Protective Effects of Extracts, Isolated Compounds from <i>Desmodium uncinatum</i> and Semi-Synthetic Isovitexin Derivatives against Lipid Peroxidation of Hepatocyte’s Membranes

Lipid peroxidation plays a pivotal role in the pathogenicity and maintenance of hepatitis. Thus, substances protecting hepatocyte membranes from lipid peroxidation are of great importance in the management of hepatotoxicity and hepatitis. The present work deals with the in vitro hepatoprotective activity of the methanol extract of Desmodium uncinatum, its sub-fractions, the major isolated compounds and some of their semi-synthetic derivatives in order to study structure activity relationships. Using hydrogen peroxide (H2O2)-induced lipid peroxidation of hepatocyte membranes as a model, the hepatoprotective-guided phytochemical survey of the methanol extract of aerial parts of D. uncinatum was carried out by successive column chromatography. One of the most active compounds (Isovitexin) was chemically transformed to yield new semi-synthetic. The identification of isolated and semi-synthetic compounds was performed using NMR techniques, mass spectrometry and by comparison of their data with those reported in the literature. The n-butanol fraction was the most effective (IC50: 22.9 μg/mL) compared to the crude methanol extract (IC50: 43.6 μg/mL) and other fractions. The n-butanol sub-fractions FA (containing non-phenolic compounds) and FB (mainly containing phenolic compounds) exhibited respective IC50 of 14.36 and 128.2 μg/ml. Purification of FA yielded 3-O-β-D-glucopyranosyl-β-sitosterol (1), 3-O-β-D- 2-acetyl-amino-2-deoxyglucopyranoxyloleanoic acid (2), (2S, 3S, 4R, 7R, 8Z)-1-O-β-D-glucopyranosyl-2-[(R)-2'-hydroxyarachidoylamino]-docosan-8-ene-3,4,7-triol (4), spiraeamide (5), mannitol (6), while FB afforded essentially three C-glycosylflavonoids namely isovitexin (7), vitexin (8) and vicenin-3 (9). Chemical transformations (methylation, allylation and prenylation) of isovitexin afforded five new semi-synthetic derivatives: 4',5,7-O- trimethyli-sovitexin (10), 4'-O-allylisovitexin (11), 4',7-O-diallylisovitexin (12), 4'-O-prenylisovitexin (13) and 8-C-prenyl-4',7-O-diprenylisovitexin (14). The screening of these derivatives revealed that allylation did not significantly affect the hepatoprotective activity while methylation, prenylation, number and position of sugar moieties on the A ring of flavonoids significantly reduced it. Results demonstrated that the n-butanol fraction obtained from the methanol extract of Desmdium uncinatum may possess hepatoprotective activity due to its content in C-glycosylflavonoids and cerebrosides. Hydroxyl groups in C-glycosylflavonoids are important for their lipid peroxidation inhibitory activity.


Introduction
The genus Desmodium is a large member of the Papilionaceae (Fabaceae) family. It contains about 350 species mainly distributed in tropical and subtropical regions of the world [1]. Most of Desmodium species are widely used in African traditional medicine and well explored in the treatment of neurological imbalances by the traditional Indian medicinal system [2]. Many of them are known for their hepatoprotective effects [3]. The positive effect of Desmodium adscendens against hepatic infections has been verified in vivo [4] and assigned to its major secondary metabolites including C-glycosylflavonoids identified as vitexin, isovitexin and saponins, mainly soyasaponins [5]. From these results, a number of herbal preparations based on D. adscendens, reputed to have hepatoprotective activity are available on the market.
Currently, lipid peroxidation (LPO) is considered as the main molecular mechanism involved in the oxidative damage of cell structures and in the toxicity process that leads to cell death. In fact, lipid peroxidation is the cornerstone mechanism of the hepatotoxicity exerted by many liver-damaging agents [6] [7] [8]. The major reactive aldehyde resulting from the peroxidation of biological membranes is malondialdehyde (MDA) [9] used as an indicator of tissue damage by a series of chain reactions [10]. Concordantly, in most cases, plant secondary metabolites with antioxidant properties have been found responsible for medicine containing a mixture of flavonolignans. The primary mechanism of its hepatoprotective effect is the inhibition of cell membrane lipid peroxidation [11]. Moreover, many plant extracts with lipid peroxidation inhibitory effects exhibited hepatoprotective activities [12] [13]. Although there are several methods for measuring the ability of plant extracts to show their hepatoprotective effect by inhibiting lipid peroxidation, the best method remains the monitoring of malondialdehyde, the most studied degradation product of polyunsaturated fatty acids peroxidation [14].
D. uncinatum is one of the Desmodium species commonly found in the western highlands of Cameroon. It is a large perennial legume with stems that may grow several meters long and trail over surrounding vegetation. Its cylindrical or angular stems are covered with short, hooked hairs that stick to hair or clothing [15]. Previous phytochemical investigations of this plant revealed the presence of flavonoids as uncinanone A, B, C, D and E, uncinacarpan and triterpenoids [16] [17] [18]. Based on the potent hepatoprotective effects of plants from the same genus, we hypothesized that the entire aerial part of D. uncinatum may possess bioactive compounds with hepatoprotective activities resulting from their ability to inhibit lipid peroxidation.
The present study was therefore designed to evaluate the hepatoprotective activitiy of the crude methanolic extract of the aerial part of D. uncinatum and to isolate its bioactive secondary metabolites based on the bioactivity-guided fractionation. Moreover some semi-synthetic derivatives of one of the major active isolated compound (isovitexin) were prepared, tested and the structure activity relationship (SAR) discussed.

