Antitumour and antioxidant potential of some selected Pakistani honeys

Food Chemistry 143 (2014) 362–366

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Antitumour and antioxidant potential of some selected Pakistani honeys Nadia Noor a, Raja Adil Sarfraz a,b,⇑, Shaukat Ali a, Muhammad Shahid a a b

Department of Chemistry and Biochemistry, University of Agriculture, 38040 Faisalabad, Pakistan Central Hi-Tech Laboratory, University of Agriculture, 38040 Faisalabad, Pakistan

a r t i c l e

i n f o

Article history: Received 22 April 2013 Received in revised form 8 July 2013 Accepted 18 July 2013 Available online 27 July 2013 Keywords: Honey Antioxidant potential Antitumour activity Agrobacterium tumefaciens Potato disc assay

a b s t r a c t Antitumour potential of honey is attributed to its excellent antioxidant activity which in turn depends on the geographical origin. The present study focuses on exploration of antioxidant and antitumour potential as well as total phenolic contents (TPC) of 58 Pakistani honeys involving spectrochemical techniques and potato disk assay. Agrobacterium tumefaciens was used to induce tumours in potato disks. All analysed honey samples exhibited 1.33 ± 0.00–155.16 ± 0.98 mg/100 g of TPC, 50% 2,2-diphenyl picryl hydrazyl (DPPH) inhibition, P7.36 ± 0.43–39.86 ± 2.34 mg/100 g qurecitin equivalent antioxidant contents, P13.69 ± 0.91–65.50 ± 1.37 mg/100 g ascorbic acid equivalent antioxidant contents, 64.65 ± 0.43– 1780.74 ± 11.79 mM ferric reducing antioxidant power and 60% peroxide inhibition. Antitumour activity observed for 43 natural and 10 commercial samples was P20%. Two samples from Faisalabad region showed 87.50 ± 5.50% and 79.00 ± 5.56% antitumour activity which were reference standard. It was concluded that Pakistani honeys possessed excellent antioxidant and antitumour potential overall. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Antioxidants play a vital role against the deteriorating action of free radicals in the organisms. Deficiency of antioxidants in living organisms leads to oxidative stress. The natural products rich in antioxidants are of great importance for scientists (Amarowicz et al., 2010; Chan, Khong, Iqbal, Mansor and Ismail, 2013; Craft, Kosinska, Amarowicz, & Pegg, 2010). Honey a sweet natural product produced by honey bees form nectar and other plant juices is also a good source of antioxidants. Antioxidant and antimicrobial properties of honey are due to presence of variety of compounds like phenolics, ascorbic acid, a-tocopherol, proline, vitamins, catalase and glucose oxidase (Al et al., 2009; Lachman, Orsak, Hejtmankova, & Kovarova, 2010; Liu, Ye, Lin, Wang, & Peng, 2012). Several studies evidenced that different types of honeys from various countries have shown antioxidant capacity; dependent on the concentration of different bioactive compounds (Mariod, Ibrahim, & Ismail, 2009; Vit et al., 2009). Honey is important not only due to its unique composition but also due to its therapeutic properties (Blasa, Candiracci, Accorsi, Piacentini & Piatti 2007; Silici, Uluozlu, Tuzen, & Soylak 2008; Tuzen, Silici, Mendil, & Soylak, 2007). Therefore it is largely produced and

⇑ Corresponding author at: Department of Chemistry and Biochemistry, University of Agriculture, 38040 Faisalabad, Pakistan. Tel.: +92 41 9200349; fax: +92 41 9201083. E-mail addresses: [email protected], [email protected] (R.A. Sarfraz). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.07.084

