The leaves of Bauhinia forficate L. (Fabaceae) are used in Brazilian folk medicine mainly as antidiabetic agent. They are rich in polyphenols, and kaempferitrin and other flavonoids are currently considered as basic chemical criteria for the quality and the effectiveness of extracts and phytopharmaceuticals derived from them. Aiming to expand the scope of current data of the chemical profile of B. forficate subsp. forficate , the present study employed HPLC-DAD and HPLC-DAD-ESI/MS to compare the flavonoid profile and kaempferitrin content in the leaves of specimens collected seasonally during one year, in two different locations in Southeastern Brazil: Rio de Janeiro (RJ) and São Paulo (SP) states. The data showed a variation in the flavonoid profile and in the kaempferitrin content depending on the place of collection and the season of the year. The target compound, kaempferitrin, was found to be only a minor constituent in RJ (0.21 - 1.02 μg/mg), and was mostly absent or occurred as trace in SP location (0.00 - 0.12 μg/mg). Some degree of similarity was observed in the flavonoid profiles according to the region of collection. In agreement with some published reports, the low and quite variable kaempferitrin content as well the wide variability of the flavonoid profiles brings into question the dubious matter of using these compounds as chemical markers for this medicinal species.
Bauhinia forficata Link (Fabaceae) is a medium-sized tree found in Southeastern and Southern Brazil, popularly known as pata-de-vaca (cow’s hoof) due to the bilobed shape of its leaves [
A number of studies have demonstrated the antidiabetic property of aqueous, alcoholic and aqueous-alcoholic extracts from the leaves of Bauhinia species [
Since it was firstly reported as a marked component in the methanolic extract of B. forficata leaves [
Ferreres et al. [
There is nowadays a growing concern onto considering a polyphenols/flavonoid more unfolded profile to authenticate and control the quality of B. forficata samples. Efforts have been made to develop and apply analytical methods to establish the qualitative and/or quantitative polyphenolic compounds [
Previous studies involving the seasonal and/or location influence in the kaempferitrin content and the flavonoid profile and content of B. forficata leaves have been done with a single exemplar from each location or specific season [
Analytical grade methanol (MeOH), formic acid (HCOOH) and HPLC grade acetonitrile (CH3CN) were purchased from Tedia-Brazil. Milli-Q grade water was used for HPLC and UPLC. The kaempferitrin used as reference compound was previously isolated from the leaves of Uncaria guianensis (99% purity) [
Leaves of Bauhinia forficata were collected from different trees in two different Brazilian sites: Valinhos city (SP, trees nº 1 - 3) from July-2009 to February-2010 and Rio de Janeiro city (RJ, tree 4) from September-2009 to March-2011. The geographic coordinates are tree nº 1 and tree nº 2: 22˚57'44.6''S, 46˚59'39.8''W, alt. 692 m; tree nº 3: 22˚58'40.8''S, 46˚57'23.0''W, alt. 807 m (SP, Valinhos); and tree nº 4: 22˚56'29.3''S, 43˚24'20.16''W, alt. 25 m (RJ―Rio de Janeiro). All specimens were identified as Bauhinia forficata subsp. forficata by the specialist Dr. Angela S. F. Vaz from the Research Institute of Rio de Janeiro Botanical Garden (JBRJ). Voucher specimens are deposited in the JBRJ Herbarium under registrations RB-537536, RB-537541, RB-537544, RB-537545 (SP samples) and RB-511138 (RJ samples). Authorization for legal access to the genetic heritage was protocoled under nº A66DDE3.
The extracts were prepared using a modified procedure previously developed to assess the kaempferitrin content from the leaves of Uncaria guianensis [
The HPLC analyses were performed using Shimadzu (VP) equipment with two pumps LC-10AD, auto-sampler SIL-10AD, column oven CTO-10A, vacuum degasser DGU-14A and diode-array detector Shimadzu SPD-M10A. A reverse-phase column Lichrocart Lichrospher 5 µm, 250 × 4 mm i.d. (Merck) coupled to a pre-column LiChrospher 100 RP18e 5 µm, 4 × 4 mm was used. Elution was performed in a gradient mode at a 1.0 mL/min flow with CH3CN (solvent B) and H2O/HCOOH, pH 3 (solvent A): 0 - 30 min 95% - 75% solvent A, 30 - 50 min 75% - 20% solvent A, 50 - 55 min 20% - 95% solvent A and finally 55 - 60 min 95% solvent A. The diode-array detector was set at an acquisition range of 200 - 600 nm. The chromatograms were monitored at 265 nm, the injected volume was 40 µL and the oven column was set at 30˚C. MeOH solutions of the crude extracts at c = 15 mg/mL were filtered through 0.45 µm Minisart RC25 membranes (Sartorius) and then injected in duplicate. The kaempferitrin quantification was done through an external calibration curve using aprevious isolated sample (Section 2.1). Five concentration levels of the reference compound were prepared in methanol (1.30, 2.60, 9.75, 32.30 and 65.00 µg/mL). Each level was injected in triplicate to yield the calibration curve (R2 = 0.9999) derived from the equation y = 87272x − 33684, where y is the peak area and x the concentration (μg/mL). Kaempferitrin in the extracts was quantified against this calibration curve. The results were later converted to μg/mg extract. The linearity and validity of the calibration curve were supported by ANOVA test. The quantitation limit (QL) was considered as the lowest point of the curve, i.e. 1.30 µg/mL (0.09 µg/mg). The confirmation of the kaempferitrin peak in the extracts was performed by fortification of three selected samples (SP-T1-JUL2009, SP-T2-DEC2009 and RJ-NOV2009) with 1 - 3 µL of the reference compound in methanolat c = 130 µg/mL. This value was calculated in order to enhance the original area by about three times. Two injections of each sample (original and fortified) were carried out and the respective area’s integrations were used to corroborate the peak identity.
