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Journal of Ethnopharmacology 131 (2010) 511–521

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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review of the antibacterial activity of Hypericum perforatum L.

eb Saddiqe ∗, Ismat Naeem, Alya Maimoonaepartment of Chemistry, Lahore College for Women University, Jail Road, Lahore 54600, Pakistan

r t i c l e i n f o

rticle history:eceived 18 December 2009eceived in revised form 16 July 2010ccepted 16 July 2010vailable online 24 July 2010

eywords:ypericum perforatumypericaceaentimicrobial activityyperforinypericin

a b s t r a c t

Hypericum perforatum L. (Hypericaceae) is a perennial herb that is commonly known as St. John’s Wort.The plant has been valued for its important biological and chemical perspectives and its use in the treat-ment of infectious diseases has been documented in ethnobotanical reports. Most recent interest in H.perforatum has focused on its antidepressant effects, and only recently has its antimicrobial activity beenevaluated against a number of bacterial and fungal strains. The present review gives a comprehensivesummary of the ethnobotanical uses, chemical constituents and biological effects (antibacterial and anti-fungal) of this species. A comprehensive account of the chemical constituents including anthraquinonederivatives (naphthodianthrones), flavonoids, prenylated phloroglucinols, tannins and volatile oils is alsoincluded. Various types of preparations, ointments, creams and extracts prepared with and compoundsisolated from this species have been found to possess a broad spectrum of biological and pharmaco-logical effects such as antidepressant effects, wound-healing, antiviral and antimicrobial activity. Theantibacterial activity of crude extracts can be related to the use of the herb as a wound healer in ancient

times. The sole antibacterial principle isolated to date is a tetraketone, hyperforin, also thought to beresponsible for the antidepressant activity of the herb. The available literature indicates that it has ahigher antibacterial activity against Gram-positive than Gram-negative bacteria, and alcoholic extracts(methanolic/ethanolic) were shown to possess more pronounced activity than aqueous extracts. Basedon the chemical and pharmacological characteristics of H. perforatum, we concluded that this species hasbeneficial therapeutic properties and has the potential for use as an effective adaptogenic herbal remedy.

© 2010 Elsevier Ireland Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5121.1. Taxonomy and description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5121.2. H. perforatum in history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512

2. Biologically active compounds of H. perforatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5132.1. Naphthodianthrones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5132.2. Phloroglucinols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5132.3. Flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5142.4. Biflavones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5152.5. Phenylpropanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5162.6. Proanthocyanidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5162.7. Volatile oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5162.8. Other chemical constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516

3. Wound-healing effects of H. perforatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5164. Antibacterial-activity studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517

4.1. Antibacterial preparations of H. perforatum . . . . . . . . . . . . . . . . . . . . . . . .4.2. Antibacterial creams and ointments containing H. perforatum extr4.3. In vitro antibacterial activity of plant extracts of H. perforatum . . .4.4. Anti-MRSA activity of extracts of H. perforatum . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +92 42 99203801-9x245.E-mail addresses: zeb rukhsana@yahoo.com, zebrukhsana@gmail.com (Z. Saddiqe).

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.07.034

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517acts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518

512 Z. Saddiqe et al. / Journal of Ethnopharmacology 131 (2010) 511–521

4.5. Antibacterial activity of oil of H. perforatum (Oleum Hyperici) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5184.6. In vivo studies of the antibacterial activity of H. perforatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5184.7. Hyperforin: an antibacterial principle from H. perforatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518

5. Antifungal activity of H. perforatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5185.1. Antifungal activity of essential oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5185.2. Antifungal activity of ethanolic extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5195.3. Antifungal activity of flavonoids isolated from H. perforatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519. . . . .. . . . . .

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Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Hypericum perforatum L. (St. John’s Wort, Hypericaceae) is aember of the genus Hypericum, of which there are 400 speciesorldwide (Mabberley, 1987). It is native to Europe, West Asia,orth Africa, Madeira and the Azores and is naturalized in manyarts of the world, notably North America and Australia. The plantpreads rapidly by means of runners or from the prodigious seedroduction and can invade pastures, disturbed sites, dirt roads, theides of roads and highways, and sparse woods. In recent years,he consumption of H. perforatum-derived products has increasedramatically, and it is presently one of the most consumed medic-

nal plants in the world (Wills et al., 2000). The plant has a wideange of medicinal applications, including skin wounds, eczema,urns, diseases of the alimentary tract and psychological disordersButterweck, 2003).

The present review encompasses the literature on the antimi-robial activity of crude extracts of H. perforatum and antimicrobialompounds isolated from these extracts. For the antibacterial activ-ty, only reports describing extracts with minimum inhibitoryoncentrations (MIC) of 1 mg mL−1 (1000 �g mL−1) or less wereelected. The literature abounds with papers describing extractsisplaying antibacterial activity with MIC values over 1 mg mL−1,hich has little relevance from a clinical perspective, as it is likely

hat a number of relatively inert substances may display antibac-erial activity at such a high concentration.

