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Food Chemistry 104 (2007) 1586–1593 www.elsevier.com/locate/foodchem
Antioxidant and antibacterial activity of leaves of Etlingera species (Zingiberaceae) in Peninsular Malaysia
E.W.C. Chan, Y.Y. Lim *, Mohammed Omar
School of Arts and Sciences, Monash University Malaysia, Bandar Sunway, 46150 Petaling Jaya, Selangor, Malaysia Received 30 November 2006; received in revised form 28 December 2006; accepted 2 March 2007
Abstract Methanolic extracts from fresh leaves of ﬁve Etlingera species were screened for total phenolic content (TPC), antioxidant activity (AOA), and antibacterial activity. Analysis of TPC was done using the Folin–Ciocalteu method. Evaluations of AOA included 1,1-diphenyl-2-picrylhydrazyl free radical-scavenging ability, ferric-reducing antioxidant power (FRAP), ferrous-ion chelating (FIC) ability, and b-carotene bleaching (BCB) activity. Antibacterial activity was screened using the disc-diﬀusion method. Highest TPC, ascorbic acid equivalent antioxidant capacity (AEAC), and FRAP were found in leaves of E. elatior and E. rubrostriata. Leaves of E. maingayi, with the lowest TPC, AEAC, and FRAP, had the highest FIC ability and BCB activity. Ranking of TPC and AOA of diﬀerent plant parts of E. elatior was in the order: leaves > inﬂorescences > rhizomes. Leaves of highland populations of Etlingera species displayed higher values of TPC and AEAC than those of lowland counterparts. Leaves of Etlingera species exhibited antibacterial activity against Grampositive but not Gram-negative bacteria. Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Etlingera; Leaves; Highland; Lowland; Total phenolic content; Antioxidant activity; Antibacterial activity
1. Introduction Etlingera Giseke of the family Zingiberaceae are tall forest plants, with larger species reaching 6 m in height (Khaw, 2001). In the Phaeomeria group, inﬂorescences are borne on erect stalks protruding from the ground and, in the Achasma group, inﬂorescences are subterranean with ﬂowers appearing at soil level (Lim, 2000, 2001). The varying shades of pink and red colours of bracts and ﬂowers make Etlingera species very attractive plants. A total of 15 Etlingera species has been recorded in Peninsular Malaysia (Lim, 2001). Plants of Etlingera have various traditional and commercial uses. In Sabah, Malaysia, the hearts of young shoots, ﬂower buds, and fruits of E. elatior, E. rubrolutea, and E. littoralis are consumed by indigenous communities
Corresponding author. Tel.: +60 3 56360600; fax: +60 3 56360622. E-mail address: Lim.Yau.Yan@artsci.monash.edu.my (Y.Y. Lim).
as condiment, eaten raw or cooked (Noweg, Abdullah, & Nidang, 2003). In Thailand, fruits and cores of young stems of E. littoralis are edible, and ﬂowers of E. maingayi are eaten as vegetables (Sirirugsa, 1999). There are no reports on the use of rhizomes of Etlingera species. Inﬂorescences of E. elatior are widely cultivated throughout the tropics as spices for food ﬂavouring and as ornamentals. They are commonly used as ingredients of dishes such as laksa asam, nasi kerabu, and nasi ulam in Peninsular Malaysia (Larsen, Ibrahim, Khaw, & Saw, 1999). Farms in Australia and Costa Rica are cultivating the species and selling its inﬂorescences as cut ﬂowers (Larsen et al., 1999). In Malaysia, fruits of E. elatior are used traditionally to treat earache, while leaves are applied for cleaning wounds (Ibrahim & Setyowati, 1999). Leaves of E. elatior, mixed with other aromatic herbs in water, are used by post-partum women for bathing to remove body odour. Flavonoids in the leaves of E. elatior have been identiﬁed as kaempferol 3-glucuronide, quercetin 3-glucuronide,
0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2007.03.023
Elettariopsis. 1977). 100%).1. elatior. fulgens (Ridl. paper discs (Oxoid. were transferred back into the extraction vessel and extracted again each time with 50 ml methanol. and E. Type 1: synthetic). 