Zingiber zerumbet (L.) Roscoe ex Sm. from northern India: Potential source of zerumbone rich essential oil for antiproliferative and antibacterial applications

Industrial Crops & Products 112 (2018) 749–754

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Zingiber zerumbet (L.) Roscoe ex Sm. from northern India: Potential source of zerumbone rich essential oil for antiproliferative and antibacterial applications

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Rajendra C. Padaliaa, , Ram S. Vermaa, Amit Chauhana, Ved R. Singhb, Prakash Goswamia, Shilpi Singhb, Sajendra K. Vermab, Suaib Luqmanb, Chandan S. Chanotiyab, Mahendra P. Darokarb a CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Research Centre, Pantnagar, P.O.-Dairy Farm Nagla, Udham Singh Nagar, Uttarakhand, 263149, India b CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, 226015, Uttar Pradesh, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Zingiber zerumbet Rhizome Essential oil Zerumbone Antiproliferative activity Antibacterial activity

Zingiber zerumbet (L.) Roscoe ex Sm. (Zingiberaceae), commonly known as bitter ginger, has long been used in traditional herbal medicine for the treatments of inflammations, rheumatism, sprains, colic pain, diarrhea, tonsillitis and various other ailments. The aim of the present study was to assess the chemical composition of the rhizome essential oil of Zingiber zerumbet grown in the foothills of northern India using GC-FID, GC–MS, IR and NMR, and to evaluate the antibacterial and antiproliferative potential of the rhizome essential oil and its major constituent. Altogether, thirty-four constituents were identified representing 98.0% of the essential oil composition. Zerumbone (72.86%), α-humulene (7.09%), camphene (5.04%), humulene oxide I (2.45%), humulene oxide II (1.8%), and camphor (1.41%) were the major constituents. The potential of the rhizome essential oil of Z. zerumbet and zerumbone was tested against nine pathogenic bacterial strains. The results showed that both essential oil and zerumbone, possessed significant antagonist activity against Staphylococcus aureus-96 (MIC: 52.0–166.6 μg/mL), Streptococcus mutans (MIC: 62.5–208.0 μg/mL), and Escherichia coli (MIC: 104.1–208.0 μg/ mL). Zerumbone was found more active compared to the essential oil. Moreover, the antiproliferative potential of Z. zerumbet oil and zerumbone was evaluated against various human cancer and normal cell lines (A549, MDAMB-231, A431, K562, WRL-68, COLO-205, HaCaT, and HEK-293). Results showed that, both essential oil and zerumbone possessed antiproliferative activity against tested cell lines, where zerumbone was more competent then essential oil.

1. Introduction Plants synthesise and preserve a variety of secondary metabolites useful for human being for diverse applications. Among them, essential oils and aroma constituents extracted from aromatic plants, represents a major component of various industrial products such as food and beverages, perfume, fragrance, nutraceutical and pharmaceuticals (Hadian et al., 2014; Padalia, 2012). Zingiberaceae, the largest monocotyledonous family in India, includes various rhizomatous plants of economic importance characterised by the presence of essential oils and oleoresins of export value. It included 52 genera and 1400 species distributed in Indo-Malaysian region of Asia. Among them, 22 genera and 178 species were reported in north eastern and peninsular region of India (Jen and Ved, 1995). Several members of this family were used as ⁎

spices, perfumes, medicines, flavouring agents, as well as the source of certain dyes and other economic uses (Burkill, 1966; Dai et al., 2013). Zingiber zerumbet (L.) Roscoe ex. Sm. considered as one of the important member of this family. It is the native of Southeast Asia, but has been now widely cultivated in tropical and subtropical regions around the world. It is mainly distributed in India, Bangladesh, Malaysia, Nepal and Sri Lanka. Traditionally this plant is known as ‘Asian ginger’ or ‘bitter ginger’ that grows naturally in damp, shaded parts of the lowland or hill slopes (Baby et al., 2009; Madegowda et al., 2016). It is also known as ‘Pinecone ginger’ and ‘Shampoo ginger’ because of the foaming properties of its pine like inflorescence (Tushar et al., 2010; Yob et al., 2011). Z. zerumbet is widely used in foods, beverages and for ornamental purposes (Koga et al., 2016; Singh et al., 2014). All plant parts of Z. zerumbet are utilised in traditional medicines for treatments

Corresponding author. E-mail address: [email protected] (R.C. Padalia).