characterised by many countries of world. Natural antioxidants are effective against inflammation, cancer, coronary diseases, burns, aging, wound healing, gastrointestinal and heart diseases. Anticancer potential of honey from different origins have been characterised by cell line assays (Jaganathan & Mandal, 2009). To the best of our knowledge, honey has never been characterised for its antitumour activities induced by Agrobacterium tumefaciens in potato previously. Potato disk assay is a simple, rapid, robust, easily available and sophisticated method to primarily evaluate the antitumour activity of any natural product. There was a need to analyse the antitumour activities of Pakistani honeys. Extensive data is available for antioxidant properties of honeys from different origins of world which evidenced that bioactivities of honeys vary from each other due to botanical and geographical variations (Al et al., 2009; AL-Waili et al., 2013). Very limited data about antioxidant properties and total penolic contents of honey indigenous to Pakistan is available, therefore; it became imperative to characterise antioxidant properties and total phenolic contents of honey samples from different origins of Pakistan. Hence, the aim of present study was to evaluate the total phenolic contents, antitumour and antioxidant properties of Pakistani honeys as well as to find a correlation of total phenolic contents and antioxidant properties with antitumour activities parameters. The second objective of our investigation was to compare antitumour activities, total phenolic contents and antioxidant potential of local natural honey samples of different localities and commercial honey samples.

N. Noor et al. / Food Chemistry 143 (2014) 362–366

2. Materials and methods 2.1. Materials All the reagents and chemicals used were of analytical grade. Nutrient broth, agar powder were supplied by OXOID (Hamsphire, UK), 2,2-diphenyl picryl hydrazyl (DPPH), ascorbic acid, qurecitin, gallic acid, trichloroacetic acid, hydrogen peroxide, ferric chloride and pottasium ferricynaide were supplied by Sigma (ALDRICH, Germany). Sodium carbonate, HPLC grade methanol and ethanol, Folin Ciocalteus reagent, monopotassium phosphate, dipotassium phosphate, potassium iodide, iodine and mercuric chloride from Applichem (Darmstadt, Germany), vinracine from UNITED PHARM. INC., KOREA. A.tumefaciens was obtained from Nuclear Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan. Red skinned fresh healthy potatoes were purchased from local market of Faisalabad, Pakistan. 2.2. Samples Total forty five natural honey samples from seven localities of the Central and Southern Punjab, Pakistan were extracted directly from beehives and filtered with cheese cloth. Beekeeper’s honey samples were purchased from different localities of Pakistan. Whilst commercial honey samples of different brands were purchased from local markets of Faisalabad, Pakistan. Samples were stored at room temperature in plastic bottles. 2.3. Antioxidant activities 2.3.1. Total phenolic content (TPC) Standard Folin–Ciocaltue method followed by Al et al. (2009) & Saxena, Gautam, and Sharma (2010) was used with some modifications to determine the total phenolic contents. Honey solution 1 mL (10% w/v in methanol) was added to 5 mL of 0.2 N Folin Ciocalteu reagent and mixed well for 10 min by using rotary shaker. Sodium carbonate 4 mL (75 g/L) was added and incubated for 2 h. Absorbance of reaction mixture was measured at 760 nm. Methanol was used as blank. Gallic acid (0–200 mg/L) was used for standard curve (R2 = 0.99). The collected data was presented as Gallic acid equivalent mg TPC per 100 g of honey. 2.3.2. Total antioxidant contents Total antioxidant contents were determined by following the method of Saxena et al. (2010) with some modifications. Methanolic honey solution 3 mL (4 g/10 mL) was mixed with 1.5 mL methanolic DPPH solution (.02 mg/mL). Reaction mixture was incubated for 15 min at room temperature. Absorbance was determined at 517 nm against methanol blank. Ascorbic acid (R2 = 0.98) 0– 16 lg/mL and Qurecitin (R2 = 0.99) 0–8 lg/mL were used as standards. Results are expressed as ascorbic acid equivalent (AEAC) and qurecetin equivalent antioxidant contents (QEAC). Experiment was performed in triplicate.

