Phytochemical analysis with the antioxidant and aldose reductase inhibitory capacities of Tephrosia humilis aerial parts’ extracts

Abstract The aerial parts of Tephrosia humilis were tested about their antioxidant potential, their ability to inhibit the aldose/aldehyde reductase enzymes and their phenolic content. The plant material was exhaustively extracted with petroleum ether, dichloromethane and methanol, consecutively. The concentrated methanol extract was re-extracted, successively, with diethyl ether, ethyl acetate and n-butanol. All extracts showed significant antioxidant capacity, but the most effective was the ethyl acetate extract. As about the aldose reductase inhibition, all fractions, except the aqueous, were strong inhibitors of the enzyme, with the n-butanolic and ethyl acetate fractions to inhibit the enzyme above 75%. These findings provide support to the ethnopharmacological usage of the plant as antioxidant and validate its potential to act against the long-term diabetic complications. The phytochemical analysis showed the presence of 1,4-dihydroxy-3,4-(epoxyethano)-5-cyclohexene(1), cleroindicin E(2), lupeol(3), methyl p-coumarate(4), methyl 4-hydroxybenzoate(5), prunin(6), 5,7,2ʹ,5ʹ-tetrahydroxyflavanone 7-rutinoside(7), protocatechuic acid(8), luteolin 7-glucoside(9), apigenin(10), naringin(11), rhoifolin(12) and luteolin 7-glucuronate(13). Graphical Abstract


Introduction
Tephrosia spp. (Fabaceae) genus contains about 400 species of shrubs indigenous in tropical and sub-tropical regions. Species of this genus are used in many traditional remedies as antimicrobial, diuretic and laxative, useful in treating cough and tightness of the chest, disorders of the liver, spleen and kidneys and recommended as a blood purifier. Most Tephrosia species also possess insecticidal, fish-poisoning, antiulcer and hepatoprotective properties, whilst some species have found to exhibit cytotoxic capacity and anticancer properties (Bashir et al. 1992;Saleem et al. 2001). Specifically, T. humilis ( Figure S1) is used as a decoction with Indigofera hirsuta by diabetic natives in central Africa. The traditional recipe states that they have to drink 200 mL of this decoction, three times per day for 7 days in a row, which is made by 34g Yakoro (T. humilis) and 65g Mabo (I. hirsuta) in 2 L of water and then boiled for 15-20 min.
With all these in view, we have been encouraged to investigate the phytochemical composition of T. humilis as a contribution to a wider study of the phytochemistry of the African flora used in traditional medicine. The aim of this study was the detailed evaluation of their ability to inhibit the aldose reductase enzyme (AR, ALR2, E.C. 1.1.1.21), indicating a potential of this plant to act against the long-term diabetic complications. ALR2 is the first enzyme of the polyol metabolic pathway and was found to be implicated in the aetiology of the longterm diabetic complications (Nicolaou et al. 2004). Recently, Ramana and Srivastava (2010) reviewed the implication of ALR2 in inflammatory pathologies, indicating the multiple roles of ALR2 inhibitors. Aldose reductase inhibitors were found to attenuate reactive oxygen species production both in vitro and in vivo (Chatzopoulou et al. 2013). The correlation between LOX inhibition and ALR inhibition is implied by the fact that most of the LOX inhibitors are antioxidants or free radical scavengers, taking also into account the role of lipoxygenase in inflammatory pathologies (Muller 1994). Both enzymes are implicated in inflammatory pathologies. Thus, compounds inhibiting them offer as antiinflammatories. Therefore, we proceeded into the evaluation of the antioxidant capacity of several plant extracts of T. humilis (DPPH ⋅ assay and Co(II)/EDTA-induced luminol chemiluminescence test, LOX test), despite the fact that there is not any reference which correlates the antioxidant capacity of the plant with its use in traditional medicine.
This research work is the first phytochemical study of the phenolic content of the aerial parts of T. humilis as well as the first scientific establishment of the good in vitro antioxidant capacity of T. humilis extracts and its strong inhibitory activity against ALR2 enzyme.
