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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 9  |  Issue : 5  |  Page : 204-208

Antibacterial activity and bioactive compounds of 50% hydroethanolic extract of Alpinia zerumbet (Pers.) B.L. Burtt & R.M. Sm.


1 Centre for Research and Development of Medical Diagnostic Laboratories; Department of Clinical Microbiology, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand
2 Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand

Date of Submission01-Mar-2019
Date of Decision26-Mar-2019
Date of Acceptance29-Apr-2019
Date of Web Publication28-May-2019

Correspondence Address:
Natthida Weerapreeyakul
Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002
Thailand
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Source of Support: This work was supported by Khon Kaen University through the Plant Genetic Conservation Project Under the Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn (Project no. 591414 and 600626), Conflict of Interest: None


DOI: 10.4103/2221-1691.259000

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  Abstract 

Objective: To evaluate antibacterial activity and the bioactive compounds of 50% hydroethanolic extract of Alpinia zerumbet (A. zerumbet) rhizomes.
Methods: Eight reference microbial strains including two Gram-positive bacteria [Staphylococcus aureus (ATCC 29213) and Enterococcus faecalis (ATCC 29212)] and six Gram-negative bacteria [Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATTC 700603), Proteus mirabilis (DMST 8212), Salmonella enterica subsp. enterica serovar Vellore. (ATCC 15611), Shigella flexneri (ATCC 12022) and Pseudomonas aeruginosa (ATCC 27853)], were used to test antimicrobial susceptibility by the broth microdilution method. Bioactive compounds were analyzed by using HPLC.
Results: The minimum inhibitory concentration values of A. zerumbet extract were 8 mg/mL for Staphylococcus aureus, Escherichia coli and Shigella flexneri and 16 mg/mL for Enterococcus faecalis and the other four Gram-negative bacilli. HPLC chromatograms revealed that the A. zerumbet extract contained hydroxybenzoic acids, hydroxycinnamic acids and flavonoids.
Conclusions: The constituents of A. zerumbet rhizomes could be a potential source of antibacterial compounds, warranting further study of A. zerumbet extract.

Keywords: Antibacterial activity, Phenolic, Flavonoid, Alpinia zerumbet, HPLC


How to cite this article:
Tavichakorntrakool R, Lulitanond A, Sangka A, Sungkeeree S, Weerapreeyakul N. Antibacterial activity and bioactive compounds of 50% hydroethanolic extract of Alpinia zerumbet (Pers.) B.L. Burtt & R.M. Sm. Asian Pac J Trop Biomed 2019;9:204-8

How to cite this URL:
Tavichakorntrakool R, Lulitanond A, Sangka A, Sungkeeree S, Weerapreeyakul N. Antibacterial activity and bioactive compounds of 50% hydroethanolic extract of Alpinia zerumbet (Pers.) B.L. Burtt & R.M. Sm. Asian Pac J Trop Biomed [serial online] 2019 [cited 2019 Jun 19];9:204-8. Available from: http://www.apjtb.org/text.asp?2019/9/5/204/259000




  1. Introduction Top


The antibacterial resistance in pathogenic bacteria has significantly increased for the treatment. Currently, the natural products from plant extracts have been used as antibacterial agents. Alpinia zerumbet (Pers.) B.L. Burtt & R.M. Sm. (A. zerumbet) syn. Catimbium speciosum (J.C.Wendl.) Holttum.-widely grown in tropical and subtropical zones-has been exploited in traditional medicine as well as cuisine[1],[2],[3]. A. zerumbet or shell ginger is in Alpinia group and is the largest genus in the family Zingiberaceae[1],[4]. In Thailand, it is commonly known as Kha-Kom. Many studies reported that A. zerumbet possesses high potential bioactivity such as anticancer[5], antioxidant[6], antiinflammatory, analgesic, antiallergic, neuroprotective effects[4], anti-diabetes[2], anti-obesity and antiatherogenic effects[2],[7], and specifically antimicrobial activity[8]. According to these numerous health benefits, A. zerumbet may promote longevity[2].