Plant Material
The aerial part of D. uncinatum was collected in Dschang, Menoua Division,

Extraction and Bioactivity-Guided Fractionation of Aerial Part of D. uncinatum
Fresh material was cut into small pieces and dried at room temperature for 2 weeks. The powder (4 kg) obtained after grinding was extracted three times (each time for 24 h) with MeOH (15 L) at room temperature before filtration.
The combined filtrates were concentrated under reduced pressure to yield the crude MeOH extract (168 g). Part of this crude extract (162 g) was suspended in water (400 mL) and successively extracted with ethyl acetate and n-butanol. The resulting soluble fractions were concentrated to dryness under reduced pressure to give the ethyl acetate fraction (78 g), n-butanol fraction (13 g) and the aqueous residual fraction. The crude extract and its three fractions were kept in the refrigerator at 4˚C until use. After assessing the hepatoprotective activity of the crude extract and its fractions, the active fractions were further submitted to column chromatography separation in order to isolate the possible active compounds (Scheme 1) while the less active fractions were left aside.
The EtOAc fraction was then purified as previously described [18] to yield mainly compounds 1 (23 mg), 2 (12 mg) and 3 (31 mg). Part of the n-butanol fraction (11.5 g) was fractionated over silica gel column chromatography, using a gradient of MeOH in EtOAc ranging from (1:9) to (1:1) to yield five sub-fractions (G1-G5). These sub-fractions were obtained after recombination of fractions of 300 mL based on their TLC profiles. They were separately subjected to Sephadex LH-20 column chromatography using MeOH as eluent to separate non phenolic compounds (fraction A) from phenolics (fraction B). The overall recombination therefore leads to two main fractions, FA (5.5 g) and FB  [19]. Afterwards, distilled water was added in order to cool the medium. The cool mixture was extracted with ethyl acetate and the obtained EtOAc phase was washed with water, dried over anhydrous Na 2 SO 4 and the solvent was evaporated in vacuo. The obtained mixture was chromatographed over silica gel using as eluent n-hexane/EtOAc (90:10) to mainly afford compound 10 (23.7 mg, 21.8%).  [20] and Dong et al. [21]. Using the same treatment procedure as earlier described for compound 10, the reaction mixture obtained was purified using silica gel CC eluted with isocratic solvent system of n-hexane-EtOAc (98:2) that afforded compounds 11 (8.4 mg, 6.4%) and 12 (6.7 mg, 5.15%).

Biological Materials
The biological materials used for the pharmacological tests were the livers obtained from male Wistar rats, aged between two and three months and weighing 150 to 200 g. They were reared in the animal house of the Laboratory of Animal Physiology and Phytopharmacology at the University of Dschang. Animals were maintained in accordance with the internationally accepted standard ethical guidelines for laboratory animal use and care as described in the European Community guidelines (EEC Directive of 1986; 86/609/EEC). They were fed on standard rat food with water ad libitum.

