Article
Antimicrobial polyphenols from small tropical fruits, tea and spice oilseeds Sahar Aman1, Asma Naim1, Rahmanullah Siddiqi2 and Shahina Naz2
Abstract The polyphenolic fractions of fruits: Terminalia catappa, Carissa carandas, Ziziphus nummularia; spice oilseeds: thymol, mustard, fenugreek and poppy seeds; and herb: green and black teas were analyzed for their total phenolics, flavonoids and antimicrobial potential. All fractions from fruits, except anthocyanin of C. carandas, displayed substantial antibacterial activity in accordance to their phenolic contents, the difference in activity being quite significant (p < 0.05), highest for T. catappa (minimum inhibitory concentration, MIC: 7.81000 mg/mL) and lowest for C. carandas (MIC: 62.51000 mg/mL). With few exceptions, both green and black teas’ fractions inhibited the tested strains, however, green tea fractions (MIC: 15.63125 mg/mL) were more active than black (MIC: 31.251000 mg/mL) and neutral were more active than their corresponding acidic fractions. Oil fractions of all oilseeds were found to be more active than their polyphenolic fractions, their antibacterial action decreased in the order thymol > mustard > fenugreek > poppy seeds (p < 0.05). Though the fruits used for the study are underutilized and have been emphasized for processed products, they may potentially be important to fight against pathogenic bacteria in view of their MICs. The teas and oilseeds, though a small part of total food intake, are more functional and active against the tested bacterial species and may find potential applications in therapeutics and food preservation.
Keywords Flavonoids, flavanols, phenolic acids, antimicrobial activity Date received: 14 November 2012; revised: 13 February 2013
INTRODUCTION The main reasons of having fruits, herbs and spices as part of our diet do not include nutritional demand, growth and survival only. Beyond this, these are considered to be very rich sources of micronutrients, phytomedicines or neutraceuticals like alkaloids, carotenoids and polyphenols and so on (Lai and Roy, 2004; Pleczar et al., 1998). Among the neutraceuticals, polyphenols that have been proved as antimicrobial (Cowan, 1999; Naga et al., 2000; Recio, 1989), antiviral and antioxidant (Sacchetti et al., 2005), anticancer (Kandil et al., 1999) are gaining popularity and Food Science and Technology International 20(4) 241–251 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1082013213482476 fst.sagepub.com
therefore have been exploited by many scientists and researchers for their bioactivities. The antimicrobial action of added material is not only important for the shelf life of food but also for the health or safety of the consumers. The antimicrobial activity of the polyphenolics has been claimed since the prehistoric time, many research studies have been conducted to explore and identify polyphenolic-rich sources, screen them for their antimicrobial activity, quantify their antimicrobial strength and evaluate their synergistic interaction with currently used 1
Department of Microbiology, University of Karachi, Karachi, Pakistan 2 Department of Food Science & Technology, University of Karachi, Karachi, Pakistan Corresponding author: Shahina Naz, Department of Food Science & Technology, University of Karachi, Karachi 75270, Pakistan. Email:
[email protected]
Food Science and Technology International 20(4) antibiotics. The inhibition of b-lactamases (extended spectrum b-lactamase) – producing multidrug-resistant bacteria by ethanolic crude extracts and fractions of Aallium sativum, Camellia sinensis, Citrus sinensis, Magnifera indica and Punica granatum (containing alkaloids, phenols and flavonoids as active phytoconstituents) has been demonstrated by Ahmad et al. (2006) and Ahmad and Aqil (2007). Growth inhibition of the human pathogenic bacteria by berry phenolics (Puupponen-Pimia¨ et al., 2005) and the volatile oils and acetone extracts of anise, carom/thymol seeds (ajwain), bay leaf (tejpat), fennel, coriander, turmeric and star anise have also been reported by Singh et al. (2007). More than 8000 polyphenolics have been isolated so far from various sources (Lecour et al., 2011). Many of these have been extensively investigated for their bioactivities. However, many food plants are still needed to be thoroughly evaluated for their antimicrobial potential due to the following reasons: (1) diversity in the structural features of the polyphenols markedly affecting the activity, (2) complexity and qualitative and quantitative variation in polyphenolic contents with respect to growth stages and ecological conditions of the source plant and (3) emergence of currently used antibiotics has become threat to the future of these drugs in chemotherapy and thus it has become essential to explore new and potential sources of antibiotics. In this study, the antimicrobial potential of the crude polyphenolic extracts and their fractions derived from some of the tropical and underutilized fruits – Terminalia catappa (red/Indian almond), Carissa carandas (karunda) and Ziziphus nummularia (jharber or ber); the herb – Camellia sinensis (black and green teas) as well as the essential oils from the spice oilseeds namely, Trachyspermum ammi (ajwain), Brassica alba (mustard/ rai), Trigonella foenum-graecum (fenugreek) and Papaver somniferum (poppy seeds/khash khash) were investigated.