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performed in triplicates. % inhibition of DPPH radical was determined by following formula

% inhibition ¼ ½Ac  absorbance of sample or standard=Ac   100

2.3.4. Ferric reducing antioxidant power assay Reducing power of honey was determined by following the method of saxena et al. (2010) with some modifications. Honey solution 5 mL (10%W/V) in ethanol was mixed with 5 mL of 0.2 m phosphate buffer (pH 6.6). Above reaction mixture was mixed with 5 mL of potassium ferricyanide (1%) and incubated for 20 min at 50 °C. Trichloroacetic acid (10%) 5 mL was added followed by thorough mixing via vortex mixture. Reaction mixture thus obtained was centrifuged for 10 min at 3000 rpm. Supernatant 2.5 mL was mixed with 5 mL of double distiled water and 0.5 mL of FeCl3 (0.1%) solution. Absorbance was measured at 700 nm. Ascorbic acid (0–10 mg/mL) was used as reference standard (R2 = 0.95). 2.3.5. Peroxide scavenging activity Peroxide radical scavenging activity was determined by method of Olayinka & Okoh (2010) with some modifications. Honey solution 8 mL (0.05 g/mL) in deionized water mixed with 1.2 mL mM H2O2 in 0.1 M (pH 7.4) phosphate buffer. Samples were incubated for 10 min and measured absorbance at 230 nm against phosphate buffer as blank. Whilst H2O2 in 0.1 M (pH 7.4) phosphate buffer was used as negative control and ascorbic acid was used as positive control. % inhibition of peroxide was determined by following formula

% inhibition ¼ ½Abs: of  ve control  absorbance of sample or standard=Abs: of  ve control  100 2.4. Potato disc assay Antitumour activity was evaluated by procedure followed by (Hussain, Zia, & Mirza, 2007; Lellau & Liebezeit 2003) with slight modifications. Fresh culture of A.tumefaciens was prepared by inoculation of 100 mL (1.3%) autoclaved nutrient broth pH 7.4 with 10 lL of stock culture. This media was left at 28 °C for 48 h to get 5  109 cells per mL. Potatoes were surface sterilized for 20 min with 20% HgCl2 solution and cut (5  5) mm by sterilized cork borer. Seven potato disks along with positive and negative controls in the center were placed in autoclaved petri dish containing 1.5% agar medium. Honey samples 300 lL of each was mixed with 50 lL of cultured A.tumefaciens and poured 50 lL of each on the potato disks. The whole experimental work was done in laminar air flow. Petri plates were incubated at 28 °C for 21 days and sprayed with Lugol’s solution (potassium iodide 10% and Iodine 5% in distiled water). tumours were counted under microscope after 20 min. Each sample was replicated for five times. % inhibition was determined by following formula

% Inhibition 2.3.3. DPPH radical scavenging activity (%RSA) DPPH % RSA was measured by method of Meda, Lamien, Romito, Millogo, and Nacoulma (2005) with slight modifications. Honey solutions and DPPH solution used in above experiment were mixed in 2:1 proportion respectively. Reaction mixture was incubated at room temperature for 15 min followed by measurement of absorbance at 517 nm against methanol as blank. 5 mL DPPH solution mixed with 2.5 mL methanol was used as negative control (Ac) whilst ascorbic acid was used as standard. Each experiment was

¼ ½1  no: of tumours in sample=no of tumours negative control  100

3. Results and discussion In this study, we explored and compared antioxidant potential of 45 natural honeys native to Pakistan, all the samples exhibited excellent antioxidant activity as compared to available data. Total

