A Novel Compound and Biological Evaluation of Phytoconstituents Isolated from Erythrina corallodendron L. Flowers

Objective: This study aimed at phytochemical investigation of the 70% alcoholic extract of Erythrina corallodendron L. flowers and biological evaluation of the isolated compounds for their activity as antiprotozoal drugs also evaluation the binding affinity to opioid and cannabinoid receptors as well as the inhibition activity against monoamine oxidase (MAO) enzymes. Method: The 70% alcoholic extract was subjected to successive column chromatographic (CC) separations using silica gel normal phase, reversed phase RP-18, Diaion HP-20, and Sephadex LH-20. The structural elucidation of the isolated compounds was achieved using HR-ESI-MS, UV, 1D and 2D NMR spectroscopic analysis. The isolated compounds were screened in vitro for the binding affinity to opioid and cannabinoid receptors using receptor binding assay as well as the inhibition activity against MAO enzymes using kynuramine deamination assay, while their antiprotozoal activity was evaluated using parasite lactate dehydrogenase serum assay (pLDH). Results: The phytochemical evaluation of the alcoholic extract of E.corallodendron flowers, afforded the isolation of an indole alkaloid Hypaphorine 1, a new flavonoid glucoside; Kaempferol-3-O-α-sophoroside 2 and three known flavonoid Cglycosides vis;, Neoschaftoside 3, Isoschaftoside 4 and Vicenin-II 5. Compounds 3 and 4 are reported for the first time from genus Erythrina. Compounds 4 and 5 showed significant antimalarial activity both with IC50 value 1.7μg/mL against (D6) strain and with IC50 1.4 and 1.1 μg/mL against (W2) strain, respectively. Compound 3 showed selective inhibition to MAO-B with IC50 value of 32.08 μM and selective index (SI) > 3.12. Conclusion: The significant antiplasmodial activity of compounds 4 and 5 correlated the known antimalarial activity of different Erythrina species to flavonoid C-glycosides, Also compounds 3 and 4 are position isomers but exhibited different response against MAO-B which gives indication about the selectivity pattern of the flavonoid C-glycosides with MAO-B receptor subtype.


INTRODUCTION
Genus Erythrina (Fabaceae) comprises about 110 species which are distributed in tropical and subtropical areas, characterized with red/or orange flowers, and represented by trees, shrubs and herbaceous plants.In Africa there are about 30   Erythrina species and subspecies found in tropical regions 1 .The genus Erythrina is well-known for its neurological properties where the alcoholic extracts of E. velutina and E. mulungu displayed anticonvulsant 2 , tranquilizer 3 and anxiolytic activities 4,5 .
Also different Erythrina species were reported to exhibit wide range of biological activities such: as antimicrobial (E.caffra) 6 , anticancer (E.variegata) 7 , antioxidant (E.lysistemon) 8 , anti-inflammatory (E.crista-galli) 9 and anti HIV (E.abyssinica) 10 activities.In many African countries different Erythrina species comprise a potential component of the folk remedies, for example in Sudan the bark of E. abyssinica is used for the treatment of coughs, ulcers and abdominal pain, the flowers are used for the treatment of dysentery, the leaves are used for peptic ulcers, arthralgia, the roots for epilepsy, malaria and syphilis, and the fruits for asthma 11 .Several Erythrina species showed strong antiplasmodial activity against plasmodium falciparum both chloroquine sensitive and resistant strains (E.fusca 12,13 , E. burttii 14 E. abyssinica 15,16 , E. subumbrans 17,18 , E. crista-galli 19 ).E. corallodendron is known for its seeds lectins, which are carbohydrate recognizing molecules with a useful application in biomedicine and glycobiolocal systems 20 .Very few literature was traced about E. corallodendron where only three studies have been conducted concerning the leaves and flowers 21,22 .
Malaria is one of the most dangerous infectious diseases affecting tropical and subtropical areas.The incidence rate of malarial infection in Africa is about 300-500 million cases per year with 1.5-2.7 million deaths and up to 90% occurrence in children under the age of five 23 .The development of drugresistant malaria with this increasedincidence 24 creates an urgent need for more discovering and exploring of new drugs from natural sources.
One of the important metabolizing enzyme systems is monoamine oxidases (MAOs).MAOs are involved in the pathogenesis of many neurodegenerative diseases.They are responsible for the oxidative deamination of the monoamine neurotransmitters.Recent studies clarify the important role of MAO-B inhibition in the treatment of both Alzheimer's 25 and Parkinson's diseases 26 .
Recombinant Human MAO-A and MAO-B were obtained from BD Biosciences (Bedford, MA, USA), drug control, Clorgyline, Deprenyl, Phenelzine, (positive control) and DMSO (negative control) were purchased from Sigma Chemical (St.Louis, MO, USA).The MAO inhibition assay was carried on Spectra-Max M5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA).Antiprotozoal Assay: Plasmodium falciparum D6, P. falciparum W2 and Vero cells were used in the antiplasmodial assay.All organisms used were obtained from NCNPR, University of Mississippi, USA.The antiplasmodial drug control Chloroquine was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Plant material
The flowers of Erythrina corallodendron L. were collected from El-Zohria Botanical Garden in March 2012 and it was identified by Prof. Dr. Salwa Al Katuchi, Department of Systematic Plant Taxonomy, The National Research Centre (NRC), Cairo, Egypt.A voucher specimen was preserved in the herbarium of The National Research Centre (# M-125).