MATERIALS AND METHODS Sampling Sample fruits E. jambolana and Z. nummularia were purchased from local fruit market in June and July, respectively; herb: green and black teas; spices: ajwain, fenugreek, mustard and poppy seeds were procured from local super market while C. carandas fruits were collected from Karachi University campus in the month of July. Verification of the samples was done by the Taxonomy Section, Department of Botany, University of Karachi. Sample fruits were stored at 5 C after washing. The seeds were removed and pulps with peels of the fruits were lyophilized before analysis. The oilseeds were washed, dried and then stored on shelves at a cool and dry place. 242
Extraction and fractionation of polyphenolics from fruits Ten grams of each fruit were soaked in 100 mL of 80% aqueous methanol in an Erlenmeyer flask which was immersed into the ultrasonic bath and sonicated for 30 min with continual nitrogen gas purging and periodic shaking. The mixture was filtered through vacuum suction using Whatman filter paper No. 2 and a chilled Buchner funnel. The residue was reextracted with 80% aqueous methanol as above and the combined filtrates were then evaporated under vacuum at 40 C to remove methanol. The concentrate was re-solubilized in deionized distilled water to a total volume of 100 mL, flushed with nitrogen gas and stored at 4 C until analysis (Kim and Lee, 2002). Fractionation of polyphenols into anthocyanins (Fraction II) and non-anthocyanins were performed. Non-anthocyanin fraction was further divided into flavanols (Fraction 1a), flavonols (Fraction 1b) and phenolic acids (Fraction 1c) as described by Kim and Lee (2002) and adopted by Siddiqi et al. (2011) with modification in the procedure of fractionation. Quantitative analyses All the fractions were analyzed for the total phenolics and total flavonoids except Fraction 1a. Fraction II was also analyzed for total anthocyanins. All the estimation was carried out in triplicate and data were expressed as mean standard error. Total phenolics. The phenolics were estimated following the procedure of Jayaprakasha et al. (2003). First, 10 mg of each extract dissolved in 1 mL (6:4 v/v) methanol and water was added to 2 mL of one-tenth diluted Folin-Ciocalteu reagent, and 2 mL of 7.5% sodium carbonate solutions. After holding the reaction mixtures for 30 min at room temperature, the absorbance was measured at 765 nm. For calibration curve, the gallic acid was used as standard and the results were expressed as mg gallic acid/100 g. Total flavonoids. To 10 mg of each extract or standard of catechin (5–25 mg) were added 4 mL of water and 0.3 mL 5% NaNO2. After 5 min 0.3 mL of 10% AlCl3 and 2 mL of 1 M NaOH were added to each and total volume was made up to 10 mL with water. The solutions were mixed and the absorbance of each was measured at 510 nm. Total flavonoids were reported as mg catechin equivalent (CE)/100 g (Zhishen et al., 1999). Analysis of total anthocyanins. Total anthocyanin contents were determined spectrophotometrically by the pH differential method (Wrolstad et al., 2005).
Aman et al. Each extract in 0.025 M potassium chloride buffer (pH 1.0) and 0.4 M sodium acetate buffer (pH 4.5) was measured at 510 and 700 nm after 15 min of incubation at 23 C. The absorbance of the diluted sample (A) was measured as follows: A ¼ ðAmax A700 ÞpH1:0 ðAmax A700 ÞpH4:5 The concentration of anthocyanins was calculated using the following formula: Anthocyanin pigment (mg/L) ¼ ðA MW DF 1000Þ=ð" 1Þ where MW is the molecular weight, DF the dilution factor and " is the molar absorptivity. Cyanidin-3-glucoside (MW ¼ 449.2 and " ¼ 29,600) was used as standard and the concentration was expressed as mg cyanidin-3-glucoside/100 g. Extraction of essential oils and polyphenols from oilseeds In all, 100 g of each of the spice oilseeds were extracted for oil by Bligh and Dyer method (Bligh and Dyer, 1959). The sample was mixed well with three parts chloroform/methanol mixture (1:2 v/v) and kept at room temperature for about an hour. Chloroform was added further for phase separation and then centrifuged at high speed for 15 min. The lower chloroform phase containing the lipids was isolated and evaporated on a rotary evaporator at 40 C while the upper methanolic phase was concentrated to remove alcohol resolubilized in water and then stored at low temperature for the analysis of polyphenols. Fractionation and analyses of the polyphenolic extracts derived from oilseeds Polyphenolic extracts obtained above were fractionated into neutral and acidic fractions as described in the section on ‘Extraction and Fractionation of Polyphenolics from Fruits’. The total phenolics were determined in both the neutral and acidic fractions while the neutral fractions were also analyzed for total flavonoids. Fractionation and analyses of the polyphenolic extracts of green and black tea Extraction and fractionation of polyphenols from crude methanolic extract of 100 g of each green and black teas were carried out as mentioned in the section ‘Extraction and Fractionation of Polyphenolics from Fruits’.