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phenolic contents, antioxidant activities and antitumour activities of analysed honey samples are given in the Table 1. Total phenolic contents determined by Folin Ciocalteu method was expressed as mg gallic acid equevalent (GAE) per 100 g of honey. TPC values of natural and commercial Pakistani honeys were 36.01 ± 0.22–252.00 ± 1.61 and 1.33 ± 0.00–140.55 ± 0.89 GAE mg/100 g of honey respectively. TPC values of South African, Indian, Czech Republic, Portugal, Romanian, Venezuelan and Slovenian honeys were in the range of 32.59 ± 0.48–114.75 ± 1.30, 47 ± 0.2–98 ± 1.2, 8.25–24.25, 22.616 ± 0.22–72.777 ± 0.23, 2– 125.00, 38.15–182.10 and 2.77–28.57 GAE mg/100 g respectively (Al et al., 2009; Bertoncelj, Dobersek, Jamnik, & Golob, 2007; Ferreira, Aires, Barreira, & Estevinho 2009; Lachman et al., 2010; Meda et al., 2005; Saxena et al., 2010; Vit et al., 2009). Overview of total phenolic contents of honey from different countries of the globe rank the Pakistani natural honeys as richest one in phenolics. Natural Pakistani honeys contained higher total phenolics as compared to commercial honeys. TPC values of natural honeys were much higher than international honeys. Radical scavenging activity (%) of natural Pakistani honeys determined by DPPH was in the range of 30.50 ± 0.31– 77.43 ± 0.77 with excellent mean value 60.41 ± 11.63. RSA (%) of twenty four Romanian honey samples determined by Al et al. (2009) was from 35.80% to 64.83% whilst %DPPH radical scavenging activity of fifty rhododendron honeys from Turkey was from 36.11 ± 8.27 to 90.73 ± 0.00. Turkish honeys with excellent antioxidant potential were also effective against certain bacteria (Silici, Sagdic, & Ekici, 2010). In our study most of Pakistani honeys showed >50% RSA whilst RSA (%) of ascorbic acid used as standard in this study was 77.92%. All samples collected from Layyah, Dunyapur, Multan, Shorkot, and Karror have shown > 50% inhibition. Only one sample from Faisalabad and one sample from Bahawalpur have shown RSA (%) < 50%. Comparison of AEAC and QEAC give information about accuracy of results because one molecule of ascorbic acid has two reaction sites for free radicals whilst one molecule of qurecetin has only one reaction site for free radicals. In this study almost all honeys have ratios comparable to the results found by Meda et al. (2005) for Burkina Fasen (South Africa) honeys. TAC determined as AEAC and QEAC showed ratios approximately equal to 2 for their all honey samples. Total phenolic contents of Burkina Fasen honeys determined as AEAC and QEAC were in the range of 10.20 ± 0.59– 65.85±.010 and 4.27 ± .03–33.34 ± 0.21 mg/100 g respectively. AEAC and QEAC values of Pakistani honeys were in the range of 8.30 ± 0.55–-22.10 ± 1.47 and 2.85 ± 0.17–14.64 ± 0.86 mg/100 g of honey respectively. Many commercial Pakistani honeys have low % RSA and antioxidant contents as compared to most of natural Pakistani honeys. Only four commercial Pakistani honeys have % RSA more than 50%. TAC contents determined by Saxena et al. (2010) for seven Indian honeys were in the range of 15.1 ± 0.7 to 29.5 ± 1.8 mg AEAC/100 g honey. Most of Indian honeys exhibited 50% RSA values. Antioxidant profile of Pakistani honeys was found comparable to that of Indian honeys. This may be due to geographical and climatic resemblance. Maximum TAC values of Argentinian honeys were 10 lg/mL and 2.73 lg/mL through DPPH and ABTS method respectively (Isla et al., 2011). AEAC contents of Georgian honeys were found to be in the range of 139.9– 1131.3 lM (Taormina, Niemira, & Beuchat, 2001). Antioxidant profile of forty Czech Republic honeys were in the ranges of 98.73–441.98, 431.38–1026.38 and 222.98–87.12 ascorbic acid equivalent mg per kg of honey evaluated by DPPH, ABTS and FRAP methods respectively (Lachman et al., 2010). Mean IC50 of different honeys of Slovenia were in the range of 7.2 ± 1.2–53.8 mg/mL by DPPH method and 71.0 ± 10.2–478.5 ± 95.5 lM as determined by FRAP method (Bertoncelj et al., 2006). Ferreira et al. (2009) evaluated antioxidant profile of three Portugal honeys. They reported