2000. Phytochemical studies on rhizomes of E. Extracts were ﬁltered and stored at À20 °C for further use.5%). In this study. Lim. 10 lg) were used. were screened. Materials and methods 2. Plant materials Five species of Etlingera studied were E. Dried samples were then crushed in a mortar with liquid nitrogen and extracted with 250 ml of methanol three times for 1 h each time. potassium ferricyanide (Unilab. AOA. 98%). Ethanolic extracts from the ﬂower shoots of E. and rhizomes of Curcuma longa L. 2005).. In our previous study (Chan. littoralis (Koenig) Giseke and E.C. Chemicals and reagents For TPC analysis. and steroids (Habsah et al. leaves of each species were cut into small pieces and 100 g were weighed and freeze-dried.2. Muller–Hinton agar (Oxoid). in press). and dipotassium hydrogen phosphate (Merck. and antibacterial activity of leaves of ﬁve Etlingera species were analysed. & Keawpradub. Boesenbergia..) Kuntze. and anhydrous sodium carbonate (Fluka. and streptomycin susceptibility discs (Oxoid. gallic acid (Fluka.K. 2. rubrostriata (Holtt. Measurement of extraction eﬃciency was based on TPC. 6 mm). . elatior have antimicrobial activity and are cytotoxic to HeLa cells (Mackeen et al. Voucher specimens were deposited at the herbarium of Forest Research Institute Malaysia (FRIM). Folin–Ciocalteu’s phenol reagent (Fluka. Rhizomes and inﬂorescences were extracted in a similar manner. In E. Pahang.M. 98%) and ferrous sulphate hepta-hydrate (HmbG Chemicals) were used. 99%) were used. 1992. 2.) C. Results showed that leaves of Etlingera had the highest TPC and AEAC. 2005). Lim. Their rhizomes have been reported to contain antioxidants comparable to a-tocopherol. 90%) was used.. E. Plubrukarn. lipid peroxidation inhibitory activity of the isolated diarylheptanoids was greater than that of a-tocopherol. Although leaves of ginger species have been used for food ﬂavouring and as traditional medicine. For comparison as positive controls. Characteristic scent of leaves when crushed was another useful cue for species identiﬁcation. For the screening of antibacterial activity. / Food Chemistry 104 (2007) 1586–1593 1587 quercetin 3-glucoside. 99. 2. Zaeoung. with continuous swirling for 1 h at room temperature. The seven genera were Alpinia. elatior (Jack) R.M.3. elatior led to the isolation of two new and six known compounds of diarylheptanoids. Their identiﬁcation in the ﬁeld was based on taxonomic descriptions and photographic illustrations of Khaw (2001) and Lim (2000. for FIC assay. labdane diterpenoids. TPC. young leaves of the tea plant.1-diphenyl-2-picrylhydrazyl (Sigma. TPC and AOA of the diﬀerent plant parts were compared.4. Smith. Altitudinal variation in leaf TPC and AEAC of four Etlingera species was also studied. and Zingiber. mature leaves were sampled from three diﬀerent clumps. Chan et al. Flavonoid content of inﬂorescences of E. Camellia sinensis (L. and from Ulu Gombak in Selangor. Habsah et al. fresh leaves (1 g) were powdered with liquid nitrogen in a mortar and extracted using 50 ml of methanol. respectively (Miean & Mohamed. 99%). 2001). For disc-diﬀusion assay. elatior were collected from FRIM while its inﬂorescences were purchased from the supermarket. Leaves of highland populations of Etlingera species were sampled from Janda Baik and Genting Highlands in Pahang. and quercetin 3-rhamnoside (Williams & Harborne. maingayi (Bak. & Lim. nutrient broth (Oxoid). for BCB assay. 1997). trichloroacetic acid (HmbG Chemicals. Scaphochlamys. 2N). For each species. Altitude of locations where populations were sampled was measured using a Casio altimeter (Model PRG-70-1VDR). Smith of the Phaeomeria group. Samples were ﬁltered and the solvent was removed using a rotary evaporator. second and third extractions were conducted. 2. 1.W. After ﬁltration. ferric chloride hexa-hydrate (Fisher Scientiﬁc.K. total phenolic content (TPC) and ascorbic acid equivalent antioxidant capacity (AEAC) of leaves and rhizomes of ﬁve wild and six cultivated ginger species.) R. were collected from a tea plantation in Cameron Highlands. belonging to seven genera. for FRAP assay. potassium dihydrogen orthophosphate (Fisher Scientiﬁc. Methanol extraction eﬃciency To test the eﬃciency of methanol extraction. Curcuma. ferrozine (Acros Organics. linoleic acid (Fluka). along with the ﬁlter paper. hardly any research has been done on their AOA. elatior has been estimated to be 286 and 21 mg of kaempferol and quercetin (per kg dry weight). 99%) were used. Lim of the Achasma group. and Tween 40 (Fluka) were used. Preparation of extracts For the analysis of TPC and AOA. and E. 2001). 100%). AOA. residues. Past studies on the antioxidant activity (AOA) of ginger species were conﬁned to rhizomes (Jitoe et al. Etlingera. and Zingiber oﬃcinale Roscoe were purchased from the supermarket.8%).. Values were signiﬁcantly higher than those of rhizomes. This study represents the ﬁrst systematic analysis of TPC.E.) C. 99. and antibacterial activity of leaves of Etlingera species. for DPPH assay. while leaves of lowland populations were sampled from FRIM. Dried extracts were kept at À20 °C for analysis. Rhizomes of E. chloroform (Fisher Scientiﬁc. b-carotene (Sigma. Using the ferric thiocyanate method.
