Introduction 149
Methodological issues 153
among children in parts of the developing world exceeds 90% by 5 years of age, e.g. in Ethiopia, HP infection is acquired early in life, with 60% of 4-year- olds being infected, and almost 100% of 12-year-olds (Lindkvist et al., 1996). In addition to the above HP- induced findings, the infection in the developing world appears to be linked with chronic diarrhoea, malnutrition and impaired growth in children (Frenck and Clemens, 2003), as well as a predisposition to other enteric infections, including typhoid fever and cholera.
The treatment of HP has become increasingly more difficult because of the frequency of antibiotic resistance and recurrence after successful treatment. In Peru, the recurrence rate of the infection is as high as 73% even after successful eradication (Ramirez- Ramos et al., 1997). In this instance recurrence is not attributed to antibiotic resistance, but to reinfection of patients. In the United States, resistant HP is also of concern; the Helicobacter pylori Antimicrobial Resistance Monitoring Program (HARP) is a multi- centre US network that tracks HP patterns of resis- tance. HARP recently reported that 34% of 347 HP isolates tested were resistant to one or more anti- biotics commonly used to treat HP infections (Duck et al., 2004). In the US most antibiotic resistance is associated with metronidazole and clarithromycin, both standard treatment options for HP. Thus, antibiotic resistance and high reinfection rates strongly argue for the development of new therapeutic modal- ities to prevent and treat HP infections worldwide.
Natural products in HP research (in-vitro
data)
In most developing countries, plant-based medicines are commonly used to treat gastrointestinal ailments, including gastritis, peptic ulcer disease and diarrhoea (Bhamarapravati et al., 2003). Thus, considering the strong association between these conditions and HP infections, it should not be surprising that some plant-based medicines would have activity against HP in vitro.
The first investigation of the in-vitro efficacy of plant extracts against HP was published in 1991 (Cassel-Beraud, 1991). This group reported that extracts of 13 Malagasy medicinal plants were effec- tive against a number of clinical strains of HP in vitro. In 1996, Fabry and coworkers reported that a number
of east African medicinal plant extracts had inhibitory effects on the growth of HP in vitro (Fabry et al., 1996). One plant, Terminalia spinosa was reported to be the most active, with a minimum inhibitory concentration (MIC) range of 62.5–500 lg/mL. Extracts of Thymus vulgaris (aqueous extract) and Cinnamonum zeylanicum (alcohol extract) were also reported to inhibit the growth of the bacterium at concentrations of 3.5 mg/mL (Tabak et al., 1996).
In 1997, investigators discovered that common food plants such as garlic, soybean and fresh vegeta- bles may be protective against HP infections (Shinchi et al., 1997; Sivam et al., 1997). An aqueous extract of garlic (Allium sativum) inhibited the growth of HP strains at concentrations of 40 lg/mL (Sivam et al., 1997) and MICs of 8–32 lg/mL for garlic oil against several HP strains were reported (O’Gara et al., 2000).
Rabdosia trichocarpa, a traditional remedy for gastric and stomachic complaints in Japan, also inhibits the growth of HP in vitro, because of a diter- pene trichorabdal A (Kadota et al., 1997). In 1999, the anti-HP effects of seven Turkish plant extracts, used in folk medicine for the treatment of gastric ailments including peptic ulcers, were reported (Yesilada et al., 1999). Extracts of the flowers of Cistus laurifolius, cones of Cedrus libani, aerial parts of Centaurea solsti- tialis ssp. solstitialis, fruits of Momordica charantia, aerial parts of Sambucus ebulus, and flowering herbs of Hypericum perforatum were active with a MIC range of between 1.95 and 250 lg/mL. In 2003, a screening of 20 plant extracts from Thailand used to treat gastrointestinal ailments reported that over 50% of the plant species tested had anti-HP activity (Bhamarapravati et al., 2003). Methanol extracts of Myristica fragrans (aril) inhibited the growth of all HP strains with a MIC of 12.5 lg/mL; extracts from
Barringtonia acutangula (leaf) and Kaempferia
galanga (rhizome) had a MIC of 25 lg/mL; Cassia grandis (leaf), Cleome viscosa (leaf), Myristica fragrans (leaf) and Syzygium aromaticum (leaf) had MICs of 50 lg/mL. Extracts with a MIC of 100 lg/mL included Pouzolzia pentandra (leaf), Cycas siamensis (leaf), Litsea elliptica (leaf) and Melaleuca quinquenervia (leaf) (Bhamarapravati et al., 2003).