The major compounds in A. zerumbet rhizomes, stems and fresh leaves are kavalactones (i.e., dihydro-5,6-dehydrokawain, and 5,6- dehydrokawain), with other minor constituents including volatile oils, phenols, and fatty acids[2]. The other plant that contains dihydro- 5,6-dehydrokawain is Piper methysticum[9]. This compound is found in two different plant families (Zingiberaceae and Piperaceae), which have long been consumed with no toxicity to human, therefore, it is enough to warrant for possible clinical applications. However, the detected amount of those phytochemicals was varied in different parts of A. zerumbet depending on growing and harvesting season, and agricultural area. Hence, the extraction is a pivotal way to obtain high content of phytochemicals. The solvent used for extraction should be concerned to avoid the risk of toxicity besides good agricultural practice. With a long history of using A. zerumbet, the bioactive compounds of A. zerumbet according to Thai traditional folklore have not yet been completely determined. We, therefore, evaluated the antibacterial activity and the bioactive compounds of A. zerumbet extract with 50% water and ethanol.


  2. Materials and methods Top


2.1. Bacteria

The reference microbial strains included (1) two Gram-positive bacteria [Staphylococcus aureus (S. aureus) ATCC 29213 and Enterococcus faecalis (E. faecalis) ATCC 29212], and (2) six Gram-negative bacteria [Proteus mirabilis (P. mirabilis) DMST 8212, Escherichia coli (E. coli) ATCC 25922, Klebsiella pneumoniae (K. pneumoniae) ATTC 700603, Salmonella enterica (S. enterica) subsp. enterica serovar Vellore. ATCC 15611, Shigella flexneri (S. flexneri) ATCC 12022 and Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853]. The Gram-positive bacteria were grown on blood agar (Oxoid Ltd., Basingstoke, England) while the Gram-negative bacteria were grown on MacConkey agar (Oxoid Ltd., Basingstoke, England), respectively prior to testing.

2.2. Plant collection

A. zerumbet was collected from Chaiyaphum province under the Plant Genetic Conservation Project conducted with the permission of the Royal Initiative of her Royal Highness Princess Maha Chakri Sirindhorn, and The Bureau of the Royal Household and Electricity Generating Authority of Thailand. A. zerumbet was authenticated using the herbarium collection (TT-OC-SK-857) at the Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen province, Thailand by Assistant Professor Thaweesak Thitimetharoch.

2.3. Extraction of A. zerumbet

A. zerumbet rhizomes were ground and macerated with 50% hydro-ethanolic (1 kg per 6 liters) for 7 d at room temperature (25-28 °C)[5]. Filtration was conducted to get filtrate which was evaporated by a rotary evaporator with 40 C in water bath temperature. The water content was removed by using freeze-dryer to obtain the dry crude extracts. The crude extract was stored in a desiccator protected from light at room temperature until used.

2.4. Antibacterial activity by broth microdilution method

The experiment was conducted according to the standard method[10]. Eight bacterial suspensions were made up to 0.5 McFarland and diluted to 1:100. A. zerumbet extract (0.125-32 mg/mL) and gentamicin (0.125-32 μg/mL) (Oxoid, Basingstoke, England) were prepared in the 96-well plates at 37 °C for 24 h[11].

The lowest concentration of A. zerumbet extract or gentamicin that could inhibit the visible growth of bacteria was expressed as the minimum inhibitory concentration (MIC). Furthermore, the lowest concentration of A. zerumbet extract or gentamicin without growth on agar plate at 37 °C, for 24 h was expressed as the minimum bactericidal concentration (MBC).

