Pharmaceutical Biology, 37, 3, 216-220. *** Doç.Dr. Metin DIGRAK *** http://www.oocities.org/mdigrak.geo

Antibacterial and Antifungal Effects of Various Commercial Plant Extracts

METIN DIGRAK1*

M.HAKKI ALMA²,

AHMET ILCIM¹

SELIM SEN²

^{1 Department of Biology, Faculty of Arts and Science, Kahramanmaraş Sütçü İmam University, TURKEY}

² Department of Industrial Eng. of Forestry ,Kahramanmaraş Sütçü İmam University Faculty of Forestry, TURKEY

*Corresponding author

ABSTRACT

The antimicrobial activities of valex (the extract of valonia), the extracts of mimosa bark, gallnut powders, Salvia aucheri Bentham var. aucheri and Phlomis bourgei Boiss were studied. The antimicrobial activity of the above plants was evaluated by the to disk diffusion method using Bacillus brevis FMC 3, Bacillus subtilis IMG 22, Bacillus cereus EÜ, Escherichia coli DM, Pseudomanas aeruginosa DSM 50071, Staphylococcus aureus Cowan 1, Listeria monocytogenes A, Micrococcus luteus LA 2971, Klebsiella pneumoniae FMC 5, Mycobacterium smegmatus RUT, Proteus vulgaris FMC 1 bacteria, and Alternaria alternata MDC 97, Penicillium italicum MDC 101, Fusarium equiseti C, Candida albicans CCM 314 fungi. The results indicated that mimosa bark extracts had the greatest antibacterial activity, followed by the valex, gallnut powders, Salvia aucheri var. aucheri and Phlomis bourgei extracts, respectively. Furthermore, it was found that gallnut powders and the extracts of mimosa bark contained high amounts of tannins and showed antifungal activity.

KEY WORDS: Antimicrobial activity; Gallnut powders; Mimosa bark; Phlomis bourgei; Salvia aucheri var. aucheri; Valex

INTRODUCTION

Recently, the inhibitory effect of various agricultural or forestry plant extracts on the growth of many bacteria in culture have been studied in our laboratory (İlçim et al., 1997ab; Dığrak et al., 1997; İlçim et al., 1998). However, the antimicrobial activities of the extracts of various vegetable materials, e.g., valonia, mimosa bark, garden sage, Phlomis bourgei, and gallnut have not been studied. First, valonia extracts, commercially called "valex", are rich in tannins and widely used in many areas e.g., in leather trade, pharmacy, and painting (Armağan, 1988). Valex produced by the extraction of valonia (a fruit of valonia oak, naturally and largely grown in Turkey as well as in Greece, Albania, Italy, Syria, Palestine, and Jordan) are used in the leather industry as a filler material (Bozkurt & Göker, 1986).

Valonia is utilized either in the form of an extract or direct powder. In the tanning of leather, valonia can be used either alone or mixed with the other tanning materials (e.g., the bark of pine, oak or spruce) to increase its penetration into leather. The valonia extracts are able to stabilize proteins in leather, thus protecting the leather against microorganisms decaying the leather. In this process, valonia fills in the pores remaining after taking the fats and hairs out of leather (Armağan, 1988). However, the antimicrobial activities of valonia have not been studied in detail.

Valonia includes hydrolyzable tannins, e.g., castalagin and vescalagin (Toptat, 1993). The components of valex determined by a filter method are as follows: Tannins, 68-70%; non-tannins, 25-25.5%; undissolved materials, 1.10-1.15%; moisture: 4.05-5.50 %. It was also found that the quality of the valex is comparable to the world standard (Anon., 1984).

In addition, the antibacterial activities of mimosa bark obtained from Acacia mollissima species grown in the Southern Africa was studied. Mimosa bark is sold either as sticks (20-25 cm), chopped bark (2-5 cm) or ground bark. Mimosa trees reach a length of 2-3 m within one year and form a bark with a thickness of 6-11 mm; the bark can be rich in tannins within 5-6 years (Browning, 1967).

As with valonia, mimosa bark has largely been used in the leather trade as filler/antibacterial materials. Mimosa bark can speedily tan leather and give a reddish color. Varying its color between red and violet, mimosa bark includes 25-35% tannins and give a bright section while being cut. The extracts obtained from mimosa bark by using hot-water or solvents (e.g., methanol and acetone) can also be used together with other tanning materials, e.g., valonia (Browning, 1967). The tannins of mimosa bark are condensed, e.g., 7,3',4'-tri- and 7,3',4',5'-tetra-hydroxy-flavan-3,4-diols (Hathway, 1975).