.1. Taxonomy and description

In earlier classifications, Hypericum L. and other related generaere included in the family Clusiaceae (Guttiferae) under the sub-

amily Hypericoideae (Engler, 1925; Thorne, 1976 [using the nameypericaceae]; Cronquist, 1981). There is, however, growing evi-ence that the Hypericoideae do not form a monophyletic groupith other Clusiaceae (Gustafsson et al., 2002; Davis et al., 2005),

nd they are now treated as a separate family, Hypericaceae, in, e.g.,he classification system by Stevens (2006).The plant is a glabrouserennial, erect and usually woody at the base. The leaves arevate to linear, sessile, opposite, and contain translucent glandularots. The flower has five short, subequal, entire, imbricate, basallyonnate sepals, and five persistently withering yellow petals. Thevary is superior, capsicular, and three-styled whereas the stamensre many, arranged in bundles of threes. The flowers are profuse,rranged in branched cymes which bloom from June until Septem-er (Hobbs, 1998).

.2. H. perforatum in history

The plant has a long history of more than 2400 years and has

njoyed a reputation as a wound healer since the fifth century B.C. Itan be found in the herbals of Gerard (1597), and Withering (1796).number of Hypericum species were used by Amerindian tribes.

ecords of the use of H. perforatum are known from the Cherokee,roquois and the Montagnais. All these tribes seemed to have used

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519

the plant as a febrifuge/cough medicine; however, the Cherokeemade very broad use of the plant.

Gerard (1597), the famous English herbalist, referred to a St.John’s Wort salve, which he formulated using the herb, as the bestand most precious natural wound-healing therapy available. In hisown words:

“Saint John’s Wort with his flowers and seed boiled and drunken,provoketh urine, and is right good against the stone in the blad-der, and stoppeth the laske. The leaves stamped are good tobe laid upon burnings, scaldings, and all wounds; and also forrotten and filthy ulcers.”

Among the first most effective and widely spread pharmaceu-tical uses of H. perforatum in Europe after the 16th century wasthe use of the distilled oil of the herb as a therapy for wounds andbruises. It was so effective that surgeons not only used it to cleanwounds but also included it in the first official pharmacopoeia ofLondon as Oleum Hyperici (Urdang, 1944).

In 1633, Gerard’s Herbal recorded the plant’s use as a balm forburns, wounds, ulcers and bites and its oil was also popularly usedduring this time.

Culpeper (1652) pointed out the unique wound healing prop-erties of the herbal ointment. He also claimed the beneficialproperties of the herb against stings and bites of poisonous animals.According to him:

“It is a singular wound herb, healing inward hurts or bruises,it opens obstructions, dissolves swelling and closes up the lipsof wounds. It is good for those who are bitten or stung by anyvenomous creature, and for those that cannot make water.”

Other popular folk-uses recorded for St. John’s Wort included asa decoction for gravel and ulcerations of the ureter (Hill, 1808),for ulcerations of the kidneys, as a febrifuge or vermifuge, andfor jaundice, gout, and rheumatism (Greene, 1824). Griffith (1847)mentioned that the plant can be used as an oil or ointment forulcers, tumors, and as a diuretic. One of the most prominent earlyusers of H. perforatum was Dioscorides, the physicians to the Romanemperor Nero’s armies. He recommended it for sciatica, burns, as adiuretic, for promoting menstruation, and periodic fevers (Gunther,1968).

In the late 1800s, King (1876) wrote in his American Dis-pensatory about its use in urinary afflictions, diarrhea, jaundice,menorrhagia, hysteria, nervous imbalances with depression andtopical use for trauma. In a later revision of the Dispensatorya tincture of Hypericum was recommended for spinal irritation,shocks, concussions, puncture wounds and hysteria (Felter andLloyd, 1898).

In the last century, Duke (1985) recounted its use in folkmedicine in the treatment of ovarian carcinoma, uterine can-

cers, stomach cancers, and tumors of the lymph. Duke cites aRussian application in bronchial asthma and numerous others:diarrhea, dysentery, neurasthenia, nervous depression, hysteria,chronic catarrh, rabies, worms, hemorrhages, and bladder prob-lems, to name a few.

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Moerman (1998) mentioned use of the plant by the Cherokeendians as an emmanagogue, an antidiarrheal, a febrifuge (infu-ion), a treatment for sores and venereal diseases (using a milkyubstance, possibly the sap), a hemostat for nosebleed (as a snuff),snakebite remedy (root chewed and used as a poultice), and a root

nfusion as a wash to give infants strength. The Montagnais Indiansecocted the plant for a cough medicine and the Iroquois used H.erforatum as a febrifuge and to prevent sterility.

The plant has also retained a position in the contemporaryist of medicinal plants of pharmaceutical importance. Over theast 30 years, the plant has been extensively investigated in clin-

cal and laboratory studies. A plethora of important physiological,istoric, chemical, pharmacological and pharmacokinetic proper-ies were documented in the medical use of the pharmaceuticalreparations of the herb. The plant contains a wide spectrumf substances, among which anthroglycosides, flavonoids, andhloroglucin derivatives have roles in the principle pharmacolog-

cal effects. This plant has recently become very popular as anffective alternative treatment in weak/medium forms of depres-ion. Large quantities of mass-produced drugs with St. John’s Wortxtracts are sold mainly in the United States and Germany andave successfully penetrated into the markets of other Europeanountries. Some adverse side effects are also associated with thelant, the best known being photosensitivity and the lesser knowneing interactions with other drugs/foods (Kapusta and Dusek,003).

Most recent interest in H. perforatum has focused on its antide-ressant effects (Butterweck et al., 2000; Kumar et al., 2000; Cervot al., 2002); however, the herb has shown other activities includ-ng wound-healing (Fedorchuk, 1964; Rao et al., 1991) antifungalRancic et al., 2005; Milosevic et al., 2007), anti-inflammatoryMascalo et al., 1987; Kumar et al., 2001), antimycobacterialFrisbey et al., 1953; Fitzpatrick, 1954) and antiviral activitiesRicher and Davies, 1995).