2.5. Phytochemical identification by using HPLC

The presence of phenolics and flavonoids in the A. zerumbet extracts was identified and quantified using HPLC (LC-20AC, Shimadzu, Japan) as per previous method[12] with minor modification. The reference standards (purchased from Sigma-Aldrich, USA) were gallic acid, vanillic acid, p-hydroxybenzoic acid, protocatechuic acid, syringic acid, chlorogenic acid, p-coumaric acid, caffeic acid, apigenin, kaempferol, ferulic acid, sinapic acid, myricetin, rutin, and quercetin. The A. zerumbet extract was prepared in DMSO and filtered through a membrane filter (0.45 μm). The injection volume was 20 μL with flow rate of 0.8 mL/min. The reverse phase column was an Inetsil® ODS-3 C18 column (4.6 mm x 250 mm, 5 μm; Hichrom Limited, Berks, U.K.). The gradient mobile phase was a mixture of 1% acetic acid in DI water (solvent X) and acetonitrile (solvent Y), which was eluted at 38 °C following the previous study[12] with some modifications. The gradient elution started from 5%-9% solvent Y (0-5 min); 9% solvent Y (5-15 min); 9%-11% solvent Y (15-22 min); 11%-18% solvent Y (22-38 min); 18%-23% solvent Y (38-43 min); 23%-90% solvent Y (43-44 min); 90%-80% solvent Y (44-45 min); isocratic at 80% solvent Y (45-55 min); 80 isocratic -5% solvent Y (55-60 min), and 5% solvent Y was used between individual runs for 5 min for equilibration. The detection wavelength of the UV-diode array detector (SPD-M20A, Shimadzu, Japan) was set for phenolic and flavonoids compounds at 280 nm and 370 nm, respectively. The retention times and their peak area of standard phenolic compounds were determined. The contents of phenolics and flavonoids in the extracts were calculated from the linear regression equations obtained from standard curves, which were plotted between the peak area (y axis) against concentrations of standard compounds (x axis) ranged from 6.25-100 μg/mL with the correlation coefficients (R2 > 0.99). Data were expressed as mean ± standard deviation.


  3. Results Top


3.1. MIC and MBC of extract of A. zerumbet against eight microbial strains

The antibacterial activities of 50% hydro-ethanolic extract of A. zerumbet are shown in [Table 1]. The ranges of MICs and MBCs of A. zerumbet extract were 8-32 mg/mL. The MIC values of the A. zerumbet extract were 8 mg/mL for S. aureus, E. coli and S. flexneri, and 16 mg/mL for E. faecalis, and the other four Gram-negative bacilli. The MBC values of A. zerumbet extract were 8 and 32 mg/mL for S. aureus and E. faecalis, respectively. The MBC values of the A. zerumbet extract were 16 mg/mL for all Gram-negative bacilli.
Table 1: Antibacterial activities of A. zerumbet extract versus gentamicin by broth microdilution method.

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3.2. Phytochemical components of extract of A. zerumbet by HPLC

The phytochemical components of A. zerumbet extract were analyzed by HPLC. The HPLC chromatograms of A. zerumbet extract are illustrated in [Figure 1]B and [Figure 2]B in comparison with the HPLC chromatograms of phenolic [Figure 1]A and flavonoid standards [Figure 2]A. The contents of phenolic acid and flavonoid compounds of A. zerumbet extract were calculated from the peak area [Table 2]. A. zerumbet extract was found to contain hydroxybenzoic acids (i.e., gallic acid, syringic acid, protocatechuic acid and p-hydroxybenzoic acid), hydroxycinnamic acids (i.e., ferulic acid) and flavonoids (i.e., quercetin, myricetin, rutin and kaempferol). Flavonoids were predominantly found in A. zerumbet according to high detected amount.
Figure 1: HPLC chromatograms of (A) standard phenolic acids including (1) gallic acid, (2) protocatechuic acid, (3) p-hydroxybenzoic acid, (4) chlorogenic acid, (5) vanillic acid, (6) caffeic acid, (7) syringic acid, (8) p-coumaric acid, (9) ferulic acid and (10) sinapic acid and (B) HPLC chromatograms of A. zerumbet extract.

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Figure 2: HPLC chromatograms of (A) standard flavonoids including (1) rutin, (2) myricetin, (3) quercetin, (4) apigenin and (5) kaempferol and (B) HPLC chromatograms of A. zerumbet extract.

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Table 2: The phenolic and flavonoid contents in the extract of A. zerumbet based on HPLC analysis.

