FABAD J. Pharm. Sci., 33, 77–86, 2008

RESEARCH ARTICLE

Antimicrobial and Cytotoxic Activities of officinalis Extracts

Ayşe NALBANTSOY*°, Duygu AYYILDIZ TAMİŞ*, I. Hakkı AKGÜN*, Tansel ÖZTÜRK YALÇIN*, İsmet DELILOĞLU GÜRHAN*, İsmail KARABOZ**

Antimicrobial and Cytotoxic Activities of Zingiber Zingiber officinalis ekstrelerinin antimikrobiyal ve officinalis Extracts sitotoksik aktiviteleri

Summary Özet In the present study, the edible Zingiber officinalis Bu çalışmada, yenilebilir bir bitki olan Zingiber officinalis’ ethanol and chloroform extracts were prepared and their in etil alkol ve kloroform ekstraktları hazırlanmış ve bu cytotoxic effects versus human cervical cancer (HeLa) and ekstraktların servikal kanser (HeLa) ve fare fibroblast (L929) mouse fibroblast (L929) cell-lines were investigated. HeLa hücre hatları üzerine olan sitotoksik etkisi araştırılmıştır. 4 4 and L929 cell lines in 96 well microplates were cultivated HeLa ve L929 hücreleri sırasıyla 8×10 hücre/mL ve 7.4×10 for 24 h with initial concentrations of 8×04 cells/mL and hücre/mL başlangıç hücre konsantrasyonlarında 96 gözlü 7.4×104 cells/mL, respectively. After that the cultures were pleytlere ekilmiş ve 24 saat kültüre edilmiştir. Ardından treated with different dilutions of the extracts and incubated kültür, farklı dilüsyonlara sahip ekstraktlar ile 48 saat for 48 h. The growth inhibition was determined to the 50% muamele edilmiştir. Büyüme inhibisyonu, üreyen hücrelerin %50’ini inhibe eden doz (IC50) % ±95 güven aralığında inhibition concentration (IC50). IC50 values were reported as ±95 % confidence intervals (±95 % CI) by using Graph GraphPad Prism ile analizlenerek belirlenmiştir. Ekstraktların Pad Prism (San Diego, CA). The antibacterial activity of antimikrobiyal aktivitesi disk difüzyon metodu kullanılarak the extracts was tested by the paper disc diffusion farklı mikroorganizma türlerine karşı taranmıştır. Morfolojik technique. The results of the morphological observation and incelemeler ve MTT sonuçları, hazırlanan ekstraktların MTT test indicated that the cytotoxic activity of the extracts kanser hücrelerine karşı doza bağlı olarak etkili olduğunu göstermiştir. Kloroform ekstraktının IC50 değeri L929 ve were dose dependent. IC50 values versus L929 and HeLa cells were found to be 87.28 µg/mL and 74.32 µg/mL, respectively, HeLa hücreleri için sırasıyla 87.28 µg/mL ve 74.32 µg/mL, for the chloroform extract, while the ethanol extract showed etanol ekstraktının ise yine sırasıyla 101.0 µg/mL, ve 33.78 µg/mL olarak tespit edilmiştir. Ayrıca ekstraktların Klebsiella IC50 values at 101 µg/mL and 33.78 µg/mL, respectively. Moreover, the extracts were evaluated for their antimicrobial pneumoniae, Salmonella thyphimurium, Bacillus cereus, activities against Klebsiella pneumoniae, Salmonella Enterococcus fecalis, Staphylococcus aureus bakterilerine thyphimurium, Bacillus cereus, Enterococcus fecalis and karşı antimikrobiyal aktiviteleri de değerlendirilmiştir. Staphylococcus aureus. The antimicrobial activity results Antimikrobiyal aktivite sonuçları, zencefil ekstraktlarının showed that the ginger extracts inhibited the growth of five sekiz organizmadan beşinde üremeyi inhibe ettiğini ancak out of eight microorganisms but had no effect on the growth Escherichia coli ATCC11230, Pseudomonas aeruginosa ATCC of Escherichia coli ATCC11230, Pseudomonas aeruginosa 27853 ve Staphylococcus epidermidis ATCC 12228 üzerinde ATCC 27853, and Staphylococcus epidermidis ATCC 12228. herhangi bir etkisinin olmadığını göstermiştir.