. Biologically active compounds of H. perforatum

Hydroalcoholic extracts (60% ethanol or 80% methanol) oferial parts of the plant contain a spectrum of six major natu-al product groups: (1) naphthodianthrones, (2) phloroglucinols,3) flavonoids, (4) biflavones, (5) phenylpropanes, and (6) proan-hocyanidins. Additionally, lesser amounts of tannins, xanthones,ssential oils, and amino acids are present. All these compoundsepresent the main constituents in the dry crude H. perforatum herbNahrstedt and Butterweck, 1997).

.1. Naphthodianthrones

These compounds are typical of the genus Hypericum (Hegnauer,989), with an intense red color and phototoxic properties (Scheyt al., 2000). The major components of this group of compounds arehe hypericins. Two so-called protoderivatives, protohypericin andseudoprotohypericin, were isolated from the plant but due to theirnstable nature are efficiently converted to the stable products, i.e.,ypericin (I) and pseudohypericin (II), respectively.

macology 131 (2010) 511–521 513

Hypericin and pseudohypericin occur in the flowers and leavesof the crude plant material in concentrations of 0.03% to 0.3% of dryweight, with significant variation depending on the developmen-tal stage of the plant (Cellarova et al., 1994). Pseudohypericin is themain naphthodianthrone in H. perforatum, usually present in twoto four fold higher amounts than hypericin (Brantner et al., 1994;Upton, 1997). Cyclopseudohypericin, an oxidation product of pseu-dohypericin, has also been reported and is partly responsible forthe red color of H. perforatum extracts (Haeberlain et al., 1992). Thehypericin content (approximately 0.1–0.15%) includes both hyper-icin and pseudohypericin (Vanhaelen and Vanhaelen-Fastre, 1983)and is sometimes referred to as ‘total hypericins’.

The naphthodianthrones show a limited solubility in almost allsolvents; the pure compounds, especially hypericin, are almostinsoluble in water at ambient temperature. Nevertheless, morethan 40% of the naphthodianthrone content is extractable from thecrude herb when preparing a tea with water at 60-80 ◦C (approx.35% pseudohypericin and 6% hypericin) (Niesel and Schilcher,1990). The increase in solubility suggests the presence of coef-fectors in the plant material that modify the solubility of thenaphthodianthrones. The potassium salts of hypericin and pseudo-hypericin been identified as “soluble” pigments of the Hypericumspecies (Falk and Schmitzberger, 1992).

The hypericins are the most interesting compounds of H. perfo-ratum and many pharmacological effects have been described forthis class of compounds. Hypericin and pseudohypericin specifi-cally inhibit protein kinase C with IC50 values of 1.7 mg L−1 and15 mg L−1, respectively, and show antiproliferative activity againstmammalian cells. This explains the antiretroviral activity of hyper-icin and pseudohypericin, which can be attributed to the inhibitionof phosphorylation by protein kinase C during the viral infectionof cells (Takahashi et al., 1989). Hypericin was also found to havepotential for the treatment of T cell-mediated diseases due to itsinhibitory effect on the CD8+ T cell-mediated cytotoxicity reaction(Lavie et al., 2000).

Recently, it was found that irradiation of a mixture ofhemoglobin and hypericin with visible light can activate hypericinto generate reactive oxygen species which change the structure ofhemoglobin and enhance the catalytic activity of the protein forH2O2 reduction. The process depends not only on the irradiationtime but also on the concentration of hypericin. This study notonly confirmed the photosensitization by hypericin and revealedthe enhanced peroxidase activity of hemoglobin but may also findapplication in the development of a more sensitive H2O2 biosensorusing this molecule (Zhao et al., 2008).

Hypericin is the major photosensitizing constituent of H.perforatum (causing hypericism) and has been proposed as a photo-sensitizer for photodynamic cancer therapy. Pseudohypericin hasno phototoxicity (Vandenbogaerde et al., 1998).

2.2. Phloroglucinols

Phloroglucinol derivatives are widely distributed in the genusHypericum. Two closely related compounds found in H. perforatumare hyperforin (III), at 2.0% - 4.5% of the main phloroglucin contentand adhyperforin (IV) (0.2% - 1.9%) which contains an additionalmethyl group (Hobbs, 1989; Upton, 1997).

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These are found only in the reproductive parts (about 2% inhe flowers, 4.4% in the ripe fruits and 4.5% in the unripe fruit)Maisenbacher and Kovar, 1992; Tekel’ová et al., 2000). However,raditional preparations such as teas and tinctures of the herb con-ain little or no hyperforin (Meier, 2001). Furohyperforin, the majorxidation product of hyperforin, occurs in the aerial parts at about% of the hyperforin concentration (Verotta et al., 1999). In addition,xepahyperforin (Verotta et al., 2000) and other oxidation ana-ogues of hyperforin have also been isolated and elucidated (Shant al., 2001).

Although hyperforin is quite unstable – especially in aqueousolutions and when exposed to light and heat – it is present inany commercial extracts at concentrations of 0–6% (Melzer et

l., 1998). The products of its degradation are 2-methyl-3-buten--ol and two oxidation products with an intact hyperforin carbonkeleton (Trifunovic et al., 1998; Orth et al., 1999).

