Antimicrobial Activities of Fruits of Crataegus. and Pyrus. Species

Abstract

The antimicrobial activity of the ethyl acetate extracts from fruits of Crataegus tanacetifolia., Crataegus x bornmülleri., Pyrus communis. subsp. communis., and Pyrus serikensis. (Rosaceae) were investigated by the agar-well diffusion assay, and the extracts exhibited antimicrobial effect against most of the bacteria and all of the yeasts tested. Although the plants differed in their activities against the microorganisms tested, the extracts of C. x bornmülleri. and P. communis. subsp. communis. displayed no antifungal activity against the fungi tested. P. serikensis. extract inhibited the growth of all bacteria tested but not Aspergillus niger., Aspergillus parasiticus., Aspergillus citri., and Aspergillus fumigatus.. In conclusion, P. serikensis. was the most active antimicrobial plant. Particularly, P. serikensis. extract showed significant antifungal activities and could be used as an antimicrobial agent in new drug therapy.

Keywords:

• Antimicrobial activity
• Crataegus tanacetifolia
• Crataegus x bornmülleri
• endemic
• Pyrus communis. subsp.. communis
• Pyrus serikensis

Introduction

Hawthorne (Crataegus. spp.) (Rosaceae) consists of small trees and shrubs. Common names for hawthornes include May blossom, quick thorn, whitethorn, haw hazels, gazels, halves, hagthorn, and bread and cheese tree. They are usually multibranched 2–5 m shrubby trees that can reach a height of up to 10 m. The hawthorne tree prefers the forest margins of lower and warmer areas. The color of the ripe fruit ranges from yellow through green to red and on to dark purple. Most of the species ripen their fruit in early to mid-autumn (Özcan et al., 2005).

Crataegus tanacetifolia. (Lam.) Pers. fruits are yellow, sometimes suffused with red, globose, and 2 cm in diameter. C. x bornmuelleri. Zabel resembles C. tanacetifolia. in its glandular-serrate leaves but differs in its larger (3–5 cm), villous leaves. Both Crataegus. spp. are endemic to Turkey (Davis, 1972).

Pyrus communis. subsp. communis. (pear) tree is up to 20 m and is common in Europe and Turkey. Fruits are pyriform to subglobose, 2–4 cm long, and yellowish green. Pyrus communis. subsp. communis. is described from Europe and Turkey (Davis, 1972).

Pyrus serikensis. A. Güner et H. Duman are trees or shrubs up to 10 m high. The trunk is up to 60 cm in diameter. It is endemic as an East Mediterranean element and is called a “zingit” by local people. Fruits are 0.7–1.5 cm in diameter, globose, reddish brown, with white spots (Güner & Duman, 1991).

The medicinal use of hawthorne has a long tradition with written records dating back to ancient Roman times (Veveris et al., 2004). Some Crataegus. constituents are predicted to be good antioxidants. The flower and fruit constituents responsible for free radical scavenging activity are epicatechin and chlorogenic acid. They are among the best antilipoperoxidants (Bahorun & Trotin, 1994; Rakotoarison & Greisser, 1997; Škerget, 2005). Traditionally, the fruits or the berries are used for their astringent properties in heavy menstrual bleeding and in diarrhea. Both the flowers and berries act as diuretics and can be used to treat kidney problems and dropsy. Apart from their delicious flavor, hawthorne fruits have been shown to have a tonic effect on the heart. Fruits of our native species are often used in the treatment of weak heart conditions, especially if this is accompanied by high blood pressure (Baytop, 1984; Schussler & Holzl, 1995). Nowadays, Crataegus. extracts are mainly utilized for the treatment of mild forms of congestive heart failure according to the New York Heart Association functional class II (Veveris et al., 2004).

Bilia et al. (1994) isolated flavanol glycoside from the aerial parts of Pyrus bourgaeana.. Jin and Sato (2003) determined that aqueous axtracts from succulent young shoots of the pear Pyrus. spp. exhibited strong antibacterial activity against the bacterium Erwinia amylovora. bv. 4.