Another remarkable feature is that all three flavanones that have been identified as pure compounds, were isolated as (S-) and (R-) isomers in mixtures with the ratio 1 : 1. In plants, flavanones are generally present as levorotatory (2S)-isomers because the enzymatic reaction which catalyses the conversion of chalcones to flavanones is highly stereospecific with an apparent 100.000 : 1 preference for the synthesis of the (S)-isomer over the (R)-isomer. However, the (2S)-isoform tends to convert to the (2R)-form, reaching an approximate epimeric ratio of about 3:2 at fruit/plant maturity. This is attributed to the fact that plants metabolites are produced in a stereospecific way because of the involvement of enzymes (Maltese et al. 2009). Moreover, the relative percentage of (2S)-naringin to (2R)-naringin undergoes a marked change with increasing maturity, as it was shown in Citrus grandis. In very small fruits, the naringin is almost all (2S) and remains very abundant during the very early part of the season (1-5 days of collection). As the fruit matures, more (2R)-diastereomer appears until the diastereomeric percentage at ripeness is 54% in (2S) (Caccamese & Chillemi 2010). In our experiment, the relative percentage of (2S) to (2R) flavanones is calculated from the integrated areas of the peaks of the anomeric protons ( Figure S4).

Radical scavenging activity (DPPH ⋅ , CL, LOX tests), ALR2/ALR1 inhibitory activity and selection
All antioxidant results are summarised in Tables S1 and S2. Amongst the plethora of methods used for the evaluation of antioxidant activity, the DPPH ⋅ test is very useful in the micromolar range demanding minutes to hours for both lipophilic and hydrophilic samples. In the presence of an antioxidant, which can donate an electron to DPPH, the purple colour being typical of the free DPPH radical decays and the change can be measured spectrophotometrically (515 nm). This interaction indicates its radical scavenging ability in an iron-free system. In cases where the structure of the electron donor is not known (e.g. plant extract), this method can afford data on the reduction potential of the sample and hence can be helpful in comparing the reduction potential of unknown materials. Flavonoids with at least one OH group at ring B present antioxidant activity. This behaviour is associated with the combination of the C2-C3 double bond and the 5-OH. All the extracts interacted with the stable free radical DPPH. Radical scavenging activity expressed as EC 50 (DPPH ⋅ test) ranged from 0.434 to 4.486 mg dry extract/mg DPPH ⋅ . The ethyl acetate fractions possessed the greatest antiradical activity, followed by the butanol, ethyl acetate residue, the initial methanolic extract and the diethyl ether fraction. The weakest antioxidant was the aquatic fraction. Two standards, known for their good antioxidant activity, trolox (weaker) and quercetin (stronger), were used. In comparison with the standards used, only the ethyl acetate extract is the one which approaches approximately half the value of the antiradical potential of Trolox.
A wide range of antioxidant capacity is also observed from the CL results. IC 50 ranges from 0.073 to 3.663 μg dry extract/mL. According to the CL test, all fractions, except for the ethyl acetate fraction, showed low hydroxyl radical scavenging ability. As with the DPPH ⋅ results, the ethyl acetate fraction was the strongest antioxidant amongst extracts and it was almost 4.5 times stronger than quercetin. All other fractions, except for butanolic and aqueous, were more potent antioxidants than Trolox.
The results of both model systems (DPPH ⋅ test and Co(II)/EDTA-induced luminol chemiluminescence method) showed that ethyl acetate fraction possessed the most significant free radical and hydroxyl radical scavenging activity which was even greater when the capacity was evaluated with the Chemiluminescence test. The low antioxidant property of the aquatic extract could be attributed to the low concentration of active chemical compounds. It is also interesting that there is a low correlation coefficient, obtained from the two methods, between the results of each extract separately. This reveals a different trend in the free radical and hydroxyl radical scavenging activity of the extracts and may indicate a specific mechanism of antiradical activity, probably due to the physicochemical and structural characteristics of the components contained.