Determination of antimicrobial activity About 35 bacterial strains were used to test the antimicrobial activity of the test fractions. These strains included clinical isolates obtained from the Department of Microbiology, University of Karachi as well as the ATCC cultures maintained on Mueller Hinton agar medium at 4 C. The preparation of Mac Farland Turbidity Standard, preparation of inocula and its adjustment to 0.5 Mac Farland Standard (106 CFU/ mL), and antibacterial assay was followed as described by Siddiqi et al. (2011). For determining the minimum inhibitory concentration (MICs), Mueller Hinton Agar plates were overlaid with 3 mL soft agar containing 50 mL of each test organism suspension. Two-fold serial dilution of each fraction (1 mg/mL) was made in phosphate buffer saline and 100 mL of it was poured into well. The plates were incubated at 37 C for 24 h. Titer of the sample was determined by the serial dilution assay and was expressed as activity/arbitrary unit (AU/mL). One activity unit (AU) was taken as the reciprocal of the last serial dilution demonstrating inhibitory activity (Naz et al., 2006, 2007). Statistical analyses All assays including total phenolics, flavonoids, anthocyanins, were performed in triplicate and results were expressed as mean standard deviation. To identify significant differences among the mean values (at p < 0.05) analysis of variance (ANOVA) and Tukey’s tests were performed.
RESULTS AND DISCUSSION Among the fruits the total phenolics, flavonoids and anthocyanins were highest in the fractions of T. catappa and least in C.carandas (p < 0.05). Among the four fractions of the same fruit, the order regarding total phenolics and flavonoids was 1b > 1c > II > 1a (Table 1). With exception to fraction II of C. carandas, the fractions 1a, 1b, 1c and II from all three fruits displayed substantial antibacterial activity against almost all the Gram-positive strains; however, the fraction II of all three fruits and 1c of Z. nummularia and C. carandas did not show any activity against Gram-negative strains (Tables 2 and 3). In general, the activity of the fractions was higher against Gram-positive strains than Gramnegative. Compared to Gram-positive, the relative insensitivity of Gram-negative strains towards the fractions may be due to an additional layer of lipid in the cell wall acting as permeability barrier and reducing the uptake of the fractions in the cell. 243
Food Science and Technology International 20(4) Table 1. Anthocyanin, flavonoid and total phenolic contents (data expressed as milligrams per 100 g of weight) Fruit sample Z. nummularia
T. catappa
C. carandas
Black tea
Fractions
Flavonoidsb
Fraction 1a
70 0.98
Fraction 1b
198 0.07
17 0.23
Fraction 1c
177 1.90
140 0.23
Fraction II
138 0.4
78 0.90
Fraction 1a
109 0.20
Fraction 1b
412 0.21
287 0.24
Fraction 1c
350 0.41
256 1.30
Fraction II
223 0.51
151 0.34
Fraction 1a
10.0 0.14
Fraction 1b
70.0 0.20
44.0 0.092
Fraction 1c
40.0 0.40
23.0 0.01
Fraction II
23.0 0.21
14 0.660
Fraction 1a
489
296
Fraction 1b
11,060
9180
Fraction 1c
770
620
Fraction II Green tea
Total phenolicsa
Fraction 1a
556
340
Fraction 1b
12,230
11,530
Fraction 1c
980
792
Fraction II
Anthnocyaninsc 6.0
104 0.31
11.2 0.36
Note: (–) estimation not performed. Values are mean SD of triplicate assays. Values in the same column were significantly different at p < 0.05. a Concentration based upon gallic as standard. b Concentration based upon catechin as standard. c Concentration based upon cyanidin-3-glucoside as standard.
