A New Isoflavone and Other Constituents from Roots of Clitoria guianensis

A new isoflavone named pratensein-7-O-β-D-rutinoside [(−)-7-O-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranosyl-5,3’-dihydroxy-4’-methoxyisoflavone] and the known compounds biochanin A-7-O-β-D-rutinoside, 6-deoxyclitoriacetal 11-O-β-D-glucopyranoside, 6-deoxyclitoriacetal, (2S)-naringenin-6-C-β-D-glucopyranoside, (2R)-naringenin6-C-β-D-glucopyranoside, tachioside, and koaburaside were isolated from the roots of Clitoria guianensis (Aubl.) Benth var. guianensis (Fabaceae), a subshrub found in the Brazilian Cerrado biome. The structures of the compounds were identified by physical and spectroscopic data measurements (specific rotation ([α]D), circular dichroism (CD), ultraviolet (UV), infrared (IR), 1D and 2D nuclear magnetic resonance (NMR), and mass spectrometry (MS)). The EtOAc fraction of the roots exhibited high toxicity against Artemia salina with median lethal dose (LD50) value of 8.53 mg L.


Introduction
Clitoria Linn. is a genus of the Fabaceae family with about sixty species widely distributed throughout the tropical regions of Africa, Asia and Central America. 1,2 In Brazil there are 28 Clitoria species found mainly in the northern region. Clitoria guianensis, also known as "Vergateza", is a subshrub with purple flowers present on dry ground. 3,4 Several plants of Clitoria genus are traditionally used for the treatment of respiratory, neurological, urinary, and skin disorders. 5,6 Ethnobotanical studies in Brazil reported that Clitoria guianensis is used in folk medicine in the form of decoction or "garrafadas" (medicinal plants mixed with alcoholic beverages) for mental disorders and sexual stimulant. 7,8 In this genus, Clitoria ternatea is the species with the highest number of pharmacological activities reported, such as antimicrobial, antipyretic, anti-inflammatory, anticonvulsant, diuretic, anesthetic, antidiabetic, and insecticidal. 2,9 Isoflavonoids and rotenoids are naturally found in plants of the Fabaceae (Leguminosae) family. 10,11 Isoflavonoids have shown potential as anticancer, antimutagenic, antioxidant, and antimicrobial. [12][13][14] Rotenoids exhibit anti-inflammatory, antiviral, anticancer activities and are used as pesticides, being classified as biological pesticides, due to their natural origin. 15,16 The chemical constituents isolated from the Clitoria genus, mostly from C. ternatea and C. fairchildiana, are rotenoids, [17][18][19][20][21][22] flavonols, anthocyanins, 6,23,24 alkaloids, 25 triterpenoids, 26 and others. There are no reports of isoflavone isolation from Clitoria.
In this paper, we report the first phytochemical study of Clitoria guianensis (Aubl.) Benth var. guianensis, the isolation and the structural elucidation of pratensein-7-O-β-D-rutinoside as a new isoflavone, and seven known compounds including rotenoids, isoflavone, flavanones and phenolic glycosides. The EtOH crude extract, and n-hexane (Hex) and ethyl acetate (EtOAc) fractions of C. guianensis roots were evaluated for toxicity against Artemia salina.

Results and Discussion
Clitoria guianensis was collected from Brazilian Cerrado biome and the roots were extracted with ethanol and further partitioned with Hex and EtOAc. The toxicity testing using brine shrimp (Artemia salina) of EtOH crude extract, Hex, and EtOAc fractions showed median lethal dose (LD 50 ) values of 23.44, 41.16, and 8.53 mg L −1 , respectively. The samples are considered highly toxic (LD 50 < 100 mg L −1 ) 27 and the fractions presented lower LD 50 values than C. ternatea leaves extracts tested, 28 suggesting the presence of compounds with potential pharmacological activity.

General experimental procedures
One-dimensional ( 1 H, 13 C, and TOCSY) and two-dimensional ( 1 H-1 H correlation spectroscopy (COSY), HSQC, and HMBC) NMR experiments were performed on a Bruker Avance TM III 600 spectrometer (14.1 T) at 600 MHz ( 1 H) and 151 MHz ( 13 C) using deuterated solvents (CDCl 3 , CD 3 OD, and dimethyl sulfoxide (DMSO-d 6 )) (99.98% D) as internal standards for 13 C NMR chemical shifts and residual solvent as an internal standard for 1 H NMR. d values are reported relative to Me 4 Si. High-resolution mass spectra were obtained on a ESI-QTOF-MS Bruker TM Maxis Impact mass spectrometer. Fourier transform infrared (FTIR) spectra were obtained on a Bruker VERTEX 70 FTIR spectrometer using ATR (attenuated total reflectance). Optical rotations were measured on a PerkinElmer TM 341-LC polarimeter. HPLC analyses were performed using a Jasco TM LC-NetII/ADC liquid chromatograph, equipped with photodiode array

Extraction and isolation
The roots (398.5 g) were ground and exhaustively extracted three times at room temperature with ethanol (3 × ca. 300 mL). The plant material remained in contact with the solvent for 7 days and was manually shaken every 12 h for 2 min, for each extraction. The EtOH crude extract

Toxicity testing using Artemia salina
The brine shrimp lethality assay was performed by the method of McLaughlin. 39,40 Brine shrimp eggs (Artemia salina) were hatched in saline solution of NaCl (38 g L −1 ) and were incubated for 24 h. The saltwater solution was aerated continuously during incubation with an aquarium air pump. The nauplii (10 units) were added to each set of tubes containing EtOH crude extract, Hex and EtOAc fractions (solubilized in saline solution containing 1% DMSO). The samples were tested in triplicate at concentrations of 2, 5, 7, 10, 20, 35, 50, and 100 mg L −1 . For each concentration, three test tubes containing the same volume of DMSO plus seawater and brine shrimp nauplii were used as control group. Survival was measured after 24 h incubation. The collected data were computerized and LC 50 values determined by Probit analysis.