The hyperforins of H. perforatum present very interestingompounds from a pharmacological standpoint, although theirharmacological activities are as yet little known. Hyperforin itself

as shown to inhibit or modulate several neurotransmitter sys-

ems in vitro. It is a potent uptake inhibitor of serotonin, dopaminend noradrenaline, with IC50 values of about 0.5 mg L−1 in synap-osomal preparations (Chatterjee et al., 1998). This supports theossible role of hyperforin in the antidepressant activity of the

Table 1Known biological properties of major Hypericum perforatu

Constituent Activity

Amentoflavone Anti-inflaHöelzl, 19

Hyperforin Antibactewound-hMaisenbainhibitorantidepreanticarcininhibitionet al., 200

Hypericin Antiviralal., 1995)Butterwe2001; Eck(Panossia

3′ , 8′′-biapigenin ProbablyProanthocyanidins Antioxida

(NahrstedPseudohypericin AntiviralQuercitrin In vitro MXanthones Antidepre

cardiotonHölzl et aButterwe

macology 131 (2010) 511–521

herb. The mechanism of its antidepressant activity is consideredto be the inhibition of synaptic reuptake of neurotransmitters(Chatterjee et al., 2001). The compound has been investigated veryintensively as a novel antineoplastic agent (Schempp et al., 2002).Hyperforin also possesses antimalarial activity and was found tobe active against Plasmodium falciparum with an IC50 value inthe micromolar range, and the activity was not critically depen-dent on either its phenol-like sensitivity to auto-oxidation or thepresence of unsaturation on the prenyl residues (Verotta et al.,2007).

2.3. Flavonoids

Flavonoids comprise the major group of biologically active com-pounds in H. perforatum (2–4%). The flavonol aglycones identifiedso far include kaempferol, luteolin, myricetin and quercetin (V)(Nahrstedt and Butterweck, 1997; Kurth and Spreemann, 1998;Hansen et al., 1999; Naeem et al., 2010). Hyperoside (hyperin)(VI) and rutin (VII) usually dominate among the glycosides of

H. perforatum followed by quercitrin (VIII) and isoquercitrin (IX)(Pietta et al., 2001). Mártonfi et al. (2001) identified a rutin-freechemotype growing in Italy. Butterweck et al. (2000) isolatedmiquelianin (quercetin 3-O-glucuronide) and astilbin (taxifolin 3-O-rhamnopyranoside) from the herb.

m constituents.

mmatory, antiulcerogenic (Berghöefer and89)rial activity against Gram-positive bacteria,ealing (Gurevich et al., 1971; Bystrov et al., 1975;cher and Kovar, 1992), neurotransmitter(Nahrstedt and Butterweck, 1997),ssant (Chatterjee et al., 1998a), potentialogenic (Schempp et al., 2002), angiogenesis(Schempp et al., 2005) and antimalarial (Verotta

7)(Meruelo et al., 1988; Weber et al., 1994; Lavie et, antidepressant activity (Nahrstedt andck, 1997; Chatterjee et al., 1998; Muller et al.,ert and Muller, 2001), anti-inflammatoryn et al., 1996)sedative (Berghöefer and Höelzl, 1989)nt, antimicrobial, antiviral, vasorelaxantt and Butterweck, 1997)

(Meruelo et al., 1988)AO-inhibiting activity (Sparenberg et al., 1993)ssant, antimicrobial, antiviral, diuretic,ic, MAOA inhibitor (Kitanov and Blinova, 1987;l., 1989; Sparenberg et al., 1993; Nahrstedt andck, 1997)

opharmacology 131 (2010) 511–521 515

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Z. Saddiqe et al. / Journal of Ethn

Flavonol glycosides were shown to possess spasmolytic activ-ty (Morales and Lozoya, 1994). These compounds also inhibit

onoamine oxidase A, the enzyme responsible for the catabolismf biogenic amines, and catechol-O-methyltransferase (Thiedend Walper, 1994). Studies performed by Sparenberg et al.1993) showed the greatest activity for the flavonoid aglycones –uercetin, kaempferol and luteolin – whereas the glycosides were

ess active. However, the level of flavonoids present in H. perfora-

um is too low to be responsible for the therapeutic efficacy of therude Hypericum herb (Jurgenliemk and Nahrstedt, 2002). Someavonoids can bind to the benzodiazepine receptor (Medina et al.,997), and it has been argued that the flavonoids of H. perforatumay act in the same way (Haeberlain et al., 1994; Viola et al., 1994).

Rutin was shown to be essential for the antidepressant activity ofH. perforatum extracts (Nöldner and Schötz, 2002).

2.4. Biflavones

Biflavones are a rare group of dimeric flavones found in somevegetable sources. Three biflavones detected in H. perforatumare 3′, 8′′-biapigenin (0.1–0.5%), amentoflavone (X) (0.01–0.05%)(Berghöefer and Höelzl, 1987, 1989) and 6′, 8′′-diquercetin (Kurkin

and Pravdivtseva, 2007). The therapeutic significance of thesebiflavones in H. perforatum is still unknown. Amentoflavone,however, was shown to possess anti-inflammatory and anal-gesic activities (Kim et al., 1998). Nielsen et al. (1988) and

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aureithel et al. (1997) reported that amentoflavone binds to therain benzodiazepine receptors with an affinity comparable toiazepam.

