Metabolomics driven analysis of Erythrina lysistemon cell suspension culture in response to methyl jasmonate elicitation

Journal of Advanced Research (2016) 7, 681–689

Cairo University

Journal of Advanced Research

ORIGINAL ARTICLE

Metabolomics driven analysis of Erythrina
lysistemon cell suspension culture in response to
methyl jasmonate elicitation
Mohamed A. Farag a,*, Hattem Mekky b, Sawsan El-Masry b
a
b

Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Einy Street, 11562 Cairo, Egypt
Pharmacognosy Department, Faculty of Pharmacy, Alexandria University, El Khartoum Square, 21521 Alexandria, Egypt

G R A P H I C A L A B S T R A C T

A R T I C L E

I N F O

Article history:
Received 29 April 2016
Received in revised form 6 July 2016
Accepted 6 July 2016
Available online 14 July 2016

A B S T R A C T

An MS-based metabolomic approach was used to profile the secondary metabolite of the ornamental plant Erythrina lysistemon via ultra-performance liquid chromatography coupled to
photodiode array detection and high resolution q-TOF mass spectrometry (UPLC-PDA-MS).
Cultures maintained the capacity to produce E. lysistemon flavonoid subclasses with pterocarpans amounting for the most abundant ones suggesting that it could provide a resource of
such flavonoid subclass. In contrast, alkaloids, major constituents of Erythrina genus, were

* Corresponding author. Fax: +20 11 202 25320005.
E-mail address: (M.A. Farag).
Peer review under responsibility of Cairo University.

Production and hosting by Elsevier
/>2090-1232 Ó 2016 Production and hosting by Elsevier B.V. on behalf of Cairo University.
This is an open access article under the CC BY-NC-ND license ( />

682

Keywords:
Cell culture
Erythrina lysistemon
Metabolomics
Methyl jasmonate
Oleanolic acid
Ultra-performance liquid
chromatography

M.A. Farag et al.
detected at trace levels in suspension cultures. Methyl jasmonate (MeJA), phytohormone, was
further supplied to culture with the aim of increasing secondary metabolites production and
with metabolite profiles subjected to multivariate data analysis to evaluate its effect. Results
revealed that triterpene i.e. oleanolic acid and fatty acid i.e. hydroxy-octadecadienoic acid were
elicited in response to methyl jasmonate, whereas pterocarpans i.e., isoneorautenol showed a

decline in response to elicitation suggesting for the induction of terpenoid biosynthetic pathway
and concurrent with a down regulation of pterocarpans. In conclusion, a total of 53 secondary
metabolites including 3 flavones, 12 isoflavones, 4 isoflavanones, 4 alkaloids, 11 pterocarpans,
and 5 phenolic acids were identified.
Ó 2016 Production and hosting by Elsevier B.V. on behalf of Cairo University. This is an open
access article under the CC BY-NC-ND license ( />4.0/).

Introduction

Material and methods

The genus Erythrina constitutes 115 species in the pea family
‘‘Fabaceae” which are distributed worldwide in tropical and
subtropical regions growing as trees, often recognized in agriculture for its bright red flowers as coral or flame trees [1].
Alkaloids and phenolics are among the most widely distributed
constituents in these flowering trees mostly localized in stem
bark [2,3], roots [4] and seeds [5,6]. Erythrina alkaloids are
tetracyclic spiroamines possessing an erythrinane skeleton.
Over 90 Erythrina alkaloids have been isolated [7,8], often classified as dienoid or lactonic alkaloids. Interest in Erythrina
alkaloids is mostly driven by its curare-like neuromuscular
blocking effect. Moreover, Erythrina spp. possess a broadspectrum of physiological activities such as anti-plasmodial
activity due to the flavonoids and isoflavonoids [9], antioxidant and anti-inflammatory activities due to pterocarpans
[10] and fungicidal activity associated with its alkaloidal content [11].
Erythrina genus has been extensively examined in terms of
its taxonomy and chemical composition. However, very little
information is available concerning biotechnological attempts
for natural products production within that genus.
Garcia-Mateos et al., showed that an unexpected profile of
oxygenated alkaloids was observed in undifferentiated callus
of Erythrina Coralloides and Erythrina americana [12]. Furthermore, San Miguel-Chavez et al., showed that jasmonic