and Imiyabir (2004) was used to screen for antibacterial activity. FeSO4 (1 ml) was mixed with diﬀerent dilutions of extract (1 ml). elatior are entirely green.5 ml and diluted with 2. The mixture was then separated into aliquots of 2.2 ml.0148 (R2 = 0. 2.5 ml.767x (R2 = 0. Determination of total phenolic content Total phenolic content (TPC) of plant extracts was determined using the Folin–Ciocalteu assay reported by Kahkonen et al.00387 mg/ml. and BRextract and BRcontrol are bleaching rates of the extract and negative control.1588 E. diluted 10 times) and sodium carbonate (1. (2002) was adopted.2 M.3.C. and transferred onto the inoculated agar.7. 000 The IC50 of ascorbic acid used for calculation of AEAC was 0. To each diluted aliquot. Absorbance was measured at 517 nm after 30 min. Bleaching rate was expressed as AOA (%) and calculated as follows: Bleaching rate ðBRÞof b-carotene ¼ lnðAinitial =Aextract Þ=60 AOA ð%Þ ¼ 1 À ðBRextract =BRcontrol Þ Â 100 where Ainitial and Aextract are absorbances of the emulsion before and 1 h after incubation.W. elatior. 2002) as follows: AEAC ðmg AA=100gÞ ¼ IC50ðascorbateÞ =IC50ðextractÞ Â 100. Leaves of E. After incubation overnight at 37 °C. Aliquots of the emulsion (3 ml) were mixed with 10.5. 10% w/v) was added to stop the reaction. Agar cultures of Gram-positive bacteria (Bacillus cereus. Chung. Chang.6. respectively.5 ml.5 ml phosphate buﬀer (0. and Salmonella cholerasuis) were prepared. 2. moderate for inhibition 50 < 70%. Antibacterial activity was also expressed as inhibition percentage of streptomycin and arbitrarily classiﬁed as strong for inhibition of P70%. 1. 50. pH 6. 2. Solutions of 2 mM FeSO4 and 5 mM ferrozine were diluted 20 times. BCB assay The b-carotene bleaching (BCB) assay reported by Kumazawa et al. Suspensions of bacteria (100 ll) were spread evenly onto 20 ml Mueller–Hinton agar preset in 90 mm Petri dishes. and emit a pleasant sour scent when crushed. shiny. Screening for antibacterial activity The disc-diﬀusion method described by Chung. After 30 min.6) and 2.6. 500 ll of ferric chloride solution (0.4. Chan et al. and Hsu (2000) was adopted with modiﬁcations. Initial absorbance of the emulsion was measured at 470 nm. Measurement at 700 nm is needed to correct for the presence of haze.0111x À 0. respectively. sometimes ﬂushed pink when young.5% w/v) were added to the extracts (300 ll. inhibition zones were measured and recorded as mean diameter (mm).6. followed by ferrozine (1 ml). undulated. Folin–Ciocalteu reagent (1. Micrococcus luteus. After 30 min. The mixture was incubated at 50 °C for 20 min. Leaves of E. and van Beek (2004) was adopted with modiﬁcations. maingayi are red- .5 ml of potassium ferricyanide (1% w/v). / Food Chemistry 104 (2007) 1586–1593 2. Chloroform was evaporated under vacuum and oxygenated ultra-pure water (100 ml) was added and mixed well.9974). Leaves of E. and 100 ll of extract and incubated in a water bath at 50 °C for 1 h.5 ml of water. and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli.1. 