https://doi.org/10.1016/j.indcrop.2018.01.006 Received 22 September 2017; Received in revised form 2 January 2018; Accepted 3 January 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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45 °C, and minimum between 2 and 5 °C. The soil of the experimental site was sandy-loam in texture, with neutral pH. Voucher specimen and herbarium record of the plant have been maintained CSIR-CIMAP, Research Centre, Pantnagar. Fresh rhizomes were taken for the study after digging the crop in spring season (March 2016). The rhizomes were crushed and hydrodistilled in a Clevenger type apparatus for 4 h, in triplicate, for extraction of essential oil. The essential oil was measured directly in the extraction burette and content (%) was determined as volume (mL) of essential oil per 100 g of fresh biomass. The oil was dehydrated over anhydrous Na2SO4 and kept in a cool and dark place prior to analysis.

of a variety of ailments. The leaves of Z. zerumbet are used in the treatments of joint pain and skin diseases, whereas the young shoots and inflorescence are used as condiments (Devi et al., 2014; Murakami et al., 2002). The cone-shaped flowers were employed in craft arrangements for ornamental purposes, while the viscous juice present in the mature inflorescence, rich in surfactants, used as a natural shampoo (Devi et al., 2014; Murakami et al., 2002; Yob et al., 2011). However, the rhizomes of the plant are the most economic part used as food flavouring and appetizer in Malay and Indian cuisines, and as drug in Indian, Asian, Chinese, and Arabic folk medicine (Norulaini et al., 2009; Sreevani et al., 2013; Yob et al., 2011). Rhizomes are used in folk medicine for the treatment of various ailments, such as cough, cold, swelling, stomachache, ear inflammation, colic pain, diarrhea, tonsillitis, sore throat, worm infestations, and skin diseases as well as antispasmodic, antirheumatic, antiflatulent and diuretic agents (Koga et al., 2016; Rana et al., 2016; Tushar et al., 2010; Yob et al., 2011). Phytochemical studies on different plant parts of Z. zerumbet from different origins reported the presence of diverse secondary metabolites, such as polyphenols, alkaloids and terpenoids (Koga et al., 2016). Most of the earlier studies focused on rhizome essential oil showed that the essential oil was a complex mixture of terpenoids, with a predominance of sesquiterpenoids, mainly zerumbone, humulene, humulene oxides, βcaryophyllene, α-caryophyllene as major constituents; followed by varying proportions of monoterpenoids viz. camphene, sabinene, myrcene, etc. (Baby et al., 2009; Batubara et al., 2013; Bhuiyan et al., 2009; Dai et al., 2013; Koga et al., 2016; Madegowda et al., 2016; Rana et al., 2008, 2016). Contrarily to rhizome oil, the essential oil extracted from the aerial parts (leaf, stem and inflorescence) of Z. zerumbet was dominated by nerolidol, trans-phytol, β-caryophyllene, linalool, pinenes, with lesser content of zerumbone (Bhuiyan et al., 2009; ChaneMing et al., 2003; Dung et al., 1995; Rana et al., 2016). However, the quantitative composition of the essential oils (content of the reported respective constituents) of different plant parts of Z. zerumbet was found to be highly variable according to origin of plant. The rhizome essential oil and its main bioactive constituent (zerumbone) have been shown to possess significant anti-tumor, anti-inflammatory, anti-oxidant, antidiabetic, antimalarial, antisecretory, anti-microbial, anti-proliferative, antiviral, anti-allergic, anti-pyretic, analgesic and cyclooxygenase-2 suppressant properties (Abdul et al., 2008; Joseph et al., 2015; Murakami et al., 2002; Sakinah et al., 2007; Somchit et al., 2012; Sulaiman et al., 2010). Thus, with the background of antimicrobial and anticancerous potential of the essential oil of Z. zerumbet and no previous report about chemical composition of Z. zerumbet grown in foothill agroclimatic conditions in northern India, the present study was planned to explore the composition and biological activity of the rhizome essential oil of Z. zerumbet from northern India. The present study was designed to evaluate the following objectives: (i) to explore the rhizome essential oil composition of Z. zerumbet grown in the foothills of northern India; (ii) to isolate and characterise the major constituent by chromatographic and spectrometric analysis; (iii) to evaluate the antiproliferative activity against various organ specific cell lines; (iv) and to evaluate the antimicrobial activity against nine pathogenic bacterial strains to have an inclusive view to use the rhizome oil of Z. zerumbet, as alternatives, in microbial and cancer control therapy in humans.

