Assessment of the antimicrobial activity of the lipoidal

CHNAES-00576; No of Pages 6 Acta Ecologica Sinica xxx (2018) xxx–xxx

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Assessment of the antimicrobial activity of the lipoidal and pigment extracts of Punica granatum L. leaves Marwa M. Elbatanony a, Amal M. El-Feky a, Bahaa A. Hemdan b,⁎, M. Azab El-Liethy b a

Pharmacognosy Department, Pharmaceutical and Drug, Industries Research Division, National Research Center, 33 El Buhouth St, Ad Doqi, Cairo Governorate 12622, Egypt Environmental Microbiology Lab, Water Pollution Research Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, 33El Buhouth St, Ad Doqi, Dokki, Cairo Governorate 12622, Egypt

b

a r t i c l e

i n f o

Article history: Received 24 January 2018 Received in revised form 15 May 2018 Accepted 17 May 2018 Available online xxxx Keywords: Punica granatum Antimicrobial activity n-Hexane extract Carotenoids Terpenoidal compounds

a b s t r a c t Punica granatum L. is one of the famous and old species belonging to Family Punicaceae. The lipoidal and natural pigment extracts of the pomegranate were screened for their antimicrobial activity against nine different microbial pathogens. Three different doses of each extract (50, 100 and 150 μl) at three different contact times (15, 30 and 60 min) were examined. Results can be cleared that, all tested microbial pathogens were inhibited by 150 μl of both extracts at 60 min. Furthermore, the removal efficiency of n-hexane extract was powerful than the pigment extract. Also, the quantitative evaluation of the pigments in the leaves was performed using spectroscopical and HPLC analyses, carotenoids and chlorophylls content of P. granatum L. leaves were spectroscopically determined (mg/g) as 6.7 ± 0.214 and 4.9 ± 0.251, respectively. Chlorophyll a and b were 2.33 ± 0.014 and 1.51 ± 0.023, respectively. HPLC analysis of lutein and β-carotene were investigated as 3.652 and 1.915 mg/g. GC/MS analysis of the saponifiable and unsaponifiable fraction of n-hexane extract was carried out and α-amyrin acetate, ergosterol, and α-tocopherol were isolated, purified and identified using different spectroscopical methods. Toxicity assessment demonstrated their high biocompatibility since no toxic effect was recorded. © 2018 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

1. Introduction Punica granatum L. is a member of Family Punicaceae. It is one of the oldest known plants all over the world [1]. A diversity of cultures and remedies in the ancient world recommend pomegranate peel to treat several health issues [2].Where this plant has been widely used in folk medicine in America, Asia, Africa and Europe for treating different diseases [3,4].Topical application of the rind powder can aid in healing bleeding gums and plaque in patients with periodontitis [5]. Moreover, this plant was used by the Egyptians for curing of tapeworm and other parasitic infections [6]. Furthermore, this plant approved to have promising antiviral and antimicrobial effects [7]. Braga et al. [8] found that different extracts of pomegranate showed significant inhibition of Staphylococcus aureus growth and subsequent enterotoxin production at 0.01, 0.05 and 1% v/v concentrations. Kabara and Vrable and Abdallah and Ibrahim [9,10] mentioned that the lipoidal and pigment compounds have a great antimicrobial activity against Gram (+) bacteria and yeast more than Gram (−) bacteria. While sterols proved to have a good inhibitory activity against most types of bacteria and fungi [11]. Moreover, long-chain polyunsaturated fatty acids are attracting attention as potential therapeutic antimicrobial ⁎ Corresponding author at: 33 El Buhouth St, Ad Doqi, Cairo Governorate 12622, Egypt. E-mail address: [email protected] (B.A. Hemdan).

agents [12]. On the other hand, carotenoids are considered as a group of valuable compounds having various pharmaceutical and medicinal applications not only because they can act as vitamin A precursors, but also for their coloring and antioxidant effect beside to tumor inhibiting activity through the removal of oxygen radicals in addition to antibacterial effect which enable them to be used as preservative [13]. The goal of this study was to investigate the antimicrobial activity of the lipoidal and pigment extracts of Punica granatum L. leaves grown in Egypt against different types of microbial pathogens and correlate between this activity and the chemical composition of these extracts. 2. Materials and methods 2.1. Plant material Leaves of Punica granatum L. were obtained from National Agriculture Centre, cleaned off from the impurities and shade dried. It was identified by Dr. Gamal Farag, (National Agriculture Centre, Egypt). 2.2. Extraction of natural pigments The natural pigments were extracted from 100 g air-dried powdered leaves with a mixture of equal volumes of acetone and n-hexane at the room temperature until colorless according to [14]. The mixture was

https://doi.org/10.1016/j.chnaes.2018.05.003 1872-2032/© 2018 Ecological Society of China. Published by Elsevier B.V. All rights reserved.