Anti-malarial Assay
In vitro antimalarial activity was determined using pLDH serum assay, against chloroquine-sensitive (D6, Sierra Leone) and chloroquine-resistant (W2, Indo China) strains of P. falciparum by measuring

Monoamine oxidase inhibitor assay
The inhibitory effect of the isolated constituents was investigated on human recombinant MAO-A and MAO-B.The 96-well plates kynuramine deamination assay was performed as previously reported 28 .A fixed concentration of substrate (Kynuramine) for each of MAO-A and -B were 80 and 50 μM, respectively and five concentrations for constituents from 0.0001 to 1 μg/mL were used to determine the IC50 values (concentration where 50% inhibition of the enzyme catalytic activity occurred).Reactions were performed in 200 μL of 0.1 M potassium phosphate buffer, pH 7.4.The inhibitors and compounds were dissolved in DMSO, diluted in the buffer solution, and pre-incubated at 37°C for 10 min (1.0% of DMSO final).Reactions were initiated by the addition of 50 μL of MAO-A (5 μg/mL) and -B (10 μg/mL), incubated for 20 min at 37°C, and terminated immediately by the addition of 75 μL of 2 N NaOH.The enzyme product formation (4-hydroxyquinoline) was recorded fluorometrically using a Spectra-Max M5 fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA) with an excitation (320 nm) and emission (380 nm) wavelength, using the Soft Max Pro program.The assays were calculated as percent of product formation compared to the corresponding control (enzyme−substrate reaction) without inhibitors.Controls including samples where the enzyme or the substrate was added after stopping the reaction were checked simultaneously to determine the interference with the fluorescence measurements.The determination of IC50 values for MAO-A and -B inhibition by the selected samples was performed using varying concentrations of the inhibitor and fixed concentration of the substrate.The IC50 values were calculated from the concentration dependent inhibition curves using XLFit software

RESULTS AND DISCUSSION
The phytochemical evaluation of the 70% methanolic extract of E. corallodendron flowers resulted in the isolation and identification of one indole alkaloid 1, one new flavonoid glucoside 2 and three known flavonoid C-glycosides 3-5 (Figure 2).Flavonoids 3 and 4 are reported for the first time from genus Erythrina.All the isolated compounds were screened for their antiprotozoal activity against Plasmodium falciparum.Compounds (1-3) did not show antiplasmodial activity.While compounds 4 and 5 exhibited significant antiplasmodial activity.Both compounds displayed more activity against chloroquine-resistant (W2) than chloroquine-sensitive (D6) strains with IC50 values 1.1, 1.4 and 1.7, 1.7 µg/mL, respectively.For the chloroquine-resistant strain (W2) assay compound 4 showed better activity than 5 with SI= 4.3 and 3.4, respectively.The selectivity index is directly related to the activity.
The results of MAO inhibitory assay revealed that the indole alkaloid 1 did not show any activity towards both enzyme subtypes.Flavonoids 2, 4 and 5 exhibited nonselective MAO inhibitory activity against both enzymes MAO-A and MAO-B (Table 2).Compound 3 displayed selective inhibitory activity against MAO-B subtype with IC50 32.08 µM and SI > 3.2.The aglycone part is responsible mainly for the activity in this assay.The C-glycoside nature of compounds 3-5 makes the molecules geometrically rigid and the presence of pentose at position C-8 in compound 3 instead of hexose increase the selectivity towards MAO-B and demolish any activity against MAO-A, which highlight clearly the important role of the substituent size in directing the activity and also the selectivity of the flavonoids in the MAO assay.
Furthermore, compounds 1, 4 and 5 were investigated for their binding affinity against human opioid and cannabinoid receptors subtypes (δ, k, and μ) and (CB1 and CB2), respectively attest concentration 10 µM, which in turn evaluate the analgesic potency of the tested compounds towards neuropathic pain 29 .
All the evaluated compounds did not exhibit activity at the concentrations ranges of the opioid and cannabinoid binding affinity assay.