2.2. Analysis and characterization of essential oil constituents The chemical composition of essential oil was analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC–MS) techniques. GC analysis was done on a DB-5 capillary column (30 m × 0.25 mm i.d., film thickness 0.25 μm) fixed inside the oven of NUCON Gas Chromatograph (model 5765). The column oven temperature was programmed from 60 to 230 °C, at the rate of 3 °C min−1, using Hydrogen as carrier gas at constant flow rate of 1.0 mL min−1. The injection volume was 0.02 μL neat (syringe: Hamilton 0.5 μL capacity, Alltech USA) in split mode (1:40). The injector and detector (FID) temperatures were maintained at 220 °C and 230 °C, respectively. GC–MS analysis was carried on Perkin-Elmer Turbomass Mass Spectrometer (Shelton, USA) fitted with Equity-5 fused silica capillary column (60 m × 0.32 mm; 0.25 μm film thickness; Supelco Bellefonte, PA, USA). The column temperature was programmed 70 °C, with initial hold time of 2 min, to 250 °C at 3 °C min−1 with final hold time of 3 min, using helium as carrier gas at a flow rate of 1.0 mL min−1. The injector, ion source and transfer line temperatures were maintained at 250 °C. The injection volume was 0.04 μL neat with split ratio 1:30. Mass analysis was carried out in electron ionization (EI) mode at 70 eV with the mass scan range of 40–400 amu. The identification of the individual compounds was carried out using retention index (RI) determined using a homologous series of n-alkanes (C7–C30, Supelco Bellefonte, PA, USA) and by comparing mass spectra with those of authentic sample as well as mass spectra library search in NIST and WILEY Mass Spectral Library, and by comparing with the mass spectral literature data (Adams, 2007). 2.3. Isolation and characterisation of zerumbone The major constituent of the essential oil was isolated by column chromatography and crystallisation process. The essential oil (2 mL) was dissolved in hexane (5 mL), adsorbed onto silica gel (100–200 mesh, 20 g), and dried at room temperature for 1 h. The adsorbed material was then column chromatographed in glass column packed with silica gel (40 g, 230–400 mesh, Merck) by using hexane followed by ethyl acetate/hexane mixture as eluting agent. Elution of the column with 5% ethyl acetate/hexane mixture (ratio 1:19) yielded 1.0 g of pure white compound, which was purified by slow crystallisation in petroleum ether at low temperature. The purity of the isolated compound was checked using thin layer chromatography (TLC) and gas chromatography (GC) (Purity > 98.0%). The compound was characterised as zerumbone by IR, MS, 1H NMR and 13C NMR spectral data. Mass spectrum was recorded in Perkin-Elmer Turbomass Mass Spectrometer (Shelton, USA), while NMR spectra of the isolated compound was recorded on a 500 MHz NMR spectrometer (500 MHz for 1H NMR and 125 MHz for 13C NMR, Bruker, Germany) using CDCl3 as dissolving agent and tetramethylsilane (TMS) as an internal standard. IR spectrum was recorded on Perkin-Elmer Spectrum BX FT-IR spectrophotometer. A thin film of the solution made by dissolving 2 mg of isolated compound in carbon tetra-chloride (CCl4), was developed between KBr plate and the spectrum was taken at room temperature in the range between 4000 cm−1 to 650 cm−1. Spectral data of zerumbone is

2. Materials and methods 2.1. Plant material and extraction of essential oil The fresh rhizomes of Z. zerumbet were collected from the crop raised at the experimental field of CSIR-Central Institute of Medicinal and Aromatic Plants (CIMAP), Research Centre, Pantnagar (Udham Singh Nagar) Uttarakhand. The experimental site is located between coordinates 29°N, 79.38°E at 243 m above mean sea level at foothills of northern India. The maximum temperature ranges between 35 and 750

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grown in DMEM except K562 and COLO-205, which are maintained in RPMI-1640, supplemented with 10% FBS and 1% Ab/Am at 37 °C in a humidified atmosphere at 5% CO2. The MTT assay for essential oil and isolated compound was performed according to the method of Mosmann, (1983). The experiment was performed in 96-well plate. The dilution of essential oil and zerumbone was prepared in DMSO. The cell lines were seeded in the wells, by inoculating the cell suspension (100 μL) to each well of the plate at the density of 1 × 104 cells per well. After 24 h of incubation, the medium was removed from the wells by aspiration and replaced with experimental medium (100 μL/well). Then, the treatments were performed with different concentrations and incubated for 24 h. After incubation, the medium was removed and the MTT dye (0.5 mg/mL) was added and then incubated in dark for 4 h. After this, the MTT dye was removed and DMSO was added to the plates and mixed well with the help of pipette. The absorbance was measured at 570 nm in Spectramax, microplate reader (Multiscan™ Go Skanlt Software 4.0 version, Thermo Fisher Scientific, Waltham, USA). Doxorubicin was used as a positive control whereas media with phosphate buffer saline was used as a negative control. The percent survival (S) of the cells was calculated by the following formula:

as: MS EI, 70 eV, m/z (rel. int.%): 53 (23.6), 55 (11.6), 67 (22.9), 77 (10.2), 79 (13.3), 81 (11.6), 91 (22.2), 93 (12.4), 96 (25.7), 105 (14.6), 107 (60.9), 108 (23.3), 109 (14.6), 121 (18.2), 123 (10.2), 135 (100.0), 136 (13.9), 149 (11.7), 150 (12.3), 163 (14.0), 189 (8.7), 203 (7.3), 218 (18.5; M+, C15H22O); IR vmax cm−1: 3032, 2929, 2861, 1751, 1654, 1458, 1377, 1264, 1034; 1H NMR (500 MHz, CDCl3-TMS): 1.09 (3H, s, eCH3), 1.18 (3H, s, eCH3), 1.52 (3H, s, eCH3), 1.77 (3H, s, eCH3), 1.88 (2H, m, eCH2), 2.17–2.39 (4H, m, eCH2), 6.00 (1H, m, ]CH), 5.82–5.98 (2H, m, ]CH), 5.24 (1H, m, ]CH); 13C NMR (125 MHz, CDCl3-TMS): 204.2 (C), 160.6 (CH), 148.7 (CH), 137.9 (C), 136.2 (C), 127.8 (CH), 125.7 (CH), 42.4 (CH2), 39.4 (CH2), 37.8 (C), 29.4 (CH3), 24.3 (CH2), 24.1 (CH3), 15.3 (CH3), 11.8 (CH3); DEPT-135 and DEPT-90 NMR: CH3: 04 nos.; CH2: 03 nos.; CH: 04 nos.; C: 04 nos. The identity of compound was established as zerumbone by comparison of its spectral data with literature (Adams, 2007; Baby et al., 2009; Rana et al., 2016). 2.4. Antibacterial activity evaluation of the essential oil The antibacterial activity of the rhizome essential oil of Z. zerumbet and is major constituents, zerumbone was determined using disc diffusion assay (CLSI, 2006). Inoculum of the nine test bacteria viz. Staphylococcus aureus (MTCC 96), Staphylococcus aureus (MTCC 2940), Staphylococcus epidermidis (MTCC 435), Bacillus subtilis (MTCC 121), Streptococcus mutans (MTCC 890), Klebsiella pneumoniae (MTCC 109), Pseudomonas aeruginosa (MTCC 741), Escherichia coli (MTCC 723), and Salmonella typhimurium (MTCC 98) was prepared equivalent to McFarland Standard 0.5 (1 × 106 cfu/mL) obtained from the Microbial Type Culture Collection Centre (MTCC), Institute of Microbial Technology (IMT) Chandigarh, India. Uniform bacterial lawns were made using 100 μL inoculums on a nutrient agar plate. Filter paper (Whatman) discs (5.0 mm) soaked with test essential oils were placed over seeded plates. The plates were incubated at 37 °C for 24 h. Activity was measured in terms of zone of inhibition (ZI, mm). The zone of inhibition was determined by subtracting the disc diameter (i.e. 5.0 mm) from the total zone of inhibition shown by the test disc in terms of clear zone around the disc. Norfloxacin was employed as a positive control, while DMSO served as the negative control. The tests were performed in triplicate. Antibacterial efficacy of the essential oil of Z. zerumbet against tested bacterial strains was determined by Micro dilution broth assay using 96 ‘U’ bottom micro-titer plates according to CLSI guidelines (CLSI, 2012). Samples were serially diluted two folds (in the range of 1000–1.95 μg/ mL) in Mueller Hinton Broth (MHB). The broth was inoculated with 10.0 μL of diluted 24 h grown culture of test organisms with a titre equivalent to 0.5 McFarland standards. The inoculated plates incubated at 37 °C for 16–24 h and the growth was recorded spectrophotometrically at 600 nm using Spectramax 190-microplate reader (Molecular Devices, CA, USA). The MIC value was determined from the turbid metric data as the lowest concentration showing growth inhibition as compared with control. An antibiotic norfloxacin was taken as positive control, while DMSO served as negative control. Experimental observations were performed in triplicate to reduce error during the procedure. The antibacterial activity of zerumbone was determined using same procedure. Norfloxacin (10 mg/mL) is used as reference compound or positive control. DMSO used as solvent or negative control, and no zone of inhibition was observed for negative control. Experimental observations were performed in triplicate to rule out any error during the antibacterial assay