acid elicited E. americana cell culture has led to reduction in
alkaloid accumulation [13]. Among the most common and
effective elicitors used for stimulating secondary metabolites
production in plant cell culture are the carbohydrate fractions
of fungal and plant cell walls, MeJA, chitosan and/or heavy
metal salts. In particular, jasmonates have been long regarded
as transducers of elicitor signals for the production of plant
secondary metabolites. Application of elicitors results in the
induction of signaling compounds, including jasmonic acid
and or MeJA, as well as the downstream up regulation of secondary metabolites. In contrast to salicylic acid being an elicitor of limited secondary metabolite classes, jasmonates seem
to be general natural products inducer via the activation and
de novo biosynthesis of transcription factors that up regulate
genes involved in secondary metabolites production [14]. For
example, jasmonates induce the accumulation of terpenoids,
flavonoids, alkaloids and phenylpropanoids [15]. The aim of
this work was to examine MeJA elicitation effect on cell
suspension culture of Erythrina lysistemon regarding the accumulation of alkaloids, flavonoids, pterocarpans and phenolic
acids using an MS-based metabolomic approach.

Plant material
Seeds of E. lysistemon were collected in January 2012 from
trees previously authenticated by Professor Nabil El Hadidi,
College of Science, Cairo University, Egypt. Voucher
specimens of the flowers and seeds are deposited at Faculty
of Pharmacy, Alexandria University, Egypt.
Callus initiation
Seeds were surface sterilized in 20% sodium hypochlorite solution for 30 min, washed three times in sterile purified water and
placed on agarized Murashige and Skoog (MS; Caisson,
Smithfield, USA) germination medium (1962) and incubated
under 12 h light period and a temperature of 23 °C ± 1 °C

in a culture room [16]. Leaves were excised from 28 d-old seedlings. The leaves were then scored on their abaxial sides with a
sterile scalpel blade and cut into 1 cm2 pieces. Explants were
cultured on 25 mL aliquots of MS supplemented with either
1 mg LÀ1 or 2 mg LÀ1 of each of kinetin (Kin; Acros, Geel,
Belgium) and 2,4-dichlorophenoxyacetic acid (2,4D; Acros,
Belgium) in addition to 30 g LÀ1 sucrose (El-Nasr, Alexandria,
Egypt), and semi-solidified with 0.8% (w/v) agar (Roko,
Llanera – Asturias, Spain), pH 5.6, in a 9 cm diameter Petri
dishes. The explants were transferred onto fresh media, until
callus was produced [16].
Cell suspension culture initiation
Cell suspensions were established by transferring 1–2 g fresh
wt. of callus and maintained in 100 mL MS liquid medium
supplemented with same growth regulators, but no agar was
added, pH 5.6 in 100 mL Erlenmeyer flasks. Cultures were
maintained on a rotary shaker at 100 rpm and incubated under
a 12 h photoperiod, with day and night temperatures of
23 °C ± 1 °C until the stock suspension was produced.
Elicitation
Aliquots of 2.5 mL packed cell volume (PCV) with 2.5 mL
spent medium of the stock suspension were transferred to six
100 mL Erlenmeyer flasks, each containing 45 mL of fresh
media and maintained under same conditions for two weeks.
Methyl jasmonate (MeJA; Sigma Aldrich, Poole, UK) was
then added into five flasks to produce a concentration


Erythrina lysistemon cell culture metabolomics
1 mM LÀ1 MeJA. The dose 1 mM LÀ1 MeJA was previously
optimized to elicit secondary metabolic pathways in cell cultures [17,18]. Furthermore, an increase in the concentration

of MeJA resulted in retarded callus growth. The remaining
flask was used as control by the addition of the same volume
of sterile water. Cultures were kept at 23 °C ± 1 °C, with a
12 h photoperiod and maintained on a rotary shaker at
100 rpm. Cell culture samples were harvested at 0, 6, 12, 24
and 48 h post elicitation and kept at À80 °C until being
analyzed.
Extraction and UPLC-MS analysis of cell culture extracts
Metabolites extraction followed the protocol developed for
similar metabolite classes [18,19]. Briefly, lyophilized E. lysistemon cultures (20 ± 0.06 mg) were extracted with 1.8 mL
aq.80% MeOH for 10 h using an orbital shaker in the dark.
Extracts were centrifuged at 10,000g for 15 min and 1.4 mL
of the supernatant was aliquot and evaporated under nitrogen
till complete dryness. The dried residue was resuspended in
300 lL 45% aq. MeOH. For comparative analysis, the extracts
were spiked with 2 lg umbelliferone as an internal standard
(IS) and quantifications were determined from peak areas normalized based on the amount of recovered IS peak. The residue was re-suspended in 300 lL methanol and used for
UPLC-MS analysis following the exact chromatographic conditions described by Farag et al. [20].
Identification and quantification of metabolites and MS data
multivariate analysis
File Converter tool in X-Calibur software was used to convert
UPLC–MS files to NetCDF file format and then further processed by AMDIS software for background subtraction and
peak deconvolution. Metabolite identification was done via
UV-VIS spectra (220–600 nm), retention times relative to external standards, mass spectra, and comparison to both the reference literature and phytochemical dictionary of natural
product database. Quantification of alkaloids was calculated
from the calibration curve of erythraline, pterocarpans using
medicarpin standard, and for oleanolic acid using that of
oleanolic acid standard detected using MS detector. Standard
calibration curves were constructed for each standard using 4
concentrations spanning from 0.1, 1, 10 and 200 lg/mL. Assays

