima could constitute a new natural source of bioactive compounds that can be used for therapeutic purposes to fight cancer and free radical damage.


Introduction
Since prehistoric times, people have used plants for food, condiments, and therapeutic purposes. More than half of medicines placed on the market come from natural products or their derivatives [1].
The use of herbal medicine is very old, and several historical studies indicate early usage of plants for medical purposes [2]. Medicinal herbs are an inexhaustible source of natural bioactive compounds that could constitute precious tools for basic research in the treatment of various diseases. Many medicinal plants have been described for their effectiveness in curing several diseases, such as hypertension, degenerative diseases, diabetes, viral infections, and cancer [3][4][5]. Natural medicines could be used in the decoction format of the whole plant, extracts of special plant parties, or purified derivatives. The aim is no longer the same between the know-how of old civilizations and today's technology. Nowadays, purifications using chromatographic techniques allow the identification and isolation of active compounds, and eventually a synthetic production of new bioactive natural substances [6].
Stipa. tenacissima L. is a typically Mediterranean perennial herb that grows in North Africa, especially in the highlands of Morocco and Algeria. It is a xerophilic plant that constitutes one of the most representative vegetation types in the dry Mediterranean ecosystems. It represents the most available source of fiber production in the region [1]. It constitutes a natural barrier against desertification and desert encroachment in the Maghreb region, and, therefore, plays an important role in ecosystem protection [2].
In North African countries, Stipa tenacissima L. is called Esparto grass, which is widely used as a potential reinforcement of polymer matrices. Furthermore, it is mainly used in the high-quality paper industry as a noble raw material due to its specific properties, since reinforcements in composite materials require a powerful adhesion between the synthetic matrix and the fiber [3]. It increases the resistance to moisture [4], and it is used for the production of cellulose nanofibers [5]. Stipa tenacissima L. is very rich in cellulose, hemicellulose, and lignin [6]. It also contains small amounts of proteins and inorganic compounds [7]. Some unsaturated fatty acids, particularly oleic and linoleic acids, are widely used in the dietetic field [8].
To the best of our knowledge, Stipa tenacissima L. is used for textile and paper production only [9]. Nevertheless, no data about its possible application in medicine have been reported to date. With the goal of contributing to better knowledge about Stipa tenacissima L. as a new promoting source of pharmacological and biological activities, the current study was the first work that aims to investigate the phytochemical composition and antioxidant and antiproliferative properties of Esparto grass aerial parts.

Collection of Plant Material
Stipa tenacissima L. was collected in July 2017 from the local region and was identified by plant taxonomist before being studied. Aerial parts were dried at room temperature before being ground into a fine powder [10].

Extraction and Isolation of Bioactive Compound Classes
Two hundred grams of the dry plant were macerated with 3 L of methanol under reflux for 4 h. Afterward, the mixture was filtered and concentrated under reduced pressure. The organic extract (40 g) was fractionated by using column chromatography with silica gel (Kieselgel 60, 70-230 mesh ASTM). A mixture of hexane/ethyl acetate was used as eluent with different compositions (30/70, 20/80, 10/90, 0/100) of increasing polarities to get four fractions: F1, F2, F3, and F4 ( Figure 1).

Determination of Total Phenol Contents
In the present work, the total phenolic content of the extracted fractions was studied using the Folin-Ciocalteu method [11]. Briefly, 0.1 mL of each fraction was mixed with 2 mL of 10 % diluted Folin reagent before one milliliter of 7.5% Na 2 CO 3 solution was added. Next, the mixture was vigorously stirred and then kept for 30 min at 45 • C in the darkness. The absorbance reading was carried out at 765 nm using a vis-NIR spectrophotometer. The standard curve of gallic acid was performed with different concentrations (0.5-15 mg/L).

DPPH Radical Inhibition Test
The radical scavenging effect of the isolated fractions was investigated using the DPPH method [12]. Each fraction was tested with concentrations ranging from 0.5 to 5.5 mg/mL. Briefly, 0.2 mL of the sample test was added to 2.8 mL of DPPH solution (24 mg/L). After 30 min of incubation at 25 • C in the darkness, the absorbance (A) was read at 515 nm. Ascorbic acid was used as a standard reference. The inhibition of free radical DPPH was given as a percentage according to the following formula [13][14][15][16]: where A C is the absorbance of the blank control and A D is the absorbance of each fraction at different concentrations. The IC 50 value (the concentration required to inhibit 50% of DPPH) was calculated from the dose response curve [17][18][19].