S= [OD(sample) − OD(zeroday) ] ÷ [OD(control) − OD(zeroday) ] × 100 Percentcytotoxicity = 100 − Percentsurvival

2.6. Statistical analysis The data of antiproliferative and antimicrobial activity is presented as mean ± standard deviation which was calculated in MS Office Excel version 2007. Comparisons with standard/positive control were done by one way ANOVA followed by Dunnet test in GraphPad Instat version 3.06. The inhibitory concentration (IC50) in μg/mL of antiproliferative activity was calculated using 2D linear and non-linear curve fitting software table curve 2D 5.0.1/2007 (Systat software Inc., UK). 3. Results and discussions The hydrodistilled rhizome essential oil of Z. zerumbet was evaluated using GC-FID and GC–MS techniques. The essential oil yield was found to be 0.58% on fresh weight basis. Altogether, thirty-four constituents, representing 98.0% of the total oil composition were identified (Table 1). The essential oil was mainly composed of sesquiterpenoids (87.83%), represented by oxygenated sesquiterpenes (80.14%) and sesquiterpene hydrocarbons (7.69%), and followed by monoterpene hydrocarbons (7.50%) and oxygenated monoterpenes (2.67%). Major constituents of the essential oil were zerumbone (72.86%), αhumulene (7.09%), camphene (5.04%), humulene oxide I (2.45%), humulene oxide II (1.8%), and camphor (1.41%). Moreover, the identity of major constituents (zerumbone) was established by its isolation and MS, IR, 1H and 13C NMR spectrometric data (see Material and methods). The essential oil composition of Z. zerumbet was studied previously from different geographic regions (Baby et al., 2009; Batubara et al., 2013; Bhuiyan et al., 2009; Dai et al., 2013; Damodaran and Dev, 1968; Madegowda et al., 2016; Malek et al., 2005; Rana et al., 2008, 2016; Singh et al., 2014; Srivastava et al., 2000). Most of the earlier studies reported sesquiterpenoids viz., zerumbone, humulene, humulene oxides, β-caryophyllene, α-caryophyllene and curzerenone as major constituents distributed in rhizome essential oil of Z. zerumbet. The content of zerumbone found to vary from 1.2–88.5% in rhizome essential oil of Z. zerumbet from different origin. The maximal content (88.5%) was reported from Z. zerumbet grown in northeastern India (Manipur) > southern India (74.8–84.8%); and minimal (12.6%) from eastern India (Baby et al., 2009; Rana et al., 2008, Srivastava et al., 2000). However, in few other reports, the content of zerumbone was found to vary with different studies carried out by different authors,

2.5. Antiproliferative evaluation of zerumbone and essential oil The human cell lines HEK-293 (Embryonic Kidney Cells), MDA-MB231 (Breast adenocarcinoma), A431 (Skin Carcinoma), HaCaT (Keratinocytes), K562 (Leukemia), COLO-205 (Colon carcinoma), A549 (Lung carcinoma) and WRL-68 (Hepatic carcinoma) were procured from National Centre for Cell Science, Pune, India. The cells were 751

