• Sonuç bulunamadı

Antimicrobial and Cytotoxic Activities of Zingiber officinalis Extracts

N/A
N/A
Protected

Academic year: 2021

Share "Antimicrobial and Cytotoxic Activities of Zingiber officinalis Extracts"

Copied!
10
0
0

Yükleniyor.... (view fulltext now)

Tam metin

(1)

Antimicrobial and Cytotoxic Activities of Zingiber officinalis Extracts

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

Antimicrobial and Cytotoxic Activities of Zingiber officinalis Extracts

Summary

In the present study, the edible plant Zingiber officinalis ethanol and chloroform extracts were prepared and their cytotoxic effects versus human cervical cancer (HeLa) and mouse fibroblast (L929) cell-lines were investigated. HeLa and L929 cell lines in 96 well microplates were cultivated for 24 h with initial concentrations of 8×04 cells/mL and 7.4×104 cells/mL, respectively. After that the cultures were treated with different dilutions of the extracts and incubated for 48 h. The growth inhibition was determined to the 50%

inhibition concentration (IC50). IC50 values were reported as ±95 % confidence intervals (±95 % CI) by using Graph Pad Prism (San Diego, CA). The antibacterial activity of the ginger extracts was tested by the paper disc diffusion technique. The results of the morphological observation and MTT test indicated that the cytotoxic activity of the extracts were dose dependent. IC50 values versus L929 and HeLa cells were found to be 87.28 µg/mL and 74.32 µg/mL, respectively, for the chloroform extract, while the ethanol extract showed IC50 values at 101 µg/mL and 33.78 µg/mL, respectively.

Moreover, the extracts were evaluated for their antimicrobial activities against Klebsiella pneumoniae, Salmonella thyphimurium, Bacillus cereus, Enterococcus fecalis and Staphylococcus aureus. The antimicrobial activity results showed that the ginger extracts inhibited the growth of five out of eight microorganisms but had no effect on the growth of Escherichia coli ATCC11230, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus epidermidis ATCC 12228.

Keywords: Zingiber officinalis, HeLa, L929, anticancer effect, antimicrobial activity.

Received: 26.11.2009 Revised: 25.02.2010 Accepted: 05.03.2010

Zingiber officinalis ekstrelerinin antimikrobiyal ve sitotoksik aktiviteleri

ÖzetBu çalışmada, yenilebilir bir bitki olan Zingiber officinalis’

in etil alkol ve kloroform ekstraktları hazırlanmış ve bu ekstraktların servikal kanser (HeLa) ve fare fibroblast (L929) hücre hatları üzerine olan sitotoksik etkisi araştırılmıştır.

HeLa ve L929 hücreleri sırasıyla 8×104 hücre/mL ve 7.4×104 hücre/mL başlangıç hücre konsantrasyonlarında 96 gözlü pleytlere ekilmiş ve 24 saat kültüre edilmiştir. Ardından kültür, farklı dilüsyonlara sahip ekstraktlar ile 48 saat muamele edilmiştir. Büyüme inhibisyonu, üreyen hücrelerin

%50’ini inhibe eden doz (IC50) % ±95 güven aralığında GraphPad Prism ile analizlenerek belirlenmiştir. Ekstraktların antimikrobiyal aktivitesi disk difüzyon metodu kullanılarak farklı mikroorganizma türlerine karşı taranmıştır. Morfolojik incelemeler ve MTT sonuçları, hazırlanan ekstraktların kanser hücrelerine karşı doza bağlı olarak etkili olduğunu göstermiştir. Kloroform ekstraktının IC50 değeri L929 ve HeLa hücreleri için sırasıyla 87.28 µg/mL ve 74.32 µg/mL, etanol ekstraktının ise yine sırasıyla 101.0 µg/mL, ve 33.78 µg/mL olarak tespit edilmiştir. Ayrıca ekstraktların Klebsiella pneumoniae, Salmonella thyphimurium, Bacillus cereus, Enterococcus fecalis, Staphylococcus aureus bakterilerine karşı antimikrobiyal aktiviteleri de değerlendirilmiştir.

Antimikrobiyal aktivite sonuçları, zencefil ekstraktlarının sekiz organizmadan beşinde üremeyi inhibe ettiğini ancak Escherichia coli ATCC11230, Pseudomonas aeruginosa ATCC 27853 ve Staphylococcus epidermidis ATCC 12228 üzerinde herhangi bir etkisinin olmadığını göstermiştir.