The difference in the range of antibacterial activity of the fractions was quite significant (p < 0.05), the order being T. catappa > Z. nummularia > C. carandas with respect to the fruit and 1a > 1b > 1c > II for the different fractions of the same fruit, same order of antibacterial activity with respect to different fractions of the same fruit was obtained for Grewia asiatica, Eugenia jambolana and Carissa carandas (Siddiqi et al., 2011). The highest antibacterial activity of T. catappa and least of C. carandas fractions was concomitant with the total phenolics, flavonoids and anthocynin contents (Tables 1 and 3) while the relative activity difference among the fractions is concerned mainly with the varied degree of hydroxylation and nature of substituent groups attached to B and/or C ring within the structure of the constituent flavonoids in the fractions (Sato et al., 1996). Pigment namely violanaxanthin, leutein, zeaxanthin and b- crytoxanthine (LopezHemandez et al., 2001), tannins (Mustapha, 2001; Rayudu and Rajadurai, 1966; Tanaka et al., 1986) and flavones glycoside (Lin et al., 2000) are present in fruits and leaves of T. catappa and known to posses antimicrobial properties (Mitscher et al., 1987). 244
The highest antibacterial activity of the phenolic acid fractions (1a) is based on their hydrophilicity or charged structure making them more successful to access the bacterial membranes (Oroojalian et al., 2010; Siddqi et al., 2011). The cell wall of Grampositive bacteria is made of peptidoglycan interspersed with a phosphodiester polymer of glycerol joined by phosphate groups. The cell wall of Gram-negative species contains lipopolysaccharides (LPS), which confers a negative and repels hydrophobic compounds. In addition to LPS, outer membrane contains unique protein called porin, which do not allow the migration of large hydrophobic molecules into the periplasmic space for possible transport across the cytoplasmic membrane (Oroojalian et al., 2010). The higher antibacterial activity of flavanol (1b) compared to flavonol (1c) is attributed to the fact that flavanol besides having higher degree of hydroxylation (Waterhouse, 2001) is the only class of flavonoid that do not form glycosides rendering the molecule sterically hindered and inaccessible to the membrane. The least activity of the anthocyanins may be due to the methoxy substituents in B ring reducing the degree of
21 1.00 23 0.571f,1e 22 1.00
27 2.08
25 2.101e,1f
23 1.52
11 0.57
10 1.00
12 2.001r,3g
10 0.50
12 1.00
11 1.52
13 2.10
11 1.50
10 1.00
13 0.50
12 s0.00
14 1.00
11 1.10
12 1.00
Fraction II
11 0.57
16 1.00
15 1.10
15 1.10
20 1.002f,1m
18 1.572e,1l,
18 1.56
17 2.00
12 2.08
15 1.20
14 0.57
14 1.00
16 1.57
14 1.00
17 2.002d,1j
19 1.00
21 2.08
23 2.08
20 0.57
22 1.00
21 0.57
23 1.52
23 1.00
22 1.00
22 0.57
21 1.00
17 1.00
18 1.57
Fraction 1a
2b
11 1.50
10 0.50
10 0.50
14 1.002i,1q
13 1.102h,1p
12 2.08
11 2.10
11 1.67
10 2.30
10 1.57
9 2.00
9 2.10
10 2.03
12 2.002g,1n
16 0.57
14 1.00
16 1.52
18 0.57
13 1.20
16 1.00
16 0.58
16 0.58
14 2.082a,
13 1.10
14 1.50
13 1.10
12 1.50
Fraction 1b
12 0.572c,1i
11 1.00
13 1.52
15 0.57
10 1.20
11 1.00
12 0.58
12 0.58
13 2.082b,2a
10 1.10
11 1.50
10 1.10
9 1.50
Fraction 1c
Carissa carandas
Zone of inhibition (mm)
Fraction II
20 2.00
26 1.00
25 1.00
26 2.00
26 2.08
24 2.00
23 0.573e,1k
25 2.08
24 1.52
26 1.00
25 1.57
23 0.57
27 1.00
25 1.50
22 0.50
26 0.57
32 0.57
33 1.10
32 2.10
32 2.08
34 0.57
31 1.00
34 1.57
32 0.57
31 2.08
34 2.08
32 2.50
27 2.50
Fraction 1a
3d,1h
18 1.00 29 1.10
15 2.08
22 1.52
19 1.00
20 0.57
16 2.08
18 2.00
17 0.573f,1o
22 1.00
21 1.52
22 1.00
18 1.00
19 0.57
21 1.05
20 1.00
17 0.57
21 0.573c,
26 0.57
3c
11 0.57
15 1.00
14 1.10
15 1.10
11 1.00
12 1.57
11 1.563g,1f
17 2.00
12 2.08
15 1.20
14 0.57
14 1.00
15 1.57
14 1.00
12 2.00
19 1.003d,
19 2.08
24 2.08
22 0.57
25 2.08
20 1.52
23 1.00 20 0.57 27 2.10
1b
20 1.003b,1d
18 0.57
21 1.003a,
19 1.00
15 1.57
Fraction 1c
29 0.57
24 1.52
29 1.10
26 0.57
24 2.08
27 1.10
26 2.08
21 2.50
Fraction 1b
Terminalia catappa
Note: Values are presented as Mean SD of triplicate assays. Values within the same row were significantly different at p < 0.05, except where the same superscripts have been used to show no significant difference.
