Pharmacognosy Magazine

: 2010  |  Volume : 6  |  Issue : 23  |  Page : 212--218

Phytochemical investigation and antimicrobial activity of Psidium guajava L. leaves

AM Metwally, AA Omar, FM Harraz, SM El Sohafy 
 Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt

Correspondence Address:
S M El Sohafy
El Khartoum Square, Azarita, Faculty of pharmacy, Alexandria


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-b-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.

How to cite this article:
Metwally A M, Omar A A, Harraz F M, El Sohafy S M. Phytochemical investigation and antimicrobial activity of Psidium guajava L. leaves.Phcog Mag 2010;6:212-218

How to cite this URL:
Metwally A M, Omar A A, Harraz F M, El Sohafy S M. Phytochemical investigation and antimicrobial activity of Psidium guajava L. leaves. Phcog Mag [serial online] 2010 [cited 2021 Mar 1 ];6:212-218
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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.

Guava leaf extract contains flavonoids, mainly quercetin derivatives, which are hydrolyzed in the body to give the aglycone quercetin which is responsible for the spasmolytic activity of the leaves. [4] Quercetin has several pharmacologic actions; it inhibits the intestinal movement, reduces capillary permeability in the abdominal cavity [13] and possesses dose-dependent antioxidant properties, [14] anti-inflammatory activity, [15],[16],[17],[18],[19],[20],[21] antiviral and antitumor activities. [22],[23],[24],[25],[26],[27] It also inhibits the aldose reductase enzyme. [28] It should be noticed that most of the flavonoidal constituents of guava leaf are quercetin derivatives, namely, quercetin, avicularin, guaijaverin, isoquercetin, hyperin, quercitrin, quercetin 3-O-gentiobioside, quercetin 4′-glucuronoide. [4],[29],[30],[31],[32],[33]

 Materials and Methods

Plant material

P. guajava L. leaf was collected from El tahrir (Alexandria-Cairo desert road) during spring while the fruits are premature. A specimen is deposited in the department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Egypt.

Reference materials

Quercetin, glucose, galactose, l-arabinose and d-arabinose were supplied by E. Merck (Darmstadt, Germany). Quercetin-3-β-D-glucoside and quercetin-3-β-D-galactoside were supplied by Sigma-Aldrich Chemie GmbH (Steinheim, Germany).


Petroleum ether (40-60ºC), chloroform, ethyl acetate, n-butanol, methanol and ethanol were of analytical grade.

Chromatographic requirements

Precoated thin layer chromatography (TLC) plates (silica gel 60F-254) with the adsorbent layer thickness of 0.25 mm (E-Merck), silica gel (Merck) and kieselgel 60, 0.063-0.20 mm for column chromatography (E-Merck) were used.

Special apparatus

Melting points were determined using Sturat SMP heating stage microscope and were uncorrected. UV spectra were obtained on Pye Unicam SP8-100 UV/VIS spectrophotometer and Perkin-Elmer, Lambada 3B UV/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 60ºC 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 Χ 30 cm length).

Elution was started with chloroform:ethyl acetate mixture (8:2). Then, the polarity was increased using methanol gradually. Thirty-one fractions of 250 ml in each were collected, screened chromatographically using solvent system chloroform-ethyl acetate-methanol in the ratios (8:2:1) and (8:2:2).

Isolation of material "A"

Fractions 12-15 containing 4-6% methanol showed a major spot of Rf­­­­ 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). Rf 0.86 [ethyl acetate-methanol-water-acetic acid (100:2:1:4 drops)], m.p. 316-318ºC, soluble in methanol, acetone, dilute alkali and gives a canary yellow color with AlCl 3. The UV spectral data, λmax nm, are illustrated in [Table 1]; [M] + m/z 302. 1 H-NMR spectral data (300 MHz, CD 3 COCD 3 ) and 13 C-NMR spectral data (75 MHz, CD 3 COCD 3 ) are shown in [Table 2].

Isolation of material "B"

Fractions 18-20 containing 12-15% methanol showed a major spot of Rf 0.57 [chloroform-ethyl acetate-methanol (8:2:2)] that gave a yellow color with ammonia. It was purified by repeated crystallization (12 mg). Rf 0.55 [ethyl acetate-methanol-water-acetic acid (100:2:1:4 drops)], m.p. 209-211ºC, soluble in methanol, acetone, dilute alkali and gives a canary yellow color with AlCl 3, gives positive molisch's test. The UV spectral data, λmax nm, are illustrated in [Table 1]. 1 H-NMR spectral data (300 MHz CD 3 OH) and 13 C-NMR spectral data (75 MHz, CD 3 OH) are shown in [Table 2]. Long range 1 H- 13 C correlation data as determined by HMBC experiments of flavonoid "B" are shown in [Table 3].