Keywords: Zingiber officinalis, HeLa, L929, anticancer Anahtar Kelimeler: Zingiber officinalis, HeLa, L929, anti effect, antimicrobial activity. kanser etki, antimikrobiyal aktivite Received: 26.11.2009 Revised: 25.02.2010 Accepted: 05.03.2010

INTRODUCTION

Over the past decade herbal medicine has gained health-care (1). According to the World Health importance and has increased the public’s desire Organization, about three quarters of the world’s for medicinal to play a part in their own population currently uses herbs and other forms of

* Ege University, Faculty of Engineering, Department of Bioengineering, 35100 Bornova, Izmir, Turkey. ** Ege University, Faculty of Science, Department of Biology, Basic and Industrial Microbiology Section, 35100 Bornova, Izmir, Turkey. ° Corresponding author E-mail: [email protected]

77 Nalbantsoy, Tamiş, Akgün, Yalçın, Gürhan, Karaboz traditional medicine to treat diseases. Conventional anti-inflammatory activities (9). medicine is widely used in India, even in USA, use of plants and phytomedicine has increased dramatically Members of (ginger species) have in the last two decades (2). been shown to have antioxidant, anti-inflammatory, and anticancer activities. Nevertheless, of the large Many naturally occurring substances present in the number of Zingiberaceae spp. that have been used human diet have been identified as potential chemo for culinary and/or medicinal purposes, only a preventive agents; and consuming moderately few members have been studied for their potential large amounts of vegetables and fruits can prevent anticancer activity (10). the development of cancer (3). Cervical cancer is the second most common cancer among women In this study, the edible plant ginger’s ethanol worldwide as reported in 2002 (4). and chloroform extracts were prepared and the cytotoxic effects of extracts on cervical cancer (HeLa) The current investigations are generally subjected to and mouse fibroblast (L929) cells as well as their the isolated plant components, which show desirable antimicrobial activity were investigated. properties in related therapy. The present research deals with the extracts and components isolated MATERIAL and METHODS from the plant genus Zingiber and the of Preparation of the Extracts the species Zingiber officinale (also known as ginger), Extracts were prepared from Zingiber officinalis roots which have activities with broad applicability in obtained from an open market in Izmir, Turkey. All the field of inhibition and treatment of infections chemicals used during extraction process were by pathogenic microorganisms including viruses, purchased from Merck, Germany. Two portions of bacteria, protozoa and parasites (5,6). 500 mg finely ground ginger were extracted with 50 mL ethanol and chloroform at reflux (Heidolph MR Z. officinale Roscoe is a well-liked spice used Hei-Standard, Germany) for 3 hours. After cooling worldwide as a food seasoning, and has been used down to room temperature, the extracts were filtered as a folk medicine in Korea, China, and Japan for the through filter paper. Clear extracts were evaporated treatment of gastrointestinal disorders, dyspepsia, under vacuum (Heidolph Laborota 4000 efficient, nausea, vomiting, pain, common cold, diarrhea and Germany; Vacuubrand RE 2.5 vacuum pump, cough. Previous study of the chemical constituents Germany) and then freeze-dried (Chirst Alpha of Zingiber officinale dealt with volatile oils, 1-4, Germany; Vacuubrand RE 2.5 vacuum pump, diarylheptanoids (7) and phenolic ketones, Germany) (11). and -related compounds (8). Cell Culture and Maintenance Ginger extracts rich in , , and Human cervical carcinoma (HeLa) and mouse other substances (gingerdiols, diaryltheptanoids, fibroblastic (L929) cell-lines were purchased from zingiberene, arcurcumene, beta-bisabolene, neral, the HUKUK (Animal Cell Culture Collections) geranial, D-camphor, beta-phellandrene, geranial, in Foot-and-Mouth Disease Institute (Ankara) of linalool, E-alpha-farnesene, beta-eudesmol) have Ministry of Agriculture & Rural Affairs of Turkey. recently gained significant interest for their capacity The cell lines were maintained in RPMI 1640 (Gibco, to interfere with cancer at the initiation, progress and U.K.) medium supplemented with 10% heat– treatment phases. The gingerols (such as 6-, 8-, and inactivated fetal bovine serum, 1% L-glutamine 10-gingerols), a series of phenolic compounds present (Biochrome, Germany) and 1% gentamycine in ginger root (ginger containing 1.0–3.0% gingerols), (Biochrome, Germany) in a humidified atmosphere o have been shown to have chemopreventative with 5% CO2, at 37 C. The cells were subcultured effects that are related to their antioxidative and twice a week.