that IC50 of DPPH inhibition was from 106.67 ± 2.48 to 168.94 ± 19.20 mg/mL and reducing power in the range of 13.26 ± 0.20–48.95 ± 1.61 mg/mL. FRAP values of natural and commercial Pakistani honeys were 213.78 ± 1.42–1780.74 ± 11.79 and 112.91 ± 1.46–805.96 ± 10.47 lM respectively. FRAP values for natural Pakistani honeys were higher than commercial Pakistani honeys. FRAP values of Slovenian, Italian and Georgian honeys were 71.0 ± 10.20–478.5 ± 95.50, 61.75–124.50 and 120.30–1353.50 lM respectively (Bertoncelj et al., 2006; Blasa, Candiracci, Accorsi, Piacentini, and Piatti, 2007; Taormina et al., 2001). FRAP values of natural Pakistani honeys were more than reported honeys. All Pakistani honeys have good peroxide scavenging activity. Most of the honeys exhibited% peroxide inhibition > 60% whilst % peroxide inhibition of ascorbic acid (0.1 mg/mL) used as standard was found to be 63.46% ± .03. Hydrogen peroxide present in honey is produced by glucose oxidase which helps to inhibit the microbial growth. On the other hand presence of catalase in honey helps to eliminate the hydrogen peroxide. Catalase is also used to scavange hydrogen peroxide from different products and works as antioxidant enzyme (AL-Waili et al., 2013; Saxena et al., 2010). Monofloral honeys from Taiwan exhibited good peroxide scavenging activity. However, our results showed higher % peroxide scavenging activity than that of these Taiwan’s monofloral honeys (Liu et al., 2012). Although no data is available for antitumour activity of honey against A.tumefaciens, however, extensive evidence about antimicrobial potential is available in literature (Isla et al., 2011; Silici et al., 2010; Taormina, Niemira, & Beuchat, 2001). All of our honey samples were screened for their antimicrobial attribute against wide range of microbes and were proved as potential natural antimicrobial products. Antitumour activity of our investigated honeys can be paralleled with other previously reported potential antitumour natural products. All of our honey samples showed excellent antitumour potential with values more than 20% inhibition except two out of forty five samples which remained with less than 20% inhibition. Antitumour activities of analysed commercial honeys were also found quite well, values of ten samples were more than 20%. Samples number five and seven from Faisalabad region manifested antitumour activity greater than that of standard used in the study. Mazid, Nahar, Datta, Bashar, and Sarker (2011) reported that different extracts ofPolygonum barbatum and Polygonum stagninum showed good antitumour activities with IC50 from 180 to more than 400 lg/disc. Lower IC50 indicated the higher antitumour potential. Mahamuni et al. (2012) analysed fruits of Lagenaria siceraria found it as a good plant for antitumour potential. Average numbers of tumour formation in presence of L.siceraria extract (1000 ppm) of n-Butanol is only one as compared to simple solvent which has 22.05 tumours. Methanolic and aqueous extracts of garlic (Allium stivum L.) were analysed by Hosseinzadeh, Behravan, Ramezani, Sarafraz, and Taghiabadi (2011) for their antitumour study. All samples showed more than 20% tumour inhibition. Maximum antitumour activity shown by a natural honey collected from Faisalabad is 87.50 ± 5.50%. Therefore, it can be concluded that natural Pakistani honeys possess excellent antitumour potential as compared to many plant extracts. Islam, Rahman, Qayum, and Alam (2011) of Bangladesh found that Croton bonplandianum was a potential antitumour drug. More than 35% tumour inhibition was observed in the case of 1000 ppm of extract solution against three species of agrobacterium. Jamil, Mirza1, Yasmeen, and Khan (2012) analysed some Pakistani plants for antitumour activity and nearly all plants showed excellent% inhibition. Methanolic extract of Q. dilata showed 84.78% inhibition which proved it to be quite effective anticancer plant. Hadizadeh et al. (2007) determined % inhibition of their synthetic compounds.% inhibition of one compound is 80.50% as compared to standard which is only 67.24%.