Please cite this article as: M.M. Elbatanony, et al., Assessment of the antimicrobial activity of the lipoidal and pigment extracts of Punica granatum L. leaves, Acta Ecologica Sinica (2018), https://doi.org/10.1016/j.chnaes.2018.05.003

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M.M. Elbatanony et al. / Acta Ecologica Sinica xxx (2018) xxx–xxx

Table 1 HPLC analysis of carotenoids in P. granatum L. leaves.

Table 3 GC/MS analysis of saponifiable matter of P. granatum L. n-hexane extract:

Carotenoids

Rt

Content (mg/g)

Lutein β-Carotene

2.9 20.7

3.652 1.915

washed with water for many times, the n-hexane layer was collected and combined as carotenoid containing extract, filtered, evaporated to dryness in a rotary evaporator and kept in refrigerator till analysis. All steps of the extraction process were performed in dark to avoid cistrans photoisomerization and photodestruction hence carotenoids are sensitive to light and heat [15].

Compound

Mol. formula

Mol. BP Rt weight

Hexadecanoic acid methyl ester 11,14-Octadecadienoic acid methyl ester 9-Octadecenoic acid methyl ester Octadecanoic acid methyl ester Cyclopropaneoctanoic acid, 2-octylmethylester Heptadecanedioic acid, 9 oxodimethyl ester

C17H34O2 270 C19H34O2 294

74 15.95 16.64 67 19.98 36.57

C19H36O2 296 C19H38O2 298 C20H38O2 310

55 20.09 23.84 74 20.46 1.95 55 23.95 0.26

C19H34O5 342

55 24.31 0.09

Relative area %

2.3. Quantitative evaluation of the natural pigments

2.5. Investigation of lipoidal content

2.3.1. Spectroscopical determination Spectroscopical determination of carotenoids and chlorophyll was performed by [14,16] respectively. The total content of carotenoids was determined by spectrophotometry (k = 450 nm) and calculated by following formula:

Saponification of the n-hexane extract (0.5 g) was carried out according to [17]. The unsaponifiable fraction was analyzed using GC/MS technique adopting the following conditions: Capillary column of fused silica, 30 m length, 0.32 mm ID. and 0.25 μm thickness, HB-5MS as stationary phase, helium at 1 ml/min, 13 psi as the carrier gas. The programming temperature ranged between 60 and 290 °C at a rate of 4 °C/min. Ion source temperature 230 °C while the used detector was the mass spectrum detector. While the saponifiable fraction (fatty acids methyl ester) was prepared according to [18] and subjected to GC/MS analysis under the same conditions.

Chlorophyll a ðmg=gÞ ¼ 16 : 5Abs664 –8:3Abs651 Chlorophyll b ðmg=gÞ ¼ 33:8Abs651 –12 : 5Abs664 Total chlorophylls ðmg=gÞ ¼ 25:5Abs651 þ Abs664 Carotenoids amount (mg/g) = optical density value × volume (mL) × dilute times × 10 ÷ 2500 (mean value A1% 1 cm 1/4 2500 of colored carotenoids). 2.3.2. HPLC analysis HPLC analysis was carried out using Agilent Technologies 1100 series liquid chromatograph equipped with an auto sampler and a diode-array detector. The analytical column was an Eclipse XDB-C18 (150 × 4.6 μm; 5 μm) with a C18 guard column (Phenomenex, Torrance, CA). The mobile phase consisted of acetonitrile (solvent A) and 2% acetic acid in water (v/v) (solvent B). The flow rate was kept at 0.8 ml/min for a total run time of 70 min and the gradient programme was as follows: 100% B to 85% B in 30 min, 85% B to 50% B in 20 min, 50% B to 0% B in 5 min and 0% B to 100% B in 5 min.