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  4. Discussion Top


Some plants are used as traditional medicines for treatment of the urinary tract[11] and gastrointestinal tract infectious diseases. A. zerumbet is a traditional medicine used as an antibacterial agent[3]. The crude extract of A. zerumbet was used because the antibacterial action may need the combination or synergistic effects of several bioactive compounds in the extract. Our results showed that the bioactive compounds of A. zerumbet extract were the flavonoids and phenolic compounds, which were previously reported to have antibacterial activity[13],[14],[15],[16],[17]. The flavonoids found in this study were myricetin, quercetin, rutin and kaempferol, respectively. In addition, the most common types of phenolic compounds were hydroxybenzoic acids group (including gallic acid, syringic acid, protocatechuic acid and p-hydroxybenzoic acid) and hydroxycinnamic acid group (ferulic acid). These compounds in the A. zerumbet extract had some potential of the antibacterial activity.

Notably, dihydro-5,6-dehydrokavain and 5,6-dehydrokawain are the major compounds found in A. zerumbet in the amount of 80–410 mg/g of fresh weight, and ≤100 mg/g, respectively[18]. Other compounds include (i) its derivative hispidin, converted by acid hydrolysis by gastric juice and subsequently metabolized by hepatic microsomal CYP2C9[19],[20], (ii) phenolics, (iii) flavonoids and (iv) volatile oils (≤150 mg/g) such as rutin, catechins, kaempferol, flavanones, labdadienes, zerumins, camphor and methyl cinnamate, 1,8-cineol, isothymol, thymol, eugenol, chalcone cardamonin and alpinetin[1],[21],[22]. The essential oils found in A. zerumbet exerted antifungal effect on plant fungal pathogen[1]. According to the reported high amount of dihydro-5,6- dehydrokavain in A. zerumbet or minor amount of oils, it cannot be ruled out in our study that the polar solvent extraction cannot yield these bioactive compounds.

Both Gram-positive bacteria (S. aureus ATCC 29213 and E. faecalis ATCC 29212) and Gram-negative bacteria (E. coli ATCC 25922, K. pneumoniae ATTC 700603, P. mirabilis DMST 8212, S. enterica subsp. enterica serovar Vellore. ATCC 15611, S. flexneri ATCC 12022 and P. aeruginosa ATCC 27853) were selected to study because they were the reference strains that related with the urinary tract and gastrointestinal tract infectious diseases. Our data found that the A. zerumbet extract could inhibit both two Gram-positive and six Gram-negative reference bacteria in difference concentrations. S. aureus ATCC 29213 was the most sensitive strain to A. zerumbet extract. These findings corresponded to the previous study in which the A. zerumbet extract exerted inhibitory effect on S. aureus[13], This previous study showed that the flower extract obtained from methanol, hexane, dichloromethane and ethyl acetate had a greater extent of inhibition on S. aureus but showed no activity against P. aeruginosa[13] when compared to the extract of A. zerumbet rhizome in our study. A. zerumbet has been long historically used with no side effect but more benefits[2],[18]. These evidences confirm that the extract of A. zerumbet rhizome exhibits the antibacterial activity and may be potential for developing product for treatment of urinary tract infection.

In conclusion, A. zerumbet extract had antibacterial effect on two Gram-positive and six Gram-negative reference bacteria especially S. aureus ATCC 29213. However, the antibacterial activities against the clinical bacterial strains should be further evaluated in the future. Moreover, the total phenolics and flavonoids contents in the extract of A. zerumbet rhizome were 85 and ~35,000 μg/g, respectively. Thus it is necessary to conduct the solvent extraction to achieve high yield of bioactive compounds of A. zerumbet extract for further clinical use.

Conflict of interest statement

The authors declare that there is no conflict of interest.

Funding

This work was supported by Khon Kaen University through the Plant Genetic Conservation Project Under the Royal Initiative of Her Royal Highness Princess Maha Chakri Sirindhorn (Project no. 591414 and 600626).



 
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    Figures

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    Tables

  [Table 1], [Table 2]



 

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