S. crotalarioides was collected in August 2014 in “Las Comadres”, Guadalcazar Municipality of San Luis Potosí State, Mexico. Then, the taxonomist José García Pérez identified the plant and a voucher specimen (SPLM43012) was deposited in the Herbarium Isidro Favela of the Universidad Autónoma de San Luís Potosí. S. crotalarioides is not an endangered species, and for this reason, a collection permit is not required by SEGAM-SLP.

From Sigma-Aldrich was acquire indomethacin (IND), TPA, and ursolic acid, and the immunoenzymatic kits (IL-1β, IL-6, IL-10 and the TNF-α) were purchase from PrepoTech Company. All other reagents were of the highest commercial grade.

The leaves and branches were dried in the shade at room temperature, then, they were powdered. A mixture of 300 g powdered plant and 3.5 L of chloroform, was heated under reflux for 4 h. After, the solvent was eliminated under reduced pressure to give a dry residue, and the solid was washed with hot hexane.

A mixture of 1 mL of isooctane, 10 mg of CESC, and 100 µL a solution of bis (trimethylsilyl) trifluoroacetamide with 10% of trimethylsilyl chloride was heated for 10 min at 100˚C in a microwave oven (CEM Discover) at 150 watts.

The analysis of the extract was carried out on a gas chromatograph coupled to a mass spectrometer (Agilent Technology, model 6890 N); which was coupled to a selective detector of mass (model 5973). A capillary column DB-5HT of 15 m in length, 0.25 mm internal diameter and 0.10 µm film thickness was used. The injector temperature was 320˚C. The next temperature program was used: the initial temperature 100˚C was maintained for 3 min, then, the temperature was increased at a rate of 10˚C/min up to 320˚C, and this temperature was held for 5 min. The splitless injection was performed at a ratio of 1:100. The spectrum was performed at 70 eV. The identification of the compounds was carried out by comparing their spectra with the mass spectra of standard samples and the Wiley14.1/NIST11 library.

The standardization of CESC was performed by HPLC (Varian Model Pro Star 310) coupled to an evaporative light scattering detector (ELSD) (Alltech model ELSD 2000). Nitrogen was used as drying gas, a flow rate of 1.7 L/min at 67˚C evaporation temperature. A Grace Smart RP C18 column (5 µm, 4.6 × 250 mm) was used. The temperature of column oven was 40˚C. The flow rate was 0.7 mL/min. The linear gradient of the mobile phase began at 0.5% (v/v) of acetic acid in a solution of water-methanol-acetonitrile with a ratio of 1:0:99 (v/v/v) and finished 20 min later with a solution of 0.5% acetic acid in a mixture of water-methanol-acetonitrile with a ratio of 1:10:89 (v/v/v). The calibration curve was obtained with solutions of ursolic acid at six concentrations (0.06, 0.12, 0.18, 0.24, 0.30 and 0.60 mg/mL). The result for each concentration was the average of three injections of 20 µL, using a loop. The integrals of the chromatographic peaks at a retention time of 5.38 min were used to generate the standard calibration curve (R² = 0.9912). CESC (6.0 mg) was dissolved in 10 mL methanol, and the sample (20 µL) were injected in triplicate and then extrapolated to the standard calibration curve.

Male CD1 mice (20 - 25 g) were obtained from the animal facility of the Universidad Autónoma Metropolitana-Xochimilco. The animals were kept in isolated cages, and they had access to food (Lab Diet 5001) and water ad libitum, and they were housed at 24˚C under light-dark cycles of 12:12 h. The Research Bioethics Committee of the UAM-X approved all experimental (number project 140). The tests in vivo were carried out according to the official Mexican Norm (NOM-062-ZOO-1999), for the care of animals. The mice were maintained to laboratory conditions for 1 week prior to the test, which were done after 9:00 am, and finally were sacrificed in a CO₂ chamber.

The acute toxicity was determined using the methodology described in the OECD Guideline for testing chemicals [6]. Groups of 5 mice were used. One group was administered polivinilpirrolidone (PVP) and the others with CESC at doses of 5000 and 2500 mg/Kg in PVP in a 1:4 ratio. After 72 h the animals were sacrificed, and biopsies were carried out to identify possible signs of toxicity.

The cell lines were used Macrophages J774A.1 (obtained from ATCC® TIB-67^TM), HeLa (ATCC® CCL-2^TM), MCF7 (ATCC® HTB-22^TM), SKLU-1 (ATCC® HTB-57^TM), LNCaP (ATCC® CRL-1740), A549 (ATCC® CCL-185^TM), SW620 (ATCC® CCL-227^TM) and HaCat (ATCC® PCS-200-011^TM), obtained from Instituto Nacional del Cáncer of México. The cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium) with 10% fetal bovine serum (FBS), 100 IU/ mL penicillin, and 100 µg/ml streptomycin. For Macrophages were used RPMI supplemented with 10% of FBS, penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37˚C under CO₂ (5%).

Macrophages J774A.1 and the cancer cell lines were washed with a PBS buffer, then, the cells were seeded in DMEM in 96-well microplates (8 × 10⁴ cells/well), they were incubated for 24 h, after this time a solution of CESC in DMSO (1 - 200 µg/mL) was added. 48 h after the treatment, 10 µL of MTT of a solution of 5 mg/ml in PBS were added, and the cells were incubated for 4 h at 37˚C. After this time, the medium was removed, and the formazan crystals were dissolved in 100 µL of DMSO. The optical density (OD) was obtained using an ELISA plate reader from BioRad at 540 nm. Six wells were used for each concentration of CESC. The viability was determined using the following equation:

%viability = Abs treated cells Abs control cells × 100

The serum levels of TNF-α, IL-6, IL-10, and IL-1β were determined with a commercially ELISA kit following the manufacturer’s instructions. The OD was measured using a microplate reader at 405 nm with a wavelength correction set to 650 nm.