α-amyrin acetate

2.4. Preparation of the lipoidal extract Four hundred grams of the air-dried powdered leaves of Punica granatum L. was extracted with n-hexane repeatedly. The extract was filtered and evaporated under reduced pressure. The residue was weighed and the percentage yield was determined. Table 2 GC/MS analysis of unsaponifiable matter of P. granatum L. n-hexane extract. Compound

Mol. formula

Mol. BP weight

Rt

Relative area %

Phenanthrene 1(Indanyl) adamantane Pregnan-3,20-dione Ergosterol β-Sitosterol Rhodoxanthin Cantaxanthin 3,4-Didehydro-1,2,7′, 8′-tetrahydro-1-methoxy-2-oxo carotene 1-Methoxy1′-hydroxy-1,1′, 2,2′-tetrahydro-3,4-didehydro lycopene

C14H10 C19H24 C21H32O2 C28H44O C29H50O C40H50O2 C40H52O2 C41H58O2

178 252 316 396 414 562 564 582

178 135 43 363 414 124 91 370

6.11 6.93 8.66 9.28 17.66 23.92 29.44 33.17

1.63 0.82 2.00 0.65 0.95 9.06 20.06 26.10

73

40.18 10.85

C41H60O2 584

Ergosterol

α-Tocopherol Fig. 1. Chemical structures of α-amyrin acetate, ergosterol and α-tocopherol.


M.M. Elbatanony et al. / Acta Ecologica Sinica xxx (2018) xxx–xxx Table 4 MIC of n-hexane extract against tested microbial strains. Strains

Minimal inhibitory concentration (MIC)

P. aeruginosa E. coli Salmonella spp. S. aureus L. monocytogenes Ent. fecalis B. cereus C. albicans A. niger

100 μl at 30 min 100 μl at 60 min 150 μl at 30 min 50 μl at 60 min 100 μl at 30 min 150 μl at 15 min 100 μl at 30 min 100 μl at 60 min 150 μl at 30 min

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2.6. Chromatographic isolation of the main terpenoidal/steroidal compounds in the n-hexane extract Six grams of the n-hexane extract of P. granatum L. leaves were mixed thoroughly with 8 g of the silica gel and packed at the top of the silica gel column (70–230 mesh ASTM, 2.5 × 100 cm) by wet method. Elution was successively carried out by n-hexane and increasing the polarity with methylene dichloride, until reach 100% ethyl acetate. Fractions of 100 ml each were successively collected. Each fraction was concentrated and screened by TLC using silica gel alumina sheet (ready made chromatographic plates (20 × 20 cm) coated with silica gel F254), and similar fractions were collected together, evaporated to dryness then the plates were developed using benzene: ethyl acetate as solvent system in two different ratios; (70:30) and (95:05).

Fig. 2. Log10 reduction of n-hexane extract against different tested microbial pathogens at different doses in three different contact times.


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Then, they were examined under UV, sprayed using (10% sulphuric acid) and heated in an oven at 110 °C for 5–8 min [19].

2.7. Determination of antimicrobial activity 2.7.1. Preparation of tested microbial strains The lipoidal and pigment extracts of P. granatum L. leaves were screened for their antimicrobial activity against three Gram negative bacteria (Pseudomonas aeruginosa, E. coli and Salmonella typhimurium), four Gram positive bacteria (Staphylococcus aureus, Listeria monocytogenes, Enterococcus fecalis and Bacillus cereus), yeast (Candida albicans) and fungus namely Aspergillus niger. The stock culture of tested microbial strains preserved at −20 °C was inoculated into tryptase soya broth 50 ml falcon tube and incubated at 37 °C for 24 h then the tube centrifuged at 4000 rpm for 20 min (Sigma, Germany). The supernatant was discarded and the pellet was washed three times using sterile distilled water to discard any enrichment broth residue. Finally, the pellet was re-suspended in 50 ml sterile distilled water.

2.7.2. Determination of tested microbial strains counts The initial tested microbial strains cells were determined by one day before starting each experiment using both total counts and optical density at 600 nm. Tested microbial strains initial counts were obtained by ten folds serial dilution using sterile distilled water. 1 ml from each dilution was poured in sterile Petri dish then the molted standard plate count agar and malt extract agar for C. albicans and A. niger was poured. All plates were incubated at 37 °C for 24 h except C. albicans and A. niger was incubated at 30 °C for 2–4 days then the plates counted by using colony counter [20].

2.7.3. Inactivation of tested microbial strains The antimicrobial activities of the two prepared extracts were evaluated by inactivation of tested microbial strains that mentioned above. Different doses of each extract (50, 100 and 150 μl) at three different contact time (15, 30 and 60 min) were examined against nine different microbial pathogens. The counts of these pathogens were determined according to [20].

Fig. 3. Log10 reduction of pigment extract against different tested microbial pathogens at different doses in three different contact times.



