Figure 2. General chemical structure of gallic acid and proanthocyanidins identified in the bark of Stryphnodendron spp known as “barbatimão”. Numbers indicate the respective molecule in Table 2, with respective substituent (R). Dimers and the polymer identified are repetition of these monomers according to their names in the corresponding carbon positions.
Nevertheless, polar extract of the leaves of S. rotundifolium also showed a high polyphenol content but not as high as that in the bark. Gallic acid, catechin, caffeic acid, and rutin are examples of polyphenols identified in the leaves . Previously, prodelphinidins, gallic acid, and flavonols were also described in the leaves of S. adstringens and S. polyphyllum , as well as saponins and cumarins . The tannins in the leaves were identified as gallic acid, epicatechin-(4β→8)-catechin, and epicatechin 3-O-gallate . Moreover, galactomannans were extracted from the seeds of S. adstringens .
Lopes et al.  observed that S. adstringens and S. rotundifolium differ by the presence of epigallocatechin 3-O-(3,5-dimethyl)-gallate and epigallocatechin-3-O-(3-methoxy-4-hydroxy)benzoate and are chemically related as they both contain the 5-deoxyproanthocyanidins-B-ring trihydroxylated (prodelphinidins), whereas S. polyphyllum shows the presence of the dihydroxylated form (profisetenidins). However, S. adstringens is the most studied “barbatimão” species and, so, S. polyphyllum and S. rotundifolium tannin composition descriptions may be incomplete.
Therefore, the chemical composition of the Stryphnodendron spp. includes a wide variety of tannins particularly of the condensed class with diverse forms, and it was possible to identify the proanthocyanidins as monomers, dimers, and polymers (Table 2). Those molecules have interesting properties and interactions with protein among other organic compounds, which is the basis of the biological activities of the bark .
6.2. Extraction and analysis of tannin metabolites of Stryphnodendron species Because of their polarity, the most commonly described solvent for extracting tannins from the bark of Stryphnodendron spp is the polar system consisting of acetone:water at a 7:3 (v/v) ratio [42, 54, 62, 66-70]. Subsequently, the extracts were dried, partitioned with water and ethyl acetate, and then the ethyl acetate fraction was subjected to column chromatography (CC) using Sephadex LH-20. The subfractions generated by CC were chromatographically analyzed using multi-layer coil counter current chromatography (MLCCC), high-pressure liquid chromatography (HPLC), or CC until purification and identification of the tannins.
Thus, gallic acid was obtained using HPLC. The condensed tannin 4'-O-methylgallocatechin was separated using HPLC from an MLCCC (ethyl acetate:n-propanol:water, 140:8:80, v/v) subfraction using an isocratic methanol:acetonitrile:water (15:5:80, v/v/v) system, and identified using electron ionization mass spectrometry (EI-MS) and the acetylated form was identified using proton nuclear magnetic resonance (1HNMR) . Gallocatechin and epigallocatechin were separated after a second MLCCC, by HPLC (same isocratic system). Epigallocatechin 3-O-gallate in the same subfraction from the second MLCCC was purified using CC . Purification of epigallocatechin 3-O-(3,5-dimethyl)gallate and epigallocatechin 3-O-(3-methoxy-4-hydroxy)benzoate required subjecting the subfraction obtained using the multi-layer method to MLCCC, generating a subfraction that was submitted to CC, followed by HPLC (using the same isocratic methanol:acetonitrile:water system), from which the compounds in the subfraction were acetylated, semi-purified using thin layer chromatography (TLC). Furthermore, a new HPLC procedure with a normal phase column (hexane:ethyl acetate, 55:45, v/v) provided the best separation of the compounds . Epigallocatechin-(4β→8)-gallocatechin and epigallocatechin-(4β→8)-epigallocatechin were purified from the subfractions where they were concentrated using MLCCC and isocratic HPLC. However, epigallocatechin-(4β→8)-epigallocatechin3-O-gallate was separated using MLCCC and HPLC with an isocratic system of methanol:water (3:1, v/v) . In comparison, purification of epigallocatechin-(4β→8)-epigallocatechin 3-O-(4-hydroxy)benzoate was slightly different, using HPLC with an isocratic system of methanol:water at 21:79 (v/v) ratio . In contrast, subfractions containing epigallocatechin 3-O-gallate-(4β→8)-epigallocatechin 3-O-gallate and epigallocatechin-(4β→6)-epigallocatechin were subjected to MLCCC and HPLC using a gradient system of methanol:water at 25–30%. Gallocatechin-(4β→8)-epigallocatechin 3-O-gallate and gallocatechin-(4α→8)-epigallocatechin 3-O-(4-hydroxy)benzoate were purified from the subfrations following the same strategy, except that the gradient used for the HPLC were 24–27% and 28–30%, respectively . Compounds were identified using EI-MS and 1H NMR .