15 1.00
V. cholerae
12 0.57 15 0.57
15 1.00
20 0.57
S. dysenteriae
19 1.00
13 1.001q,2i
21 2.081m,2f
S. paratyphi B
21 0.57
13 2.081p,2h
20 2.081l,2e
S. paratyphi A
S. sonneie
18 1.001o,
S. flexneriae
17 1.00
22 1.52
22 1.521k,3e
S. typhi
12 1.00
10 1.00
14 0.571i
17 1.00
16 1.56
18 0.57
16 1.10
3f
18 0.50 15 1.00
11 1.00
12 1.00
S. typhi ATCC 19430
17 1.52
P. aeruginosa ATCC 27853
12 1.52
18 1.00
19 1.00
21 0.57
17 0.57
E.coli FPL5014
E. coli ATCC 25922
K. pneumoniae
14 1.00 11 2.08
19 1.00
E. coli BU40
P. aeruginosa PAO286
14 1.52
18 0.571j,
Gram-negative bacteria E. coli WT 12 1.521n,2g
20 1.001h,1g
22 0.571g,1h
C. botulinum 3502 2d
23 1.00
28 1.10
27 0.57
S. pyogenes
L. monocytogenes ATCC 35152
20 0.57
17 0.58
20 2.081d,1c
22 1.00
21 1.001c,1d
S. pneumoniae
S. epidermidis
E. faecalis 2400
27 1.10
S. aureus ATCC 25923
16 1.10
20 1.001b,1a
21 0.57
22 1.001a,1b
28 1.52
26 0.57
S. aureus
26 0.57
27 2.08
C. diphtheriae
12 1.50 12 1.10
18 0.57
Fraction 1c
21 1.10
S. saprophyticus
29 1.10
B. subtilis ATCC 6633
Fraction 1b
E. faecalis
23 2.50
26 2.08
Gram-positive species B. subtilis
Fraction 1a
Indicator organisms
Ziziphus nummularia
Table 2. Antibacterial activity of the polyphenolic fractions of the fruits
14 0.50
14 1.00
16 1.52
16 0.43
13 1.50
16 1.00
15 0.58
15 0.78
14 1.52
13 2.10
14 1.10
13 1.50
11 1.00
Fraction II
Aman et al.
245
Food Science and Technology International 20(4) Table 3. MICs of the fractions obtained from fruits, spice oilseeds, green and black teas MIC (mg/mL) Fruits, herbs, spices and their fractions
S. aureus
Z. nummulariaz Fraction 1a
31.25
Fraction 1b
62.50*
Fraction 1c Fraction II
62.50* 250.0
C. carandasc Fraction 1a
62.50
Fraction 1b
125.0*
B. subtilis
62.50 125.0
E. faecalis
E. coli
P. aeruginosa
S. typhi
62.5
250
500
250.0
500
62.50
125.0
1000
1000
500.0
1000
125.0
125.0
125.0
1000
250.0
250.0
250.0
125.0
125.0
500
1000
125.0
250.0
500
1000
62.50 125.0
125.0*
250.0
500.0
Fraction II
500.0
500.0 1000
500.0 1000
15.63
15.63
7.80
15.63
125.0
125.0
250.0
31.250
31.25
31.25
31.25
250.0
250.0
500.0
Fraction 1c
62.50
500.0
500.0
Fraction II
125
125 250
62.50 250
125.0 125.0
62.50
62.50
31.25
31.25
31.25
62.50
62.50
62.50
62.50
Acidic
15.63
7.80
7.80
31.25
31.25
15.63
31.25
62.50
7.80
4.00
4.00
15.63
15.63
15.53
15.63
31.25
125.0
125.0
125.0
250.0
250.0
125.0
125.0
62.50
250 500
62.50
B. alba (mustard/rai)M Crude
125.0
1000
125
Neutral Oil
125.0
1000
Fraction 1b
T. ammi (ajwain)A Crude
S. dysenteriae
62.50
Fraction 1c T. CatappaT Fraction 1a
L. monocytogenes
250.0
250.0 250.0
62.50
62.50
Acidic
31.25
15.63
31.25
62.50
62.50
62.50
62.50
Oil
15.63
7.80
7.80
31.25
31.25
31.25
31.25
250.0
250.0
250.0
500.0
500.0
500.0
500.0
125.0
125.0
125.0
250.0
250.0
250.0
250.0
125.0
125.0
125.0
125.0
Acidic
62.50
62.50
62.50
Oil
31.25
15.63
31.25
P. somniferum (poppy seeds)P Crude
62.50
Neutral
500.0
500.0
250.0
500.0
Acidic
250.0
125.0
125.0
250.0
250.0
Oil
125.0
125.0
125.0
62.50
62.50
62.50
1000
1000
Green teaG Crude
31.25
15.63
15.63
31.25
31.25
Neutral
62.50
31.25
31.25
62.50
62.50
125.0*
62.50
62.50
62.50
31.25
Acidic Black teaB Crude Neutral
125.0
Acidic
125.0*
62.50 125.0
31.25
125.0 62.50
62.50
125.0 62.50
125.0
125.0
125.0
125.0
125.0
500.0
62.50
250.0 62.50
62.50
1000 250.0 250.0 125.0
1000
250.0
250.0
125.0
250.0
62.50 125.0
250.0
250.0
500.0
500.0
125.0
500.0
62.50 125.0
125.0
125.0
62.50
Neutral
250.0
250.0
Neutral
T. foenum-graecum (Fenugreek)F Crude
62.50
125.0
62.50 125.0 1000 500.0 500.0
MIC: minimum inhibitory concentration. Data shown for MICs are a result of five replicates of which three to four values were identical for every organism and every tested sample. p < 0.05 for values Z,T,C. p < 0.05 for values A,M,F,P. p < 0.05 for values G,B. Values * mean no significant difference.