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Table 1 Total phenolic contents mg/100 g of honey, qurecitin equivalent antioxidant contents (QEAC) and ascorbic acid equivalent antioxidant contents (AEAC) determined as mg/100 of honey, DPPH radical scavenging activity (%RSA), Ferric reducing antioxidant power (FRAP) mM and % peroxide inhibition and % tumor inhibition. Standard deviation is given as ±SD for each determination. Location of samples

No. of samples

Natural samples Abdul 1 Hakeem 2 3 4 Bahawalpur 1 2 3 4 5 6 7 Dunya Pur 1 2 3 4 Faisalabad 1 2 3 4 5 6 7 8 9 10 11 12 Layyah 1 2 Multan 1 2 3 4 5 6 7 8 9 Shorkot 1 2 3 4 Karror 1 2 3 Commercial samples Beekeepers 1 2 3 4 5 6 1 Islamic Shahed (IS) 2 Salman’s 1 Honey(S) Marhaba 1 honey(M) China 1 honey(C) Life Style(L) 1 Honey(H) 1 Standard

Total phenolic contents (mg/100 g of honey)

QEAC ± SD (mg/ 100 g of honey)

AEAC ± SD (mg/ 100 g of honey)

%RSA

81.17 ± 0.51

02.85 ± 0.17

0 8.30 ± 0.55

30.52 ± 0.31

54.28 ± 0.35 36.76 ± 0.23 54.55 ± 0.35 64.04 ± 0.41 88.32 ± 0.56 252.00 ± 1.61 92.68 ± 0.59 225.79 ± 1.44 42.61 ± 0.27 53.82 ± 0.34 154.60 ± 0.34 64.59 ± 0.41 73.01 ± 0.46 95.43 ± 0.67 55.42 ± 0.35 71.07 ± 0.45 92.571 ± 0.39 84.42 ± 0.54 156.02 ± 0.99 106.47 ± 0.68 72.71 ± 0.46 47.50 ± 0.30 83.36±.08 90.45 ± 0.57 72.42 ± 0.46 36.01 ± 0.22 155.16 ± 0.98 70.13 ± 0.38 131.40 ± 0.83 93.23 ± 0.59 97.5 ± 0.62 91.42 ± 0.52 149.40 ± 0.95 91.58 ± 0.58 41.56 ± 0.26 91.63 ± 0.46 34.62 ± 0.09 70.28 ± 0.45 60.83 ± 0.39 89.087 ± 0.53 64.971 ± 0.51 118.67 ± 0.76 114.56 ± 0.73 98.42 ± 0.63

07.36 ± 0.43 12.96 ± 0.76 05.87 ± 0.35 11.85 ± 0.70 11.48 ± 0.68 39.86 ± 2.34 06.07 ± 0.36 12.10 ± 0.71 08.40 ± 0.49 14.62 ± 0.86 07.90 ± 0.46 11.61 ± 0.68 11.68 ± 0.69 06.45 ± 0.38 08.36 ± 0.49 14.64 ± 0.86 08.36 ± 0.49 07.60 ± 0.45 08.05 ± 0.47 14.06 ± 0.83 08.45 ± 0.50 09.96 ± 0.59 09.24 ± 0.54 11.13 ± 0.65 13.64 ± 0.80 02.90 ± 0.17 11.70 ± 0.69 14.56 ± 0.86 11.11 ± 0.65 13.18 ± 0.78 10.78 ± 0.63 13.39 ± 0.79 11.70 ± 0.69 13.12 ± 0.77 13.10 ± 0.77 10.93 ± 0.64 11.64 ± 0.68 07.92 ± 0.47 08.56 ± 0.50 07.43 ± 0.44 09.92 ± 0.58 11.89 ± 0.70 09.34 ± 0.55 13.73 ± 0.81

13.69 ± 0.91 20.15 ± 1.34 12.01 ± 0.80 19.01 ± 1.27 18.44 ± 1.23 20.50 ± 1.37 12.20 ± 0.81 19.11 ± 1.27 14.86 ± 0.99 22.06 ± 1.47 14.30 ± 0.95 18.60 ± 1.24 18.67 ± 1.24 12.63 ± 0.84 14.84 ± 0.99 22.10 ± 1.47 14.84 ± 0.99 13.96 ± 0.93 14.52 ± 0.97 21.40 ± 1.43 14.94 ± 1.00 16.69 ± 1.11 15.84 ± 1.06 18.04 ± 1.20 20.93 ± 1.40 08.54 ± 0.57 18.70 ± 1.25 21.00 ± 0.99 18.00 ± 1.20 20.41 ± 1.36 17.67 ± 1.18 20.67 ± 1.38 18.70 ± 1.25 20.32 ± 1.35 20.30 ± 1.35 17.81 ± 1.19 18.62 ± 1.24 14.35 ± 0.96 15.06 ± 1.00 13.77 ± 0.92 16.54 ± 1.10 18.91 ± 1.26 15.99 ± 1.07 21.08 ± 1.41