Prorobinetinidins were further purified from the ethyl acetate fraction by first subjecting it to CC using Sephadex LH-20, and then main subfractions were subjected to MLCCC (ethyl acetate:n-propanol:water, 35:2:2, v/v) and HPLC (reverse-phase C18). Therefore, robinetinidol-(4β→8)-epigallocatechin and robinetinidol-(4α→8)-gallocatechin were purified using HPLC and a methanol:acetonitrile:water (3:1:16, v/v/v) system, while robinetinidol-(4α→8)-epigallocatechin was purified using a methanol:water (21:79, v/v) system. The other prorobinetinidins were purified using a gradient system of methanol:water at 28–30% (robinetinidol-[4β→6(8)]-gallocatechin and robinetinidol-(4β→8)-epigallocatechin-3-O-gallate) and 25–30% (robinetinidol-(4α→6)-gallocatechin, robinetinidol-(4α→6)-epigallocatechin, and robinetinidol-(4α→8)-epigallocatechin-3-O-gallate) . Subjecting different subfractions to MLCCC allowed the purification of 4’-O-methylrobinetinidol-(4α→8)-4’-O-methylepigallocatehin, 4’-O-methylgallocatechin, 4’-O-methylrobinetinidol-(4α→8)-4’-O-methylgallocatehin, gallocatechin, and epigallocatechin . Purification of the dimer 4’-O-methylgallocatechin-(4α→8)-4’-O-methylgallocatechin followed the same purification path: after extraction with acetone:water, the subfraction was partition using CC with ethyl acetate and water, and one of the subfractions from the MLCCC process produced the molecule that was identified by chemical shifts using 1H NMR and 13C NMR [54, 70].
Metabolites from S. polyphyllum were similarly separated and analyzed, and after the extraction, partitioning, and CC, gallic acid was identified and the subfractions were subjected to other chromatographic purification procedures. Subsequently, 4’-O-methylrobinetinidol-(4β→6)-4’-O-methylgallocatechin was obtained from a second CC process using Sephadex LH-20 and gallocatechin, epigallocatechin, and 4’-O-methylgallocatechin were obtained from different subfractions following the MLCCC. A reverse and a normal-phase HPLC were used to obtain epigallocatechin-(4β→8)-gallocatechin. After the MLCCC procedure, the subfractions were subjected to a normal-phase column HPLC process, which produced profisetenidins fisetinidol-(4β→8)-gallocatechin and fisetinidol-(4β→8)-gallocatechin .
The water fraction from the partitioning step was subjected to CC using a Sephadex LH-20 column, which generated a subfraction rich in a polymer, identified using electrospray ionization (ESI)-MS and 13C NMR, as a 2114 Da polymer with six monomers of flavan-3-ols and one galoil group consisting of prodelphinidin and prorobinetinidin units .
After identifying the purified compounds from the barks of Stryphnodendron species, the presence of the compounds was evaluated in studies using straight strategies. HPLC with reverse-phase column was used to analyze the organic phase of the fraction obtain from the liquid-liquid partitioning of ethanolic extracts from the barks of S. adstringens and S. rotundifolium using ethyl acetate:butanol:i-propanol:water (3.5:0.5:1.0:4.5, v/v/v/v), which was characterized by the presence of gallic acid, gallocatechin, epigallocatechin, catechin, and epigallocatechin 3-O-gallate [56,65]. The ethanolic extract of S. adstringens bark, which was partitioned with ethanol:-propanol:-butanol (42:12:6, v/v/v) and analyzed using ultraperformance liquid chromatography (UPLC) coupled to ESI-MS, was chemically characterized by the presence of gallic acid, gallocatechin, epigallocatechin, methylepigallocatechin 3-O-gallate, and the dimers of epigallocatechin, epigallocatechin 3-O-gallate, methylepigallocatechin3-O-gallate and epigallocatechin 3-O-gallate, epigallocatechin and epigallocatechin 3-O-gallate, 4’-O-methylgallocatechin and, methylepigallocatechin and epigallocatechin . Analysis of the extract of S. adstringens obtained with acetone:water showed the characteristic chemical composition when subjected to MS and tandem MS (MS/MS): gallic acid, robinetinidol, epigallocatechin, 4′-O-methylepigallocatechin, epigallocatechin 3-O-gallate, epigallocatechin-O-methylgallate, robinetinidol-epigallocatechin, robinetinidol-4′-O-methylepigallocatechin, epigallocatechin-epigallocatechin, 4′-O-methylepigallocatechin-4′-O-methylepigallocatechin, and epigallocatechin-epigallocatechin 3-O-gallate.