Anahtar Kelimeler: Zingiber officinalis, HeLa, L929, anti kanser etki, antimikrobiyal aktivite

INTRODUCTION

Over the past decade herbal medicine has gained importance and has increased the public’s desire for medicinal plants to play a part in their own

health-care (1). According to the World Health Organization, about three quarters of the world’s 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.

(2)

traditional medicine to treat diseases. Conventional medicine is widely used in India, even in USA, use of plants and phytomedicine has increased dramatically in the last two decades (2).

Many naturally occurring substances present in the human diet have been identified as potential chemo preventive agents; and consuming moderately large amounts of vegetables and fruits can prevent the development of cancer(3). Cervical cancer is the second most common cancer among women worldwide as reported in 2002 (4).

The current investigations are generally subjected to the isolated plant components, which show desirable properties in related therapy. The present research deals with the extracts and components isolated from the plant genus Zingiber and the rhizome of the species Zingiber officinale

(

also known as ginger), which have activities with broad applicability in the field of inhibition and treatment of infections by pathogenic microorganisms including viruses, bacteria, protozoa and parasites (5,6).

Z. officinale Roscoe is a well-liked spice used worldwide as a food seasoning, and has been used as a folk medicine in Korea, China, and Japan for the treatment of gastrointestinal disorders, dyspepsia, nausea, vomiting, pain, common cold, diarrhea and cough. Previous study of the chemical constituents of Zingiber officinale dealt with volatile oils, diarylheptanoids (7) and phenolic ketones, paradol and gingerol-related compounds (8).

Ginger extracts rich in gingerols, shogaols, and other substances (gingerdiols, diaryltheptanoids, zingiberene, arcurcumene, beta-bisabolene, neral, geranial, D-camphor, beta-phellandrene, geranial, linalool, E-alpha-farnesene, beta-eudesmol) have recently gained significant interest for their capacity to interfere with cancer at the initiation, progress and treatment phases. The gingerols (such as 6-, 8-, and 10-gingerols), a series of phenolic compounds present in ginger root (ginger containing 1.0–3.0% gingerols), have been shown to have chemopreventative effects that are related to their antioxidative and

anti-inflammatory activities (9).

Members of Zingiberaceae (ginger species) have been shown to have antioxidant, anti-inflammatory, and anticancer activities. Nevertheless, of the large number of Zingiberaceae spp. that have been used for culinary and/or medicinal purposes, only a few members have been studied for their potential anticancer activity (10).

In this study, the edible plant ginger’s ethanol and chloroform extracts were prepared and the cytotoxic effects of extracts on cervical cancer (HeLa) and mouse fibroblast (L929) cells as well as their antimicrobial activity were investigated.

MATERIAL and METHODS Preparation of the Extracts

Extracts were prepared from Zingiber officinalis roots obtained from an open market in Izmir, Turkey. All chemicals used during extraction process were purchased from Merck, Germany. Two portions of 500 mg finely ground ginger were extracted with 50 mL ethanol and chloroform at reflux (Heidolph MR Hei-Standard, Germany) for 3 hours. After cooling down to room temperature, the extracts were filtered through filter paper. Clear extracts were evaporated under vacuum (Heidolph Laborota 4000 efficient, Germany; Vacuubrand RE 2.5 vacuum pump, Germany) and then freeze-dried (Chirst Alpha 1-4, Germany; Vacuubrand RE 2.5 vacuum pump, Germany) (11).

Cell Culture and Maintenance

Human cervical carcinoma (HeLa) and mouse fibroblastic (L929) cell-lines were purchased from the HUKUK (Animal Cell Culture Collections) in Foot-and-Mouth Disease Institute (Ankara) of Ministry of Agriculture & Rural Affairs of Turkey.

The cell lines were maintained in RPMI 1640 (Gibco, U.K.) medium supplemented with 10% heat–

inactivated fetal bovine serum, 1% L-glutamine (Biochrome, Germany) and 1% gentamycine (Biochrome, Germany) in a humidified atmosphere with 5% CO2, at 37oC. The cells were subcultured twice a week.