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least three independent measurements that were

reproducible and statistically significant. The IC50 In-vitro Cytotoxicity Assay values were reported at ±95% confidence intervals Screening of the extracts’ cytotoxicity, based on (±95% CI). This analysis was performed with Graph metabolic cell viability, was done using a modified Pad Prism (San Diego, U.S.A). MTT [3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H- tetrazolium bromide)] assay (12,13) that effects the Microorganisms mitochondrial reductase activity of viable cells. The Gram-positive and gram-negative bacteria and yeast survivals of viable cells after treatment of extracts in were used for antimicrobial activity studies. The monolayer culture were determined. HeLa and L929 Gram-negative bacteria used were Escherichia coli cell line cultivated for 24 h in 96 well microplates ATCC29998, Pseudomonas aeruginosa ATCC 27853, with 8 x 104 cells/mL and 7.4×104 cells/mL as initial and Salmonella thyphimurium CCM 5445. The Gram- concentrations, respectively. After that the cultured positive bacteria used were Bacillus cereus ATCC cells were treated with different dilutions of the 7064, Enterococcus fecalis ATCC 29212, Staphylococcus extracts and incubated for 48 h. The growth inhibition aureus ATCC 6538/P, and Staphylococcus epidermidis was compared with the untreated controls and it was ATCC 12228. The yeast used was Candida albicans found that the extract concentration inhibits growth ATCC 10239. The lyophilized bacteria and yeast were by 50% (IC50). obtained from the Standard ATCC bacteria strain and Standard ATCC fungi strain collection of the Ege The assay is based on cleavage of the yellow University, Faculty of Science, Department of Basic tetrazolium salt, MTT, which forms water-insoluble, and Industrial Microbiology. dark blue formazan crystals. This cleavage only takes place in living cells by the mitochondrial enzyme Antimicrobial Activity Studies succinate-dehydrogenase. The water-insoluble, The antibacterial activity of the Ginger extracts dark blue formazan crystals are solubilized by was tested by the paper disc diffusion technique using dimethyl sulfoxide. The optical density of the (2,14). The 30μL of each extract (chloroform extracts, dissolved material is measured at 570 nm (reference concentration 12.5 mg/mL; ethanol extracts, filter, 690 nm) with U.V. visible spectrophotometer concentration 2.5 mg/mL) of Zingiber were absorbed (Molecular-Devices, U.K.). onto sterile 6 mm diameter filter paper discs (Schleicher and Schul, Nr 2668, Dassel, Germany). Morphological Studies The morphological studies of the cells were done with Bacterial strains were inoculated on Mueller-Hinton inverted microscope (Olympus, Japan) comparing broth (MHB) (Oxoid), fungal strain was inoculated with the control group 24 h after treatment. on Sabouraud Dextrose Broth (SDB) (Difco) and incubated for 24 h at 37° ± 0.1°C. Adequate amounts Data Analysis of autoclaved Muller-Hinton Agar (Oxoid) and Cytotoxicity was expressed as mean percentage Sabouraud Dextrose Agar (SDA) (Difco) were poured increase relative to the unexposed control ± SD. Control into sterile plates, and allowed to solidify under values were set at 0% cytotoxicity. Cytotoxicity data aseptic conditions. Bacterial strains were adjusted to (where appropriate) was fitted to a sigmoidal curve yield approximately 1.0 × 107–1.0 × 108 cfu mL−1 and and a four parameters logistic model used to calculate fungal strains were adjusted to yield approximately 5 the IC50, which is the concentration of nanomaterial 1.0 × 10 using the Standard McFarland counting causing 50% inhibition in comparison to untreated method. 0.1 mL of the each test organism was controls. The mean IC50 is the concentration of inoculated with a sterile swab on the surface of the agent that reduces cell growth by 50% under the appropriate solid medium in the plates. experimental conditions and is the average from at

