Isolation, Characterization and Antibacterial Activity Screening of Anthocyanidine Glycosides from Alchornea Cordifolia (Schumach. and Thonn.) Mull. Arg. Leaves

: From the ethanolic extract of the leaves of Alchornea cordifolia (Schumach. and Thonn.) Mull. Arg,, 5–methyl 4 ’ -propenoxy anthocyanidines 7- O- β -D – diglucopyranoside was isolated. The structure was elucidated using NMR spectroscopy in combination with IR and MS spectral data. Antibacterial studies showed that the isolated compound successfully inhibited Pseudomonas aerguinosa, Escherichia coli, Proteus mirabilis, Klebsiella pueumonia and Staphylococcus aureus . This result authenticates the use of the plant in phytomedicine for disease prevention and treatment of infections.


Introduction
Anthocyanidines are widely distributed natural products exhibiting a broad spectrum of pharmacological profile.They were found to be potent antiplasmodial agents.Besides the cytotoxic, antioxidant and antiviral activities, anthocyanidines also possess antibacterial activities 1,2 As part of an ongoing search for biologically active secondary metabolites from the Rain Forest Biodiversity of Nigeria, Alchornea cordifolia (Schumach.and Thonn.)Mull.Arg.(Euphorbiaceae) was selected for studies because of its multipurpose utilization as fodder, food and medicine.A. cordifolia is a multi-stemmed shrub or small tree; sometimes climbing but occasionally grows as an erect spreading plant, up to 5 m high and 30 cm girth 4 .The leaves, roots and stem bark extracts are used extensively in traditional medicine in the preparation of drugs for urinary, respiratory and gastro intestinal disorders 3 .Slurry from the fruits is administered for asthma and cough.A decoction of the leaves is used as eye lotion 5 .The leaves and stem bark when powdered are used in the treatment of ringworms and other skin infections 6 .There are many convergence in the traditional use of Alchornea cordifolia through out tropical Africa as topical anti-inflammatory; chancre, yaws, wounds, ulcers, dental caries and plaques, toothache, gum inflammation and conjunctivitis [5][6][7][8] .
It is distributed sporadically in the rain forest of Southern Nigeria and the roots and leaves are used as a carminative in Nigeria folk medicine 5 .However, very little is known about the chemical constituents of A. cordifolia and so far there have been few previous phytochemical investigations which reported the isolation and structural elucidation of gallic acid, ellagic acid and protocatechic acid from the plant 9 .Phytoconstituents such as steroids, phenolic compounds, flavonoids, flavones, tannins, xanthones and alkaloids have been isolated from A. cordifolia 10 .Recently, a new flavonol glycoside isorhamnetin-3-O-β-D-xyloside was isolated from the leaves of A. cordifolia 11 .As part of our chemical studies on Nigeria medicinal plants, we describe herein the isolation of a new anthocyanidine glycoside 5'-Methyl 4' Propenoxy anthocyanidines 7-O-β-D-diglucopyranoside (1) along with an antioxidant 5'-Methyl 4', 3, 5, 7 tetrahydroxy anthocyanidine (2).In addition, we investigate the antibacterial activity of the propenoxy anthocyanidine diglucopyranoside (1) isolated from A. cordifolia leaves.

Experimental
The IR spectra were determined on a Thermo Nicolet Nexus 470 FT -IR spectrometer.The 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance 400 FT spectrometer for 1 H NMR and 75 FT spectrometer for 13 C NMR, using TMS as internal standard.Chemical shifts are expressed in parts per million.
LC-ESIMS spectra were determined in the positive ion mode on a PE Biosystem API 165 single quadruple instruments; HRESIMS (positive ion mode) spectra was recorded on a Thermo Finniga MAT 95 XL mass spectrometer.Column chromatography was carried out with silica gel (200-300 mesh) and to monitor the preparative separations, analytical thin layer chromatography (TLC) was performed at room temperature on pre-coated 0.25 mm thick silica gel 60 F 254 aluminum plates 20 x 20 cm Merck, Darmstadt Germany.

Plant materials
The fresh leaves of Alchornea cordifolia were collected from an uncultivated farmland in Umudike, Abia State, Nigeria on 6 th February, 2008.The plant samples (fruits, seeds and leaves) were identified by Dr.A Nmeregini of Taxonomy Section, Forestry Department Michael Okpara University of Agriculture, Umudike, Nigeria.A voucher Specimen No: AC/552 has been deposited at the Forestry Department, Herbarium of the University.

