Phytochemical investigation and antimicrobial activity of Psidium guajava L. leaves

Psidium guajava L. leaves were subjected to extraction, fractionation and isolation of the flavonoidal compounds. Five flavonoidal compounds were isolated which are quercetin, quercetin-3-O-α-L-arabinofuranoside, quercetin-3-O-β-D-arabinopyranoside, quercetin-3-O-β-D-glucoside and quercetin-3-O-β-D-galactoside. Quercetin-3-O-β-D-arabinopyranoside was isolated for the first time from the leaves. Fractions together with the isolates were tested for their antimicrobial activity. The antimicrobial studies showed good activities for the extracts and the isolated compounds.


INTRODUCTION
Psidium guajava L. leaf (family Myrtaceae) has a long history of folk medicinal uses in Egypt and worldwide as a cough sedative, an anti-diarrheic, in the management of hypertension, obesity and in the control of diabetes mellitus. [1][2][3][4][5][6][7] The leaf extract was found to possess anticestodal, [8] analgesic, anti-inflammatory properties, [9] antimicrobial [10] hepatoprotective [11] and antioxidant activities. [12] In addition, the leaf extract is used in many pharmaceutical preparations as a cough sedative. VIS spectrophotometer. Nuclear magnetic resonance (NMR) analyses were recorded on JOEL 500 MHz and Bruker Avance 300 MHz spectrometers. Mass spectral analyses were recorded on VG 7070 E-HF.

Extraction, fractionation and isolation
The air-dried powdered P. guajava leaves (1 kg) were exhaustively extracted with 50% ethanol at room temperature. The extract was filtered and concentrated under reduced pressure at 60°C to about 0.5 l and then successively fractionated with petroleum ether, chloroform, ethyl acetate, and n-butanol. Each extract, as well as the interface formed between the chloroform and aqueous layer, were separately concentrated and freed from solvent. Six fractions were obtained: petroleum ether (0.6 g), chloroform (2.7 g), interface formed between chloroform and aqueous phase (10 g), ethyl acetate (9.7 g), n-butanol (22.8 g) and the remaining aqueous extract (9.5 g).
A portion of the ethyl acetate extract (3.5 g) was chromatographed on a 150-g silica gel column (3.5 cm diameter × 30 cm length).

Isolation of material "A"
Fractions 12-15 containing 4-6% methanol showed a major spot of R f 0.46 [chloroform-ethyl acetate-methanol (8:2:1)] that gave a yellow color with ammonia. It was purified from the other minor spots by repeated crystallization from methanol, yielding yellow crystalline needles (21 mg Table 2.
Crystallization of fraction containing 10% methanol in chloroform-ethyl acetate (1:1) yielded a mixture of two compounds which were separated by preparative TLC using ethyl acetate-formic acid-acetic acid-water (25:2:2:4) with  Acid hydrolysis of flavonoids "B", "C", "D" and "E" Three milligrams of each compound was separately dissolved in a mixture of 0.5 ml methanol and 1 ml 2N hydrochloric acid. The solutions were then heated under reflux for 2 h, cooled, diluted with 1 ml water and the aglycones were extracted with ethyl acetate. The aglycones of "B", "C", "D" and "E" were identified to be quercetin by co-chromatography using reference compounds and chromatographic system chloroform-ethyl acetatemethanol (8:2:1). The aqueous solutions were neutralized with 5% Na 2 CO 3 solution and concentrated. The sugar moieties of "B", "C", "D" and "E" were identified by TLC in comparison with authentic reference materials, using chloroform-methanol (6:4) and visualized by methanol/ H 2 SO 4 spray reagent. The structures of the isolated compounds are given in Table 4.

Antimicrobial activity
Antibacterial and antifungal activities were determined using the agar diffusion technique [34] against the grampositive bacterium Staphylococcus aureus (S. aureus), two gramnegative bacteria, Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), and the fungus Candida albicans (C. albicans). The used organisms are local isolates provided from the Department of Microbiology, Faculty of Pharmacy, University of Alexandria.
One milliliter of 24-h broth culture of each of the tested organisms was separately inoculated into 100 ml of sterile molten nutrient agar maintained at 45°C. The inoculated medium was mixed well and poured into sterile 10-cm diameter petri dishes, receiving 15 ml. After setting, 10 cups, each of 8 mm diameter, were cut in the agar medium (Oxoid, Cambridge, England). Twelve milligrams of each extract or fraction or 3 mg of each isolate, accurately weighed, was dissolved in 1 ml dimethyl formamide (DMF). The solutions were inserted in the cups and incubated at 37°C for 24 h. The results of antimicrobial activity are shown in Tables 5 and 6.