246
Aman et al. Table 4. Antibacterial activity of the crude, acidic, neutral and oil fractions of the spice oilseeds against some Gram-positive species Zone of inhibition (mm)/indicator organisms Spices and fractions
B. subtilis
C. diphtheriae
S. aureus
E. faecalis
S. pneumoniae
S. pyogenes
L. monocytogenes
C. botulinum
Ajwain Crude
27.0 1.7
28.0 0.8
26.0 1.4
24.0 0.8
22.6 1.7
19.0 1.1
22.2 2.4
18.0 1.7
Neutral
35.2 1.2
33.5 1.3
32.3 1.8
30.1 0.6
28.5 0.6
25.0 1.6
27.1 0.7
24.6 2.0
Acidic
42.3 2.0
38.5 1.7
37.2 0.6
36.3 0.6
32.3 0.9
31.2 0.8
32.0 0.6
31.5 1.2
Oil
46.6 2.0
43.7 1.7
42.0 1.2
40.6 0.8
36.5 2.3
38.6 0.2
40.0 0.3
37.5 0.8
Mustard/rai Crude
24.2 1.5
22.0 2.0
22.0 1.0
20.0 2.1
18.0 0.8
15.1 2.0
17.3 0.5
14.0 1.1*
Neutral
30.0 1.5
27.0 2.3
27.0 1.1
26.0 1.3
22.0 0.8
20.2 1.8
23.3 0.5
20.2 1.2
Acidic
36.0 0.5
32.4 1.8
32.5 1.2
32.7 1.4
27.1 1.2
26.2 1.7
28.6 0.8
26.0 1.2
Oil
42.3 0.5
38.8 1.6
37.5 1.2
36.7 0.9
31.2 1.2
32.3 1.7
35.6 0.7
32.0 1.5
Fenugreek Crude
22.5 1.5
18.8 2.0
18.5 1.0
16.5 2.1
15.3 0.8
13.7 2.0
12.3 0.5
14.0 1.1*
Neutral
26.4 1.5
23.6 2.3
22.4 1.1
21.7 1.3
17.3 0.8
15.6 1.8
17.2 0.5
15.4 1.2
Acidic
31.4 0.5
28.6 1.8
28.5 1.2
27.4 1.4
21.2 1.2
21.5 1.7
23.2 0.8
21.5 1.2
Oil
37.0 0.5
33.0 1.6
31.6 1.2
30.4 0.9
26.0 1.2
27.5 1.7
31.0 0.7
27.5 1.5 10.0 1.1
Poppy seeds Crude
17.0 1.5
13.0 2.0
12.0 1.0
13.5 2.1
11.0 0.8
9.5 2.0
8.0 0.5
Neutral
21.0 1.5
19.1 2.3
17.0 1.1
17.5 1.3
12.1 0.8
10.5 1.8
10.0 0.5
9.5 1.2
Acidic
26.2 0.5
23.0 1.8
23.2 1.2
22.3 1.4
16.5 1.2
14.5 1.7
15.3 0.8
16.5 1.2
Oil
32.3 0.5
28.0 1.6
27.3 1.2
25.2 0.9
21.5 1.2
21.7 1.7
25.3 0.7
22.4 1.5
Values are presented as Mean SD of triplicate assays. Values for the different fractions of the same oilseed and for the same fraction of different oilseeds were significantly different at p < 0.05 except where the same superscript ‘*’ has been used to show no significant difference.