(3)

In-vitro Cytotoxicity Assay

Screening of the extracts’ cytotoxicity, based on metabolic cell viability, was done using a modified MTT [3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H- tetrazolium bromide)] assay (12,13) that effects the mitochondrial reductase activity of viable cells. The survivals of viable cells after treatment of extracts in monolayer culture were determined. HeLa and L929 cell line cultivated for 24 h in 96 well microplates with 8 x 104 cells/mL and 7.4×104 cells/mL as initial concentrations, respectively. After that the cultured cells were treated with different dilutions of the extracts and incubated for 48 h. The growth inhibition was compared with the untreated controls and it was found that the extract concentration inhibits growth by 50% (IC50).

The assay is based on cleavage of the yellow tetrazolium salt, MTT, which forms water-insoluble, dark blue formazan crystals. This cleavage only takes place in living cells by the mitochondrial enzyme succinate-dehydrogenase. The water-insoluble, dark blue formazan crystals are solubilized by using dimethyl sulfoxide. The optical density of the dissolved material is measured at 570 nm (reference filter, 690 nm) with U.V. visible spectrophotometer (Molecular-Devices, U.K.).

Morphological Studies

The morphological studies of the cells were done with inverted microscope (Olympus, Japan) comparing with the control group 24 h after treatment.

Data Analysis

Cytotoxicity was expressed as mean percentage increase relative to the unexposed control ± SD. Control values were set at 0% cytotoxicity. Cytotoxicity data (where appropriate) was fitted to a sigmoidal curve and a four parameters logistic model used to calculate the IC50, which is the concentration of nanomaterial causing 50% inhibition in comparison to untreated controls. The mean IC50 is the concentration of agent that reduces cell growth by 50% under the experimental conditions and is the average from at

least three independent measurements that were reproducible and statistically significant. The IC50 values were reported at ±95% confidence intervals (±95% CI). This analysis was performed with Graph Pad Prism (San Diego, U.S.A).

Microorganisms

Gram-positive and gram-negative bacteria and yeast were used for antimicrobial activity studies. The Gram-negative bacteria used were Escherichia coli ATCC29998, Pseudomonas aeruginosa ATCC 27853,

and Salmonella thyphimurium CCM 5445. The Gram- positive bacteria used were Bacillus cereus ATCC 7064, Enterococcus fecalis ATCC 29212, Staphylococcus aureus ATCC 6538/P, and Staphylococcus epidermidis ATCC 12228. The yeast used was Candida albicans ATCC 10239. The lyophilized bacteria and yeast were obtained from the Standard ATCC bacteria strain and Standard ATCC fungi strain collection of the Ege University, Faculty of Science, Department of Basic and Industrial Microbiology.

Antimicrobial Activity Studies

The antibacterial activity of the Ginger extracts was tested by the paper disc diffusion technique (2,14). The 30μL of each extract (chloroform extracts, concentration 12.5 mg/mL; ethanol extracts, concentration 2.5 mg/mL) of Zingiber were absorbed onto sterile 6 mm diameter filter paper discs (Schleicher and Schul, Nr 2668, Dassel, Germany).

Bacterial strains were inoculated on Mueller-Hinton broth (MHB) (Oxoid), fungal strain was inoculated on Sabouraud Dextrose Broth (SDB) (Difco) and incubated for 24 h at 37° ± 0.1°C. Adequate amounts of autoclaved Muller-Hinton Agar (Oxoid) and Sabouraud Dextrose Agar (SDA) (Difco) were poured into sterile plates, and allowed to solidify under aseptic conditions. Bacterial strains were adjusted to yield approximately 1.0 × 107–1.0 × 108 cfu mL−1 and fungal strains were adjusted to yield approximately 1.0 × 105 using the Standard McFarland counting method. 0.1 mL of the each test organism was inoculated with a sterile swab on the surface of the appropriate solid medium in the plates.

(4)

Agar plates containing the test organisms were incubated for 1 h before placing the extract impregnated paper discs on the plates. Then, the sterile discs impregnated with different extracts were placed on the agar plates and incubated at 37º ± 0.1 ºC for 24 h.

Antibacterial activity was determined by measuring the inhibition zone (in mm) against the test organisms (15,17). All experiments were done under sterile conditions in duplicate. The standard antibacterial agent ceftazidime (20 mg/disc) was used as a positive control for bacteria, and the standard antifungal agent nystatin (20 mg/disc) was used as the positive control for yeast. All experiments were repeated three times.