As summarized here, the purification of condensed tannins from Stryphnodendron was a particularly laborious task, involving several fractionations using suitable chromatographic methods. However, the chemical fingerprint of the bark can currently be obtained in a straightforward manner. To confirm the identity of “barbatimão” species, the Brazilian Pharmacopeia refers to the extract preparation with the acetone:water mixture and the characteristic presence of gallic acid, epigallocatechin, and 4’-O-methylgallocatechin .
7. Biological activities correlated
According to previous reports, the efficacy of the main ethnopharmacological uses of the bark of “barbatimão” were confirmed for the treatment of wounds, gastric wounds, and infectious and inflammatory disorders. The activity of the bark extract on other disorders such as cancer, pain, diabetes, blood pressure, and as a diuretic were evaluated but the results obtained were insufficient to scientifically prove its usefulness for these indications. Other activities not correlated to the ethnopharmacological applications such as anti-protozoal and antiviral (not against influenza), were also analyzed with promising preliminary results. For those activities, improved, established methods identified the compounds present in the plant sample tested, which could be associated with the confirmed activity (Table 3), paying more attention to the epigallocatechin 3-O-gallate and proanthocyanidin polymer of 2114 Da. A general description of the biological activities tested for in “barbatimão” from the Stryphnodendron genus is presented as follows.
Table 3.Ethnopharmacological uses scientifically studied and correlated compounds (identified by their numbers in Table 2).
Antioxidant activity has been extensively evaluated, but anticancer activity is not conclusive
1, 2, 6, 42 and 43
Antimicrobial – oral and genitourinary infections
Yes – against gram positive bacteria and Candida species
1 and 41
7.1. “Barbatimão” bark promotes wound healing
The most common ethnopharmacological use stated for “barbatimão” stem bark is wound healing. Studies on animals and humans using only the extract or a pharmaceutical formulation in which it was incorporated, confirmed this biological activity. One of the earliest studies reported the wound healing activity of S. adstringens bark decoction at 1% on incisions in mice , which was also reported in similar studies performed subsequently [75-77]. Then, ointments containing the “barbatimão” bark extract were prepared and the wound healing activity was analyzed in rats and in humans. An ointment containing 10% of the aqueous bark extract was used to treat cutaneous wounds on rats and showed complete epithelization after 14 days with better inflammation and neovascularization process recovery than that of the control group treated with a physiological solution . Subsequently, a 3% aqueous extract showed potential angiogenic activity, corroborating the previously observed neovascularization, which is an important step in the wound healing process [79, 80]. Another ointment with 3% of the extract also healed patients with cutaneous wounds of decubitus position [77, 81]. Based on those results, a topical preparation was developed with a composition of 1–6% total phenols from a dry extract of S. adstringens or S. polyphyllum and a patent was administered to the preparation . An ointment containing 1% of a lyophilized ethyl acetate fraction (obtained from an acetone:water extract) of the bark of S. adstringens was similarly tested against wounds in rats, and its topical application stimulated epithelization .
In addition to reepithelization, the production of collagen fibers was also observed in wounds of diabetic rats treated with a gel formulation with a 1% acetone:water extract , and similar results were obtained with a 10% glycolic extract . Further characterization of the acetone:water extract of the bark of S. adstringens indicated it contained almost 40% total polyphenol content and hydrolysable tannin gallic acid, prorobinetinidins robinetinidol, robinetinidol-epigallocatechin, robinetinidol-4′-O-methylepigallocatechin, prodelphinidin epigallocatechin, epigallocatechin 3-O-gallate, 4′-O-methyl-epigallocatechin, epigallocatechin-O-methylgallate, epigallocatechin-epigallocatechin, 4′-O-methyl-epigallocatechin-4′-O-methyl-epigallocatechin, and epigallocatechin-epigallocatechin 3-O-gallate . It is interesting to note that the reepithelialization and angiogenesis effects have been described for epigallocatechin 3-O-gallate, which is one of the characteristic tannin metabolite in Strpynhodendron acetone:water bark extract [85, 86].