Ginger root (Zingiber officinale), a plant well known worldwide to treat gastrointestinal ailments, also has activity against HP (Mahady et al., 2003a). Methanol extracts of ginger rhizome inhibited the
growth of 19 HP strains in vitro with a MIC range of 6.25–50 lg/mL. The 6-, 8- and 10-gingerols all had varying degrees of activity, with a MIC range of 0.78 to 12.5 lg/mL and interestingly had significant activity against the CagA cancer-causing strains (Mahady et al., 2003a). Curcumin, a polyphenolic constituent isolated from turmeric (Curcuma longa), and a methanol extract of the dried, powdered turmeric rhizome were both active against 19 strains of HP, including five CagA strains. The MIC range was 6.25–50 lg/mL (Mahady et al., 2002). In addi- tion, red wine extract (Vitis vinifera) and resveratrol inhibited the growth of HP in vitro (Mahady and Pendland, 2000; Mahady et al., 2003b). Resveratrol, a stilbene present in red wine had a MIC of 25 lg/mL, while the red wine extract had a MIC range of 25–50 lg/mL (Mahady and Pendland, 2000). Interestingly, resveratrol was more active against CagA strains of HP than CagA⫺ strains (Mahady et al., 2003b).
Two indigenous American plants, Sanguinaria canadensis and Hydrastis canadensis, used tradition- ally by the Native American Indians for the treatment of gastrointestinal ailments, have also been shown to be active against HP (Mahady et al., 2003c). Methanol extracts of the rhizome or suspension cell cultures of S. canadensis had a MIC range of 12.5–50.0 lg/mL. Three isoquinoline alkaloids were identified in the active fraction. Sanguinarine and chelerythrine, two benzophenanthridine alkaloids, inhibited the growth of the HP, with a MIC of 50 and 100 lg/mL, respec- tively. Protopine, an alkaloid, also inhibited the growth of bacterium, with a MIC of 100 lg/mL. A crude methanol extract of H. canadensis rhizomes was very active, with a MIC of 12.5 lg/mL (range 0.78 to 25 lg/mL). Two isoquinoline alkaloids, berberine and b-hydrastine, were identified as the active constituents, and having a MIC of 12.5 and 100.0 lg/mL, respec- tively (Mahady et al., 2003c).
The in-vitro susceptibility of 15 HP strains to natural products that had a history of traditional use in the treatment of GI disorders was recently assessed (Mahady et al., 2005). Methanol extracts of Myris- tica fragrans (seed) had a MIC of 12.5 lg/mL and Rosmarinus officinalis (rosemary leaf) had a MIC of 25 lg/mL. Methanol extracts of botanicals with a MIC of 50 lg/mL included Achillea millefolium (aerial parts), Foeniculum vulgare (seed), Passiflora incarnata (aerial parts), Origanum majorana (herb)
and a (1 : 1) combination of Curcuma longa (root) and ginger rhizome. Botanical extracts with a MIC of 100 lg/mL included Carum carvi (seed), Elettaria cardamomum (seed), Gentiana lutea (roots), Juniper communis (berry), Lavandula angustifolia (flowers), Melissa officinalis (leaves), Mentha piperita (leaves) and Pimpinella anisum (seed). Methanol extracts of Matricaria recutita (flowers) and Ginkgo biloba (leaves) had a MIC of 100 lg/mL (Mahady et al., 2005). A traditional medicine from Iceland, the lichen Cetaria islandica, also used to treat gastro- intestinal ailments has anti-HP activity (Ingolfsdottir et al., 1997). Protolichesterinic acid, an aliphatic a-methylene-c-lactone, was identified as one of the active constituents. The MIC range of proto- lichesterinic acid, in free as well as salt form, was 16–64 lg/mL.
The antibacterial activity of Gosyuyu, a crude extract from the fruit of Evodia rutaecarpa, a Chinese herbal medicine, has also been tested for activity against HP in vitro (Hamasaki et al., 2000). Two compounds were identified as the active constituents and were the quinolone alkaloids, 1-methyl-2-[(Z)-8- tridecenyl]-4-(1H)-quinolone and 1-methyl-2-[(Z)-7- tridecenyl]-4-(1H)-quinolone. The MIC of these compounds against reference strains and clinically isolated HP strains was ⬍0.05 lg/mL, similar to that of amoxicillin and clarithromycin (Hamasaki et al., 2000). Finally, in a recent report, screening of 70 medicinal plants from Greece, led to the discovery of a number of plants with anti-HP activity. Extracts of Anthemis melanolepis, Cerastium candidissimum, Chamomilla recutita, Conyza albida, Dittrichia viscosa, Origanum vulgare and Stachys alopecuros were active against one standard strain and 15 clinical isolates of HP (Stamatis et al., 2003).
In addition to antibacterial activity, tea (Camellia sinensis) and rosemary (Rosmarinus officinalis L.) extracts inhibit HP urease in vitro. Green tea extract showed the strongest inhibition of HP urease, with a MIC of 13 lg/mL. Active principles were identified as catechins, with the hydroxyl group of 5⬘ position appearing important for urease inhibition (Matsubara et al., 2003).