As for the inhibition of the lipoxygenase, the fractions of the ethyl acetate and its residue proved to be the strongest inhibitors of the enzyme. The results are summarised in Table  S2. The extracts (100 μL) at a final volume of 1 mL showed inhibitory activity of 41-82%. Comparing with the standards used [NDGA (nordihydroguaiaretic acid): 83% and coumarin: 36%], it occurs that all extracts exceed the percentage of coumarin's LOX inhibition, and the ethyl acetate extract, and its residue, appears to have almost the same inhibitory activity against LOX. These promising results can be attributed to the ability of some compounds of the extracts to act as ligands for the Fe in the active site of LOX. Chelating agents are effective as secondary antioxidants because they reduce the redox potential, thereby stabilising the oxidised form of the metal ion.
The evaluation of the ALR2 inhibitory activity was conducted on partially purified rat lenses ALR2. Human ALR2 and rat ALR2 exhibit 85% sequence homology, whilst the catalytic active sites of both the enzymes are considered identical. ALR2 inhibitory activity is expressed as % inhibition. Aldehyde reductase (ALR1) is a cytosolic enzyme, member of the aldoketoredouctase superfamily, such as ALR2. It also catalyses several physiological functions, amongst them, detoxification of toxic aldehydes. Furthermore, the two enzymes share a high level of sequence (65%) and structural homology, as well as physical and chemical properties. As a result, there is an increased tendency of many substrates or ligands to bind with both ALR1 and ALR2. The inhibition of both the enzymes is thought to lead to toxic phenomena, due to the resulting insufficient detoxification capacity of the living cell (Chatzopoulou et al. 2011). In the present study, ALR1 inhibitory activity was evaluated on partially purified ALR1 obtained from rat's kidney. It is expressed as % inhibition accompanied with the selectivity index (SI = ALR2 inh./ALR1 inh.). All samples were dissolved in DMSO. The final concentration of DMSO in all incubations was 0.3%, and the results are mean values from 3 measurements with SD < 10%. Sorbinil was used as a positive control and showed 45% inhibition at the same concentration. As about the ALR2 inhibition, all fractions, except the aqueous, were strong inhibitors of the enzyme, with the n-butanolic and the ethyl acetate fractions to inhibit the enzyme above 75%, results probably attributed to their flavonoids content. The estimated values of ALR1 inhibition ranged from 41% to 76% for all the methanolic fractions and 12% for the dichloromethane fraction. From the obtained results (Table S3), we can see that all fractions of the methanolic extract present a relatively medium selectivity, except for the dichloromethane extract which shows low ALR2 inhibitory activity. So, considering the fact that the dichloromethane extract shows low ALR2 inhibitory capacity, it is assumed that the ethyl acetate extract proved to attribute the best aldose reductase inhibitory activity, since it is amongst the best extracts with the maximum ALR2 inhibition and with the highest selection.

Conclusions
In the perspective of chemotaxonomy, this is the first report of the occurrence of cycloexylethanoids in the Fabaceae family. Despite the ample literature in the variety of uses of the genus Tephrosia in traditional medicine, the pharmacological investigation of the distinct species is comparatively little. This research work constitutes the first phytochemical study of the phenolic content of the aerial parts of T. humilis as well as the first scientific establishment of the good in vitro antioxidant capacity of T. humilis extracts and its strong inhibitory activity against ALR2 enzyme. The isolated compounds will be tested with their pharmacological activities in a future experiment.
This study has clearly shown that T. humilis is an effective antioxidant in different in vitro antioxidant assay when it is compared with standard antioxidant compounds (quercetin, trolox). All this information may be useful for the promotion of use of T. humilis as a natural antioxidant in food and medicinal products and can be an answer to the continual demand for new natural antioxidants. Moreover, the results also revealed a promising potential of T. humilis as a possible anti-inflammatory agent by inhibiting soybean lipoxygenase. To our knowledge, this is the first report on the inhibitory capacity of the genus Tephrosia on ALR2 activity in vitro. The data obtained add new information and highlight the plant's significance for its use in the diet of patients suffering from diabetes mellitus as a potential protective agent against the long-term diabetic complications, especially at early stages. As a result, the use of Tephrosia may be considered as a natural ALR2 inhibitor in food and medicinal products with potent pharmacological activity and therapeutic role against the long-term diabetic complications, justifying the traditional use of the plant as food beneficial to health.