Phytochemical Composition, Anti-Inflammatory and Cytotoxic Activities of Chloroform Extract of Senna crotalarioides Kunth

Senna crotalarioides is used 
in traditional medicine to treat inflammation. The aim of this work was to investigate 
the anti-inflammatory and cytotoxic activities and the possible mechanism of 
action of the chloroform extract washed with hexane of S. crotalarioides (CESC). The 
anti-inflammatory effect was tested on 
12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema in mice. 
The levels of TNF-α, IL-1β, IL-6 and IL-10 were determined in 
macrophages J774A.1 stimulated by lipopolysaccharide (LPS). The cytotoxic 
activity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl 
tetrazolium bromide (MTT) assay against six human cancer cell lines: HeLa 
(cervical cancer), SKLU-1 and A549 (lung cancer), LNCaP (prostate cancer), 
SW620 (colon cancer) and MCF7 (breast cancer). The composition of the CESC was 
determined by GC-MS analysis, and standardized by HPLC-ELSD with ursolic acid 
as the phytochemical marker. CESC inhibited ear edema 61.45%. In chronic ear 
edema, CESC diminished the inflammation by 53.77%. CESC decreased TNF-α, IL-1β and IL-6 concentrations, and increased the concentration of 
IL-10. The extract showed IC50 values on HeLa, SKLU-1, A549, LNCaP, 
SW620 and MCF7 by 48, 21, 8.16, 6.82, 1.81, 4.06 and 12.5 μg/mL, respectively. 
The main components were ursolic acid, 1-octacosanol, stigmasterol, β-sitosterol, 1-triacontanol, (Z, Z) 
hexadec-9-enoic acid octadec-9-enyl ester. CESC might be useful for developing 
a phytomedicine with anti-inflammatory and cytotoxic activities.


Introduction
Inflammation is a protective response to an attack on the body. However, if inflammation is persistent, it can cause several pathological conditions, such as arthritis, cancer, among others. The inflammation is induced by chemical and other mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) [1]. These mediators are pro-inflammatory cytokines.
The biological functions of TNF-α, IL-1β and IL-6 are similar, and they are secreted as a response to inflammatory stimuli.
Current therapies for inflammation include non-steroidal anti-inflammatory drugs (NSAIDs) and corticosteroids; however, both of these approaches have severe side effects [2] such as gastric ulcers and an increased risk of heart attack [3]. Because of these risks, new sources of anti-inflammatory compounds are of great interest.
Products from medicinal plants, such as pure compounds or standardized extracts, are good sources for new drugs.
Senna crotalarioides Kunth (Fabaceae) is a puberulent plant that grows in the Nuevo León, Coahuila, and San Luís Potosí States of Mexico, and this species is used in traditional medicine to treat some types of inflammation [4].
Previously, we reported the antioxidant potential and anti-inflammatory effect of the chloroform extract of this plant on TPA-induced ear edema and carrageenan-induced rat paw edema. However, there have been no studies of the bioactive compounds in this plant or their mechanisms of action.
In the present study, the composition and standardization of the CESC were determined, and we assessed both the in vivo and in vitro anti-inflammatory effects of this extract and the possible mechanism of its anti-inflammatory properties. Additionally, its acute toxicity and cytotoxicity on six cancer cell lines were determined.

Chemicals
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.

CESC Preparation
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.

Derivatization
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.

CESC Analysis
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.

Standardized Extract
The

Experimental Animals
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 CO 2 chamber.

TPA-Induced Mouse Ear Edema
The TPA-induced mouse ear edema procedure was described previously by de Young [5]. A solution of TPA (2.5 µg) in acetone (25 µL) was applied topically to the inner and outer surfaces of the right ears of the mice (groups of 8 mice), and acetone (25 µL) was administered to both surfaces to the left ear. Thirty minutes after a solution of CESC (2.0 mg/ear) or IND in acetone was topically applied to the right ear. After six h. the mice were sacrificed, and 6 mm plugs of the central portion of both ears were obtained and weighed. The following formula was used to determine the percent inhibition of the edema.

Determination of Anti-Inflammatory Activity on Induced Mouse
Ear Edema by Multiple Application of TPA Groups of 8 mice were topically applied to the right ear on both the inner and outer surfaces with a solution of 2.5 µg TPA in 25 µL acetone. After thirty min, IND (0.5 mg/ear) or CESC (2 mg/ear) were administered topically. TPA and the treatments were applied on day 1, 3, 5, 7 and 9 after the treatment. After 6 h, the animals were sacrificed, and 6 mm plugs of the central portion of both ears of each mouse were obtained and the circles were weighed. The above formula was used to determine the percent inhibition of the edema.

Acute Toxicity
The acute toxicity was determined using the methodology described in the 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.

Cell Culture
The

Determination of the Levels of Cytokines
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 ×

Statistical Analysis
The results are expressed as the means ± SE, and statistical analyses were per-American Journal of Plant Sciences formed using Student's t-test and ANOVA followed by Tukey's test. Differences were deemed significant at p < 0.05.

Standardized and Composition of the CESC
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.

Anti-Inflammatory Activity in Vivo
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 inflamma-tion 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.

Anti-Inflammatory Activity in Vitro
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].

Cytotoxic Effect
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 IC 50 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.

Conclusion
The results showed that CESC possesses anti-inflammatory and cytotoxic activities; therefore, standardized CESC might be useful for developing a phytomedicine.