Ointments containing 2.5% lyophilized acetonic extract or the ethyl acetate fraction from the barks of S. rotundifolium and S. polyphyllum were also tested on rat wounds and were shown to induce reepithelization. However, the epidermal growth was faster with the extract of S. polyphyllum and the ethyl acetate fraction of S. rotundifolium than with the other fractions. In that case, the first extract contained approximately 50% total phenolic content and 25% tannin content while the second fraction contained 89% and 36% phenolic and tannin contents, respectively . The authors suggested that the differences were likely attributable to the presence of a variety of prodelphinidins, prorobinetinidins, and profisetenidins in S. polyphyllum although higher phenolic and tannin contents were observed in S. rotundifolium, whereas only prodelphinidins were observed in this last species. Prodelphinidins, prorobinetinidins, and a polymer of these two condensed tannins were also described in the bark of S. adstringens, indicating the diversity of its tannin composition that significantly corroborated the wound healing activity reviewed here. Therefore, the diverse condensed tannin contents could be more useful for wound healing than the content of a few structures, as was also observed for S. adstringens.
Treatment of gastric ulcer wounds with the bark of “barbatimão” has been widely reported as well and this application has been demonstrated using animal models (Table 3). Lesions induced by acute stress or acidified ethanol were significantly reduced after treatment with aqueous and butanolic fractions (obtained from an acetonic bark extract) of S. adstringens . These observations indicate a concentration of specific polyphenols since the proanthocyanidins previously described were identified in the extract with wound healing activity. Another study showed the reduction of ulcer lesions induced by ethanol and hypothermic restraint-stress in rats pre-treated with an acetonic fraction (obtained from a methanolic extract) from S. adstringens . The fraction also decreased the gastric secretory volume and elevated pH, showing an antisecretory effect . However, both studies reported that the fraction did not have activity on indomethacin- or acetic acid-induced ulcers that could be caused by the indomethacin-induced inhibition of prostaglandin biosynthesis and not a potent antisecretory activity by the extract, as observed with cimetidine [88, 89]. Tannins interact with proteins and, therefore, it has been proposed that the complex forms a protective layer for the recovery of the stomach endothelium and inhibition of the H+/K+ ATPase by hydrolysable tannin has also been observed, which reduces acid secretion and supports the results of S. adstringens on gastric ulcer treatment [90-92]. Although impressive results have been reported, the scientific studies of the internal use of these extracts against gastric ulcer were performed in rat models. Furthermore, one study reported a toxic effect that was comprehensively characterized , and should be considered.
7.2. Anti-inflammatory activity of “barbatimão” and correlation to antinociception
Another claimed ethnopharmacological property of the barks of these species is anti-inflammation. The acetonic fraction of S. adstringens inhibited rat paw edema with reduction of the exudate volume and migration of leukocytes. In addition, inhibition of paw edema was observed in a model with induced arthritis and decreased vascular permeability mediated by intraperitoneal administration of acetic acid in mice . Antiedema results were similarly observed with a 1% solution of S. adstringens . The aqueous and organic (ethanol:isopropanol:butanol) fractions (obtained from an ethanolic bark extract) of S. adstringens reduced the accumulation of neutrophils in the joint cavity of rats with arthritis induced by lipopolysaccharide (LPS) but this effect was not mediated by a decrease in C-X-C motif chemokine ligand 1 (CXCL1), the major chemokine recruiter of neutrophils . The same study showed that tumor necrosis factor (TNF)-α production decreased in human monocytes THP-1 cells stimulated with LPS, which supports the anti-inflammatory claim for S. adstringens . The organic fraction reduced neutrophil accumulation considerably more than the dexamethasone control did. Phytochemical evaluation of the organic fraction identified the presence of gallic acid and 11 different monomers and dimers of prodelphinidins, including epigallocatechin 3-O-gallate . The attenuation of inflammatory process by some of those tannins, especially to epigallocatechin 3-O-gallate, was previously described [95-97], indicating a correlation between the anti-inflammatory activity of the stem bark of S. adstringens and the prodelphinidin constituents.