High-performance liquid chromatography-diode array detection/electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS) analyses were performed using a Shimadzu Prominence-Nexera (Shimadzu, Japan) composed by a high-pressure binary pump LC-30AD and column oven CTO-30A. The system was equipped with a photodiode array detector (model SPD-M20A) and coupled to a quadrupole mass spectrometer MS ZQ (Micromass-Waters) equipped with an ESI source and an ion trap mass analyzer. Data were acquired using a CLASS-VP software, which was controlled by a CBM-20A interface. The following parameters were set in the negative ion mode: capillary voltage 3.0 KV, cone voltage 40.0 V, extractor voltage 1.0 V, RF lens 1.0 V, source block temperature 150˚C, desolvation temperature 350˚C, cone N2 flow 10 L/h, desolvation N2flow 300 L/h. Samples (10 µL injection volume) were injected into a RP-18 reversed-phase LiChrospher® column (125 × 2.0 mm × 5 μm particle size) (Merck) maintained at 35˚C. The elution conditions were as follows: 0.25 mL/minflow rate; solvent A, water pH 3.0 adjusted with formic acid and solvent B acetonitrile. The gradient was: 0 - 15 min, linear 5% - 25% B; 15 - 25 min, linear 25% - 80% B; 25 - 27.5 min, linear 80% - 5% B; 27.5 - 30 min, isocratic 5% B. The DAD was programmed in the range 190 - 600 nm with resolution at the level of 4 nm. Mass acquisition was set in the range m/z 100 - 1000 (resolution 0.4). The system was calibrated with standard kaempferitrin (m/z 577.0). The two more representative samples from each locale, in terms of kaempferitrin concentration, were analyzed.
In our study, evaluation of the flavonoid profile and kaempferitrin content in B. forficata subsp. forficata leaves was carried out with plants collected seasonally during approximately one year in two different places of the Brazilian Southeast region, with differing altitude and climate (seaside town of Rio de Janeiro, RJ and Valinhos in the interior of São Paulo, SP]. The extract yields, although not-statistically considered, pointed to lower amounts and greater variation in the samples from SP than those from RJ (
Sample code | Extract weight (mg) | Extract yield (%)1 | KF content in the extract (µg/mg) | |
---|---|---|---|---|
Collection in Valinhos (SP state) | ||||
SP-T1-JUL2009 | 29.5 | 11.8 | 0.12 ± 0.00 | |
SP-T1-AUG2009 | 15.3 | 6.0 | ||
SP-T1-SEP2009 | 15.1 | 6.0 | ||
SP-T1-NOV2009 | 11.3 | 4.5 | ||
SP-T1-DEC2009 | 20.6 | 7.9 | ||
SP-T1-JAN2010 | 26.3 | 10.5 | ||
SP-T2-AUG2009 | 10.0 | 4.0 | ||
SP-T2-SEP2009 | 25.3 | 10.0 | 0.09 ± 0.01 | |
SP-T2-NOV2009 | 25.7 | 10.3 | 0.12 ± 0.03 | |
SP-T2-DEC2009 | 15.2 | 6.0 | ||
SP-T2-JAN2010 | 36.3 | 14.3 | ||
SP-T3-AUG2009 | 27.2 | 10.9 | 0.10 ± 0.01 | |
SP-T3-SEP2009 | 21.3 | 8.4 | ||
SP-T3-NOV2009 | 30.8 | 12.2 | ND | |
SP-T3-DEC2009 | 16.1 | 6.4 | ND | |
SP-T3-JAN2010 | 36.0 | 14.0 | ||
SP-T3-FEB2010 | 42.7 | 16.9 | ||
Collection in Rio de Janeiro (RJ state) | ||||
RJ-SEP2009 | 45.9 | 18.4 | 0.21 ± 0.02 | |
RJ-NOV2009 | 44.7 | 17.8 | 0.22 ± 0.00 | |
RJ-DEC2009 | 54.1 | 21.1 | 0.57 ± 0.05 | |
RJ-JAN2010 | 44.6 | 17.4 | 0.32 ± 0.00 | |
RJ-MAR2010 | 42.2 | 16.4 | 0.24 ± 0.01 | |
RJ-FEB2011 | 30.0 | 12.0 | 0.59 ± 0.04 | |
RJ-MAR2011 | 45.0 | 18.0 | 1.04 ± 0.01 |
1Related to 250 mg of leaves; KF = kaempferitrin; QL = Quantitation Limit = 0.09 µg/mg. ND = notdetected; T1, T2 and T3 = trees 1, 2 and 3, respectively (see item 2.2).