1.33 ± 0.00 14.72 ± 0.94 27.30 ± 0.17 48.35 ± 0.31 7.50 ± 0.48 25.48 ± 0.16

9.47 ± 0.56 7.95 ± 0.47 3.34 ± 0.20 11.9 ± 0.70 7.03 ± 0.41 0.86 ± 0.05 07.5 ± 0.44

58.92 ± 0.38 122.54 ± 0.88

FRAP (mM)

Peroxide inhibition(%) ± SD

Tumor inhibition (%) ± SD

240.30 ± 1.59

62.65 ± 0.01

31.98 ± 05.45

48.84 ± 0.49 70.76 ± 0.71 43.11 ± 0.43 64.90 ± 0.65 64.75 ± 0.65 72.02 ± 0.72 43.78 ± 0.44 67.18 ± 0.67 52.78 ± 0.53 77.23 ± 0.77 50.92 ± 0.51 65.70 ± 0.66 65.75 ± 0.66 55.23 ± 0.45 52.74 ± 0.53 77.43 ± 0.77 52.73 ± 0.53 50.11 ± 0.50 51.63 ± 0.52 75.01 ± 0.75 53.11 ± 0.53 59.02 ± 0.59 56.11 ± 0.56 63.57 ± 0.64 73.83 ± 0.74 31.36 ± 0.31 65.74 ± 0.66 77.00 ± 0.77 63.49 ± 0.63 71.62 ± 0.72 62.33 ± 0.62 72.43 ± 0.72 65.83 ± 0.66 71.36 ± 0.71 71.29 ± 0.71 62.79 ± 0.63 65.28 ± 0.65 51.08 ± 0.51 52.53 ± 0.54 50.11 ± 0.49 58.84 ± 0.59 66.56 ± 0.67 56.68 ± 0.57 73.89 ± 0.74

251.61 ± 1.67 587.26 ± 3.89 261.17 ± 1.73 818.13 ± 5.42 1084.65 ± 7.18 1539.43 ± 10.19 1544.22 ± 10.23 1334.22 ± 8.84 307.70 ± 2.04 376.83 ± 2.50 805.96 ± 5.34 521.61 ± 3.45 623.35 ± 4.13 732.91 ± 4.85 213.78 ± 1.42 258.13 ± 1.71 232.04 ± 1.54 453.78 ± 3.01 402.04 ± 2.66 1119.43 ± 7.41 649.43 ± 4.30 589.43 ± 3.90 865.96 ± 5.73 697.26 ± 4.62 960.30 ± 6.36 64.65 ± 0.43 847.26 ± 5.61 791.61 ± 5.24 568.13 ± 3.76 556.39 ± 3.68 1158.13 ± 7.67 1678.57 ± 11.12 722.04 ± 4.78 781.61 ± 5.18 652.04 ± 4.32 560.74 ± 3.71 309.87 ± 2.05 288.57 ± 1.91 376.39 ± 2.49 331.17 ± 2.19 392.91 ± 2.60 1043.78 ± 6.91 1780.74 ± 11.79 1517.26 ± 10.05