.5. Phenylpropanes

These compounds mainly occur as esters of hydroxycinnamiccids, such as p-coumaric acid and caffeic acid. Chlorogenic acidXI) has been detected in H. perforatum extract at concentrationselow 1% (Nahrstedt and Butterweck, 1997). Its role in the pharma-ological effects of H. perforatum is unknown. Besides chlorogeniccid, caffeic, p-coumaric, ferulic, isoferulic, gentisic (Hobbs, 1989;pton, 1997), and shikimic acids (Bilia et al., 2001) have also been

eported.

.6. Proanthocyanidins

These compounds are represented by tannins. Their total con-entration ranges from 2 - 4% with a maximum concentration at thereflowering stage (Brantner et al., 1994). The dimeric procyani-in (XII) has been isolated from the plant together with additionalimeric, trimeric, and tetrameric procyanidins (Melzer et al.,

989).

The various biological effects of proanthocyanidins includentioxidant (Bagchi et al., 2000), antiviral (De Bruyne et al., 1999),nd antimicrobial (Scalbert, 1991), but no antidepressant effectas ever been reported.

macology 131 (2010) 511–521

2.7. Volatile oils

The essential oil of H. perforatum contains aliphatic com-pounds (2-methyl octane, n-nonane, n-decane, n-undecane,n-tetradecanol, 2-methyl-decane, and 2-methyl-dodecane) alongwith terpenoids (�-pinene, �-pinene, geraniol, �-caryophyllene,�-farnesene, humulene, and germacrene D) (Brondz et al., 1983;Gudzic et al., 1997; Mockuté et al., 2003; Akhbari and Batooli, 2009).Differences in the biosynthesis of sesquiterpene and aliphatichydrocarbons, and in oxygenated aliphatics, in flowers and leaveshave been indicated. The data has shown that concentrations of�-caryophyllene and caryophyllene oxide in essential oils fromthe leaves are higher than those from the flowers, whereasdodecanol, spathulenol, viridiflorol, carotol and tetradecanol arepresent in higher quantities in the flowers (Radusiene et al.,2005).

2.8. Other chemical constituents

Other typical constituents are xanthones (1,3,6,7-tetrahydroxyxanthone and kielcorin C (0.01%) (Nielsen andArends, 1978), acids (isovalerianic, nicotinic, myristic, palmitic, andstearic), carotenoids, choline, nicotinamide, pectin, �-sitosterol,pectin, fatty acids, amino acids, vitamin C, tannins (Duke, 1985),hydroperoxycadiforin (in stems and leaves) (Rücker et al., 1995)and bisanthraquinone glycosides (Wirz et al., 2000) (Table 1).

3. Wound-healing effects of H. perforatum

H. perforatum has long been used successfully as a remedy forwound-healing. Hypericum oil infusion has been used tradition-ally to speed healing of burns and wounds. Part of its effect mightbe due to its antibacterial activity (Upton, 1997). A tincture of theflowering tops and fresh leaves of the herb proved more effectivewhen orally administered to albino rats than the topical applica-tion of an undiluted tincture of flowering tops and fresh leavesof Calendula (another wound-healing herb). In treating incisionwounds, epithelization occurred in 15 days with H. perforatumtreatment and in 16.5 days in the Calendula treated group (Rao et al.,1991).

Research in humans also supports the effectiveness of the plantin this arena. In a study of 24 female patients who had recently hadcaesarean sections during childbirth, the use of a combination oilextract of Calendula and Hypericum oils (30:70) in healing the inci-sion site was examined and compared to wheat-germ-oil extract.

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atients treated with the Hypericum/Calendula mixture exhibitedsignificant decrease (approximately 38%) in the SPA (surface

erimeter area) of the surgical wound compared to patients in thelacebo group (approximately 16%) (Lavagna et al., 2001).

In another study, an olive-oil extract of the flowering aerial partsf H. perforatum was assessed for wound-healing activity by apply-ng it to in vivo excision and incision model wounds. An in vivo

odel, based on the inhibition of acetic acid-induced increase inapillary permeability was used for the anti-inflammatory activity.he results proved that the olive-oil extract of H. perforatum hadsignificant wound-healing effect on excision (5.1–82.6% inhibi-

ion) and circular incision (20.2–100.0% inhibition) model wounds.xtraction of the aerial parts with ethanol and further fractionationhowed that the ethyl-acetate subextract was the most active one,nhibiting wounds between 17.9% and 100.0% in the excision modelnd between 9.4% and 100.0% in the incision model (Süntar et al.,010).

The wound-healing activity of H. perforatum extracts seems toe mainly due to the increase in the stimulation of fibroblast colla-en production and the activation of fibroblast cells into polygonalhapes, which plays a role in wound repair by closing the damagedrea (Oztürk et al., 2007).

. Antibacterial-activity studies

.1. Antibacterial preparations of H. perforatum

The widely prescribed Russian preparations of Hypericum,ovoimanine and imanine (antibacterial preparations from H. per-

oratum), have been tested in vivo and in vitro against Staphylococcusureus infections and have been found to be more effective thanulfanilamide. Novoimanine had an MIC value of 0.1 �g mL−1

Derbentseva and Robinovich, 1968; Aizenman, 1969).

.2. Antibacterial creams and ointments containing H.erforatum extracts

A German patent mentions that an ointment containing anxtract of H. perforatum flowers shortened the healing time ofurns and showed antiseptic activity (Saljic, 1975). According tohe report, first-degree burns healed in 48 h when treated with theintment, whereas second- and third-degree burns healed withouteloid (a type of scar tissue) formation three times faster than burnsreated by conventional methods.