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which includes 74.8% of zerumbone in Z. zerumbet from Manipur, and 74.8% from Meghalaya of northeastern India; and 21.2–49.8% from Calicut and Kerala of south Indian origin (Damodaran and Dev, 1968; Madegowda et al., 2016; Rana et al., 2016; Singh et al., 2014). Besides these, variable content of zerumbone was reported in Z. zerumbet grown worldwide, viz. Malaysia (68.9–73.1%), Vietnam (72.3%), French Polynesia (63.0–65.3%), Fiji (59.0%), Japan (48.0–71.0%), China (48.13%), Thailand (41.7%–56.5%), Bangladesh (46.8%), Reunion Island (37.2%), France (37.0%), Philippines (35.5%), Indonesia (11.0%), and Vietnam (1.2%) (Batubara et al., 2013; Bhuiyan et al., 2009; Chane-Ming et al., 2003; Dai et al., 2013; Dung et al., 1993; Duve, 1980; Madegowda et al., 2016; Malek et al., 2005; Vahirua et al., 1993; Yu et al., 2008). Considering no previous report on essential oil quality of Z. zerumbet grown in agroclimatic conditions of foothills of northern India, the results of present studies compliments significant content of zerumbone (72.8%) for the first time from northern India. Moreover, the essential oil yield of Z. zerumbet grown in foothills of northern India was found to be significantly higher (0.58%) as compared with previous reported yield (0.23-0.41%) from different regions of India (Baby et al., 2009; Rana et al., 2008, 2016; Singh et al., 2014; Srivastava et al., 2000) and other countries (0.12-0.41%), viz. Vietnam, French Polynesia, Fiji, Japan, China, Thailand, Reunion Island, France, Philippines, and Indonesia (Batubara et al., 2013; Chane-Ming et al., 2003; Dai et al., 2013; Dung et al., 1993; Duve, 1980; Vahirua et al., 1993; Yu et al., 2008). However, the examined oil yield was relatively lower than that reported from Malaysia (1.2%) and Bangladesh (1.1%) (Bhuiyan et al., 2009; Malek et al., 2005). The rhizome essential oil of Z. zerumbet and zerumbone was tested for antagonist activity against nine pathogenic bacteria. The results in terms of zone of inhibition (ZI) and minimum inhibitory concentration (MIC) are presented in Table 2. Results showed that both essential oil and zerumbone exhibited varying degree of antibacterial activity against tested strains. The zone of growth inhibition and MIC for the essential oil against tested bacterial strains ranged from 6 to 10 mm and 166.6 to 833.0 μg/mL, respectively. The essential oil of Z. zerumbet showed maximal activity against Staphylococcus aureus-96 (166.6 μg/ mL) followed by Streptococcus mutans (208.0 μg/mL) and Escherichia coli (208.0 μg/mL), and moderate antagonist activity against other tested strains viz., Staphylococcus aureus-2940, Bacillus subtilis, and Staphylococcus epidermis (833.0 μg/mL), Klebsiella pneumoniae (666.0 μg/mL), and Salmonella typhimurium (666.0 μg/mL). The zone of growth inhibition and MIC for zerumbone against tested bacterial strains ranged from 3 to 13 mm and 52.0 to 666.0 μg/mL, respectively. Staphylococcus aureus-96 (52.0 μg/mL) and Streptococcus mutans (62.5 μg/mL), and Escherichia coli (208.0 μg/mL) were most sensitive to zerumbone followed by Staphylococcus aureus-2940 (208.0), Klebsiella pneumoniae (208.0 μg/mL), Salmonella typhimurium (208.0 μg/mL). Zerumbone was found to possess higher activity as compared to essential oil. Earlier studies on rhizome essential oil of Z. zerumbet revealed moderate to low

Table 1 Composition of rhizome essential oil of Zingiber zerumbet (L.) Roscoe ex Sm. from foothills of north India. No.

RICal

RILit

Compounds

Content (%)

Identificationa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

924 932 945 971 975 990 1002 1006 1022 1026 1028 1086 1099 1143 1146 1155 1168 1174 1184 1188 1419 1452 1532 1556 1586 1602 1608 1618 1634 1642

921 932 946 969 974 988 1002 1008 1020 1024 1026 1083 1095 1141 1145 1155 1165 1174 1186 1284 1417 1454 1531 1561 1582 1596 1608 1622 1630 1639

0.13 1.18 5.04 t t t 0.23 0.35 0.10 0.47 0.77 t 0.26 1.41 t t 0.10 t 0.10 t 0.60 7.09 0.11 0.10 0.97 2.45 1.80 t t 0.40

RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI, RI,

MS MS, MS, MS MS MS MS MS MS MS MS MS MS MS, MS MS MS MS MS MS MS MS, MS MS MS MS, MS, MS MS MS

31 32 33 34

1652 1656 1710 1728

1649 1652 1713 1732

Tricyclene α-Pinene Camphene Sabinene β-Pinene Myrcene α-Phellandrene δ-3-Carene p-Cymene Limonene 1,8-Cineole Fenchone Linalool Camphor Camphene hydrate Isoborneol Borneol Terpinen-4-ol α-Terpineol Bornyl acetate β-Caryophyllene α-Humulene (Z)-Nerolidol (E)-Nerolidol Caryophyllene oxide Humulene oxide I Humulene oxide II 10-epi-γ-Eudesmol γ-Eudesmol allo-Aromadendrene epoxide β-Eudesmol α-Eudesmol 14-Hydroxy-α-humulene Zerumbone

RI, RI, RI, RI,

MS MS MS MS, IR, NMR

Class composition Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Total identified Oil content (fresh weight basis)