As there are vast, renewable resources containing diverse natural products, the search for novel defense compounds from plants continues. Development of such plant-based compounds for microbial control is of interest to countries where there is an extensive indigenous flora. There is not much work on the antimicrobial activity of hawthorne and Pyrus. fruits as indicated above. Therefore, attempts were made in this study to determine the antibacterial and antifungal activity of matured endemic hawthorne and Pyrus. fruit collected from Turkey.

Materials and Methods

Plant materials and extraction procedure

Fresh wild hawthorne and Pyrus. fruits (Davis, 1972; Güner & Duman, 1991) were collected from typical localities in Turkey. Crataegus tanacetifolia. fruits were collected from Civan harmani, Kalabak-Eskişehir (October, 2003; 1510 m), Crataegus x bornmülleri. fruits were collected from Dalli yayla, Kalabak-Eskişehir (October, 2003; 1520 m); Pyrus communis. subsp. communis. and Pyrus serikensis. fruits were collected from Bati, Serik-Antalya (November 2003; 20 m). The specimens collected were identified with the help of Flora of Turkey and the East Aegean Islands. (Davis, 1972) and The Karaca Arboretum Magazine. (Güner & Duman, 1991). The voucher specimens were deposited in the Herbarium of the Faculty of Science, Anadolu University (ANES). The fruits were transported in polypropylene bags and held at room temperature. Fruits were cleaned manually and rinsed with water; 20 g of fresh fruit sample was ground in a mortar and then extracted with 150 ml of ethyl acetate (Fluka, Italy) for 24 h by using Soxhlet equipment. The extracts were filtered using Whatman filter paper (no. 1) and then concentrated in vacuo. at 70°C. The residues were stored in a refrigerator until subsequent use. For the bioassay, the extracts were suspended in ethyl acetate at a concentration of 10 mg/ml.

Microorganisms

A total of 40 microbial genera including 28 bacteria, 3 yeasts, and 9 mold species were used in this study. Table 1 shows the isolates used in the study and their source of origin.

Table 1 Strains used in the study and their source.

Microorganism	Source
Bacillus cereus. NRRL B-3711	USDA, Agricultural Research Service, Peoria, IL, USA
Bacillus subtilis. NRRL 744
Salmonella typhimurium. NRRL B-4420
Microccoccus luteus. NRRL B-4375
Proteus. vulgaris NRRL B-123
Xanthamonas campestris. pv. campestris. NRRL-B1459
Enterobacter aerogenes. NRRL B-3567
Streptococcocus feacalis. NRRL B-14617
Rhodotorula rubra. NRRL Y-2505
Fusarium oxysporum. NRRL 5836	Ege University, Faculty of Science, Department of Biology, İzmir, Turkey
Fusarium moniliforme. NRRL 1866
Penicillium notatum. NRRL 807
Aspergillus fumigatus. NRRL 163
Aspergillus parasiticus. NRRL 2999
Aspergillus niger. NRRL 321
Escherichia coli. ATCC 25922	Ankara University, Faculty of Veterinary, Ankara, Turkey
Pseudomonas aeroginosa. ATCC 27853
Staphylococcus aureus. ATTC 6538 T
Aeromonas hydrophila.	J.B. Jones, University of Florida, IFAS, Plant Pathology Department
Listeria monocytogenes.
Klebsiella pneumoniae.
Yersinia enterocolitica.
Pseudomonas fluorescens. B 130
Bacillus pumilus. B122
Pseudomonas syringae pv.. tomato. 32
Pseudomonas tobacco. 8
Xanthamonas campestris pv.. vesicatoria. 75-3
Pseudomonas lachrymans.
Pseudomonas syringae. -1
Pseudomonas syringae pv. glycine. PG1-T
Pseudomonas syringae pv. phaseolicola. NCPPB 52	Vidaver, A. K. University of Nebraska Lincoln, Institute of Agricultural and
Pseudomonas gingeri. 3146	Natural Resources
Pseudomonas gladioli pv.. agricola. RR3	J.G. Turner, University of East Anglia, UK
Brucella spp..	Osmangazi University Medical Faculty
Pseudomonas maltophila.
Candida albicans.
Candida globrata.
Geotrichum graminis.
Cladosporum herbarum.	Anadolu University Faculty of Science
Aspergillus citri.