63.96 ± 0.02 62.01 ± 0.37 63.83 ± 0.03 60.34 ± 0.05 54.80 ± 0.06 57.57 ± 0.11 59.28 ± 0.06 55.25 ± 0.27 61.91 ± 0.28 61.53 ± 0.14 94.48 ± 0.01 60.15 ± 0.03 60.44 ± 0.06 57.38 ± 2.47 63.15 ± 0.04 56.91 ± 0.04 64.00 ± 0.05 63.08 ± 0.03 63.59 ± 0.03 62.42 ± 0.62 62.96 ± 0.01 61.65 ± 0.03 63.03 ± 0.04 61.89 ± 0.02 59.83 ± 0.03 63.09 ± 0.01 60.47 ± 0.04 60.96 ± 0.06 60.10 ± 0.06 61.85 ± 0.03 62.24 ± 0.02 61.46 ± 0.02 61.24 ± 0.04 60.18 ± 0.06 61.77 ± 0.03 61.70 ± 0.04 61.01 ± 0.06 63.38 ± 0.04 62.01 ± 0.03 62.71 ± 0.01 63.03 ± 0.02 59.73 ± 0.02 56.55 ± 0.02 55.83 ± 0.04

44.91 ± 08.58 26.38 ± 10.45 23.11 ± 08.68 28.05 ± 03.54 35.33 ± 07.47 52.00 ± 10.78 61.00 ± 15.83 41.57 ± 08.01 44.00 ± 09.80 31.55 ± 10.48 60.00 ± 10.00 43.00 ± 09.00 37.10 ± 11.14 55.00 ± 19.50 23.33 ± 10.55 15.00 ± 10.21 57.00 ± 08.33 45.00 ± 09.50 87.50 ± 05.50 30.02 ± 10.20 79.00 ± 05.66 52.50 ± 11.82 52.26 ± 13.33 45.00 ± 05.60 26.00 ± 10.00 28.07 ± 14.73 44.71 ± 12.80 50.17 ± 12.67 46.75 ± 11.82 40.75 ± 11.31 22.75 ± 12.02 65.50 ± 09.09 20.34 ± 05.40 07.63 ± 03.78 27.00 ± 09.57 59.50 ± 08.08 24.00 ± 15.00 25.34 ± 08.89 21.88 ± 05.77 37.08 ± 08.12 28.10 ± 11.75 59.50 ± 05.66 27.79 ± 11.02 42.62 ± 09.47

16.14 ± 1.08 14.36 ± 0.96 09.05 ± 0.60 18.92 ± 1.26 13.31 ± 0.89 6.185 ± 0.41 13.93 ± 0.93

57.14 ± 0.57 51.12 ± 0.51 33.09 ± 0.33 66.57 ± 0.67 47.56 ± 0.48 23.37 ± 0.23 49.60 ± 0.50

445.96 ± 5.80 396.39 ± 5.15 192.48 ± 2.49 112.91 ± 1.46 132.91 ± 1.72 119.44 ± 1.55 449 ± 5.83

62.18 ± 0.06 63.74 ± 0.06 62.58 ± 0.06 64.27 ± 0.25 64.30 ± 0.02 64.94 ± 0.03 63.54 ± 0.06

42.87 ± 13.68 43.96 ± 09.75 42.78 ± 15.46 25.76 ± 07.40 12.50 ± 05.00 22.73 ± 12.14 13.25 ± 08.86

05.5 ± 0.32 03.5 ± 0.21

11.54 ± 0.77 9.225 ± 0.62

41.52 ± 0.42 33.68 ± 0.34

193.78 ± 2.51 344.65 ± 4.48

63.67 ± 0.06 65.06 ± 0.06

21.20 ± 10.49 30.38 ± 06.61

131.28 ± 1.35

5.24 ± 0.31

11.25 ± 0.75

40.55 ± 0.41

464.22 ± 6.02

63.17 ± 1.97

34.78 ± 08.09

123.28 ± 0.03

6.35 ± 0.37

12.52 ± 0.83

54.85 ± 0.45

62.86 ± 0.05

58.28 ± 12.98

140.55 ± 0.89 3.34 ± 0.02

08.54 ± 0.50 10.43 ± 0.61

15.04 ± 1.00 17.22 ± 1.15

53.48 ± 0.53 60.81 ± 0.61 77.92 ± 1.45

63.01 ± 0.02 63.09 ± 0.05 63.46 ± 0.03

54.47 ± 21.33 13.83 ± 10.53 70.00±13.43

222.913± 805.96 ± 10.47 314.22 ± 4.08

366

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