2.9998). Bleaching rate of b-carotene was measured at 470 nm and 700 nm. Diﬀerent dilutions of the extract (1 ml) were added to 2. FRAP assay The ferric-reducing antioxidant power (FRAP) assay reported by Chu. absorbance was measured at 765 nm. Diﬀerent dilutions of the extract (1 ml. They emit a pleasant sour scent similar to those of E. triplicate). Streptomycin susceptibility discs (10 lg) and methanolimpregnated disc were used as positive and negative controls. fulgens are dark green. Paper discs (6 mm diameter) were impregnated with 1 mg of plant extract dissolved in 100 ll solvent.2. and their underside is bright red when young. Pseudomonas aeruginosa. Absorbance was measured at 562 nm after 10 min.6.1% w/v) were added. b-Carotene/linoleic acid emulsion was prepared by adding 3 ml of b-carotene (5 mg/50 ml chloroform) to 40 mg of linoleic acid and 400 mg of Tween 40. Trichloroacetic acid solution (2.6. 7. Goh. Ngeow. DPPH assay The DPPH free radical-scavenging (FRS) assay used by Miliauskas. TPC was expressed as gallic acid equivalents (GAE) in mg per 100 g. Descriptions of plant specimens Photographs of plants of the ﬁve species of Etlingera studied are shown in Fig. 2.1. Venskutonis. respectively. Results and discussion 3. 3. (1999). absorbance was measured at 700 nm.9 mg/100 ml methanol). Determination of antioxidant activity 2. FRS ability was calculated as IC50 and expressed as ascorbic acid equivalent antioxidant capacity (AEAC) in mg AA/100 g (Leong & Shui. The calibration equation for gallic acid was y = 0. The ability of extracts to chelate ferrous ions was calculated as follows: Chelating effect % ¼ ð1 À Aextract =Acontrol Þ Â 100 where Aextract and Acontrol are absorbance of the extract and negative control. FRAP of extracts was expressed as mg GAE/ g. FIC assay The ferrous-ion chelating (FIC) assay used by Singh and Rajini (2004) was adopted. and weak for inhibition <50%. The calibration equation for gallic acid was y = 16. triplicate) were added to 2 ml of DPPH (5.
sometimes ﬂushed pink when young.6 ± 1. sinensis (Chan.e. exhibited the highest FIC ability (Wong. / Food Chemistry 104 (2007) 1586–1593 1589 Fig.6 ± 2. elatior rubrostriata littoralis fulgens maingayi 3550 ± 304a 3480 ± 390ab 2810 ± 242bc 2540 ± 91c 1110 ± 93d Values of TPC. Leaves of E. respectively. leaves of Alpinia galanga. and do not have any scent.05. fulgens. Leaves of E. It is evident that Etlingera species with high leaf TPC also have high AEAC and FRAP. respectively. 3. Lowest values were found in the leaves of E. Moderately high AEAC and FRAP were found in the leaves of E.2% in E. AEAC. rubrostriata to 88.0b 9. Methanol extraction eﬃciency of leaves Methanol showed high extraction eﬃciency of leaves of Etlingera species. elatior > E. translucent when young. which could alter antioxidant activity (Yao et al.6 ± 2. respectively. and FRAP have low FIC ability and vice versa. Leaves of E. dish below. elatior and E. It is a suitable solvent due to its ability to inhibit polyphenol oxidase. which are generally better chelators than are phenols. Lim. sinensis (positive control). TPC (mg GAE/100 g) Antioxidant activity (AOA) AEAC (mg AA/100 g) 3750 ± 555a 3540 ± 401a 2930 ± 220b 2030 ± 126c 963 ± 169d FRAP (mg GAE/g) 19.3.8%. Of the Etlingera species analysed.. Activity was expressed in mg AA/100 g. 3. Values were 3750 ± 555 mg AA/100 g and 19. For each column. the trend was reversed with leaves of E. 2006). maingayi. E. ascorbic acid equivalent antioxidant capacity (AEAC). rubrostriata are green with distinctive purplish-brown bars on upper surface and do not have any scent.8d E. maingayi. A second extraction yielded 12. and a third yielded 4. with the lowest TPC. and leaves of E. and 3540 ± 401 mg AA/100 g and 16. and 2030 ± 126 mg AA/100 g and 9. rubrostriata had the highest TPC (Table 1). maingayi. rubrostriata.4a 11. FRAP.7% in E. Among the species of Etlingera studied. elatior and E.9 ± 0. 