hydroxylation, increasing hydrophobicity and partly due to the glycosidic links (Puupponen-Pimia¨ et al., 2008; Sato et al., 1990). The crude, acidic and neutral fractions of both green and black teas were found to be effective against all the tested strains of Gram-positive bacteria (Tables 3 and 4). Presence of polyphenols in tea contributes for its antimicrobial properties (Kumar et al., 2012). During this study the fractions of green tea showed higher activity than that of black tea which may be due to the loss of polyphenolics during conversion of green into black via fermentation process (Almajano et al., 2008; Tiwari et al., 2005). Crude fraction was most effective and the acidic least in green tea while in black the activity of neutral fraction was not significantly different or higher than acidic. The key compounds of green tea are flavanols or catechins of epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC; Chan et al., 2011; Graham 1999; Sharma et al., 2011). The higher activity of the crude and neutral fractions of green tea may be accounted on the basis of biologically active catechins which constitute up to 30% of the dry leaf weight (Taylor et al., 2005). Bancirova (2010) and Hamilton (1995) reported
EGCG and ECG are mainly responsible for the antibacterial activity of green tea against both Grampositive and Gram-negative bacteria. Antibacterial activity of black tea has also been reported (Bancirova, 2010; Tiwari et al., 2005). As in case of fruits, the activity of all the fractions of green and black tea against Gram-negative species was relatively low, the activity order was crude > neutral > acidic for green tea while crude > acidic > neutral for black tea. All oilseeds polyphenolic extracts, their fractions and essential oil significantly masked bacterial growth. For all oilseeds, the oil fraction was found to be more active than the crude extracts and their fractions (p < 0.05). Further, the acidic fraction showed activity more than neutral and neutral more than crude (Figure 1), while the antibacterial action of the oilseeds decreased in the order ajwain > mustard > fenugreek > poppy seeds (p < 0.05; Tables 3, 5 and 6). Individual compounds derived from essential oil of some oilseeds like anethole from anise, fennel from star anise, thymol from ajwain, eugenol from tejpat, cinnamaldehyde from cassia bark, turmerone from turmeric, carvone from dill and linalool from coriander have already been reported to have antimicrobial activities (Srinivas et al., 2009). Phenolic compounds 247
Food Science and Technology International 20(4)
Figure 1. Antibacterial activity of the crude extract and fractions of the ajwain against C. botulinum.
like thymol, carvacrol, c-terpinene and q-cymene in the essential oils have been found to possess significant antibacterial activity (Burt, 2004). The higher antibacterial activity of ajwain as compared to other oilseeds is possibly related to the higher amount of thymol and cterpinene (Friedman et al., 2002). The activity of the oils, polyphenolics extracts and their fractions were more against Gram-positive as compared to Gramnegative. Many studies have reported greater sensitivity of Gram-positive bacteria to essential oils than Gramnegative bacteria. The phospholipid bilayer of the cell membrane in Gram-positive bacteria allow greater accessibility to the oils’ hydrophobic components, where these components work effectively resulting either increase of permeability and leakage of vital intracellular constituents, or impairment of bacterial enzyme systems (Sandri et al., 2007). Investigation of the mode of action of some essential oils revealed that these constituents increase membrane permeability by dissolving in the membrane which ultimately results in swelling, decline in membrane function and death of cell (Holly and Patel, 2005; Oroojalian et al., 2010).
Table 5. Antibacterial activity of the crude, acidic, neutral and oil fractions of the spice oilseeds against some Gram-negative species Zone of inhibition (mm)/indicator organisms Spice and fractions
E. coli
K. pneumoniae
P. aeruginosa
S. typhi
S. dysenteriae
S. sonneie
S. flexneriae
Ajwain Crude
22.4 1.2
21.0 0.8
19.0 1.0
18.0 0.5
16.5 2.1
13.0 1.1
16.0 2.4
11.0 1.78
V. cholerae
Neutral
30.0 1.4
25.0 1.3
2 4.3 1.0
26.1 0.4
21.7 0.4
20.0 1.1
21.1 0.7
a
Acidic
36.2 2.0
32.5 1.7
32.2 0.5
32.3 0.6
27.5 0.5
26.5 0.8
26.0 0.4
26.0 1.2
Oil
18.0 2.0
40.4 2.0
38.0 1.7
37.2 1.0
38.5 0.4
32.5 2.3
32.2 1.0
34.1 0.2
34.4 1.1
Mustard/rai Crude
18.2 1.5
16.0 2.0
17.2 2.0
15.5 2.1
13.0 0.8
11.6 2.0
11.6 0.5
10.6 1.18a
Neutral
24.0 1.5
21.0 2.0
21.0 1.1
20.5 1.3
16.4 0.8
15.5 2.1
17.4 0.5
14.6 1.2
Acidic
310 0.5
26.4 1.2
32.0 1.2
26.5 1.2
21.3 1.1
19.7 1.7
22.5 0.8
21.0 1.2
Oil
35.6 0.5
32.8 1.0
31.5 1.2
33.6 0.9
27.4 1.2
20.3 2.36a
28.4 1.7
27.0 1.5
16.5 1.5
14.8 2.0
13.5 1.0
12.5 2.0
21.1 1.51a
17.6 2.3
16.0 1.13a
15.5 1.3
11.2 0.85a
11.6 1.86b
12.2 0.5
10.4 1.28b
20.5 1.3
5b
17.5 1.0
18.2 0.8
15.5 1.28c
Fenugreek Crude Neutral Acidic Oil
1b
26.4 0.5 32.0 0.5
Poppy seeds Crude Neutral
21.0 1.51a 1b
2a
22.4 1.8
21.6 1.2
28.0 1.6
26.6 1.2
19.1 2.3 2a
16.2 1.6
6a
26.4 0.5
21.0 1.5
19.5 1.0
22.0 0.7
21.5 1.5
17.0 1.13a
17.5 1.3
12.1 0.85a
10.5 1.86b
10.0 0.5
9.5 1.28b
5b
Acidic
26.2 0.5
23.0 1.8
23.2 1.2
22.3 1.4
16.5 1.2
14.5 1.7
15.3 0.8
16.5 1.28c
Oil
25.3 0.5
21.0 1.6
22.3 1.2
19.2 0.9
17.5 1.2
17.7 1.7
17.3 0.7
16.4 1.5
Values are presented as Mean SD of triplicate assays. Values for the different fractions of the same oilseed and for the same fraction of different oilseeds were significantly different at p < 0.05 except where the same superscripts have been used to show no significant difference.