The use of “barbatimão” against pain could be related to its anti-inflammatory property. One study evaluated the aqueous and ethyl acetate fractions of the acetonic extract of S. adstringens against three pain models but an antinociceptive effect was only observed in the acetic acid- and formalin-induced writhing models . It is interesting to note that the extract and aqueous fraction reduced the number of writhing compared to the saline control in two models of pain induced by inflammatory process. Specifically, 1) acetic acid induces capillary permeability, liberating substances that cause pain in nerve ends  and 2) the late phase of the formalin test, which is mediated by an inflammatory process, is reported as a return of the nociception minutes after formalin injection, and is used to elucidate pain and analgesia mechanisms [100, 101]. The aqueous fraction contained concentrated levels of the proanthocyanidin polymer of 2114 Da, whereas the dimers where found in the ethyl acetate fraction, indicating that the peripheral antinociceptive effect of S. adstringens is partly due to the polymer of the condensed tannin . Interestingly, epigallocatechin 3-O-gallate was also associated with antinociception in bone cancer due to the attenuation of inflammation by the reduction of TNF-α expression .
The reactive oxygen species (ROS) scavenging activity of the bark extract of S. adstringens prepared with 50% and 70% ethanol, acetone:water (7:3, v/v), and chloroform was comparatively evaluated based on the reduction of the reagent 1,1-diphenyl-2-picrylhydrazyl (DPPH) to DPPHH. Polar extracts showed scavenging capacity as high (95%) as that of the controls rutin (97%), gallic acid (97%), and vitamin C (98%) at the same concentration. However, the chloroformic extract, which showed irrelevant levels of total phenolic content, exhibited <75% scavenging capacity. TLC plates stained with DPPH presented a spot representing reduction at same retention time as that of the tannin spots . The scavenging capacity of the acetonic extract, aqueous and ethyl acetate fractions, and CC subfractions of the stem bark of S. rotundifolium was similarly evaluated using TLC stained with DPPH and all samples reduced the free radicals . Subsequently, the DPPH scavenging potential of hydroalcoholic bark extracts and aqueous extracts of the barks and leaves of S. rotundifolium was measured and both bark extracts showed better activity than that of vitamin C, whereas the leaf extract showed comparable scavenging capacity to that of the control at higher concentrations . According to the authors, the bark extract contents of gallic acid, catechin, caffeic acid, and rutin were higher than those in the leaf extract, which explained the different antioxidant activity of the samples.
Comparison of the acetonic extract and ethyl acetate fraction of S. rotundifolium and S. polyphyllum revealed the greater scavenger capacity of the second species, similar to the results of the wound healing potential comparison . Therefore, the proanthocyanidin constituent of the barks of Stryphnodendron spp and the potent scavenging capacity of the extracts, justify their ethnopharmacological use as anticancer agent; however, scientific studies need to be performed to prove that application and establish the best extract, compound, and concentration for use.
Furthermore, an ethyl acetate fraction obtained from the acetone:water extract of the leaves of S. adstringens was evaluated for antioxidant and anticancer potential. It showed good antioxidant activity by reducing iron, inhibiting protein oxidation and scavenging DPPH at same 50% radical inhibition concentration as vitamin C. The fraction did not show cytotoxicity for non-cancerous rat primary bone marrow but was cytotoxic to MCF-7 and MDA-MB-435 human breast cancer cell lines, and altered their morphology in addition to inducing DNA cleavage, apoptosis, and autophagy . A tannin, epigallocatechin 3-O-gallate, present in the leaf fraction has already been designated as an anticarcinogenic compound . The oxidation of epigallocatechin 3-O-gallate by O2- species was observed in the galloyl group, thereby modulating ROS production, which in combination with the inhibition of nuclear factor-κB, activation of mitogen-activated kinases, and inhibition of DNA methyltransferases, suggests prodelphinidin is a strong anticancer agent [104, 105].
S. adstringens saline extract also reduced the technetium-99m (used in nuclear medicine) labeling of red blood cells probably because of the redox or chelating properties of tannins .
7.4. Antimicrobial activity corroborates oral and genitourinary use
It is notable that the ethnopharmacological use of Stryphnodendron spp for infection treatment has been scientifically demonstrated mostly against gram-positive bacteria and pathogenic yeasts, as described in Table 4.