± 0.93

± 0.27

± 0.68

± 0.56 ± 0.37

0.93 0.30 0.22 72.86 ± 5.44

Cs Cs

Cs

Cs

Lit Cs

7.50 2.67 7.69 80.14 98.00 0.55 ± 0.05

a Identification mode: RI: Retention Index (GC-RI), MS; Mass spectra (GC–MS), Cs: Coinjection/comparison with the RI and Mass spectra of standard/known reference essential oils; Lit: Reported literature; IR: Infra Red spectral data; NMR: 1H and 13C NMR spectral data; t = trace (< 0.05%); Compounds higher than 1.00% are main constituents with ± Standard deviation (SD); Content (%) of individual constituents is an average of three replicate; RIExp: Retention index determined in DB-5 (30 m × 0.25 mm) using nalkanes; RILit: Retention index from Literature (Adams, 2007).

Table 2 Antibacterial potential of essential oil of Zingiber zerumbet (L.) Roscoe ex Sm. and zerumbone. Bacterial Strains

Staphylococcus aureus (MTCC 96) Staphylococcus aureus (MTCC 2940) Bacillus subtilis (MTCC 121) Staphylococcus epidermidis (MTCC 435) Streptococcus mutans (MTCC 890) Klebsiella pneumoniae (MTCC 109) Escherichia coli (MTCC 723) Salmonella typhimurium (MTCC 98) Pseudomonas aeruginosa (MTCC 741)

Essential oil

Standarda

Zerumbone

ZI (mm)

MIC (μg/mL)

ZI (mm)

MIC (μg/mL)

ZI (mm)

MIC (μg/mL)

10 ± 1 7±2 Na na 7±1 Na 6±1 6±2 na

166.6 ± 41.6 833 ± 333 833 ± 167 833 ± 333 208 ± 41.6 666 ± 166 208 ± 42 666 ± 166 833 ± 167

13 ± 2 9±1 5±2 4±1 10 ± 1 7±2 8±1 8±2 3±1

52.08 ± 10.41 208 ± 41.6 500 ± 0 666 ± 166 62.5 ± 0 208 ± 41.6 104.1 ± 20.8 208 ± 41.6 666 ± 166.6

26 20 25 21 29 24 22 27 20

0.39 ± 0.0 1.3 ± 0.26 0.78 ± 0.0 2.6 ± 0.52 0.52 ± 0.13 0.19 ± 0.0 0.19 ± 0.0 0.25 ± 0.06 1.56 ± 0.0

ZI: zone of inhibition; MIC: minimum inhibitory concentration. a Standard: Norfloxacin; Data are expressed as mean ± standard deviation.

752

± ± ± ± ± ± ± ± ±

2 1 3 2 3 2 2 1 2

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Table 3 Antiproliferative potential of zerumbone and rhizome essential oil of Zingiber zerumbet (L.) Roscoe ex Sm. IC50 (μg/mL) of antiproliferative activity assay against tested cell linesa Cell linesb Zerumbone Essential oil Doxorubicin

A549 24.38 ± 0.97 39.49 ± 1.02 3.46 ± 0.03

MDAMB-231 24.41 ± 1.04 33.37 ± 0.92 2.79 ± 0.03

A431 23.75 ± 0.86 35.17 ± 1.24 4.25 ± 0.04

K562 20.60 ± 0.57 45.10 ± 1.21 0.06 ± 0.01

WRL-68 31.32 ± 0.98 42.16 ± 1.13 2.63 ± 0.02

COLO-205 25.16 ± 1.16 – 2.93 ± 0.01

HaCaT 43.13 ± 1.07 46.02 ± 0.93 0.42 ± 0.01

HEK-293 32.37 ± 1.05 – 3.93 ± 0.05

The inhibitory concentration (IC50) in μg/mL was calculated using 2D linear and non-linear curve fitting correlation (Systat software Inc., UK). Organ specific cancer and normal cell lines, viz. A549 (Lung carcinoma); MDAMB-231 (Breast adenocarcinoma); A431 (Skin Carcinoma); K562 (Leukemia); WRL-68 (Hepatic carcinoma); COLO-205 (Colon carcinoma); HaCaT (Keratinocytes); HEK-293 (Embryonic Kidney Cells). a

b

Fig. 1. Percent cytotoxicity of essential oil and zerumbone against human cancer and normal cell lines.