In Vitro Assessment of D. uncinatum Extracts and Compounds on Lipid Peroxidation of Hepatocytes's Membranes
Rats were sacrificed by cervical dislocation. The entire liver was rapidly collected and homogenized at 10% in an icy phosphate buffer solution (20 mM; pH: 7.4). The homogenates obtained were centrifuged at 3000 rpm at 4˚C for 10 minutes and the supernatant was discarded. The pellet (200 µL) made of hepatocyte's membranes were mixed with 200 µL of phosphate buffer (control tubes) or 200 µL of the testing substances (methanol extract, fractions, isolated compounds, semi-synthetic derivatives or silymarin) at the concentrations of 1, 3, 10, 30, 100 or 300 µg/mL. When the testing substance was not water-soluble, the adequate control made of vehicle was conducted. After 10 minutes of incubation at 37˚C, phosphate buffer (200 µL, neutral control) or H 2 O 2 (200 µL, 120 mM, for negative control and testing substances) was added and the mixture incubated for additional 50 minutes. Thereafter, the reaction milieu was thoroughly crushed using an electric tissue grinder. The tubes were then homogenized with Tris buffer (100 µL) and centrifuged at 10,000 rpm for 10 minutes at 4˚C. To a portion of the supernatant (100 µL), was added a solution of trichloroacetic acid (500 µL, 5%). The mixture was incubated at ambient temperature for 15 minutes and centrifuged at 6000 rpm for 10 minutes at 15˚C. Then, 400 µL of a solution of thiobarbituric acid (1% in 10% of orthophosphoric acid) was added to a portion of the supernatant (400 µL). The mixture was heated at 100˚C for 10 minutes. After cooling, optic densities were spectrophotometrically read at 532 nm to assess the malondialdehyde (MDA) level, indicating of lipid peroxidation. The percentage of inhibition of lipid peroxidation was calculated by the following formular:

Statistical Analysis
Statistical analysis of the data was performed using Graph Pad prism version 5.01. Data were expressed as mean ± standard error of the mean. The IC 50 of each extract was calculated from the non-linear regression curve. To classify tested substances based on the activity, their efficiency index (EI) was calculated with the following formula: EI = E max /IC 50 , where E max is the average of the maximal activity.

In Vitro Effects of D. uncinatum Crude Extract and Its Fractions on Lipid Peroxidation of Hepatocytes's Membranes
The crude methanol extract of the aerial part of D. uncinatum exhibited a concentration dependent inhibition of lipid peroxidation with an IC 50 of 43.6 µg/mL that was lower than that of silymarin (112.2 µg/mL). Although silymarin showed a highest Emax (88.2%), its EI (0.8) was lower than that of the methanol extract (IC 50 : 43.6 µg/mL, EI: 1.3) (Figure 1).
Fractionation of this crude extract led to three main fractions including the ethyl acetate, n-butanol and residual aqueous fractions which were evaluated. The n-butanol (IC 50 : 22.9 µg/mL, EI: 3.4) and EtOAc (IC 50 : 33.8 µg/mL, EI: 1.9) fractions revealed better activity as compared to the crude extract while the residual aqueous fraction was almost inactive (IC 50 : 370.6 µg/mL, EI: 0.043) ( Figure   1). The residual aqueous extract was then kept aside in the further studies.

Characterisation and Effects of Compounds Isolated from the n-Butanol Fraction
The n-butanol soluble fraction was separated over a column chromatography into two main sub-fractions FA and FB, which were respectively constituted of non-phenolic and phenolic compounds. These two sub-fractions were also evaluated for their hepatoprotective effect and thereafter subjected to repeated silica gel and sephadex LH-20 column chromatography to afford eight known compounds (1, 2, 4-9). These compounds were respectively identified as: (1)  The least active was mannitol (6). However, the effects of compounds 4, 5 and 6 at concentrations above 10 µg/mL were higher than that of FA ( Figure 5).
All the isolated compounds from the phenolic sub-fraction (FB) showed concentration-dependent hepatoprotective activity. Isovitexin (7) and vitexin (8) were more efficient than the sub-fraction B from which they were isolated with respective IC 50 of 78.0 and 97.0 µg/mL and EI of 0.8 and 0.7. Compound 9 has a lower inhibitory activity with IC 50 of 98.5 µg/mL and EI of 0.2 ( Figure 6). It was established from these results that the most active and major compound was isovitexin. Thus it was therefore submitted to further chemical transformations in order to study its structure-activity relationship.