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Agar plates containing the test organisms were The similar cytotoxic effect was shown with ethanol incubated for 1 h before placing the extract impregnated extract on L929 cells. Cytotoxicity level depends on paper discs on the plates. Then, the sterile discs the concentration of ethanol extract. The cytotoxicity impregnated with different extracts were placed on was determined at 80 µg/mL and increases gradually the agar plates and incubated at 37º ± 0.1 ºC for 24 h. at higher concentrations. The calculated IC50 value Antibacterial activity was determined by measuring was 101.0 µg/mL. the inhibition zone (in mm) against the test organisms (15,17). All experiments were done under sterile Cytotoxic effect on human cervical cancer (HeLa) conditions in duplicate. The standard antibacterial cells agent ceftazidime (20 mg/disc) was used as a positive The cytotoxic effects of the ginger extracts showed control for bacteria, and the standard antifungal agent similarity on HeLa cells at different concentrations nystatin (20 mg/disc) was used as the positive control with L929 cells. There are distinctions in the for yeast. All experiments were repeated three times. IC50 values of the extracts on HeLa cells. The DMSO was used as a negative control. determined cytotoxic level of chloroform extract

and the IC50 value were 80 µg/mL and 74.32 µg/ RESULTS mL, respectively. Extraction of Samples At the end of the freeze drying 41.75 mg ethanol The cytotoxic effect of the ethanol extract on HeLa extract and 22.85 mg chloroform extract were was found to be 20 µg/mL and the effect amplified at obtained. The yields were 8.35% and 4.57% for higher concentrations, 5-500 µg/mL. The IC50 value ethanol and chloroform extracts, respectively. These was calculated as 133.78 µg/mL. extracts were prepared as a stock solution in DMSO and then they were diluted in culture medium for Morphological studies further experiments. In this part of the experiment, the morphological changes were obtained for L929 (Figure 1 and 3) and Result of Cytotoxicity Test HeLa cells (Figure 2 and 4), which were growing in

In this study, the cytotoxic effect and IC50 value of logarithmic phase throughout the treatment with ginger extracts on cervical cancer (HeLa) and mouse extracts. After the 48 h post-treatment of extracts, fibroblast (L929) cells were investigated by using an increased number of rounded cells and growth different concentration of the extracts and the results inhibition were observed when compared with the are shown in Table 1. untreated control cells.

Cytotoxic Effect on Mouse Fibroblast (L929) Antimicrobial Activity Studies Cells Results from the antimicrobial activity screening The MTT assay result showed that chloroform tests are shown in Table 2. As clearly seen in Table 2, extract inhibits cell proliferation in a dose-dependent the ginger extracts inhibited the growth of five out of manner. The IC50 value was found to be 87.28 µg/mL. eight microorganisms but had no effect on the growth of Escherichia coli ATCC11230, Pseudomonas aeruginosa

Table 1. The IC50 value of ginger extracts

Cell line Extract IC50 (μg/mL)

Chloroform 87.28 L929 Ethanol 101.0 Chloroform 74.32 HeLa Ethanol 33.78

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A A

B B

C C

Figure 1. Morphological changes towards L929 as viewed Figure 2. Morphological changes towards HeLa as viewed under the inverted microscope after the post-treatment of under the inverted microscope after the post-treatment of ethanol extract. (a) Untreated cells (b) treated cells with 500 ethanol extract. (a) Untreated cells (b) treated cells with μg/mL (c) treated cells with 100 μg/mL (Magnification 500 μg/mL (c) treated cells with 40 μg/mL (Magnification 10X). 20X).