24 h revealed that the proliferation of tested cells were strongly inhibited by the zerumbone, followed by the essential oil (Table 3). Zerumbone decreased the proliferation of lung, breast, colon, hepatic and leukaemia cancer cells after 24 h incubation with an IC50 ranged 20.60–31.15 μg/mL as well as the growth of keratinocytes (43.13 μg/ mL). Essential oil affect the proliferation of breast, hepatic, leukaemia and lung cancer cells and growth of keratinocytes with an IC50 range 33.37–46.02 μg/mL but moderately changes the viability of colon and kidney cell lines (percent inhibition 48.14 ± 0.47% and 44.68 ± 1.06%, respectively, Fig. 1). Zerumbone and essential oil showed antiproliferative potential but found non-significant compared to Doxorubicin (p > 0.05). Results showed that zerumbone was more effective with low IC50 values with broad spectrum antiproliferative potential against all tested cell lines compared to essential oil. Essential oil is a mixture of various constituents which might have antagonistic properties responsible for overall low activity of essential oil compared to zerumbone. Earlier studies showed significant activity of zerumbone in different cancer cell lines of colon, breast, pancrease, liver and leukaemia via different pathways. Zerumbone was found to induce apoptosis in ovarian and cervical cancer cell lines, and human renal carcinoma. The most compelling antiproliferative potential of zerumbone was found to be mainly due to its apoptosis-inducing and antiproliferative influences due to the presence of four-methyl group in a cross-conjugated dienone system (Abdul et al., 2008; Baby et al., 2009; Koga et al., 2016; Murakami et al., 2002; Rana et al., 2016; Sakinah et al., 2007; Yodkeeree et al., 2009). Zerumbone, a sesquiterpene alcohol, has exclusive presence in the essential oils of some aromatic plants of family Zingiberaceae. Zerumbone possessed significant biological activities viz., anticancer,

activity against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Mycobacterium intracellular, Salmonella choleraesuis (Abdul et al., 2008; Devi et al., 2014; Singh et al., 2014; Yob et al., 2011). However, zerumbone and its synthesized structural analogues were found to possess higher sensitivity against various tested bacterial strains (Kumar et al., 2013; Sivasothy et al., 2012; Vishwanatha et al., 2012). The activity potential of essential oils was found to vary as per their composition (content of the constituents) that varied depending upon various exogenous and endogenous factors. Essential oils are usually complex mixture constituents having synergistic or antagonistic properties to represent the overall activity of the essential oil (Lopes-Lutz et al., 2008). In light of this, it not judicious to assign a single or few constituent responsible of the overall antimicrobial activity of essential oils. However, present results revealed tested strains are more sensitive to zerumbone compared to essential oil. The lower activity of the essential oil as compared to zerumbone might be due to antagonistic effect of other constituents present in the rhizome essential oil. The antiproliferative activity of the essential oil of Z. zerumbet and its major constituent, zerumbone, was evaluated against various human cancer and normal cell lines (A549, MDAMB-231, A431, K562, WRL68, COLO-205, HaCaT, and HEK-293). The results of antiproliferative potential of rhizome essential of Z. zerumbet and zerumbone represented in Table 3 and Fig. 1. Principally, the ability of viable cells with active mitochondria able to cleave the tetrazolium ring was observed via MTT assay, where optical density is proportional to the number of viable cell. In the present investigation hepatic, breast, colon, leukaemia, lung, skin and keratinocytes were treated with different concentration of essential oil and zerumbone (10–50 μg/mL) for 753

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anti-inflammatory, anti-HIV, anti-bacterial, fungitoxic, consequently the potential of this bioactive molecule was studied against several diseases, viz. Alzheimer’s disease, suppressing different type of tumors, and as anti-inflammatory and analgesic agent (Abdul et al., 2008; Joseph et al., 2015; Koga et al., 2016; Murakami et al., 2002; Singh et al., 2014). Moreover, it is used as a versatile starting material for conversion to other derivatives and useful compounds such as paclitaxel possessing interesting bioactivities (Kitayama et al., 2002; Ruslay et al., 2007). The rhizome essential oil of Z. zerumbet is one of the best sources of the zerumbone. Besides Z. zerumbet oil, it is also reported in considerable amount in the essential oils of some other plants of family Zingiberaceae viz., Alpinia galanga (44.4%), Zingiber spectabile (59.1%), Zingiber ottensii (25.6–37.0%), and Globba ophioglossa (22.2%) (Lakshmi et al., 2007; Raj et al., 2010; Sirat and Nordin, 1994; Sivasothy et al., 2012). The diverse reported biological activities of zerumbone make it useful to develop natural derived therapeutics. Therefore, Z. zerumbet grown in foothills of north India with significant essential oil yield and zerumbone content could be exploited for this phyto-molecule having widespread use in the food-flavor and pharmaceutical products.

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