were carried out in triplicate.
MS data processing for multivariate analysis
Relative quantification and comparison of metabolites profiles
after UPLC-MS were performed using XCMS data analysis
software, which can be downloaded for free as an R package
from the Metlin Metabolite Database (http://137.131.20.
83/download/) [21].
Results and discussion
E. lysistemon cell culture metabolite profile

683
for the elution of cinnamates, flavonoids, alkaloids and fatty
acids within 13 min (ca. 800 s). The elution order of secondary
metabolites followed a sequence of decreasing polarity,
whereby cinnamates and alkaloids eluted first, followed by flavonoid glycosides, free aglycones, prenylated aglycones and
finally triterpenes and fatty acids. Simultaneously acquired
UPLC–PDA and UPLC–MS total ion chromatograms of
E. lysistemon cell culture extracts in positive and negative ionization mode are presented in Fig. 1. The identities, retention
times, UV and MS spectral data observed for secondary
metabolites are presented in Table 1 with a total of 53 identified
metabolites. It is worth noting that this is the first comprehensive metabolite profile of E. lysistemon plant. Identified metabolites belonged to various classes (Table 1, Suppl. Fig. 1)
including phenolic acids (cinnamates) i.e. N-caffeoyl aspartic
acid (2), alkaloids i.e. erysotrine (6), pterocarpans i.e. isoneorautenol (30), isoflavones i.e. lysisteisoflavone (44), triterpenes
i.e. oleanolic acid (53) and fatty acid i.e. hydroxy-9,11octadecadienoic acid (45), with isoflavones and pterocarpans
as the most abundant classes in cell culture extract. The structures of major metabolites identified in E. lysistemon and discussed throughout the manuscript are shown in Suppl. Fig. 1.
Flavonoids
Photodiode array detection provided an overview of the main
flavonoid constituents (Fig. 1A). UV spectra (200–600 nm)
were measured for flavonoid sub-classes including 12 isoflavones, 3 flavones, 4 isoflavanones and 11 pterocarpans. Each
sub-class exhibits a characteristic UV spectrum. For example,

flavones have a maximum absorbance near 265 nm with a second maximum between 320 and 340 nm (peak 9), whereas pterocarpans have k max around 280–290 nm (42). Extracts were
analyzed in positive and negative ion electrospray ionization
(ESI) MS modes to provide a comprehensive overview of the
metabolite composition. Compared to the positive-ion ESI
mode (Fig. 1C), negative-ion MS spectra (Fig. 1B) revealed
better sensitivity than in positive mode, especially in the elution range of flavonoids (200–500 s). In addition, negativeion MS spectral characteristics showed strong [M À H]À ions
and lower chemical noise and consequently better sensitivity
[22]. The positive ion ESI mass spectra were characterized by
cations corresponding to [M + H]+, [M + Na]+ and fragment ions attributed to the sequential losses of isoprenyl
(69 amu), malonyl (86 amu) and hexosyl (162 amu) groups.
Few minor isoflavone peaks 13, 15, 18, 32 and 43 were only
detected in positive ionization mode warranting the importance of acquiring data in both ionization modes. Two major
flavone glycosides including dihydroxyflavone hexoside
(m/z 415.102, [M À H]À peak 9) and apigenin hexosylmalonate
(m/z 517.1702, [M À H]À peak 11) were identified in cell
culture. With regard to isoflavanone subclass, vogelin A
(m/z 369.0999, [M À H]À peak 25) and 5-deoxyglyasperin
F/5-deoxylicoisoflavanone (m/z 337.1085, [M À H]À peak 27)
exhibiting UV max around 310–320 nm typical for flavanones
were measured.
Pterocarpans

Callus was produced from cut ends of scored E. lysistemon
explants after 3 weeks. Chemical constituents of callus extracts
were analyzed via UPLC/PDA/(À)ESI-qTOF-MS that allowed

Among flavonoid subclasses, pterocarpans amounted for the
major forms in cell culture (11 peaks), exhibiting k max around

684

M.A. Farag et al.