Cancer Cell Lines and Culture Conditions
The potential antiproliferative effect of stipa tenacissima L. fractions was tested on two cancerous cell lines, including human colorectal adenocarcinoma (HT-29) and human breast cancer cell lines (MDA-MB-231). Cancer cell lines were cultivated in Dubelco's Modified Eagle's Medium (DMEM) with 10 % (v/v) of FBS (heat-fetal bovine serum, PAA Laboratories, Austria), and 1% penicillin/streptomycin (10,000 IU/mL). Afterward, cells were cultured at 37 • C, 95% humidity, 5% carbon dioxide, and 80% confluency [20]. In this work, mitomycin was used as a drug reference for comparison purposes.

MTT Assay
The antiproliferative effect of different fractions of Stipa tenacissima L. extract on tumor cell lines was determined by the MTT colorimetric assay. In summary, when the culture reached exponential growth at a cellular density of 8000 cells/well, concentrations of each fraction ranging from 15.6 to 500.0 µg/mL were added to this culture. Cells were incubated for 48 h at 37 • C. At the end of the experimental period, 10 µL of MTT with 5 mg/mL was added to multiwall plates. Next, these plates were incubated again for an additional 4 h at 37 • C. Afterward, the reduced MTT was read at 570 nm using a microplate reader. Experiments were carried out in duplicate assays [21].
The percentage of antiproliferative effect (cell death) was calculated using the following Equation: Cell where control OD is the absorbance of the non-treated cells and sample OD is the absorbance of the treated cells.

Gas Chromatography-Mass Spectrometry (GC-MS)
Stipa tenacissima L. extract fractions were characterized using GC-MS. A Claus 580 chromatography apparatus with a capillary column of 5% phenyl and 95% methyplisyloxane (30.0 m × 250 µm), coupled with a mass spectrometer (MS), was used in this assay. Helium gas was involved as a carrier. The split was chosen to 1/75, while the injection volume was 1 µL. In this sense, the injection and detection temperatures were, respectively, set to 250 and 280 • C. Moreover, the temperature of the regulating furnace was programmed as follows: from 50 • C to 200 • C with a rate of 11 • C/min and then from 200 • C to 240 • C with a rate of 6 • C/min. The identification was conducted by comparing the spectrum along with the retention time of compounds with those of the NIST library [22].

Statistical Analysis
The present data were expressed as the means of triplicate assays ± SD (standard deviation). The significance values of the difference between the averages were deemed using ANOVA and GraphPad Prism 7 software. The averages of the investigated data were compared using the Holm Sidak Test. Statistically, a significant difference was considered at p < 0.05.

Extraction Yield and Fractionation of Natural Organic Compounds from Stipa tenacissima L. Extract
The yield of the extract obtained from Stipa tenacissima L. after the maceration with methanol was 8.2%. Fractionation of the organic extract of Stipa tenacissima L. produced four fractions: F1, F2, F3, and F4, with yields of 53%, 2.9%, 3%, and 3.5%, respectively. Analysis of variance used for statistical purposes showed no significant difference between fractions F2, F3, and F4 concerning the extraction yield (p > 0.05). However, a significant difference was observed between F1 and F2, F3, and F4 (p *** < 0.05).

Determination of Total Phenolic Compounds
The results of the total phenolic compounds contained in the isolated fractions F1, F2, F3, and F4 are summarized in Table 1, where the total phenolic content is expressed in gallic acid (GA) equivalent per 100 g of dry extract. The phytochemical analysis revealed the presence of phenolic compounds in the four evaluated fractions. As reported in Table 1, F3 and F4 fractions showed high phenolic content (31.790 ± 0.008 and 31.2600 ± 0.0005 g GA/100 g, respectively) when compared to F1 and F2 fractions (5.530 ± 0.003 and 18.120 ± 0.005 g GA/100 g, respectively). Regarding the total polyphenolic content, the statistical analysis showed no significant difference between fractions F3 and F4 (p > 0.05). However, a significant difference was observed between fraction F1 and fractions F2, F3, and F4 (p ** < 0.05). The phenolic compounds are the major active classes responsible for antioxidant properties [13]. In the last few decades, research for natural antioxidant substances has become interesting, owing to their role in the protection of health. In this sense, polyphenols have been widely reported to have great antioxidant potential, along with radical scavenging ability [23].