Schempp et al. (2003) assessed the efficacy of a cream containing. perforatum (extract standardized to 1.5% hyperforin) in compar-

son with the corresponding vehicle (placebo) for the treatment ofubacute atopic dermatitis. The study showed a significant superi-rity of the Hypericum cream compared to the vehicle alone in theopical treatment of mild to moderate atopic dermatitis.

Peeva-Naumovska (2010) evaluated the antibacterial effects ofhree formulations of lipophilic ointments containing H. perforatumil (30%, 40% and 50% Hyperici oleum), suitable for dermal or vagi-al application, against six bacterial strains: Streptococcus pyogenestwo strains), Streptococcus viridans, Micrococcus luteus ATCC9341,oraxella catarrhalis and Lactobacillus acidophilus using the method

f diffusion on blood-agar plates. Inhibition of bacterial growth wasegistered for all the tested strains with exception of L. acidophilus,

hich is a part of the normal vaginal bacterial flora. This shows

hat vaginal application of the prepared ointments will not distorthe normal vaginal flora. As expected, by increasing the quantityf Hyperici oleum in the ointment formulation, antibacterial effectncreased correspondingly.

macology 131 (2010) 511–521 517

4.3. In vitro antibacterial activity of plant extracts of H.perforatum

A number of studies are available in the literature regarding thein vitro antibacterial activity of crude plant extracts of the aerialparts of H. perforatum, supporting the use of these plants in tra-ditional medicine to treat wounds, skin and infectious diseases(Brondz et al., 1982; Grauds, 1997; Keles et al., 2001; Tolkunovaet al., 2002).

The extracts are reported to be more active against staphylo-cocci, shigellae and Escherichia coli than decoctions (Kolesnikova,1986) and exhibit more pronounced activity against Gram-positivebacteria than Gram-negative bacteria (Reichling et al., 2001; Avatoet al., 2004). Mazandarani et al. (2007) reported the higher antibac-terial activity of ethanolic extracts of flowering aerial parts of H.perforatum against Gram-positive bacteria (Enterococcus faecalisand S. aureus PTCC 1112), with growth-inhibition zones in therange of 25–26 mm, than of Gram-negative bacteria (Salmonellatyphi PTCC 1595, Shigella dysenteriae PTCC 1188, Yersinia entero-colitica PTCC 1151, E. coli PTCC 1330 and Pseudomonas aeruginosaPTCC 1074), for which no or only minimal activity was observed. Ina study by Conforti et al. (2005), the methanolic extracts of H. per-foratum evidenced the best performance against the Gram-positivebacteria, with an MIC value of 50 �g mL−1.

The antibacterial activity of different extracts of H. perfora-tum, when tested against Gram-positive (Staphylococcus oxford, S.aureus, Streptococcus mutans and Streptococcus sanguis) and Gram-negative bacteria (Proteus vulgaris, P. aeruginosa and E. coli), showedvariations in activity among different extracts. The petroleum etherextract, on agar plates, was active on P. aeruginosa (0.04 mL/disc)and in broth culture was active against S. aureus, S.oxford, S. mutansand S. sanguis, with an MIC of 310 �g mL−1, and E. coli and P. vul-garis, at 1250 �g mL−1. The chloroform extract was active against S.oxford, S. aureus and S. mutans (0.04 mL/disc). The methanol extractshowed activity against E. coli, P. vulgaris, S. mutans and S. sanguisand in broth culture against S. oxford (MIC 62 �g mL−1) and S. aureus(MIC 1250 �g mL−1). The water extract was active only on S. oxford.The results showed that the organic solvents were more suitable forextracting antibacterial plant components than water (Barbagalloand Chisari, 1987).

The anti-staphylococcal activities of H. perforatum extractsin different solvent combinations (water/alcohol/glycerol, WAG;water/alcohol, WA; and alcohol/glycerol, AG extracts) have beenanalyzed against reference strains of S. aureus, S. epidermidis, and S.saprophyticus and those isolated from patients with pyoinflamma-tory diseases of the skin; the WAG extracts were the most active(Molochko et al., 1990).

H. perforatum preparations made by extraction into 10% ammo-nia solution and 70% ethanol were studied for their antimicrobialeffects on the growth of Gram-positive (S. aureus and S. pyogenes)and Gram-negative (Pasteurella haemolytica, E. coli, and P. vulgaris)bacteria discharged from diseased animals, manifesting activity atdilutions of 1:4–1:160 (Danilov et al., 1999).

The extracts and fractions obtained from the flowering aerialparts of H. perforatum were assayed for anti-Helicobacter activ-ity against standard and clinical isolates of H. pylori. The extractsshowed activity with MICs between 1.95 and 250 �g mL−1 (Yesiladaet al., 1999). In a similar study, the butanol fraction of the herbextract revealed anti-H. pylori activity, with MIC values rangingbetween 15.6 and 31.2 �g mL−1 (Reichling et al., 2001).

Meral and Karabay (2002) reported the antibacterial activity of

methanolic extracts of H. perforatum against both Gram-positive (S.aureus ATCC 29213, S. aureus 65381/P, S. epidermidis ATCC 12228and E. faecalis ATCC 29212) and Gram-negative (P. aeruginosa ATCC27853, E. cloacae ATCC 13047, E. coli ATCC 29998 and E. coli ATCC8737) bacteria at a concentration of 1000 �g mL−1.