Isolation of materials "C", "D" and "E"

Fractions 23-29 (0.3 g) containing 17.5-20% methanol showed four spots (giving a yellow color with ammonia) of Rf values 0.61, 0.49, 0.4, 0.35 [ethyl acetate-formic acid-acetic acid-water (25:2:2:4)]. It was rechromatographed on 60 g silica gel column (2.5 cm diameter Χ 30 cm length). The column was eluted with chloroform, with increasing concentrations of ethyl acetate (0-50%) and then increasing concentrations of methanol. Fractions 13 (chloroform-ethyl acetate (1:1) containing 4% methanol) was crystallized to give 8 mg. Meanwhile, fraction containing 6% methanol in chloroform-ethyl acetate (1:1) was purified by crystallization to give compound "C" (19 mg).

Material "C": Rf 0.41 [ethyl acetate-methanol-water-acetic acid (100:2:1:4 drops)], m.p. 264-267C, soluble in methanol, acetone, dilute alkali and gives a canary yellow color with AlCl 3, gives positive molisch's test. The UV spectral data, λmax nm, are illustrated in [Table 1]. 1 H-NMR spectral data (300 MHz CD 3 OH) and 13 C-NMR spectral data (75 MHz, CD 3 OH) are shown in [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 double run to give compound "D" (10 mg) and compound "E" (11 mg).

Material "D": Rf 0.31 [ethyl acetate-methanol-water-acetic acid (100:2:1:4 drops)], m.p. 240-243ºC, soluble in methanol, acetone, dilute alkali and gives a canary yellow color with AlCl 3 , gives positive molisch's test.

Material "E": Rf 0.24 [ethyl acetate-methanol-water-acetic acid (100:2:1:4 drops)], m.p. 237-239ºC, soluble in methanol, acetone, dilute alkali and gives a canary yellow color with AlCl 3 , gives positive molisch's test.

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 acetate-methanol (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 gram-positive bacterium Staphylococcus aureus (S. aureus), two gram-negative 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 45ºC. 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 37ºC for 24 h. The results of antimicrobial activity are shown in [Table 5] and [Table 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 6.4 (d, J = 2.1 Hz), assigned for H-6 and H-8, respectively, confirming a 5,7-disubstituted ring A. 3′,4′-disubstituted ring B was deduced by the appearance of three protons at δ 7.71 (d, J = 2.15 Hz,), 6.86 (d, J = 8.5 Hz) and 7.57 (dd, J = 8.5, 2.15 Hz) assigned for H-2′, H-5′ and H-6′, respectively. The 13 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 0β-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 structure of flavonoids "B" and "C" could be identified as quercetin-3-O-α-l-arabinofuranoside and quercetin-3-O-β-d-arabinopyranoside, 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.

Flavonoids "D" and "E" were identified to be quercetin-3-O-β-d-glucoside and quercetin-3-O-β-d-galactoside through comparison with reference compounds using m.m.p. and co-chromatography using ethyl acetate-formic acid-acetic acid-water (25:2:2:4) as the mobile phase.

The results of antibacterial and antifungal screening [Table 5] and [Table 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.


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.