2.8. Cytotoxicity assay In-vitro cytotoxicity studies of the lipoidal and natural pigment extracts of the pomegranate were investigated against normal skin fibroblast normal cells (BJ1), hepatocellular carcinoma (HEPG2) and breast cancer (MCF7) cell lines by MTT assay 3-[4, 5-dimethylthiazol-2-yl]-3, 5-diphenyltetrazolium bromide dye. Cells were inserted in a 96-well plate at a density of 1 × 104 cells per well. The media were inoculated with 1% antibiotic–anti-mycotic mixture which is composed of 10,000 U mL−1 potassium penicillin, 10,000 μg/mL streptomycin sulfate, 25 μg/mL amphotericin B and 1% L-glutamine (Bio west, USA). Further, the media were incubated at 37 °C in a humidified atmosphere with 5% CO2. Post-cells attachment, the media were replaced by the hydrogel samples for 72 h. The cells were incubated with the MTT solution (5.0 mg/mL) at 37 °C for 4 h. The purple formazan crystals developed were dissolved in 100 μL dimethyl sulfoxide (DMSO) and recorded (ELISA reader). The cell viability % was calculated applying the following equation:

Viability% ¼

OD test−OD blank 100 OD control−OD blank

OD test: Mean value of sample absorbance measured at 570 nm, OD blank: Mean value of blank absorbance measured at 570 nm and OD control: Negative control.

3. Results and discussion 3.1. Identification and characterization of natural pigments by UV-Vis spectroscopy UV spectrum of the natural pigments in n-hexane/acetone extract was scanned from 200 to 800 nm and maximum absorption was obtained at 455 and 665 nm for carotenoid and chlorophyll resp., this was in good agreement with that reported in [21] (carotenoids have a broad absorption 428 to 453 nm while spectral range of total chlorophyll lies between 661 and 642 nm). This confirmed that the extraction procedure was valid.

3.2. Quantitative evaluation of the natural pigments Total carotenoids and chlorophylls contents (mg/g) of P. granatum L. leaves were spectroscopically determined and the contents of the total carotenoids and chlorophyll were 6.7 ± 0.214 and 4.9 ± 0.251 (mg/ g), respectively. Chlorophyll a and chlorophyll b were 2.33 ± 0.014 and 1.51 ± 0.023 (mg/g), respectively. HPLC analysis of lutein and βcarotene were determined and results are illustrated in Table 1.

3.3. Investigation of the lipoidal content The chemical components of P. granatum hexane extract was identified by GC/MS technique, Tables (2&3) revealed the presence of nine compounds from the unsaponifiable matter representing 72.12% of the total content consisting of 2.45% unoxygenated compounds, 66.07% oxygenated compounds and 3.60% steroidal and triterpenoidal compounds. The major compound in the unsaponifiable matter was3,4-didehydro-1,2,7′, 8′-tetrahydro-1-methoxy-2-oxo- carotene (26.10%), followed by cantaxanthin (20.06%). On the other hand seven compounds were recognized from the saponifable matter amounting 79.35% classified as 18.59% saturated fatty acids, and 60.76% unsaturated fatty acids from which 11,14-Octadecadienoicacid, methyl ester was the main identified compound (36.57%).

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3.4. Isolation and identification of the main terpenoidal/steroidal compounds (1, 2 & 3) from n-hexane extract of P. granatum L. The conventional column chromatography technique as mentioned before was done aiming to isolate the main active compounds. The resulting similar fractions from the column chromatography were collected together according to Rf values where the isolated compounds were identified by different spectral analyses (IR, Mass spectrometry and H1-NMR). Compound 1 was isolated from (100% CH2Cl2), while compound 2 was isolated from CH2Cl2:Ethyl acetate (50%:50% v/v). As for compound 3, it was isolated from CH2Cl2:Ethyl acetate (20%:80% v/v). The isolated compounds were further purified several times with preparative TLC technique. The three isolated compounds were chromatographed on TLC alongside with available authentic references (Tables 2 and 3). Compound 1: α-amyrin acetate was in a form of pearl white needles, yielding 25 mg, melting point 227–228 °C (226–227 °C [22,23]. Its Rf value 0.70 in benzene-ethyl acetate (95:5 v/v) as solvent system. The H1−NMR spectrum show the presence of signals at δ 0.88 to δ 1.2 (m, 18H, 6 xCH3), δ 1.1 to δ 1.20 (m, 18H, 9 xCH2). At δ 1.9 to δ 2.4 (8H, methine protons), δ 3.6 (1H, CHOH), δ 5.38 (1H, vinylic proton), δ 5.01 and δ 5.12) that's a distinguishing from β amyrinolefinic protons which appear at δ 5.16. Mass spectrum gave M+ at m/z 468 for molecular formula C32H52O2 and 218 as the base peak. The other fragments at m/z: 408 (1.92), 393 (2.65), 365 (10), 273(3.71) 249(2), 218 (100), 203 (61), 189 (58). Compound 2: ergosterol was in a form of white crystals, yielding 23 mg, melting point 160 °C (160–163 °C). Its Rf value 0.98 in benzene-ethyl acetate (95:05 v/v) as solvent system. Mass spectrum gave M+ at m/z 396 for molecular formula C28H44O and 377, 379 for [M–H3O] + and [M−HO] + fragment ions, 363 for [C27H39]+ ion which is also very distinctive for ergosterol, 299.24 was consistent with the mass of a [C21H31O]+ ion, 269 and 271 as a result of breaking of the C-17/C-20 bond. The isolated compound was compared with an authentic reference and the spectral data was confirmed with the previously published data [24]. Compound 3: α-tocopherol was obtained as oily residue, yielding 20 mg. the Rf value recorded 0.5 in benzene-ethyl acetate (95:05 v/v) as solvent system. Mass spectrum gave M+ at m/z 430 for molecular formula C29H50O2 and 415 resulted from loss of a terminal CH3 group, 203 and 205 were consistent with fragments generated by loss of the side chain. A very intense peak at m/z 165 and a weaker peak at m/z 151were probably produced by fragmentation in the B-ring. These data were in convenience with the published data in [25] as well as comparison against authentic reference.