2004). elatior. Of four species studied. Values were 3550 ± 304 and 3480 ± 390 mg GAE/ 100 g. AEAC.9% and 7. littoralis > E. Antioxidant activity of leaf extracts Antioxidant activity (AOA) of leaf extracts from Etlingera species was evaluated using the DPPH. 3. maingayi and E. AEAC. 2007). & Chew. littoralis were comparable to the FIC ability of young leaves of C.8 mg GAE/g were found in the leaves of E. and emit an unpleasant sour scent. Methanol has been recommended for the extraction of phenolic compounds from fresh plant tissues. rubrostriata had high AEAC and FRAP (Table 1). leaves of E. littoralis are entirely green. . respectively. fulgens. E. The compounds responsible could be nitrogen-containing compounds. maingayi. FIC. This would mean that phenolic compounds in extracts responsible for antioxidant activities of scavenging free radicals and reducing ferric ions might not be directly involved in ferrous ion chelation. and BCB assays. 1. elatior and E.4 mg GAE/g for E. mg GAE/g.4% and 4. fulgens > E. leaf AEAC and FRAP shared the same order of ranking as leaf TPC i.1 mg GAE/g for E.4 ± 0. maingayi and E.0%. fulgens having the highest values (Fig. values followed by the same letter (a–d) are not statistically different at P < 0.6 ± 2. Photographs of plants of ﬁve species of Etlingera studied. Values were 2930 ± 220 mg AA/100 g and 11. E. respectively. Lowest values of 963 ± 169 mg AA/ 100 g and 4. as measured by the Tukey HSD test. rubrostriata > E. and FRAP.4 ± 0. In terms of FIC ability. Total phenolic content of leaf extracts Total phenolic content (TPC) of leaf extracts was determined using the Folin–Ciocalteu method and expressed in mg GAE/100 g.4c 4. It can therefore be seen that leaves of Etlingera species with high TPC.. Chan et al.6 ± 2. and for young leaves of C.6 ± 1. Leaves of E.4 mg GAE/g for E. and FRAP are means ± SD (n = 3). E. and ferric-reducing antioxidant power (FRAP) of leaves of Etlingera species (fresh weight) Etlingera sp. High methanol extraction eﬃciency has been reported for leaves and ﬂowers of Alpinia species (Wong. littoralis and E. Yields of the ﬁrst extraction ranged from 82. 2006). Table 1 Total phenolic content (TPC). Similar observations were made with leaves of Alpinia.E. fulgens had the lowest TPC of 1110 ± 93 and 2540 ± 91 mg GAE/100 g.4.C.9 ± 0.W.0 mg GAE/g for E. Results showed that leaves of E. E. fulgens were superior.1a 16. chelating ability (%) and AOA (%). 2). maingayi and E.2. rubrostriata. respectively.
I. Values were 3550 ± 304 mg GAE/100 g. Results are means ± SD (n = 3). Although leaves of E. Outstanding leaf TPC and AEAC of both E. screening of ﬁve wild and six cultivated ginger species showed that leaf TPC and AEAC were generally higher than those of rhizomes. elatior and E. sinensis (L) E.1590 100 E. Amomum. High BCB activity of leaves of Etlingera species reﬂects their ability to strongly inhibit lipid peroxidation. and Zingiber rhizomes were comparable to a-tocopherol. Its leaf BCB activity was better than that of rhizomes of C. BCB activity of leaves was much higher than that of rhizomes but slightly lower than that of inﬂorescences. rubrostriata. E. maingayi had the highest values. In our previous study (Chan et al. 2. In terms of BCB activity. 268 ± 45 mg AA/100 g. TPC.6 ± 2. Elzaawely. 