The third material examined in this study is the nodules occurring on the leaves of Quercus infectoria, which is also known as Turkish tannin or gallotannin (commercial name). It can directly be used after grinding (i.e.,in the form of powder). Since it contains high amounts of tannic acids such as gallic and egallic acids and is expensive, it is exported and used in the production of tannic acid. It makes leather open colored and is ideal tanning material. Its tannins are also included to the hydrolyzable tannin groups and consists of polygallol and m-digallolyl derivatives of glucose or polysaccharides (Bozkurt & Göker, 1986).

Gallnuts consist of gallic tannin (50-75%), gallic acid (2-3%), and ellagic acid (2%), and glucose, starch, and etheric fats (3%) (Hut, 1969). Since tannins of gallnuts are very valuable, it can be used in the production of ink, textile painting (as a fixator), and blue dye, rather than in processing leather. Among the materials having tannins, the highest amounts of tannins (64.18 and 69.70%) are found in gallnuts (Bozkurt & Göker, 1986).

The antibacterial of the extracts of some additional plant species, i.e., Salvia aucheeri var. aucheri and Phlomis bourgei, were also investigated. Salvia aucheeri var. aucheri is generally known as "garden sage" in Turkey. It has been used externally as an antiseptic (especially in the ear), gas remover, power-giving, and stimulator. It can be taken internally either in the form of an indusion (1-5%) or as a chemical component of gargle (Hathway, 1975). The Salvia species used in this study are mainly camphor and 1,8-cineol (Baytop, 1984). Phlomis bourgei species have traditionally been used for curing stomach ache in the city of Isparta-Turkey (Baytop, 1984). However, there is not any information about its chemical contents.

Table 1. Evaporation conditions as a function of stages.

Based on these uses, we considered to study the antibacterial activities of extracts of these vegetable materials and to extend their usages to new areas such as the wood industry. Therefore, as reported in this study, we determined the antimicrobial activities of extracts of valonia, mimosa bark, Salvia aucheeri var. aucheri, Phlomis bourgei, and gallnut powders, using a variety of common bacteria which have not been evaluated previously.

MATERIALS AND METHODS

Materials

The materials tested as bactericides in this study are valex that was obtained from the extraction valonia, an essential fruit of valonia oak (Quercus macrolepis Ky-Q. aegilops L.), grown in the Western Anatolia region (Turkey). Valonia consists of gland (camata; fruit in cupula), hoof (trillo; sharp and stubby points covered with cupula), and cup (cupula; outside of valonia). The extracts of mimosa barks provided from Acacia mollissima species were provided by Sümerbank Co. in Turkey, and gallnut powder was obtained from Quercus infectoria grown in the Southern Anatolia (Turkey). In addition, Salvia aucheri Bentham var. aucher (Adana-Pozantı) and Phlomis bourgei Boiss were collected in the city of K.Marat, Turkey.

Furthermore, standard antibiotic discs such as penicillin G, ampicillin, cefataxime, vancomycine, oflaxacin and tetracycline used for comparision were provided by the Microbiology Division of Medicine Faculty of Fırat University in Elazığ-Turkey. Bacillus brevis FMC 3, Bacillus subtilis IMG 22, Bacillus cereus EÜ, Escherichia coli DM, Pseudomanas aeruginosa DSM 50071, Staphylococcus aureus Cowan 1, Listeria monocytogenes A, Micrococcus luteus LA 2971, Klebsiella pneumoniae FMC 5, Mycobacterium smegmatus RUT, Proteus vulgaris FMC 1 bacteria, and Alternaria alternata MDC 97, Penicillium italicum MDC 101, Fusarium equiseti C, Candida albicans CCM 314 fungi were also provided by the Microbiology Division of Science Faculty of Fırat University in Elazığ-Turkey. In addition, choloroform was used as solvent for the extraction of the plants.

Methods

Extraction of the Plants

The extraction method used in the production of valex is a classic reverse current method. Valonia was firstly broken into small pieces 2-6 mm by using a cylinderic crasher. Then, the valonia pieces were extracted in hotwater at 60-70oC by using an extractor made of copper resistant to oxidation. The obtained valonia solution was acidic (i.e., pH 3-3.5). The solution with tannin was transfered into a diffussion tank, mixed with distilled water and filtered to remove inert materials. On the other hand, in order to increase the quality of valex, sodium bisulphide was added to the solution. The solution thus collected was indirectly heated by steam in a tank, charged into evaporation tanks (Armağan, 1988), and evaporated at three stages as summarized in Table 1.