DMSO was used as a negative control.

RESULTS

Extraction of Samples

At the end of the freeze drying 41.75 mg ethanol extract and 22.85 mg chloroform extract were obtained. The yields were 8.35% and 4.57% for ethanol and chloroform extracts, respectively. These extracts were prepared as a stock solution in DMSO and then they were diluted in culture medium for further experiments.

Result of Cytotoxicity Test

In this study, the cytotoxic effect and IC50 value of ginger extracts on cervical cancer (HeLa) and mouse fibroblast (L929) cells were investigated by using different concentration of the extracts and the results are shown in Table 1.

Cytotoxic Effect on Mouse Fibroblast (L929) Cells

The MTT assay result showed that chloroform extract inhibits cell proliferation in a dose-dependent manner. The IC50 value was found to be 87.28 µg/mL.

The similar cytotoxic effect was shown with ethanol extract on L929 cells. Cytotoxicity level depends on the concentration of ethanol extract. The cytotoxicity was determined at 80 µg/mL and increases gradually at higher concentrations. The calculated IC50 value was 101.0 µg/mL.

Cytotoxic effect on human cervical cancer (HeLa) cells

The cytotoxic effects of the ginger extracts showed similarity on HeLa cells at different concentrations with L929 cells. There are distinctions in the IC50 values of the extracts on HeLa cells. The determined cytotoxic level of chloroform extract and the IC50 value were 80 µg/mL and 74.32 µg/

mL, respectively.

The cytotoxic effect of the ethanol extract on HeLa was found to be 20 µg/mL and the effect amplified at higher concentrations, 5-500 µg/mL. The IC50 value was calculated as 133.78 µg/mL.

Morphological studies

In this part of the experiment, the morphological changes were obtained for L929 (Figure 1 and 3) and HeLa cells (Figure 2 and 4), which were growing in logarithmic phase throughout the treatment with extracts. After the 48 h post-treatment of extracts, an increased number of rounded cells and growth inhibition were observed when compared with the untreated control cells.

Antimicrobial Activity Studies

Results from the antimicrobial activity screening tests are shown in Table 2. As clearly seen in Table 2, the ginger extracts inhibited the growth of five out of 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)

L929 Chloroform 87.28

Ethanol 101.0

HeLa Chloroform 74.32

Ethanol 33.78

(5)

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

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

A

B

C

A

B

C

ATCC 27853, and Staphylococcus epidermidis ATCC 12228. In this study, the antimicrobial activities of Ginger extracts of 20 mL/discs were compared with

DISCUSSION

Since cervical cancer is the second most common cancer worldwide, it continues to be one of the deadly health problems. The chemotherapeutic drugs

(6)

Figure 3. Morphological changes towards L929 as viewed under the inverted microscope after the post-treatment of chloroform extract. (a) Untreated control (b) treated cells at 250 μg/mL(c) treated cells at 10 μg/mL (Magnification 20X).

Figure 4. Morphological changes towards HeLa as viewed under the inverted microscope after the post- treatment of chloroform extract. (a) Untreated cells (b) treated cells with 80 μg/mL (c) treated cells with 40 μg/mL (Magnification 20X).

A

B

C

A

B

C

destroy cancer cells by interfering the cell division and growth. The affected cells become damaged and eventually die. Nevertheless, apart from affecting the

use of natural compounds extracted as an antioxidant and functional foods from fruits, vegetables, oil seeds and herbs has recently become a global tendency. The

(7)

world’s population relies on conventional medicine for their main health care requirements and most of this therapy involves the use of plant extracts or their active components. Recently, twenty five percent of modern drugs prescribed worldwide are currently used in the treatment of various illnesses (4).

Since Turkey has a broad plant diversity, herbal therapy is a method people trust in. However, even though the activities against diseases of some plants have not been confirmed yet, the people are using them unconsciously. Ginger is also one of the most commonly used plants in Turkey to cure some of the diseases. On the other hand, it can easily be found all around the world. Consequently, the activities of ginger and its related species have prominently been

Some compounds present in ginger may exert cancer preventive effects by inducing apoptosis in cancerous or transformed cells. The oleoresin from the root of ginger contains [6]-gingerol, the major pharmacologically active component and lesser amounts of a structurally related vanilloid, [6]-paradol (18).