3.5. Determination of antimicrobial activity The lipoidal and pigment extracts of P. granatum L. leaves were screened for their antimicrobial activity against different types of bacteria, yeast and fungus, results illustrated graphically in Fig.1 which showed that, 100 μl of n-hexane extract at 30 min able to completely inhibition of P. aeruginosa, L. monocytogenes and B. cereus. While, S. aureus was killed by 50 μl at 60 min. In case of S. typhimurium and Aspergillus niger, results showed that, the effective dose which can be fully reduced the bacterial cell numbers was 150 μl at 30 min. Additionally, the densities of E. coli and C. albicans counts were decreased with100 μl of nhexane extract at 60 min. While, Ent. fecalis was inhibited by 150 μl at 15 min from these obtained results, it can be cleared that, all tested microbial pathogens can be removed by 150 μl of n-hexane extract at 60 min. The results of MIC of n-hexane extract against tested microbial strains was calculated and illustrated in Table 4. Accordingly, 150 μl at 60 min dose of the pigment extract can be taken as optimum extract dose in comparative with other doses, and the removal efficiency of this extract was less than n-hexane extract (Fig. 2).


6


From these results, it was illustrated that the significant antimicrobial properties of P. granatum L. are attributed to the presence of various secondary metabolites such as steroids, terpenoids and natural pigments, in addition to other constituents like alkaloids, phenols etc. distributed all over the plant parts which are found to be potent antibacterial agents as mentioned in [26]. The mechanism of antimicrobial activity of pomegranate leaves involves precipitation of membrane proteins resulting in microbial cell lysis [2]. In 2013, [27] proved that sterols alongside with other phytoconstituents inhibited the growth of Salmonella typhi (Fig. 3). Previous findings showed that the aqueous leaf extract of P. granatum L. exerted efficient antibacterial effect against some bacteria such as Bacillus subtilis, Staphylococcus aureus and Salmonella typhi [28]. In conclusion, the significant antimicrobial effect of the lipoidal extract of P. granatum L. may be attributed to the dominant presence of the steroidal and terpenoidal compounds [11] (α-amyrin acetate, ergosterol, and α-tocopherol) in the n-hexane extract, in addition to the complexity of the fatty acids compositions and the bacterial skeletons characteristics [29]. Also, the existence of unsaturated fatty acids in the n-hexane extract has a powerful effect on the antimicrobial activity [9] due to increase cell membrane permeability [30]. Nevertheless, the pigment extract exerted noticeable antibacterial effect mainly due to presence of carotenoids and chlorophyll [10].

[7]

[8]

[9] [10]

[11] [12]

[13]

[14]

[15]

[16]

3.6. Cytotoxicity assay [17]

The in-vitro cytotoxicity indicates cell death due to biomaterials application and recognizes their limitations [31]. The viability percentages of three cell lines being the Skin fibroblast normal cells (BJ1), hepatocellular carcinoma (HEPG2) and breast cancer (MCF7) against the lipoidal and natural pigment extracts of the pomegranate were evaluated. Their values ranged between 90 and 83 at 100% ppm. Therefore, their safety feature for its intended application is proved. Acknowledgement The authors would like to thank the Pharmacognosy Dept., and Environmental Microbiology Lab., Water Pollution Research Dept., Lab., National Research Centre for their financial support to this research work.

[18] [19] [20] [21]

[22]

[23]

[24]

Declaration of interest [25]

The authors have not declared any conflict of interests. [26]

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