Antioxidant Activity by DPPH
The antioxidant capacity of the four fractions was evaluated using the DPPH method. The IC 50 values of DPPH scavenging activities of each fraction were compared to IC 50 of ascorbic acid as a positive reference. Table 2 reports the DPPH radical scavenging activity results of the assessed fractions of Stipa tenacissima L. extract. The present fractions showed interesting antioxidant properties, with IC 50 values ranging from 1.26 to 1.85 mg/mL. Regarding the investigated antioxidant activity, the analysis of variance showed no significant difference between fractions F2, F3, and F4 (p > 0.05). However, a significant difference was observed between all tested fractions F1, F2, F3, and F4 and the ascorbic acid (p *** < 0.05). The antioxidant properties reported in these fractions could be due to the high amount of polyphenols determined by the chemical analysis, which were consistent with the results reported in the literature [24,25]. The characterization of fractions by GC-MS showed the presence of interesting bioactive compounds contained in each fraction. The analyzed fractions showed different bioactive constituents, which differed according to the extraction solvent. The major identified compounds in each fraction (F1, F2, F3, and F4) are presented in Tables 3-6

GC-MS Analysis
The GC-MS analysis of fraction F1 from Stipa tenacissima L. extract revealed the presence of four major compounds, including γ-sitosterol and lupeol (Table 3; Figure 2). It was reported that γ-sitosterol and lupeol are biologically active. γ-Sitosterol belongs to the plant sterols, which were previously discovered in Girardinia heterophylla extracts [26]. γ-Sitosterol can reduce hyperglycemia and inhibits glycogenesis [27]. The anticancer properties of γ-sitosterol and beta-sitosterol (an epimer of γ-sitosterol) against various cancerous cell lines were also reported elsewhere [28]. Plant species that contain sitosterols are recommended for cancer treatment [29]. γ-Sitosterol induced an antiproliferative effect on cancerous cells through the downregulation of c-myc expression, along with the induction of the apoptotic pathways [30]. Another major compound detected by GC-MS in the F1 fraction was lupeol, a phytosterol widely common in medicinal plants. The latter currently arouses great interest due to its biological and therapeutic potential. Lupeol possesses anti-inflammatory and anti-tumor activities, as reported in a study published by Hata et al. [24].
GC-MS analysis of fraction F4 revealed the presence of three major compounds, including methyl sulfolane, coniferyl alcohol, and 2-(benzyloxy)-3, 6-difluorophenol (Table 6; Figure 5). Presently, no additional data have been reported on these compounds in terms of pharmacological activities.

Antiproliferative Activity of Fractions of Stipa tenacissima L. Extract on Cancer Cells
Despite some advances in cancer treatments, some types of cancer resist several synthesized drugs. In this sense, it was reported that natural drugs derived from plants can be effective against cancer. Even though several natural anti-cancer compounds have been tested, plants are still a non-limited source of bioactive compounds. Thus, a large unexplored plant species deserves to be studied in terms of medicinal potential [14].
Stipa tenacissima L. is a cultivated species used in textile production. It has never been investigated in terms of medicinal potential. Therefore, the current research work was the first report on the antiproliferative effect of Stipa tenacissima L. extract fractions. Using cell viability indices, the MTT test revealed that the four fractions reduced cell viability in both cancer cell lines in a dose-dependent manner at 72 h post-treatment (Figures 6-9).    Table 7.  Regarding the IC 50 values of the antiproliferative effect induced by fractions F1 and F2 on both MDA-MB-231 and HT-29 cell lines, the statistical analysis showed a significant difference between the IC 50 values of fractions F1 and F2 towards both MDA-MB-231 and HT-29 when compared to F3 and F4 (p * < 0.05). The mitomycin used as a drug reference for comparison purposes showed a strong antiproliferative effect towards all cancer cell lines tested with IC 50 , scoring 0.05 µg/mL (Figure 8).
The GC-MS analysis of the F1 fraction showed the presence of sterols, such as γsitosterol and lupeol. These compounds could be responsible for the antiproliferative effects exhibited by the fraction. The present findings were in agreement with those reported in earlier work, which showed that γ-sitosterol and lupeol possess potential cytotoxic effects [27,28]. The antiproliferative effect of the F2 fraction may be due to loliolide and diacetone alcohol, which might have antiproliferative effects [18]. Regarding the bioactive compound classes detected in the F3 and F4 fractions, no previous literature has reported their potential antiproliferative effects, hence, it seems that new compounds from Stipa tenacissima L. exhibit antiproliferative effects on MDA-MB-231 and HT-29 cancer cell lines.

Conclusions
The present research work provides data about newly bioactive fractions from the organic extract of Stipa tenacissima L., which is currently used for textile production. The plant fractions showed high antioxidant properties, along with high antiproliferative effects on human colorectal adenocarcinoma cell MDA-MB-231 and human breast adenocarcinoma cell HT-29. Stipa tenacissima L. could constitute a new way of conceptualizing effective drugs against cancer and free radical damage.