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The extracts of H. perforatum prepared with water and 10% and0% water/ethanol (v/v) solutions were evaluated for their antag-nistic properties against four test bacteria, Enterococcus faecium,ifidobacterium animalis, Lactobacillus plantarum and E. coli isolatedrom the human large intestine. The highest inhibitory propertiesere observed with the 30% ethanol solution (Lasik et al., 2007).

In an attempt to determine the effects of seasonal changesn the antibacterial activity of the extracts of H. perforatum, thentimicrobial activity of aqueous ethanolic extracts of H. perfora-um prepared from frozen plant material was studied against oneram-positive (S. aureus, ATCC 12600) and two Gram-negative (E.oli, ATCC 8677 and P. aeruginosa, ATCC 9721) bacterial strains.he extracts showed activity against all the tested strains. Thetudy showed that the degree of antimicrobial activity seems toe affected by the date of collection. The samples collected in Julyid not display antimicrobial activity, whereas samples collected

n August were active, indicating that seasonal variation of phyto-hemical production probably occurs in this species (Borchardt etl., 2008).

.4. Anti-MRSA activity of extracts of H. perforatum

Hydrous solutions of H. perforatum teas were found to be antimi-robially effective against Gram-positive bacteria, with particularctivity towards methicillin-resistant strains of S. aureus, with MICalues of 1.3–2.5 mg herb mL−1 (Reichling et al., 2001).

Dadgar et al. (2006) assessed the antibacterial activity of aque-us and ethanolic extracts of H. perforatum obtained by percolationethods against 14 clinical and standard strains of methicillin-

esistant and sensitive S. aureus. The results showed that thethanolic extract had a better antibacterial effect than the aque-us extract, and the anti-staphylococcal activity of ethanolic extractas higher against MRSA than MSSA (methicillin-sensitive S.

ureus) strains.Quave et al. (2008) investigated plants from different ethnob-

tanical usage groups from Southern Italy for the inhibition ofrowth and biofilm formation in MRSA. A total of 168 extracts rep-esenting 104 botanical species were screened for activity againstRSA (ATCC 33593). A broth-dilution method was employed to

etermine the MIC after 18 h of growth using optical-densityOD600nm) readings. H. perforatum extracts showed an MIC50 valuef 256 �g mL−1 and an IC50 (for biofilm formation) of 128 �g mL−1.

.5. Antibacterial activity of oil of H. perforatum (Oleum Hyperici)

H. perforatum oil (Oleum Hyperici) is a widely used herbaledicine in the treatment of bruises, injuries and burns. The most

ommon procedure for obtaining the oil is maceration of the freshowers of H. perforatum L. in oil exposed to sunlight for severaleeks. The oil extract is brilliant red with an orange-red fluores-

ence.Gudzic et al. (1997) tested the microbiological activity of the

ssential oil of H. perforatum using the method of diffusion on disc.he essential oil showed microbiological activity with the testedicroorganisms (K. pneumoniae, Salmonella enteridis, S. lutae 9341,

. aureus 6538, E. coli 95, B. subtilis “S”, and B. subtilis 841); no activityas observed against P. aeruginosa.

In a number of studies, the essential oil of the herb was shown toave potent antibacterial activity against a panel of bacterial strainsRancic et al., 2005; Radulovic et al., 2007; Saroglou et al., 2007).

.6. In vivo studies of the antibacterial activity of H. perforatum

In a study by Zakharova et al. (1986) on the action of plantxtracts on the natural immunity indices of animals, a leaf extract

macology 131 (2010) 511–521

of H. perforatum was found to enhance the immunity of mice (ren-dering them more resistant) to S. aureus and Bordetella pertussis.

4.7. Hyperforin: an antibacterial principle from H. perforatum

Studies of the antimicrobial activity of the various parts of H. per-foratum in India (Gaind and Ganjoo, 1959) led to the isolation of anantibacterial principle named hyperforin (a tetraketone) (Schemppet al., 1999), which is currently recognized as an antidepressantprinciple of St. John’s Wort (Chatterjee et al., 1998). Its chemicalstructure was elucidated in 1975 (Bystrov et al., 1975). Hyperforinwas also reported to be the main antibiotic constituent of novoima-nine (Gurevich et al., 1971).

In a study by Brondz et al. (1982) on the antibacterial activityof petroleum ether extract of the herb, the extract was found tocontain hyperforin.

The inherent instability of hyperforin in its pure form hin-dered the studies of antimicrobial activity until an investigationby Schempp et al. (1999). Using the agar-dilution method,the researchers reported that hyperforin showed no antibacte-rial activity at concentrations of 0.1–100 �g mL−1 in cultures ofGram-negative bacteria. With Gram-positive bacteria, hyperforininhibited the growth of all strains tested, including methicillin-and penicillin-resistant S. aureus (1–100 �g mL−1). The methicillin-resistant strain was also resistant to a number of penicillins and tocephalosporins, clindamycin, erythromycin, gentamicin, ofloxacin,and piperacillin/tazobactum. Against Corynebacterium diptheriae (E6040), as little as 0.1 �g mL−1 was required to inhibit growth, and1.0 �g mL−1 was needed to inhibit Streptococcus agalactiae B (D595)and S. pyogenes A (E12449). However, it has been emphasized thatthe antibacterial effects of hyperforin are only observed at highconcentrations (Fiebich et al., 1999; Voss and Verweij, 1999).