In this research ethanol and chloroform extracts of ginger were studied. This study confirmed that the extracts applied on HeLa and relatively more resistant cells L929 lead to death. This effect was demonstrated with both MTT test and the morphological appearances (Figure 1,2,3 and 4) of the cultures under microscope after 24 h of post- treatment with the extracts. Considering the IC50 Table 2. Antibacterial activity of the ginger extracts by the disc diffusion method

Microorganism

Zingiber Standards

Clorofom

extract Ethanol

extract CF 20 NS 20 DMSO

Inhibition Zone ( mm )*

Enterıcoccus faıcalis

ATCC 29212 G(+) 10 9 24 Nt -

Staphylococcus aureus

ATCC 6538/P G(+) 9 9 17 Nt -

Staphylococcus epidermidis

ATCC 12228 G(+) - - 12 Nt -

Bacillus cereus

ATCC 7064 G(+) 10 9 11 Nt -

Salmonella typhimurium

CCM 5445 G(-) 10 9 20 Nt -

Pseudomonas aeruginosa

ATCC 27853 G(-) - - 25 Nt -

Escherichia coli

ATCC 29998 G (-) - - 21 Nt -

Candida albicans

ATCC 10231 yeast 10 9 Nt 19 -

CF20: Ceftazidime (20 mg); NS 20: Nystatin (20 mg) ; Nt.: not tested; -: not active.

* Includes diameter of disc (6 mm).

(8)

was less than the effect of the ethanol extract on L929 mouse fibroblast cells. However, the reverse effect has been acquired for the HeLa cells. On the contrary, this result proved the sensitivity of HeLa cells compared to L929, which are not cancer cell-line. A study about apoptogenic effects of zerumbone (ZER) on HeLa cells was carried out by Wahab et al. (19). In that study examine, the results of MTT assay showed that ZER has less effect on normal cells compared to cancer cells (19). As a consequence, this observed result showed a resemblance with our results.

Natural compounds that inhibit or suppress the proliferation of cancer cells are called as anti-tumor agents. The anti-cancer agents derived from the edible plants present a big advantage due to their low toxicity. And in this study, we showed that ginger can be used as an efficient cytotoxic agent and causing the death of cervical cancer cells. According to the obtained outcomes from the morphological observation and MTT test, the prepared extracts showed cytotoxic activity against cancer cells dependent on doses. Kirana et al., (7) have reported the anticancer properties of zerumbone (2,6,9 humulatriene-8-one, a sesquiterpenoid) from Zingiber aromaticum that were analyzed with in vitro MTT tetrazolium salt assay using HT-29, CaCo-2, and MCF-7 cancer cells and showed that Zerumbone is effective as an anticancer agent, possibly by its apoptosis-inducing and anti-proliferative influences (11). Similarly, Abdul A.B.H et al., (1) extracted, isolated and purified from the rhizomes of edible plant Zingiber zerumbet by using methanol extraction and column chromatography (CC) method. The study investigated the purified zerumbone crystals for its anticancer properties on human cervical cancer cell line (HeLa); on the other hand, cisplatin was used as a positive control. As a result, important growth retardation was identified in the HeLa cancer cells, after the treatment with both zerumbone and cisplatin compounds (4). Wei et al. showed that many diarylheptanoids and gingerol-related compounds obtained from the rhizome of ginger (Zingiber officinale) possess significant anti-proliferation activity on HL-60 cells, likely via induction of the cell apoptosis (20). In a similar manner, [6]-Gingerol

et al., who used MDA-MB-231 human breast cancer cells (21). And, also another study by Brown et al., (9) was carried out on a different cancer cell line (YYT colon cancer cells); in vitro results showed that ginger extract and 6-gingerol both directly interfered with colon cancer cell proliferation, and interfered indirectly by blocking the delivery of angiogenic signals to the endothelial cells that supplied blood to the colon cancer cells. These results indicate that ginger’s phytochemicals have distinct potentials for chemoprevention and chemotherapy strategies (9).

In the present study, the ethanol and chloroform extracts were found to possess antibacterial properties against five out of eight microorganisms but had no effect on the growth of Escherichia coli ATCC11230, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus epidermidis ATCC 12228 in our study.

Our results show similarity with the studies which were carried out by Akoachere et al., (22), Singh et al., (23) and Chen et al., (24). They also showed that crude extracts of the ginger rhizome inhibit the growth of certain Gram-negative and Gram-positive bacteria and showed antifungal activities as well as respiratory tract pathogens Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae and Haemophilus influenza.