Next, the concentrated solutions were filtered and transferred into the upper section of a drying tower. The solution was sprayed towards the middle of the tower in a thin form. Solution sprayed in pulverized form became powder after air drying at 185-200oC.

Finally, the dried valex was ground in a mill to get smaller particle sizes and collected in small plastic bags. On the other hand, mimosa bark directly provided from the company had been extracted by using hot-water at 100^oC (Armağan, 1988).

The collected Salvia and Phlomis species, and gallnut, were identified and brokeninto pieces under sterilized conditions. The pieces (20 g) were extracted with chloroform (150 ml) (Merck, Darmstadt) for 24 h by using a Soxhlet equipment (Erol & Tuzlacı, 1996; Dülger et al., 1997).

Preparation of Microorganism Culture

All the extracts thus obtained and the standard antibiotics were injected into empty sterilized antibiotic discs having a diameter of 6 mm (Schleicher & Shüll No: 2668, Germany) in the amount of 20 ml. The discs injected with only chloroform were used as a control. All the bacteria mentioned above were incubated at 30±0.1°C for 24 h by inoculation into Nutrient Broth (Difco), and the fungi studied were incubated in Malt Extract Broth (Difco) for 48 h. Mueller Hinton Agar (oxoid) sterilized in a flask and cooled to 45-50⁰C was distributed to sterilized Petri dishes having a diameter of 9 cm, by using pipettes in the amount of 15 ml after injecting cultures of bacteria prepared as mentoined above and mold for 24 h in the amount of 0.01 ml (10⁵ bacteria per ml and 10³ fungi spores per ml), and providing the distribution of food medium in Petri dishes homogenously. Dishes injected with extracts were located on the solid agar medium by pressing slightly (Özçelik, 1992; Collins et al., 1989; Bradshaw, 1992). After Petri dishes so-obtained were placed at 4⁰C for 2 h, plates inoculated with fungi were incubated at 25 ± 0.1⁰C for 7 days. At the end of the period, inhibition zones formed on the food medium were evaluated is millimeters. These studies works were performed in triplicate.

RESULTS AND DISCUSSION

Table 2 shows the in vitro antibacterial and antifungal activities of the extracts of valonia, mimosa bark, gallnut, salvia, and phlomis. As can clearly be seen from Table 2, the extracts of mimosa barks have the qreatest antibacterial efficiency, followed by valex, the extracts of Salvia sp., gallnut, and Phlomis sp., respectively. Moreover, it was determined that the extracts of gallnut and mimosa bark have antifungal effects, while the others (e.g., valex and phlomis) do not (Table 2).

Table 2. Antimicrobial activities of some palnt extracts

Inhibition zone, mm

A: Valex, B: Mimosa bark C: Gallnut D: Salvia aucheeri var. aucheri

E : Phlomis bourgei, F: Control (Chloroform)

Inactive (-); moderately active (7-13); highly active (>14).

*İncludes diameter of disc (6 mm).

The extracts obtained from valex inhibits considerably the growth of L. monocytogenes, P. vulgaris and B. brevis, having an inhibition zone of 22-31 mm. The growth of S. aureus and B. subtilis is inhibited by the whole extracts used in the study and an inhibition zone varying between 12 and 26 mm is formed. When the results obtained with valex were compared to those of standard antibiotics, it was determined that K. pneumoniae is more resistant, P. aeruginosa, M. luteus, and B. cereus are similarly resistant, and the other species is more susceptible to the valex. On the other hand, S. aureus and B. subtilis are more susceptible, L. monocytogenes, B. cerus and P. vulgaris are similarly resistant, and the others (except for K. pneumoniae) are more resistant to the gallnut extracts, as compared to VA 30, OFX 5 and TE 30 standard antibiotics.

B. brevis and S. aureus are similarly resistant and the other bacteria species are seen to be much more resistant to Phlomis extracts when compared to penicillin G. All the bacteria are found to be more susceptible to the extracts obtained from mimosa bark when compared to the standard SAM 20 and CTX 30 antibiotics. Similarly, in comparison to VA 30 standard, it was observed that P. vulgaris is more resistant and S. aureus is more susceptible to Salvia extracts.

Recently, İlçim et al. (1998) reported that the extracts of Parmelia furfuraceae, Myrtus communis subsp. communis and Eugenia coryophyllata showed antibacterial activities. Especially, E. caryophyllata was found to be very effective on all the bacteria tested (except for K. pneumoniae and E. aerogenes). In a similar study, B. megaterium was determined to be similarly resistant and the other bacteria (except for K. pneumoniae and S. aureus) were resistant to the extracts of Morus nigra, showing an inhibition zone of 7-9 mm. In addition, the extracts of Juniperus drupacea inhibited the growth of some bacteria at different ratios.