185 ± 59 mg AA/100 g. 20. L. and 19. and E. littoralis. longa (Fig. Xuan. 2007). fulgens (L) E.05 (Table 2). in press). AEAC.. & Gargova. Rhizomes of cultivated species have been reported to possess radical-scavenging compounds comparable to commercial antioxidants on a weight per weight basis. 3750 ± 555 mg AA/100 g. 3a) and young leaves of tea (Fig. Jitoe et al. elatior reaﬃrmed that TPC and AOA of leaves were significantly higher than those of rhizomes at P < 0. 3b). respectively. Recently.5. fulgens. (1992) reported that AOA of extracts of Alpinia. and 22 times higher in leaves than in rhizomes. Leaves of wild and cultivated Etlingera species therefore contain more antioxidants than do other plant parts. leaves of E. rhizomes. longa and superior to that of young tea leaves and rhizomes of Z. Curcuma. Similarly. Extracts of Z. maingayi (L) E. This implies that rhizomes would have higher AOA than would other plant parts. Young leaves of Camellia sinensis were used as positive control. values were higher than that of young leaves of C. There appears to be no correlation between BCB activity and AOA. elatior having slightly lower values (Fig. inﬂorescences. comparable with that of rhizomes of C. elatior showed superiority over inﬂorescences and rhizomes in terms of FIC ability (Fig. oﬃcinale rhizomes (Fig. Out of the 11 species screened. elatior (L) E. 3. Denev.. Stoyanova.C. maingayi were seven and eight times higher than those of rhizomes. In Zingiberaceae. eight species had signiﬁcantly higher leaf TPC and/or AEAC. rubrostriata (L) C.2 mg GAE/g for rhizomes. / Food Chemistry 104 (2007) 1586–1593 80 Chelating ability (%) 60 E. Chan et al. leaves of E. With the exception of E. Results of this study on the diﬀerent plant parts of E. oﬃcinale. leaves of all Etlingera species studied showed high BCB activity.. respectively. littoralis (L) E. 3b). R. 295 ± 24 mg GAE/100 g. fulgens showed the lowest BCB activity. oﬃcinale rhizomes had better radical-scavenging ability than had butylated hydroxytoluene and quercetin (Stoilova. and 187 ± 46 mg GAE/ 100 g.1 mg GAE/g for leaves. Ferrous-ion chelating (FIC) ability of leaves of Etlingera species and diﬀerent plant parts of Etlingera elatior (fresh weight). and FRAP than had inﬂorescences and rhizomes at P < 0. BCB activities of inﬂorescences and rhizomes were comparable to those of rhizomes of C. FIC ability of leaves was comparable to that of young leaves of C. Abbreviations: E.9 ± 0. as measured by the other assays. longa with leaves of E. and 1. Camellia. 2). Etlingera. TPC and AOA of extracts from diﬀerent plant parts Analyses of diﬀerent plant parts of E. and Tawata (2007) reported that ethyl acetate extracts from leaves of Alpinia zerumbet .5 ± 0. AEAC and FRAP were 19. respectively. elatior (I) E. and 0.W. Krastanov.2 mg GAE/g for inﬂorescences. leaves. it is generally believed that antioxidants and other secondary metabolites are transported to the rhizomes where they are accumulated. elatior showed that leaves had signiﬁcantly higher TPC. C. This is supported by ﬁndings of Lim and Quah (2007) that methanolic extracts of six cultivars of Portulaca oleracea showed that TPC correlated well with AEAC and FRAP but not with BCB activity. sinensis but lower than that of Z. 3a). sinensis. Ranking of TPC and AOA (AEAC and FRAP) was in the order: leaves > inﬂorescences > rhizomes.05. elatior (R) 40 20 0 0 5 10 15 20 25 Mass (mg in 3 ml) Fig. respectively.