Figure 2. Structures of isolated compounds (1-5) from E.corallodendron flowers
Furthermore, compounds 1, 4 and 5 were investigated for their binding affinity against human opioid and cannabinoid receptors subtypes (δ, k, and μ) and (CB1 and CB2), respectively attest concentration 10 µM, which in turn evaluate the analgesic potency of the tested compounds towards neuropathic pain 29 All the evaluated compounds did not exhibit activity at the concentrations ranges of the opioid and cannabinoid binding affinity assay.

Identification of isolated compounds: Compound 2
Obtained as amorphous yellow precipitate (3 mg), the molecular formula C27H30O16 was calculated from the 13 C NMR and HR-ESI-MS which showed quasi-molecular ion peak at m/z 609.15580 [M-H] - (calculated 609.14547).
. The flavonoid nucleus was deduced from the color change to yellow upon exposure to ammonia vapor, also the UV spectrum of MeOH showed two major absorption peaks at 268, 337 which indicated the presence of flavone or 3-O-substituted flavonol nucleus 30 .The diagnostic UV reagents revealed the presence of free OH function groups at position C-4` and C-7, where both shifting reagents NaOMe and NaOAc displayed a bathochromic shift characterized with increase in intensity in case of NaOMe.The 1 HNMR (DMSO, 400MHz) showed distinctive features of AA`, BB` spinning system, δH 8 (2H, d, J=8.4Hz, H-2`,6`), δH 6.9 (2H, d, J=8.4Hz, H-3`,5`) denoting a mono substituted B ring, it displayed also two singlet signals at δH6.4 (1H, s, H-8) and δH 6.1 (1H,s, H-6), indicating the absence of any C-glycosidic linkage.The appearance of two anomeric protons resonating at δH 5.6 (1H, d, J = 2.8 Hz, H-1``) and δH 4.6 (1H, d, J=8Hz, H-1```) accompanied with absence of singlet signal characteristic of H-3 indicate that position C-3 is occupied with sugar moieties.The 13 C NMR spectrum showed twelve carbon signals characteristic of two glucose units in the sugar region.It displayed also a carbonyl carbon resonated at δc 177.8 (C-4) characteristic of flavonol moiety.In addition to the downfield shift of (C-2) at δc 156.6 and the small upfield shift of (C-3) at δc 133.2 which verify that glycosylation occurred at position C-3 31 .

CONCLUSION
The phytochemical investigation of E. corallodendron flowers resulted in isolation of new flavonoid glycoside compound 2 in addition to two flavonoid C-glycosides compounds 3 and 4, which were isolated for the first time from genus Erythrina.The isolated flavonoid C-glycosides compounds 4 and 5 showed significant antimalarial activity over the flavonoid O-glycoside compound 2, which could correlate the antiplasmodial activity of Erythrina plants to flavonoid C-glycosides.All the isolated flavonoids compounds (2-5) exhibited inhibition activity against both MAO enzymes which in turn confirm the folk medicinal uses of Erythrina in treatment of the neurological disorders.
The selective inhibition of compound 3 against MAO-B provides important conclusion about the significant role of the substituent's size in orienting the structure selectivity.

ISSNFigure 1 .
Figure 1.Flow chart of fractionation and isolation of compounds from 70% methanolic extract of E. corallodendron flowers.