DISCUSSION ATCC 27853, and Staphylococcus epidermidis ATCC Since cervical cancer is the second most common 12228. In this study, the antimicrobial activities of cancer worldwide, it continues to be one of the deadly Ginger extracts of 20 mL/discs were compared with health problems. The chemotherapeutic drugs standard antibiotics ceftazidime (CF20) and nystatin (NS20) used as positive controls. 81 Nalbantsoy, Tamiş, Akgün, Yalçın, Gürhan, Karaboz

A A

B B

C C

Figure 3. Morphological changes towards L929 as Figure 4. Morphological changes towards HeLa as viewed under the inverted microscope after the viewed under the inverted microscope after the post- post-treatment of chloroform extract. (a) Untreated treatment of chloroform extract. (a) Untreated cells control (b) treated cells at 250 μg/mL(c) treated cells (b) treated cells with 80 μg/mL (c) treated cells with at 10 μg/mL (Magnification 20X). 40 μg/mL (Magnification 20X). destroy cancer cells by interfering the cell division use of natural compounds extracted as an antioxidant and growth. The affected cells become damaged and and functional foods from fruits, vegetables, oil seeds eventually die. Nevertheless, apart from affecting the and herbs has recently become a global tendency. The cancer cells, these drugs also affect normal cells. The World Health Organization estimated that 80% of the

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Table 2. Antibacterial activity of the ginger extracts by the disc diffusion method

Zingiber Standards

Clorofom Ethanol Microorganism extract extract CF 20 NS 20 DMSO

Inhibition Zone ( mm )*

Enterıcoccus faıcalis G(+) 10 9 24 Nt - ATCC 29212 Staphylococcus aureus G(+) 9 9 17 Nt - ATCC 6538/P Staphylococcus epidermidis G(+) - - 12 Nt - ATCC 12228 Bacillus cereus G(+) 10 9 11 Nt - ATCC 7064 Salmonella typhimurium G(-) 10 9 20 Nt - CCM 5445 Pseudomonas aeruginosa G(-) - - 25 Nt - ATCC 27853 Escherichia coli G (-) - - 21 Nt - ATCC 29998 Candida albicans yeast 10 9 Nt 19 - ATCC 10231

CF20: Ceftazidime (20 mg); NS 20: Nystatin (20 mg) ; Nt.: not tested; -: not active. * Includes diameter of disc (6 mm). world’s population relies on conventional medicine Some compounds present in ginger may exert for their main health care requirements and most of cancer preventive effects by inducing apoptosis this therapy involves the use of plant extracts or their in cancerous or transformed cells. The oleoresin active components. Recently, twenty five percent of from the root of ginger contains [6]-gingerol, the modern drugs prescribed worldwide are currently major pharmacologically active component and used in the treatment of various illnesses (4). lesser amounts of a structurally related vanilloid, [6]-paradol (18). Since Turkey has a broad plant diversity, herbal therapy is a method people trust in. However, even In this research ethanol and chloroform extracts though the activities against diseases of some plants of ginger were studied. This study confirmed have not been confirmed yet, the people are using that the extracts applied on HeLa and relatively them unconsciously. Ginger is also one of the most more resistant cells L929 lead to death. This effect commonly used plants in Turkey to cure some of the was demonstrated with both MTT test and the diseases. On the other hand, it can easily be found morphological appearances (Figure 1,2,3 and 4) of all around the world. Consequently, the activities of the cultures under microscope after 24 h of post- ginger and its related species have prominently been treatment with the extracts. Considering the IC50 in research for a long time. value the anticancer effect of the chloroform extract