Table 2 Antimicrobial activity of Crataegus bornmülleri., C. tanacetifolia., Pyrus serikensis., and P.. C.. Subsp. communis. ethyl acetate extracts against the bacterial, yeast, and fungal strains tested based on agar well-diffusion method.

	Inhibiton zone in diameter (mm/sensitive strains)
Microorganism	C. tanacetifolia.	C. x bornmülleri.	Pyrus serikensis.	P. c.. subsp. communis.	Standard antibiotic
B. cereus.	12	12	13	10	30
B. subtilis.	7	9	9	8	40
S. typhimurium.	—	10	15	—	38
M. luteus.	12	12	10	10	50
P. vulgaris.	9	12	13	8	31
E. coli.	9	10	12	8	37
P. aeroginosa.	12	11	14	8	23
P. maltophila.	9	9	12	8	38
S. aureus.	9	9	11	9	35
E. aeroginosa.	12	10	12	12	36
Y. enterocolitica.	12	—	12	12	33
Brucella spp..	10	10	15	—	25
S. feacalis.	10	12	10	—	35
A. hydrophila.	8	9	10	12	30
L. monocytogenes.	14	18	19	10	30
K. pneumoniae.	12	12	17	7	30
P. fluorescens.	—	—	8	—	32
B. pumilus.	10	12	13	9	25
P. s. pv.. tomato.	14	17	20	14	35
P. tobacco.	11	13	17	—	38
X. campestris pv.. campestris.	12	14	15	8	43
X. campestris pv.. vesicatoria.	10	12	15	8	42
P. lachrymans.	12	14	14	—	31
P. syringae.	13	14	18	1	36
P. s. pv.. glycine.	14	15	19	8	33
P. s. pv.. phaseolicola.	12	14	17	—	38
P. gingeri.	—	9	9	—	35
P. gladioli pv.. agricola.	10	16	16	8	28
C. albicans.	13	14	14	10	34
C. globrata.	12	14	10	10	34
R. rubra.	13	13	15	11	8
G. graminis.	—	—	10	—	25
F. oxysporum.	—	—	15	—	20
F. moniliforme.	—	—	15	—	25
C. herbarum.	—	—	15	—	30
P. notatum.	10	—	10	—	24
A. parasiticus.	—	—	—	—	40
A. niger.	—	—	—	—	20
A. citri.	—	—	—	—	15
A. fumigatus.	—	—	—	—	13

Antimicrobial activity

The antimicrobial activities of the ethyl acetate extract of the samples were evaluated by means of the agar-well diffusion assay (Rojas et al., 2003; Şahin et al., 2003) with some modifications. Twenty milliliters of the specified molten agar (45°C) were poured into 9-cm, sterile Petri dishes. A suspension (100 µl) containing 10⁸ cfu/ml bacteria, 10⁶ cfu/ml yeasts, or 10⁴ spore/ml of fungi was spread on the plates of Nutrient agar (Merck, USA), Mueller-Hinton agar (Oxoid), or Sabouraud dextrose agar (Oxoid, UK) medium, respectively. Once the plates were dried aseptically, 6-mm wells were bored using a sterile cork borer. Extracts (50 µl) were placed into the wells and the plates were incubated for 37°C for 24 h for bacterial strains, 48 h for yeast, and at room temperature for 72 h for fungi. Chloramphenicol (10 mg/ml) for bacteria and ketoconazole (10 mg/ml) for yeast and fungi were used as standard antibiotics. The tests were carried out in triplicate. Antimicrobial activity was evaluated by measuring zone of inhibition against the test organism.

Results and Discussion

The microorganisms tested in this study comprised a panel of human pathogens, plant pathogens, food toxicants, and saprophytes (Table 1). The antimicrobial activities of Crataegus tanocetifolia., Crataegus x bornmüllerii., Pyrus serikensis., and P. communis. subsp. communis. ethyl acetate extracts against microorganisms were examined in the current study and their potency was qualitatively assessed by the presence or absence of inhibition zones and zone diameter (Table 1).