1Karawya MS, Abdel Wahab SM, Hifnawy MS, Azzam SM, El Gohary HM. Essential oil of egyptian guajava leaves. Egypt J Biomed Sci 1999;40:209-16.
2Abdelrahim SI, Almagboul AZ, Omer ME, Elegami A. Antimicrobial activity of Psidium guajava L. Fitoterapia 2002;73:713-5.
3Begum S, Hassan S, Ali S, Siddiqui B. Chemical constituents from the leaves of Psidium guajava. Nat Prod Res 2004;18:135-40.
4Lozoya X, Meckes M, Abou-Zaid M, Tortoriello J, Nozzolillo C, Arnason JT. Calcium-antagonist effect of quercetin and its relation with the spasmolytic properties of Psidium guajava L. Arch Med Res 1994;25:11-5.
5Ishihara T, Han R. Manufacture of guava leaf extracts. Japanese Kokai Tokkyo Koho Patency number JP 10202002, Kind A2, Date 19980804, Application number JP 1997-46858, Date 1997.01.23; 4 pp. (Japan).
6Sunagawa M, Shimada S, Zhang Z, Oonishi A, Nakamura M, Kosugi T. Plasma Insulin concentration was increased by long-term ingestion of guava juice in spontaneous non-insulin-dependant diabetes mellitus (NIDDM) rats. J Health Sci 2004;50:674-8.
7Tanaka T, Ishida N, Ishimatsu M, Nonaka G, Nishioka I. Six new complex tannins, guajavins, psidins and psiguavin from the bark of Psidium guajava L. Chem Pharm Bull (Tokyo) 1992;40:2092-8.
8Tangpu TV, Yadav AK. Anticestodal efficacy of Psidium guajava against experimental Hymenolepis diminuta infection in rats. Indian J Pharmacol 2006;38:29-32.
9Ojewole JA. Anti-Inflammatory and analgesic effects of Psidium guajava Linn. (Myrtaceae) leaf aqueous extracts in rats and mice. Methods Find Exp Clin Pharmacol 2006;28:441-6.
10Nair R, Chanda S. In-vitro antimicrobial activity of Psidium guajava L leaf extracts against clinically important pathogenic microbial strains. Braz J Microbiol 2007;38:452-8.
11Roy K, Kamath V, Asad M. Hepatoprotective activity of Psidium guajava L leaf extract. Indian J Exp Biol 2006;44:305-11.
12Hui-Yin Chen, Gow-Chin Yen. Antioxidant activity and free radical-scavenging capacity of extracts from guava (Psidium guajava L.) leaves. Food Chem 2007;101:686-94.
13Zhang W, Chen B, Wang C, Zhu Q, Mo Z. Mechanism of quercetin as antidiarrheal agent. Di Yi Jun Yi Da Xue Xue Bao 2003;23:1029-31.
14Nakamura Y, Ishimitsu S, Tonogai Y. Effects of quercetin and rutin on serum and hepatic lipid concentrations, fecal steroid excretion and serum antioxidant properties. J Health Sci 2000;46:229-40.
15Havsteen B. Flavonoids, A class of natural products of high pharmacological potency. Biochem Pharmacol 1983;32:1141-8.
16Middleton E, Drzewieki G. Flavonoid inhibition of human basophil histamine release stimulated by various agents. Biochem Pharmacol 1984;33:3333-8.
17Middleton E, Drzewieki G. Naturalty occurring flavonoids and human basophil histamine release. Int Arch Allergy Appl Immunol 1985;77:155-7.
18Amella M, Bronner C, Briancon F, Haag M, Anton R, Landry Y. Inhibition of mast cell histamine release by flavonoids and bioflavonoids. Planta Med 1985;51:16-20.
19Pearce F, Befus AD, Bienenstock J. Mucosal mast cells: III Etfect of quercetin and other flavonoids on antigen-induced histamine secretion from rat intestinal mast cells. J Allergy Clin Immunol 1984;73:819-23.
20Busse WW, Kopp DE, Middleton E. Flavonoid modulation of human neutrophil function. J Allergy Clin Immunol 1984;73:801-9.
21Yoshimoto T, Furukawa M, Yamamoto S, Horie T, Watanabe-Kohno S. Flavonoids Potent inhibitors of arachidonate 5-lipoxygenase. Biochem Biophys Res Commun 1983;116:612-8.
22Murray MT, Pizzorno JE. Flavonoids-Quercetin, citrus favonoids, and HERs (hydroxylethylrutosides). In: Pizzorno JE, Murray ME, editors. Text book of Natural Medicine. 2 nd ed. London: Harcourt Brace and company Ltd; 1999. p. 745-50.
23Mucsi I, Pragai BM. Inhibition of virus multiplication and alteration of cyclic AMP level in cell cultures by flavonoids. Experientia 1985;41:930-1.
24Kaul T, Middleton E, Ogra P. Antiviral effects of flavonoids on human viruses. J Med Virol 1985;15:71-9.
25Farkas L, Gabor M, kallay F, Wagner HI. Flavonoids and Bioflavonoids. New York: Elsevier; 1982. p. 443-50.
26Guttner J, Veckenstedt A, Heinecke H, Pusztai R. Effect of quercetin on the course of mengo virus infection in immunodeficient and normal mice: A histological study. Acta Virol 1982;26:148-55.
27Kaneuchi M, Sasaki M, Tanaka Y, Sakuragi N, Fujimoto S, Dahiya R. Quercetin regulates growth of Ishikawa cells through the suppression of EGF and cyclin D1. Int J Oncol 2003;22:159-64.
28Chaundry PS, Cabrera J, Juliana HR, Varma SD. Inhibition of human lens aldose reductase by flavonoids, sulindac and indomethacin. Biochem Pharmacol 1983;32:1995-8.
29Abdel Wahab SM, Hifawy MS, El Gohary HM, Isak M. Study of carbohydrates, lipids, protein, flavonoids, vitamin C and biological activity of Psidium guajava L growing in Egypt. Egypt J Biomed Sci 2004;16:35-52.
30El Khadem H, Mohamed YS. Constituents of the leaves of Psidium guajava L: Part II: Quercetin, avicularin, and guajaverin. J Chem Soc 1958;32:3320-3.
31Arima H, Danno G. Isolation of antimicrobial compounds from guava (Psidium guajava L.) and their structural elucidation. Biosci Biotechnol Biochem 2002;66:1727-30.
32Seshadri T, Vasishta K. Polyphenols of the leaves of Psidium guava; quercetin, guaijaverin, leucocyanidin, amritoside. Phytochemistry 1965;4:989-92.
33Kandil FE, El-Sayed NH, Micheal HN, Ishak MS, Mabry TJ. Flavonoids from Psidium guajava. Asian J Chem 1997;9:871-2.
34Jian FR, Kar A. The antibacterial activity of some essensial oils and their combinations. Planta Med 1971;20:118-22.
35Harbone JB, Mabry TJ. The Flavonoids: Advances in research. London: Chapman and Hall; 1982.
36Agrawal PK. Carbon-13 NMR of flavonoids. Amsterdam: Elsevier; 1989.
37Markhan KR, Terani B, Stanley R, Geiger H, Mabry TJ. Carbon-13 NMR studies of flavonoid glycosides and their acylated derivatives. Tetrahedron 1978;34:1389-97.