83 Nalbantsoy, Tamiş, Akgün, Yalçın, Gürhan, Karaboz was less than the effect of the ethanol extract on L929 et al., who used MDA-MB-231 human breast cancer mouse fibroblast cells. However, the reverse effect cells (21). And, also another study by Brown et al., has been acquired for the HeLa cells. On the contrary, (9) was carried out on a different cancer cell line this result proved the sensitivity of HeLa cells (YYT colon cancer cells); in vitro results showed that compared to L929, which are not cancer cell-line. A ginger extract and 6-gingerol both directly interfered study about apoptogenic effects of zerumbone (ZER) with colon cancer cell proliferation, and interfered on HeLa cells was carried out by Wahab et al. (19). In indirectly by blocking the delivery of angiogenic that study examine, the results of MTT assay showed signals to the endothelial cells that supplied blood that ZER has less effect on normal cells compared to the colon cancer cells. These results indicate that to cancer cells (19). As a consequence, this observed ginger’s phytochemicals have distinct potentials for result showed a resemblance with our results. chemoprevention and chemotherapy strategies (9).

Natural compounds that inhibit or suppress the In the present study, the ethanol and chloroform proliferation of cancer cells are called as anti-tumor extracts were found to possess antibacterial agents. The anti-cancer agents derived from the properties against five out of eight microorganisms edible plants present a big advantage due to their but had no effect on the growth of Escherichia coli low toxicity. And in this study, we showed that ATCC11230, Pseudomonas aeruginosa ATCC 27853, and ginger can be used as an efficient cytotoxic agent and Staphylococcus epidermidis ATCC 12228 in our study. causing the death of cervical cancer cells. According Our results show similarity with the studies which to the obtained outcomes from the morphological were carried out by Akoachere et al., (22), Singh observation and MTT test, the prepared extracts et al., (23) and Chen et al., (24). They also showed showed cytotoxic activity against cancer cells that crude extracts of the ginger rhizome inhibit the dependent on doses. Kirana et al., (7) have reported growth of certain Gram-negative and Gram-positive the anticancer properties of zerumbone (2,6,9 bacteria and showed antifungal activities as well humulatriene-8-one, a sesquiterpenoid) from as respiratory tract pathogens Staphylococcus aureus, Zingiber aromaticum that were analyzed with in vitro Streptococcus pyogenes, Streptococcus pneumoniae and MTT tetrazolium salt assay using HT-29, CaCo-2, Haemophilus influenza. and MCF-7 cancer cells and showed that Zerumbone is effective as an anticancer agent, possibly by its Conclusion apoptosis-inducing and anti-proliferative influences Today, medicinal ginger is used mainly for the pre- (11). Similarly, Abdul A.B.H et al., (1) extracted, vention of the symptoms of travel sickness and in isolated and purified from the of edible cancer chemoprevention. Due to its abundance, low plant by using methanol extraction cost and safety in consumption, ginger has been the and column chromatography (CC) method. The subject of concentrated scientific research over the study investigated the purified zerumbone crystals past two decades investigating its anticarcinogenic, for its anticancer properties on human cervical antibacterial, antifungal, hypoglycemic, and antia- cancer cell line (HeLa); on the other hand, cisplatin therosclerotic activities. This study showed a prom- was used as a positive control. As a result, important ising way towards ginger’s chloroform and ethanol growth retardation was identified in the HeLa cancer extracts for their potential antimicrobial and antican- cells, after the treatment with both zerumbone and cer effects. Further studies on determining the anti- cisplatin compounds (4). Wei et al. showed that many cancer activity of ginger and its active components diarylheptanoids and gingerol-related compounds should ideally include human intervention trials to obtained from the rhizome of ginger (Zingiber investigate its effectiveness against human cancers officinale) possess significant anti-proliferation and other diseases. activity on HL-60 cells, likely via induction of the cell apoptosis (20). In a similar manner, [6]-Gingerol ACKNOWLEDGEMENT inhibits metastasis of cancer cells was shown by Lee We would like to thank Prof. Dr. Erdal Bedir for his

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