ascorbic acid equivalent antioxidant capacity (AEAC). There is increasing evidence that enhanced UV-B radiation induces production of phenolic compounds in plants (Bassman. 3. 2004). such as higher UV-B radiation and lower air temperature. Camellia.1a 1.2c Leaves Inﬂorescences Rhizomes 3550 ± 304a 295 ± 24b 187 ± 46c Values of TPC. elatior. R. and 2. cereus and S. cereus. aureus (Table 4). 3a: C.C. Rhizomes of Curcuma longa and Zingiber oﬃcinale. Leaves of E. which have antioxidant ability (Jansen. 3b: Z. 1998). Phenylalanine ammonia lyase (PAL) is up-regulated. Abbreviations for Fig. Higher altitudes seem to trigger an adaptive response in Etlingera species. 3. Etlingera.E. 3. longa (R) E. rubrostriata. Mean diameter of the zone of inhibition of streptomycin was 23 mm for M. fulgens showed signiﬁcantly higher values with greater altitude at P < 0. littoralis was marginally higher (Table 3). 2004). E. rubrostriata (L) E. and by the leaves of E. while E. Etlingera. fulgens. & Greenberg. elatior. AEAC. and ferric-reducing antioxidant power (FRAP) of diﬀerent plant parts of Etlingera elatior (fresh weight) Plant part of E. elatior (I) E. Results are means ± SD (n = 3). L. Gaba.. aureus. rubrostriata on B. fulgens had the lowest values of 1280 ± 143 mg GAE/100 g and 845 ± 159 mg AA/100 g. elatior (L) b 100 80 AOA (%) 60 40 20 0 Z. Abbreviations for Fig. as measured by the Tukey HSD test. Leaves of E. with values of 3480 ± 390 mg GAE/100 g and 3540 ± 401 mg AA/100 g.6. respectively. leaves of all ﬁve Etlingera species were found to inhibit Gram-positive B. left. L. leading to increased production of ﬂavonoids and other phenolics (Chalker-Scott & Scott. 2004). and right bars represent extract concentrations of 0. fulgens (L) E. elatior. and young leaves of Camellia sinensis were used as positive controls. b-Carotene bleaching (BCB) activity of leaves of Etlingera species (fresh weight). E. maingayi exhibited moderate inhibition of the three bacteria. M. respectively.05. Table 2 Total phenolic content (TPC). Curcuma. rubrostriata. I. littoralis on S. littoralis (L) E. respectively. Moderate inhibition was shown by the leaves of E. For each column. Altitudinal variation in TPC and AEAC of leaf extracts Leaves of all Etlingera species sampled from highland populations were found to have higher TPC and AEAC than those of lowland counterparts. with values of 3550 ± 304 mg GAE/100 g and 3750 ± 555 mg AA/100 g. elatior TPC (mg GAE/100 g) Antioxidant activity (AOA) AEAC (mg AA/100 g) 3750 ± 555a 268 ± 45b 185 ± 59c FRAP (mg GAE/g) 19. E. inﬂorescences. showed higher inhibition of b-carotene oxidation and scavenging activity of free radicals than did rhizomes. and E. and E. middle. R. resulting in the accumulation of ﬂavonoids and anthocyanins. elatior (R) C. rhizomes. C.W.6 ± 2. maingayi (L) E. Antibacterial activity of leaf extracts Using the disc-diﬀusion method. 1. and of E.0 lg in 3 ml.2b 0. leaves.7.. This further supports our result that leaves have free-radical scavengers that are more eﬀective than those found in rhizomes. Highest leaf TPC and AEAC were found in highland populations of E. and S.0. and FRAP are means ± SD (n = 3). Methanol showed no inhibitory eﬀect on the three bacteria. values followed by the same letter (a–c) are not statistically different at P < 0..9 ± 0.05. rhizomes. aureus (Table 4). Chan et al. / Food Chemistry 104 (2007) 1586–1593 1591 a 100 80 AOA (%) 60 40 20 0 C. leaves. Zingiber. luteus. E. Low temperatures have also been shown to enhance PAL synthesis in a variety of plants. sinensis (L) Fig. cereus and S. Enzymes associated with the synthesis of phenolics are produced in greater quantities or show increased activity (Chalker-Scott & Scott.. Higher leaf TPC and AEAC of highland populations over those of lowland counterparts might be due to environmental factors. Streptomycin was used as positive . aureus. Lowland populations of E. For each species. luteus. officinale (R) E. and 17 mm for B.5 ± 0..2.