Also, Dığrak et al. (1997) found that extracts of Ajuga orientalis, Smyrinum olusatrum, Astragalus schizopterus, and Salvia viridis inhibited the growth of some bacteria and fungi. Especially, it was found that A. shizopterus was effective against Bacillus species with an inhibition of 23 mm and it inhibited the growth of Enterobacter aerogenes. The findings obtained from this study are almost the same as those stated above.

The extract of mimosa bark and gallnut powder inhibited the development of A. alternata, having inhibition zones of 21 and 15 mm, respectively. However, it was also determined that they were effective on the other fungi (P. italicum, F. equiseti, C. albicans). When the results were compared to standard antifungal nystatin, the extracts of mimosa bark and gallnut powder were found to be more effective.

It is not surprising that there are differences in the antibacterial effects of plant groups, due to phytochemical properties and differences among species. For the evaluation of plants which are naturally grown in Turkey and are potential useful resources, additional studies will be beneficial from medicinal and economic standpoints. In conclusion, whole extracts, especially the extracts of mimosa bark, valex, and gallnut powders can be used for protection against bacteria and, in some cases, against fungi.

REFERENCES

Anon (1984): Reports on Valex. Sümer Holding Co-Turkey.

Armağan M (1988): Evaluation of Valonia, Production of Valex and Its Usage Areas. Undergraduation Thesis, Pub. No: 8534, KTÜ Publication, Trabzon.

Baytop T (1984): Curing with the Plants in Turkey. İstanbul University, Pub. No: 3255, İstanbul.

Bozkurt Y, Göker Y (1986): Orman ürünlerinin kullanılması. İstanbul Üniversitesi Orman Fakültesi Yayınları, Tas Basımevi. 401 sayfa, İstanbul.

Bradshaw LJ (1992): Laboratory of Microbiology. Fourth Edition, Saunders College Publishing, Harcourt Brace Javanovich College Publishers. Printed in USA, pp. 435.

Browning BL (1967): Methods of Wood Chemistry. A Division of John Wiley Sons, New York, pp. 240.

Collins CH, Lyne PM, Grange JM (1989): Microbiological Methods. Sixth Edition, Butterworths & Co. Ltd. London, pp. 410.

Dığrak M, İlçim A, Tanış H, Bağcı E (1997): Kahramanmaraş yöresinde yetişen bazı bitkilerin antimikrobiyal aktivitesi. II. Spil Fen Bilimleri Kongresi, 23-25 Ekim 1997, Celal Bayar Üniversitesi, Manisa.

Dülger B, Gücin F, Kara A, Aslan A (1997): Usnea florida (L.) Wigg. likeninin Antimikrobiyal Aktivitesi. Turkish J Biol 21: 103-108.

Erol MK, Tuzlacı E (1996): Plants used as traditional folk medicine in Eğirdir (Isparta). Proceedings of the XI^th Symposium on Plant Originated Crude Drugs, May 22-24, Ankara.

Hathway DE (1975): Condensed Tannins; In: The Chemistry of Wood,. Browning BL, Robert E, Krieger Publishing Co. New York, pp. 202.

Huş S (1969): Orman Ürünleri Kimyası. Kurtuluş Yayınevi, İstanbul, sayfa 78.

İlçim A, Dığrak M, Bağcı E (1997a): Juniperus drupacea lab., Morus nigra ve Jasminum fruticans L.’nin antimikrobiyal etkisi. I. Kızılırmak Fen Bilimleri Kongresi, 14-16 Mayıs 1997, Bildiriler Kitabı, Sayfa 116-121. Kırıkkale Üniversitesi, Kırıkkale.

İlçim A, Dığrak M, Bağcı, E (1997b): Bazı bitki ekstraktlarının antimikrobiyal etkilerinin araştırılması. Kükem Dergisi, 10. Kükem Kongresi Özel Sayısı 20:18-19.

İlçim A, Dığrak M, Bağcı E, (1998): Bazı bitki ekstrelerinin antimikrobiyal etkilerinin araştırılması. Turkish J Biol 22:119-125.

Özçelik S (1992): Gıda Mikrobiyolojisi Laboratuvar Kılavuzu. Elazığ Fırat Üniversitesi Fen-Edebiyat Fakültesi Yayınları, Yayın No: 1, sayfa 135.

Toptaş A (1993): Leather Technology. Sade Ofset Matbaacılık, İstanbul, sayfa 237.