Characterisation of Semi-Synthetic Derivatives of Isovitexin (7)
The See Table 1 and Table 2.  Table 1 and Table 2.     Table 1 and Table 2.  Table  1 and Table 2.  Table 1 and Table 2.

Discussion
Oxidative stress, mainly lipid peroxidation (LPO) has been recognised to be involved in the etiology of several liver diseases [29] including viral and non-viral hepatitis [30]. As such, targeting LPO can be a valuable way of managing liver   [31]. In a biological system, hydrogen peroxide (H 2 O 2 ), a reactive oxygen species generated during oxidative stress, is known to cause damage to proteins, nucleic acids, and cell membranes [32]. It is the most effective specie for cellular injury [33], a well-known oxidant often used as a model compound to induce acute oxidative stress and subsequent lipid peroxidation (LPO) in vitro and in vivo. In the present study, a phytochemical survey of the aerial part of D. uncinatum was con- The cerebrosides enrich sub-fraction (FA) and C-glycosylflavonoids enriched sub-fraction (FB) obtained from the n-butanol fraction were less effective than the n-butanol fraction itself. This suggests that the hepatoprotective effect of the n-BuOH extract might be due to a synergistic action between polar phenolic and non-phenolic compounds present in the whole fraction. Sub-fraction FA was more potent than FB and these findings are somehow surprising as flavonoids are known as reputed antioxidants. The great activity of the cerebrosides (4,5) which are the main constituents of sub-fraction FA could be attributed to the presence of the alkene functions present on their side chains. Alkene groups can  (7) which had the highest LPO inhibitory and subsequent hepatoprotective activity differed from the other flavonoids (8,9) by the position of the glucosyl moiety. Thus, the C-glycosylation of flavonoids at C-6 position in the A-ring tends to increase the LPO inhibitory activity than the glycosylation at C-8 position in the same ring. The C-glycosylation at both sites C-6 and C-8 as in compound 9 significantly reduced the activity. These results might be due to steric hindrance imposed by the presence of a sugar moiety at C-8 which can inhibit the proton donation through the hydroxyl group at C-7. In order to verify this hypothesis, five semi-synthetic derivatives of isovitexin (7) were prepared and their activities also evaluated. The combination of the structures and the activities seems to support our proposal.
All the semi-synthetic derivatives of isovitexin (7) namely 10, 11, 12, 13 and 14 inhibited lipid peroxidation of hepatocyte's membranes more or less in a concentration-dependent manner. It is worth noticing that all semi-synthetic derivatives of isovitexin were less active than isovitexin, thus suggesting that substitution of hydrogens in hydroxyl groups at C-4', C-7 and C-5 by alkyl groups significantly reduced the hepatoprotective effect. It is also clearly established that the activity depends on the nature of the alkyl group present at this position. Although the O-alkylation at C-4' position in B ring reduced the activity, the allyl group (compound 11) has a lower effect on the LPO inhibitory activity of 7 as compared to the prenyl group (compound 13). It can therefore be assumed that the higher the alkyl group is substituted the more the steric hindrance and the less effective the protons of the remaining phenolic hydroxyl groups can be donated thus leading to a reduced activity. Compound 14 was found to be the most efficient, and compounds 12 and 13 the less active. It is also established that compound 12 containing one more allyl group at position Advances in Biological Chemistry C-7 is less active than compound 11. It could therefore be deduced that the activities of C-glycosylflavonoids are mainly attributed to their free hydroxyl groups. This is corroborated with the activity of compound 10 which is drastically reduced compared to that of compound 7.

Conclusion
The inhibitory effect of the methanol crude extract of aerial part of D. uncinatum on lipid peroxidation of hepatocyte's membranes is mainly attributed to compounds of high to medium polarity. These compounds are mainly cerebrosides and C-glycosylflavonoids namely vitexin and isovitexin. From the structure-activity study, it has been established that the number of C-sugars, their sites of fixation on the aglycone and the number of the free hydroxyl groups on the aglycone are capital features for the activity. This study reveals that D. uncinatum possesses potent LPO inhibitory substances that may result to its hepatoprotective activity. It however appears that the n-butanol fraction showed a higher activity as compared to other fractions and to the isolated compounds. Therefore it could be advisable for the development of phytomedicine against hepatitis from D. uncinatum to focus on this particular fraction. The advantage of the present study is that using bio-guided fractionation; we have streamlined the active extract.