The results showed that the extract of Crataegus tanacetifolia. mediated some degree of activity against bacteria and yeasts (Table 1). Penicillium notatum. was the only fungus that was inhibited by the. C. tanacetifolia. extract. Salmonella typhimurium., P. fluorescens., and Pseudomonas gingeri. isolates were not inhibited by the ethyl acetate extract of C. tanacetifolia.. The most significant result was that yeast isolate Rhodotorola rubra. was inhibited by all of the plant extracts, and the inhibition zone diameter was higher than the standard antibiotic ketoconazole.

The extract of Crataegus x bornmülleri. showed some degree of activity against some bacteria and all yeasts (Table 1) but not fungi. C. x bornmülleri. did not inhibit the growth of Yersinia enterocolitica. and P. fluorescens.. None of the fungi showed any inhibition zone against C. bornmülleri. extracts. Inhibiton zone diameters of C. bornmülleri. were generally higher than C. tanacetifolia. extract.

The extract of Pyrus serikensis. exhibited some degree of activity against all the bacteria tested and some yeasts and fungi. Although inhibition zone diameters were lower than the standard antibiotics, P. serikensis. extract had higher activity degree than P. communis subsp.. communis. extract (Table 1). The most significant results with P. serikensis. extract were that P. serikensis. extract was the only test material that inhibited the growth of P. fluorescens.. However, Aspergillus niger., A. parasiticus., A. citri., and A. fumigatus. were not inhibited by the extract, although the other fungi were susceptible.

The Pyrus communis. subsp. communis. extract inhibited the growth of many bacteria and all the yeasts but not the fungi (Table 1). S. typhimurium., Brucella. spp., Streptococcus feacalis., P. fluorescens., Pseudomonas tobacco., Pseudomonas lachrymans., P. s.. pv. phaseolicola., Pseudomonas gingeri., and R. rubra. were not inhibited by the P. communis. subsp. communis. extract.

We have evaluated plants that are naturally grown in Turkey and preferably endemic and are potential useful resources. Therefore, these studies will be beneficial from medicinal and economic standpoints.

It is well-known that an extract of succulent young shoot tissue of pear exhibits strong antibacterial activity, and this activity was found in 12 cultivars of Chinese pears (P. ussuriensis.), Japanase pears (P. pyrifolia.), and European pears (P. communis.) (Jin & Sato, 2003). However, this is the first study that shows the antimicrobial activities of ethyl acetate extract of fruits of Pyrus communis. subsp. communis. and endemic Pyrus serikensis., Crataegus tanacetifolia., and Crataegus x bornmulleri..

Vierling et al. (2003) investigated the pharmaceutical and pharmacological equivalence of different Hawthorne extracts. They concluded that all extracts had a relaxant effect on aorta, although aqueous-alcoholic extracts were characterized by similar procyanidin, flavonoid, total vitexin, and total phenols content, and aqueous extract showed lower concentration of procyanidins, flavonoids, and total phenols but a similar total vitexin content.

Škerget et al. (2005) determined phenols, proanthocyanidins, quercetin, and apigenin in extracts of hawthorne (C. laevigata.) and concluded that these groups were responsible for the antioxidative activity.

Bilia et al. (1994) isolated a new flavanol glycoside, isorhamnetin 5-O.-β.-D galactopyranoside from the aerial parts of Pyrus bourgaeana., but there is no record on the effect of this substance in pharmaceutical and pharmacological use.

Jin and Sato (2003) determined that aqueous extracts from succulent young shoots of the pear Pyrus. spp. exhibited strong antibacterial activity against the bacterium Erwinia amylovora. bv. 4. The substance was identified as benzoquinone (2,5-cyclohexadiene-1,4-dione) by NMR spectra, mass spectra, and HPLC analysis. However, there is no record on the chemical composition of fruits of Pyrus. spp.

In conclusion, C. tanacetifolia., C. bornmülleri., P. serikensis., and P. communis. subsp. communis. collected from Turkey exhibited a broad range of antimicrobial activity to varying degrees. Particularly, P. serikensis. extract showed significant antifungal activities and could be used as antimicrobial agent in new drug therapy. Investigations on the chemical composition of the ethyl acetate extracts of fruits of Pyrus. spp. and Crataegus. spp. should be carried out in further studies to reveal their antimicrobial effect.

Acknowledgment

The authors are grateful to Dr. Alaattin Güven for his effort in preparation of extract of the plants by using Soxhlet apparatus.