248
Aman et al. Table 6. Antibacterial activity of the crude, acidic, and neutral fractions of the green and black teas Zone of inhibition (mm) Green tea
Black tea Gram-positive bacteria
Indicator organisms
Crude
Acidic
Neutral
Crude
Acidic
Neutral
Bacillus subtilis ATCC 6633
33.5 0.8
15.2 0.6
28.5 0.8
24.5 0.6
18.2 0.5
21.0 0.5
Corynebacterium diphtheriae
36.5 1.4
16.5 1.0
30.5 1.4
28.5 1.2
19.5 1.2
24.2 1.2
Staphylococcus aureus ATCC 25923
27.0 2.1
13.1 2.0
21.0 2.1
20.0 2.1
17.1 2.0b1
17.3 2.0b1
Enterococcus faecalis 2400
31.0 1.4
13.0 1.0
26.0 1.4
22.0 1.2
15.5 1.1b2
16.5 1.0b2
Streptococcus pneumoniae
29.4 2.0
12.0 1.1
22.4 2.0
19.4 2.0
15.2 2.0b3
16.2 2.1b3
Streptococcus pyogenes
29.6 1.6
11.0 1.0
22.6 1.6
18.5 1.6
14.0 1.0b4
14.5 1.1b4
Listeria monocytogenes ATCC 35152
28.4 1.1
15.1 0.5g1
22.4 1.1
19.2 1.1
15.1 0.5b5,g1
15.4 1.0b5
Clostridium botulinum 3502
24.3 1.0
12.0 1.0g2
18.3 1.0
16.1 1.0
12.0 1.0b6,g2
12.3 1.2b6
Gram-negative bacteria Escherichia coli ATCC 25922
26.0 0.6
12.0 0.4
21.3 0.4
17.3 0.2
14.1 0.5b7
13.4 0.1b7
Klebsiella pneumoniae
28.5 1.4
12.0 1.0
23.5 1.0
15.6 1.2
15.0 1.5
11.5 1.0
Pseudomonas aeruginosa ATCC 27853
24.5 1.5
19.0 1.5
15.0 2.1
12.4 1.0
Salmonella typhi ATCC 19430
22.5 2.0
17.0 2.1
13.0 1.2
12.5 1.0
Shigella dysenteriae
25.4 1.5
19.4 1.6
15.4 2.0
11.2 1.0b8
11.4 1.0b8
Shigella sonneie
21.0 1.2
16.5 1.4
12.5 1.6
10.0 0.5
Shigella flexneriae
22.0 1.5
12.0 0.5
15.2 1.0
11.2 1.1
14.0 1.0
Vibrio cholerae
17.5 2.3
13.1 1.0
10.1 1.0
11.0 0.5
Values are presented as Mean SD of triplicate assays. Values for the different fractions of the same tea and for the same fraction of different type were significantly different at p < 0.05 except where the same superscripts have been used to show no significant difference.
CONCLUSION Tea, after water, is the most frequently consumed beverage and contain fairly high amount of polyphenols. So, it is one of the very important sources of polyphenols in our diet. Though spices contribute little to our diet, they may still be very important contributors to our polyphenols intake as far as their strength is concerned. The small fruits used for this study are underutilized and have been focused for their utilization in product development such as polyphenolic-rich neutraceuticals. The broad spectrum in vitro antibacterial potential demonstrated by these fruits, spices and teas lead us to conclude that the high inherent polyphenolics contents of these contribute to their medicinal and food preserving qualities. The active ingredients indigenous to these fruits, spices and teas may provide better alternatives to conventional drug for combating infections and diseases.
FUNDING This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
CONFLICT OF INTEREST None declared.
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