The presence of kaempferitrin in the extracts was indicated by matching its retention time (Rt = 30.2 min) and identity of the UV-spectrum with the reference compound, moreover than being thereafter confirmed by the fortification procedures. The kaempferitrin content in B. forficata leaf extracts varied from 0.21 to 1.04 μg/mg for RJ specimens, and was mostly absent or occurred as trace compounds in the SP samples. Most of these latter collections resulted extracts containing amounts of kaempferitrin below the quantitation limit (0.09 μg/mg). In these cases, the contents of kaempferitrin could be inferred from the calibration curve; however, such an extrapolation was eventually considered worthless (
Overall, our data corroborates some previously reports on the chemical variability of the flavonoid profile in B. forficata leaves [
Also in accordance with the literature, most of flavonoid constituents―herein emphasizing the major ones―were kaempferol derivatives. The degree of the aglycones glycosylation was suggested by the quasi molecular ion [M-H]− obtained from the UPLC-ESI-MS operating in the negative mode (
Some researchers have also caught the attention to the unavailability of reports on antidiabetic activity of other kaempferol glycosides that are usually present in the B. forficata extracts [
Peak1 | Rt (min) | λmax (nm) | [M-H]? (m/z)3 | Suggested Structure4 |
---|---|---|---|---|
1 + 2 | 24.6 | 265,346 | 755.3 | K-triglycoside |
2 | 26.2 | 265,346 | 739.2 | K-triglycoside |
3 | 26.6 | 265,346 | 739.2 | K-triglycoside |
4 | 27.9 | 255,351 | 609.1 | Q-diglycoside |
5 | 28.9 | 253,353 | 463.1 | Q-glycoside |
(*)2 | 30.2 | 266,352 | 577.3 | Kaempferitrin |
6 | 30.9 | 266,346 | 593.3 | K-diglycoside |
7 | 31.5 | 255,352 | 623.2 | I-diglycoside |
8 | 32.2 | 254,348 | 447.1 | Q-glycoside |
Standard KF | 30.3 | 264,346 | 577.3 | K-diglycoside |
1For peak numbering see
compounds by the plant [
Finally yet importantly, most of the early reports in the literature on chemical and biological properties of B. forficata Link have approached such a species as a particular botanical entity, disregarding possible morphological distinctions that encompass, for instance, subspecies or possible varieties. In this context, taxonomic studies have been gradually evolve toward consolidating two subspecies growing in Brazil: Bauhinia forficata Link subsp. forficata and Bauhinia forficata subsp. pruinosa (Vogel) Fortunato & Wunderlin [
Recent compilation in the literature reveals that researchers have been alert to considering such a taxonomic detail [
The data presented in this study show the complex question of considering the phenolic content of Bauhinia forficata leaves―be it the flavonoid profile or another phenolic group profile, or even an individual compound―as a tool to the quality control of the vegetal raw material, herbal and phytopharmaceutical derived from this species. Additionally, the spontaneous growth of two B. forficata subspecies regarded as anti-hyperglycemic also demonstrates the need for a precise botanical classification when approaching medicinal properties of this species. Further studies on the correlation between the chemical composition and therapeutic property of this important antidiabetic species are necessary.
VFM and JOC thank CNPq-Brazil for their fellowships. The authors are indebted to Dr. J. L. Mazzei for his valuable suggestions.
The authors declare no conflicts of interest concerning the present publication.
Tostes, J.B.F., Siani, A.C., Monteiro, S.S., Melo, V.F., Costa, J.O. and Valente, L.M.M. (2019) Seasonal Flavonoid Profile and Kaempferitrin Content in the Leaf Extracts of Bauhinia forficata Subspecies forficata from Two Locations in Southeastern Brazil. American Journal of Plant Sciences, 10, 208-220. https://doi.org/10.4236/ajps.2019.101016