Reichling et al. (2001), in a study of the antibacterial activity of apetroleum ether extract of the aerial parts of H. perforatum, reportedthat hyperforin was the antimicrobial principle. Hyperforin exhib-ited an excellent effect against methicillin-resistant strains of S.aureus, with an MIC value of 1.0 �g mL−1.

Avato et al. (2004) investigated the antimicrobial properties ofthe aerial parts of H. perforatum in different extracts (methanol,petroleum ether, chloroform and ethyl acetate) against selectedmicroorganisms; the ethyl acetate fraction was the most active.The main constituents of this extract, as determined by high-performance liquid chromatographic analysis, were flavonoids,hypericins and hyperforins. Incubation of the selected microorgan-isms with the pure chemicals resulted in significant inhibition oftheir growth by hypericin, hyperforin and its stable dicyclohexil-ammonium salt, whereas the flavonoids appeared to be completelyinactive.

5. Antifungal activity of H. perforatum

5.1. Antifungal activity of essential oil

Khosa and Bhatia (1982) studied the antifungal activity ofthe essential oil and water-soluble fraction of an alcohol extractof H. perforatum. Both the fractions exhibited antifungal activ-ity against Microsporum gypseum, Trichophyton rubrum, Aspergillusflavus, Curvularia lunata and Fusarium vasiinfectum. The inhibi-tion index of the hot-water extract was 10 mg mL−1 (MIC) for theM. gypseum and T. rubrum and 6 mg mL−1 for the others. The oil

demonstrated inhibition in all species tested with a 4% solution in25% alcohol.

In a study by Gudzic et al. (1997), the essential oil of the herbshowed antifungal activity against Aspergillus niger but had noeffect on Candida albicans.

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.2. Antifungal activity of ethanolic extracts

Milosevic et al. (2007) investigated the antifungal activity of anthanolic extract of H. perforatum against the fungi Fusarium oxys-orum and Penicillium canescens at concentrations of 5–45 mg mL−1

sing a spore counting method. A concentration of 45 mg mL−1

howed the greatest fungi-static activity, with a decrease in theumber of spores to five spores for F. oxysporum and to 15 spores

or P. canescens with initial inoculations of 1 × 102 CFU mL−1 spores.Maskovic and Solujic (2009) estimated the effectiveness of the

thanol extract of H. perforatum in inhibiting the growth of P.anescens and F. oxysporum using the dilution (MIC) and micro-copic methods. The MIC of the extract for the two fungal strainsas 10 mg mL−1. The microscopic method showed that an increase

n the extract concentration induced a decrease in the number ofpores in both fungi, by 62% in P. canescens (within the extractoncentration range of 0–45 mg mL−1, the spore number decreasedrom 104 to 40 CFU mL−1) and by 72% in F. oxysporum (within thextract concentration range of 0–45 mg mL−1, the spore numberecreased from 100 to 24 CFU mL−1); i.e., the higher concentrationsf the H. perforatum extract showed antifungal effects (with highxtract concentrations yielding higher antifungal effects).

.3. Antifungal activity of flavonoids isolated from H. perforatum

Yan-Hua et al. (2002) assessed the antifungal activity of theix known flavonoids quercitrin, hyperoside, avicularin, rutin,uercetin and kaempferol and a new flavonol glycoside, 6′′-O-acetyluercetin 3-O-�-D-alloside, isolated from the aerial parts of H.erforatum. Antifungal assays of all the compounds showed that′′-O-acetyl quercetin 3-O-�-D-alloside, quercitrin and quercetinere inhibitory to the growth of the phytopathogenic funguselminthosporium sativum, with MICs of 25, 50 and 100 �g mL−1,

espectively. Moreover, 6′′-O-acetyl quercetin 3-O-�-D-allosidend quercitrin were also shown to inhibit the growth of Fusariumraminearum Schw., with an MIC of 100 �g mL−1.

. Conclusions

H. perforatum has been used as a medicinal herb since ancientimes. The plant has a long history of wound-healing which is veryell supported by the demonstrated antibacterial activity of crude

xtracts of the herb. A number of reports are available regardinghe antibacterial activity of crude plant extracts and creams andintments containing these extracts. The extracts were found toe more active against Gram-positive bacteria than Gram-negativeacteria. Additionally, the alcoholic extracts were found to be morective than aqueous solutions. Regarding antifungal activity, littleata is available on the antifungal activity of the plant extracts, anduch research remains to be carried out on this aspect. The plant

ontains a broad spectrum of biologically active compounds. Soar, however, only the compounds hyperforin and hypericin, haveeen identified as the major antimicrobial components of the herb.ecause the traditional preparations of the plant (teas and tinc-ures) do not contain hyperforin, there is still a need to study thesereparations and to determine the active ingredients found therein.he extracts have also shown activity against resistant bacteriauch as MRSA, which necessitates further research on the isola-ion of novel anti-MRSA lead agents from this plant. The extractsrom this species may be evaluated against a number of character-

zed isolates of MRSA, and may provide significant benefits to theharmaceutical industry and to public health. The medicinal prop-rties derived from various extracts of the plant vary considerablyepending on the specific chemical composition of each extract.he phytochemical analysis and the biological-activity data sug-

macology 131 (2010) 511–521 519

gest the possible use of H. perforatum extracts in the alimentary,cosmetic and pharmaceutical fields.

Acknowledgement

The authors wish to thank the Higher Education Commission ofPakistan for providing funding for the completion of this work.

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