Extraction and isolation of plant materials
Plant materials were treated and analyzed at the Chemistry Laboratory, Michael Okpara University of Agriculture, Umudike, Nigeria.The leaves (1 kg) were dried on the laboratory bench for 10 days.The dry sample was milled and ground into powder (910 g) using Thomas Wiley Machine (model 5 USA).
The powdered plant sample (500 g) was packed into a soxhlet apparatus (2 L) and extracted exhaustively with 1000 mL ethanol for 24 h.The ethanol extract was concentrated using a rotary evaporator at room temperature, and left on the laboratory bench for 2 days to obtain dark green pigment (50.3 g).The column was packed with silica gel and eluted with methanol, chloroform and petroleum ether (20:30:50) to afford a green amorphous solid (0.83 g).The green amorphous solid was recrystallized from hexane to afford compound 1; green amorphous solid (0.36 mg).Thin layer chromatography (chloroform: methanol (7: 3)  iodine vapor shows the presence of one spot.(R f 0.62).IR ν max 34.01 cm -1 (OH) 2926 cm -1 (CH 2 ), 1616 cm -1 (C=C aromatic) and 1097 cm -1 (C-O ether).HREIMS m/z 647.5367 calculated for C 31 H 37 O 15 m/z 648) and base peak m/z 57.0709 calculated for C 3 H 5 O. (m/z 57). 1 H NMR and 13 C NMR of compound 1 were presented in Table 1.

Bioassay procedure
The in vitro antibacterial activity of compound 1 was carried out for 24 h culture of five selected bacteria.The bacteria organism used were Escheria coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis and Klebsiella pneumonia.All the test organisms are clinical isolates of human pathogens obtained from the Federal Medical Centre (FMC) Umuahia, Nigeria.
Cultures were brought to laboratory conditions by resuscitating the organism in buffered peptone broth and thereafter agar medium and incubated at 37 o C for 24 h.The antibacterial activity was performed by filter paper disc diffusion technique.The medium (7 g nutrient agar in 250 mL distilled water, autoclaved at 115 o C for 15 min) was cooled to 50 o C. The medium (20 mL) was poured into a sterile Petri-dish and allowed to solidify.It was allowed to stay for 8 h and observed for contamination.The sterility of the medium was tested. 1 g of compound 1 was dissolved in 1 mL of absolute ethanol and made up to 10 mL with distilled water to give a concentration of 100 mg/mL (10% dilution).A colony of each test organism was sub-cultured on nutrient broth and incubated at 37 o C for 8 h.This was then used to flood the agar plates.Sterilized filter paper disc soaked in compound 1 was placed on the plates with test organisms.The plates were incubated at 37 o C for 24 h.After incubation, plates were observed for zones of inhibition (in mm diameter).The minimum inhibitory concentration was determined by comparing the different concentrations of compound 1 having different zones of inhibition and selecting the lowest concentration.
The sensitivity susceptibility of the test bacteria to the standard drug was tested using inoculated agar plate and ciprofloxacin.The zones of inhibition were measured and compared with those of compound 1.