RESULTS AND DISCUSSION
The UV spectra of flavonoid "A" [ Table 1] in different shift reagents showed the pattern of 5,7,3′,4′-tetrahydroxy flavonol aglycone, where the presence of free 5-hydroxy and 3′,4′-ortho dihydroxy groups was deduced from NaOMe spectrum, AlCl 3 and AlCl 3 /HCl spectra, while the presence of a free 7-hydroxy group was found from the NaOAc spectrum. [35] The electron impact-mass spectrometry (EI-MS) of flavonoid "A" illustrated the presence of molecular ion peak [M] + at m/z 302, suggesting the presence of five hydroxyl groups.
1 H-NMR spectra [ Table 2] showed two meta coupled aromatic protons at δ 6.13 and δ 6. C-NMR spectrum [ Table 2] indicated the presence of 15 signals corresponding to 15 carbons. Thorough study of the homonuclear correlation spectroscopy (COSY) and the heteronuclear multiple quantum coherence (HMQC) spectra helped in the full assignment of all protons to their carbon signals. All the spectral data of flavonoid "A" were found to be identical to those reported for quercetin. [31,36] The identification of the flavonoid was further confirmed by direct comparison with reference sample through mixed melting point (m.m.p.) and co-chromatography.
NMR spectra of flavonoids "B and C" [ Table 2] showed similar pattern to those of flavonoid "A", whereas they showed in addition, the appearance of five additional signals in the 13 C-NMR spectra matching those of arabinose, [36,37] along with the appearance of signals of one sugar moiety in 1 H-NMR spectra. Therefore, flavonoid "A" was probably the flavonol aglycone and flavonoids "B and C" were its arabinosides. These data were further supported by the results of the acid hydolysis through comparison of flavonoid "A" and the aglycones resulting from the acid hydrolysis of flavonoids "B and C" with a reference quercetin sample by co-chromatography. Similarly, the sugar moieties were established to be arabinose by comparison with reference sample.
The anomeric proton of flavonoid "B" was observed at δ 5.48 (1H, br s) with its corresponding carbon atom at δ 107.9, while the anomeric proton of flavonoid "C" was observed at δ 5.17 (1H, d, J = 6.5 Hz) with its corresponding carbon atom at δ 105, indicating that the arabinose moiety possessed α-configuration in flavonoid "B" and β-configuration in flavonoid "C". The ring size of the sugar moiety in both flavonoids was deduced from inspection of the chemical shift values for C-1'' and C-4'' where they appeared at δ 107.9 and 86.4 for flavonoid "B" and at δ 105 and 69.5 for flavonoid "C", thus revealing the presence of α-arabinofuranoside and β-arabinopyranoside moieties in flavonoids "B" and "C", respectively. [36,37] 3-O-glycosylation was confirmed from the study of the HMBC spectrum of flavonoid "B" [ Table 3], which showed the correlation between the carbon at δ 133.3 (C-3) and the proton at δ 5.48 (H-1''). 2D HMQC, 2D HMBC and 2D COSY allowed the assignment of all protons to their carbons. Also, the UV absorption of band I at 354.5 and 358 nm (371 nm for quercetin aglycone) indicates the absence of free 3-OH.
From the previous discussion, the str ucture of flavonoids "B" and "C" could be identified as quercetin-3-O-α-l-arabinofuranoside and quercetin-3-O-β-darabinopyranoside, respectively. The observed data were found to be similar to those published for these materials. [36,37] It is worth mentioning that this is the first report for the isolation of quercetin-3-O-β-d-arabinopyranoside from the species P. guajava L.
The results of antibacterial and antifungal screening [Tables 5 and 6] showed that quercetin and its glycosides have strong antibacterial activity against the gram positive S. aureus, and the gram negative E. coli and P. aeruginosa. They also showed antifungal activity against C. albicans. It is worth mentioning that the minimum inhibitory concentrations (MIC) of quercetin-3-O-β-d-arabinopyranoside and that of quercetin-3-O-α-l-arabinofuranoside glycosides against the tested organisms were even lower than quercetin itself.
All the extracts showed antibacterial and antifungal activities, whereas the chloroformic fraction of the aqueous-alcoholic extract possessed a strong activity against S. aureus.

CONCLUSION
The above results revealed that quercetin is the main flavonoidal nucleus of guava glycosides. Meanwhile, the antimicrobial testing showed that the extracts and the isolated compounds possess antibacterial and antifungal activities. These findings explain the folkloric use of the extracts as bactericide, in cough, diarrhea, gargles to relieve oral ulcers and inflamed gums wound.