Adding 2 mM EDTA to the agar caused P. The FIC ability of leaves of E. Wong. maingayi and E. Conclusion Results showed that methanolic extracts from fresh leaves of Etlingera species had high values of TPC. applicable to the food and nutraceutical industries. In this study. S.C. Ranking of TPC and AOA of diﬀerent plant parts of E. Malaysia. elatior E. Bacillus. coli and S. elatior E. and S.W. fulgens E. Furr. aureus appeared to be more sensitive. Baluja. This suggests a diﬀerent mode of action for EDTA. 2005. About 52% of the extracts inhibited S. M. Leaves of Etlingera species exhibited high BCB activity. 2004). 1974). Initial ﬁndings warrant further investigations. Gram-negative bacteria have an outer membrane consisting of lipoprotein and lipopolysaccharide. E. littoralis E. elatior was in the order: leaves > inﬂorescences > rhizomes. Screening for antibacterial activity of 191 plant extracts belonging to 30 families of plants from Sabah. moderate ++ for inhibition 50 < 70%. together with the antibiotic. rubrostriata E. P. & Chanda. Species with the highest leaf TPC. Unlike the commercial use of rhizomes. Leaves of Etlingera showed no antibacterial activity on Gram-negative bacteria of E.05 measured by the Tukey HSD test. aureus 11(65)++ 11(65)++ 9(53)++ 11(65)++ 10(59)++ 17 – ria was carried out. and FRAP. control because it has been used as the antibiotic for Grampositive and Gram-negative bacteria. EDTA has been reported to permeabilise the outer membrane of P. This renders the Gram-negative bacteria generally less susceptible to plant extracts than the Gram-positive bacteria. and FRAP possessed the lowest leaf FIC ability and vice versa. leaves of Etlingera species have great potential to be developed into natural preservatives and herbal products. & Russell. rubrostriata Streptomycin Methanol 10(59)++ 11(65)++ 8(47)+ 9(53)++ 9(53)++ 17 – M. or weak + for inhibition < 50%. Chan et al. aeruginosa. Leaves of highland populations of Etlingera species had higher values of TPC and AEAC than had those of lowland counterparts. making it susceptible to antibiotics and certain antiseptic agents (Haque & Russell. Abbreviations: B. values followed by the same letter (a–b) are not signiﬁcantly diﬀerent at P < 0. With promising antioxidant and antibacterial properties. the harvesting of leaves does not result in destructive sampling of plants. AEAC. Mean diameter of the zone of inhibition is in millimetres.. 4. longa and superior to that of young tea leaves. matching that of rhizomes of C. aeruginosa. ANOVA does not apply between species. Among the Gram-positive bacteria. luteus 13(57)++ 12(52)++ 9(39)+ 14(61)++ 11(48)+ 23 – S. aureus. It is the ﬁrst time the method has been used for testing antibacterial activity of plant extracts. For columns of each species. cholerasuis. Bacteria can be either exposed to the permeabiliser. possibly synergistic with plant extracts. Staphylococcus. S. sinensis. / Food Chemistry 104 (2007) 1586–1593 Table 3 Total phenolic content (TPC) and ascorbic acid equivalent antioxidant capacity (AEAC) of leaves of Etlingera from highland and lowland locations (fresh weight) Etlingera sp. cholerasuis. . showed similar results (Chung et al. Leaves of Etlingera species inhibited Gram-positive but not Gram-negative bacteria. Figures in parentheses are inhibition percentages compared to streptomycin. aeruginosa to be susceptible to all leaf extracts of Etlingera species but inhibited the growth of E. the pre-treatment method was not eﬀective. fulgens Location Janda Baik FRIM Ulu Gombak FRIM Genting Highlands FRIM Janda Baik FRIM Altitude (m asl) 400 100 300 100 800 100 400 100 TPC (mg GAE/100 g) 3550 ± 304a 2390 ± 329b 3480 ± 390a 2430 ± 316b 2810 ± 243a 2340 ± 386a 2270 ± 31a 1280 ± 143b AEAC (mg AA/100 g) 3750 ± 555a 2280 ± 778b 3540 ± 401a 2640 ± 508a 2930 ± 220a 2220 ± 913a 2030 ± 126a 845 ± 158b Values of TPC and AEAC are means ± SD (n = 3).. 1999). fulgens was superior to that of young leaves of C. Zone of inhibition in mm (inhibition %) B. maingayi E. For all ﬁve Etlingera species. Antibacterial activity is categorized as strong +++ for inhibition P 70%. AEAC. Micrococcus. leaves showed stronger antibacterial activity than did rhizomes. littoralis E. or pre-treated with the permeabiliser prior to introduction of the antibiotic (Ayres. coli.1592 E... cereus E. 2001). 2006). Preliminary investigation on the use of ethylenediamine tetraacetic acid (EDTA) to improve the eﬃcacy of leaf extracts of Etlingera species against Gram-negative bacteTable 4 Antibacterial activity of leaves of Etlingera species against Gram-positive bacteria using the disc-diﬀusion method Etlingera sp. Antibacterial studies of extracts from various ginger species also showed no inhibition of Gram-negative bacteria (Chandarana. which is selectively permeable and thus regulates access to the underlying structures (Chopra & Greenwood.
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