Results and Discussion
The molecular formula of compound 1 was established as C 31 H 37 O 15 based on its HREIMS and NMR data.The IR spectrum revealed hydroxyl, aliphatic, aromatic and ether bands at (3401, 2926, 1616 and 1097 cm -1 ) respectively.Compound 1 was identified as 5 ' methyl 4' propenoxy anthocyanidine 7-O-β-Ddiglucopyranoside was assigned the molecular formula m/z 647.5367 calculated for C 31 H 37 O 15 (m/z 648) with base peak at m/z 57.0709 on the basis of HERIMS.The relative molecular mass of 647.5367 with base peak at m/z 57.0709 confirmed compound 1 as 5' -methyl 4' propenoxy anthocynoidine 7-O-β-D-diglucopyranoside. The pattern of fragmentation (Figure 1) showed that compound 1 undergoes cleavage or detachment of the propenoxy group C 3 H 5 O (m/z 57.0709) to produce the base peak.Further fragmentation occurred at C 7 to remove the diglucoside moiety while the detachment at C 4 ' removes the propen fragment from the anthocyanidine system producing the peak C 16 H 12 O 5 (m/z 284).Proton migration and hydrolysis produces the peak at (m/z 285.2430) calculated for C 16 H 13 O 5 (m/z 285).This seems to be a new compound 2. Also detachment of propenoxy group at C 4 ' and a methyl group at C 5 ' from compound 1 afforded the peak at m/z 577.5172 calculated for C 27 H 29 O 14 (m/z 577).
Analysis of the 1 H NMR spectrum is shown in Table 1.The 1 H NMR spectrum showed the presence of the aromatic protons at δH 7.2609 (1Hs), 7.5155 (1Hs), 7.5380 (1Hs), 7.6938 (1Hs), 7.5158 (1Hs), and 7.5155 (1Hs).The 1 H NMR spectrum also contained two methyl signals at δH 0.8803 (3H d) and 0.8962 (3Hs).The protons of the two hydroxyl groups attached to the anthrocyanidine nucleus showed broad singlet peaks at δH 4.1213 and 4.1510 respectively.In addition, the olefinic protons showed the signals at δH 5.3360 and δH 5.3266 respectively confirmed by 13  Hydrolysis of Compound 1 produce maltose and pelargonidin derivative characterized as 5'-methyl 4', 3, 5, 7-tetrahydroxy anthocyanidines 2. The conjugation of the electrons of the heterocyclic ring within the aromatic system may be responsible for the yellow color of the flavonoids.The presence of this phenolic compound indicates that this plant may be antimicrobial agent since phenol and phenolic compounds are extensively used in disinfections and remain the standard with which other bactericides are compared 12 .These phenolic compounds in A. cordifolia may be responsible for the therapeutic, antiseptic, antifungal or bactericide, as well as anti-viral and anti-tumor activities of A. cordifolia 3 .The bioactive phenolic compounds act as radical scavengers and singlet oxygen quenchers.They react with peroxyl radicals and thus bringing about the termination of the radical reaction generated within the system.
The antibacterial activity of compound 1 was evaluated in vitro.The results are reported in Table 2.The compound showed a broad diversity regarding growth inhibitory activity (Table 2).The anthocyanidine diglucoside isolated from the leaves of A. cordifolia has shown inhibition activity against Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirabilis and Escherichia coli (Table 2).This agreed with the findings of Lamikanra et al. 13 who reported that extracts from the leaves of A. cordifolia exhibited inhibition on Staphylococcus aureus and Escherichia coli.The observed inhibiting role on the microorganism explains the reason behind the utilization of A. cordifolia extract in traditional medicine as cough suppressant, anti-tumor agent, ulcer treatment and wound healing activity in traditional medicine 5,7 .These findings shows that phenolic flavonoids are potent water soluble super antioxidant and free radical scavengers which prevent oxidative cell damage, have strong anticancer activity and anti-inflammatory properties 12 .This bioactive compound isolated is responsible for the marked medicinal properties of the plant.The mechanism of inhibiting action of this phenolic compounds on these microorganism may be due to impairment of a variety of enzyme systems, including those involved energy production, interference with the integrity of cell membranes and structural component synthesis 14,15 .
The minimum inhibitory concentration (mic) of Compound 1 on the microorganisms was 6.50-12.50mg/mL (Table 3).E. coli, P. aeruginosa, S. aureus, P. mirabilis and K. pneumonia are human commensals and have been incriminated in the infection of wounds 5,16 .The inhibition of these bacteria justifies the use of A. cordifolia in healing wounds, ulcers and toothache in traditional medicine 5 .The ability of this Compound to inhibit these micro-organisms may be the reason behind the use of the leaves of A. cordifolia in the treatment of gonorrhea and urogenital infections in herbal medicine in Nigeria.The isolation and characterization of 5' methyl 4'-propenoxy anthocyanidine 7-O-β-D diglucopyrancoside lend credence to the common use of A. cordifolia as antibacterial agent.These findings justify the traditional use of this plant for wound treatment in traditional medical practice.The plant offer wide-scope for utilization as raw material by pharmaceutical industries for drug formulation.

Table 2 .
Diameter of zones of inhibition (mm) of Compound 1 isolated from A. cordifolia leaves and ciprofloxacin

Table 3 .
Minimum inhibitory concentration of Compound 1 Isolated from leaf of A. cordifolia on the pathogens, mg/mL.Data are means of triplicate determinations-No Zone of Inhibition.