Conclusion

Today, medicinal ginger is used mainly for the pre- vention of the symptoms of travel sickness and in cancer chemoprevention. Due to its abundance, low cost and safety in consumption, ginger has been the subject of concentrated scientific research over the past two decades investigating its anticarcinogenic, antibacterial, antifungal, hypoglycemic, and antia- therosclerotic activities. This study showed a prom- ising way towards ginger’s chloroform and ethanol extracts for their potential antimicrobial and antican- cer effects. Further studies on determining the anti- cancer activity of ginger and its active components should ideally include human intervention trials to investigate its effectiveness against human cancers and other diseases.

ACKNOWLEDGEMENT

(9)

technical advice, assistance and helpful discussions and also Melis Kuban for her kindly help from Ege University, Department of Bioengineering. We also thank Ezgi Ozturk and Rıza Berker Ozbek from Izmir Fen Lisesi for their participations during the experiment.

REFERENCES

1. Abdul A.B.H., Al-Zubairi A.S., Tailan N.D., Wahab S.I.A., Zain Z.N.M., Ruslay S. And Syam M.M. Anticancer Activity of Natural Compound (Zerumbone) Extracted from Zingiber zerumbet in Human HeLa Cervical Cancer Cells. Int. J.

Pharm 4 (3): 160-168, 2008.

2. Akoachere J.F., Chenwi E.B., Njock T.E., Anong D.N. Antibacterial effect of Zingiber officinale and Garcinia kola on respiratory tract pathogens.

East Afr. Med J 79: 588–592, 2002.

3. Atlas RM, Parks LC, Brown AE. Laboratory Manuel of Experimental Microbiology, Mosby- Year Book Inc., St. Louis, Missouri, 1995.

4. Bradshaw L.J. Laboratory Microbiology, 4th ed.

New York USA, Emeritus California State University, Saunders College Publishing, Fullertons, 1992.

5. Brown A. C., Shah C., Liu J., Pham J. T. H., Zhangb,J.

G., Jadus M. R. Ginger’s (Zingiber officinale Roscoe) Inhibition of Rat Colonic Adenocarcinoma Cells Proliferation and Angiogenesis In Vitro.

Phytother. Res 23, 640–645, 2009.

6. Chen I.N., Chang C.C., Ng C.C. , Wang C.Y., Shyu Y.T., Chang T.L. Antioxidant and Antimicrobial Activity of Zingiberaceae Plants in Taiwan. Plant Foods Hum Nutr 63:15–20, 2008.

7. Collins CM, Lyne PM. Microbiological Methods.

Butterworths and Co. Ltd., London, 1987.

8. Doyle A., Griffths J. B. Cell and Tissue Culture:

Laboratory Procedures in Biotechnology. John Wiley and Sons Inc; 1998.

9. Jung H. W., Yoon C.H., Park K. M, Han H. S., Park Y.K. Hexane fraction of Zingiberis Rhizoma Crudes extract inhibits the production of nitric oxide and proinflammatory cytokines in LPS-stimulated BV2 microglial cells via the NF-kappa B pathway.

Food Chem Toxicol 47: 6, 1190-1197, 2002.

10. Kim J.S., Lee S. I., Park H. W., Yang J. H., Shin T.Y., Kim Y.C., Baek N. I., Kim S.H., Choi S. U., Kwon B. M., Leem K. H., Jung M. Y., and Kim D. K.

of Zingiber officinale Roscoe. Arch Pharm Res 31:4, 415–418, 2008.

11. Kirana, C., Mcintosh, G. H., Record, I. R. And Jones, G. P. Anticancer Activity of Extract of Zingiber aromaticum and Its Bioactive Sesquiterpenoid Zerumbone. Nutr Cancer 45:2, 218-225, 2003.

12. Kotiranta, A., Lounatmaa, K., Haapasalo, M.

Epidemiology and Pathogenesis of Bacillus cereus Infections. Microbes Infect Vol. 2, Issue 2, 189-198, 2000.

13. Lee H. S., Seo E. Y., Kang N. E., Kim W. K.

[6]-Gingerol inhibits metastasis of MDA-MB- 231human breast cancer cells. J Nutr Biochem 19;

313–319, 2008.