In plates of 6 wells macrophages J774A.1 were seeded at a density of 1 × 10⁶/well and treated with CESC (25 μg/mL) or IND at a concentration of 17 µg/mL (50 μM) and incubated for 2 h. After this time, 5 µg/mL of LPS was added and the cells were incubated for 24 h, the supernatants were collected and stored at −80˚C until they were analyzed. The levels of IL-6, IL-1β, IL-10 and TNF-α in the supernatants of the cultures of macrophages were determined using a commercial immunoenzymatic kit (PeproTech).

The results are expressed as the means ± SE, and statistical analyses were performed using Student’s t-test and ANOVA followed by Tukey’s test. Differences were deemed significant at p < 0.05.

The CESC was standardized by HPLC using ursolic acid, because this compound is the main component of the extract, which makes it an excellent fingerprint to carry out the standardization of the extract by this analytical method.

The difference of percentages of ursolic acid in CESC obtained by the HPLC method with ELSD detector (17.05%) and the GC-MS (16.39%), might be due to that in the last method the extract was derivatized and the reaction could be not complete.

After two h of topical application of TPA there are vasodilation, edema and platelet aggregation, and three h later the ear edema is increased by exudate action and after 6 h is observed the maximum expression of edema. The inflammation produced by topical administration of TPA is mediated the phospholitase A2 and cyclooxygenase stimulation [7]. Then, one way to control inflammation is the inhibition of the stimulation of phospholipase A2. CESC significantly diminished the edema, which suggests that it inhibits phospholipase A2 production, vasodilation, and platelet aggregation.

The multiple applications of TPA cause ear edema, epidermal hyperplasia [8], and infiltration of inflammatory cells, such as polymorphonuclear leukocytes. The results showed that CESC (2 mg/ear) significantly diminished TPA-induced ear edema, these facts suggested that the extract decreased the cellular infiltration and epidermal hyperplasia.

Inflammation is a response to irritation or infection, and macrophages play an important role in the inflammation process [9]. These cells show a vigorous response to LPS, this compound increases the production of inflammatory modulators such as TNF-α, IL-6 and IL-1β.

TNF-α, also known as cachectin, is involved in immune response and can prevent infections and keep inflammation localized, but inappropriate or excessive production of this mediator can be harmful [10].

IL-6 is an inflammatory interleukin [11], and it is responsible for the induction and perpetuation of inflammation, and it can amplify the inflammatory cascade and cause injury.

IL-1β is also a mediator of inflammatory response and exacerbates damage during chronic and acute tissue injury [12].

IL-10 possesses anti-inflammatory activities and plays a crucial role in preventing inflammatory and autoimmune pathologies. IL-10 inhibits the production of IL-1β and TNF-α in LPS-activated macrophages. Therefore, compounds that regulate cytokines may have therapeutic effects. The inhibition of the mediators occurs by acting on NF-κB (nuclear factor kappa B), which regulates the release of inflammatory cytokines [13]. This fact suggests that CESC acts on transcription factors, but further studies are required to confirm this suggestion. Thus, the agents that inhibit the production of pro-inflammatory mediators and promote the synthesis of anti-inflammatory cytokines may be useful in treating inflammatory conditions.

The anti-inflammatory activity of ursolic acid has been attributed to its ability to suppress NF-ƙB activation [14]. Additionally, this compound suppresses the expression of COX-2 and iNOS [15] and inhibits the production of NO [16].

1-Octacosanol [17], triacontanol [18], stigmasterol [19], and β-sitosterol [20] also show anti-inflammatory activities, which suggests that the anti-inflammatory activity of CESC might be due to the presence of these five compounds.

The cytotoxic activity of CESC and ursolic acid, the main component of this extract, were evaluated against HeLa, MCF7, SKUL-1, LNCaP, SW620 and A549, and HaCat (keratinocytes). CESC exhibited the highest activity on SKUL-1, LNCaP, SW620 and A549, which was higher than that obtained with ursolic acid on these four cell lines.

According to the National Cancer Institute, pure compounds or extracts with IC values lower than 4 µg/mL and 30 µg/mL, respectively, are considered as cytotoxic [21], and CESC showed IC₅₀ values lower than 30 μg/mL in 5 human cancer cell lines, and its IC50 value (80.3 μg/mL) in HaCat, a non-cancerous human cell line, was greater than those obtained for the cancer cell lines. The results suggest that the cytotoxic activity of CESC might be specific to cancer cell lines.

There are reports that indicate that ursolic acid shows cytotoxic activity against different cancer cell lines [22] [23]. This triterpene acts by inhibiting cell proliferation and inducing apoptosis by activating caspase-3. Cellular apoptosis is always accompanied by the disruption of the mitochondrial membrane, resulting in rapid collapse and the activation of downstream caspases, which induces cell apoptosis. In the case of MCF-7, this compound has a different mechanism action, including inhibition of growth and suppression of migration, and in this cell line, the signaling pathway is triggered by NFƙB [24].

The cytotoxic effect of ursolic acid [25], 1-octacosanol [26], and β-sitosterol has been reported. Thus, the cytotoxic activity of CESC on SKUL-1, LNCaP, SW620, and A549 might be due to the mixture of these three compounds. More studies are requested to know the mechanism of action of CESC.