14. National Committee for Clinical Laboratory Standards Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically. Approved Standard- 8th ed. NCCLS Wayne NCCLS document, Pennsylvania USA, M7- A6, 2003.

15. Pithayanukul, P., Tubpraset, J. and Wuthi- Udomlert, M. In Vitro Antimicrobial Activity of Zingiber cassumunar (Plai) Oil and a 5% Plai Oil Gel. Wiley InterScience, 365-368, 2006.

16. Ranga R.S., Girija R., Nur-e-alam M., Sathishkumar S., Akbarsha M.A., Thirugnanam S., Rohr J., Ahmed M. M. and Chendil D.

Rasagenthi lehyam (RL) a novel complementary and alternative medicine for prostate cancer.

Cancer Chemother Pharmacol 54: 7–15, 2004.

17. Rao K. V. K., Stanley S. A., Nair H. K., Aalinkeel R., Mahajan S., Chawda R. and Madhavan P. N.

N. Plant derived products as a source of cellular growth inhibitory phytochemicals on PC-3M, DU-145 and LNCaP prostate cancer cell lines.

Current science 87,2004.

18. Ruslan K., Bos R., Kayser O., Woerdenbag H.

J., Quax W. J.. Jamu: The Indonesian traditional herbal medicine. Chapter 2. Available from: URL:

http://dissertations.ub.rug.nl/FILES/faculties/

science/2006/elfahmi/02_c2.pdf

19. Shukla Y., Singh M. Cancer preventive properties of ginger: A brief review. Food Chem Toxicol 45;683–690, 2007.

20. Singh G., Kapoor I.P.S., Singh P., Heluani C.S.,

(10)

antioxidant and antimicrobial investigations on essential oil and oleoresins of Zingiber officinale.

Food Chem Toxicol 46: 3295–3302, 2008.

21. Surakka, A., Sihvonen, M.L., Lehtimaki, M. J., Wahlsten, M., Vuorela. P, Sivonen, K. Benthic cyanobacteria from the Baltic Sea contain cytotoxic Anabaena, Nodularia and Nostoc strains and an apoptosis inducing Phormidium strain.

Environ. Toxicol 20: 285-292, 2005.

22. Wahab S.I.A., Abdul A.B., Alzubairi A. S., Elhassan M.M., Mohan S. In Vitro Ultramorphological

Assessment of Apoptosis Induced by Zerumbone on (HeLa). J Biomed Biotechnol Vol. 2009, Article ID 769568, doi:10.1155/2009/769568, 2009.

23. Wei Q.Y., Ma J. P., Cai Y.J., Yang L., Liu Z.L.

Cytotoxic and apoptotic activities of diarylhep- tanoids and gingerol-related compounds from the rhizome of Chinese ginger. J Ethnopharmacol 102; 177–184, 2005.

24. Wu T. Zingiber Plant Extract. Chemical Abstracts 105: 224-787, 2002.

Referanslar

Benzer Belgeler

高膽固醇血症之飲食原則 返回 醫療衛教 發表醫師 劉如濟主任 發佈日期 2010/01/15 高膽固醇血症之飲食原則 1.維持理想體重。

spinosa methanolic and aqueous extracts demonstrated the presence of tannins, flavonoids, terpenoids, glycosides, and alkaloids in this plant.. Antileishmanial effects

The aim of the present study is to evaluate the total amount of the phenolic and flavonoid contents of methanol extract obtained from flowers and leaves of E.. campestre and to

Çalışmamızda, vokal kord displastik lezyonu (displazi, karsinoma in situ) saptanan hastalar retrospektif olarak incelenerek uzun dönem takiplerinde invaziv karsinom gelişme oran

Results of antibacterial screening of plant extract addition yogurt samples determined by the agar diffusion method (Inhibition zone in mm).. Plant species- yogurt

In the present study, the total antioxidant activity, the total phenolic content, and the antimicrobial activity of methanol extracts obtained from the female cones of Pinus

The aim of the present investigation was to evaluate the effect of Taraxacum serotinum and Heliotropium europaeum extracts on reproductive organs and fertility of male rats..

Therefore, the ether extracts of some selected Turkish liverworts such as Riccia fluitans L., Porella cordaeana (Huebener) Moore, Porella platyphylla (L.) Pfeiff., Corsinia