UV response

mAU
500
400

(A)

32

IS

300
200

2

1

9

42

100

x105

0
4

(B)

3

IS

2

MS response

30
33

3

1
0
4

17

5

23

34

42
53

27
35

31

(C)

42

33

3

x105 2

18

1

47

34

11 13

52

22

0

200

300

400

500

600

700

800

rt (sec)
Fig. 1 UPLC–UV at 250 nm (A), UPLC–negative-ion ESI–MS (B) and UPLC–positive-ion ESI–MS chromatogram of E. lysistemon cell
culture extracts (C). Chromatographic conditions are described under Material and methods. Insets 1 and 2 represent UV spectra of peak
9 (dihydroxyflavone hexoside) and peak 42 (sandwicensin, a pterocarpan), respectively. The identities, rt-value, UV and MS spectra of all
peaks are listed in Table 1. IS = spiked internal standard (umbelliferone). Chromatographic conditions followed that were described in
[20].

280–290 nm with isoneorautenol (m/z 321.1147, [MÀH]À peak
30) as the most abundant (Table 1). Other identified pterocarpans include erythribyssin B (m/z 283.0598, [M À H]À
peak 19), eryvarin D (m/z 335.1264, [M À H]À peak 20),
dihydroisoneorautenol (m/z 323.1288, [M À H]À peak 31)

and sandwicensin (m/z 337.1445, [M À H]À peak 42). The predominant loss of 69 amu (–C5H9, prenyl group) in the MSn
spectrum of pterocarpans is diagnostic for the presence of
the isoprenyl group; a total of 6 peaks showed this pattern.
For example, erystagallin B (m/z 437.1993, [M À H]À peak
38) showed 2 mass fragments at m/z 368 and 299 indicative
for 2 isoprenyl losses (À2 Â 69 amu). The abundance of
isoprenylated pterocarpans in cell culture suggests for the presence of isoprenyl transferase enzyme with higher affinity
toward pterocarpans. This is the first report for the accumulation of pterocarpans in E. lysistemon cell culture and suggests
that it could provide a resource of that flavonoid subclass.
Alkaloids
With an increased sensitivity for detection of nitrogenous
metabolites in positive mode, alkaloids could only be detected
in that mode. Alkaloids that are known to predominate
E. lysistemon plant extracts were almost absent in cell culture,
except for few alkaloid peaks present at trace levels including
erysotrine (m/z 314.1756 [M + H]+, peak 6), erythrartine/11methoxyerysodine (m/z 330.1696, [M + H]+, peak 10) and
erysotramidine (m/z 328.1534 [M + H]+, peak 12). In contrast, DOPA methyl ether (m/z 226.1073 [M + H]+, peak 8)
was present as the major nitrogenous secondary metabolite
identified in culture. No UV absorbance could be traced for
alkaloid peaks, except for DOPA methyl ether showing distinct UV max at 270 nm. In tandem MS, alkaloids showed
methyl losses from methoxy group (À15 Da).

Phenolic acid (cinnamates)
The most abundant nitrogenous compounds detected in cell
culture were amino acyl hydroxycinnamic acid conjugates. A
total of 5 peaks (2–5, 16) not previously reported in E. lysistemon plant tissue were identified in cell culture suggesting for an
activation toward the production of acylcinnamates in cell culture. The predominant fragment of cinnamic acid derivatives
in the MSn spectrum and characteristic UV max values at
298 and 325 nm are diagnostic for cinnamates; a total of 5
peaks showed similar UV (Table 1). MS/MS analysis confirmed the structure of N-p-coumaroylaspartic acid (3) m/z

278 and N-feruloylaspartic acid (5) m/z 308 from their respective product ions at m/z 163 and 193 indicative of a
p-coumaroyl and feruloyl moieties, respectively, whereas
N-caffeoylaspartic acid (2) gave a [M–H]À at m/z 294 with
product ions m/z 132 for the aspartic acid moiety.
Differences in metabolites composition observed in E. lysistemon callus from its native plant are likely to be the result of
genetic variation and/or lack of differentiation [22,23]. It is
worth mentioning that there was no obvious qualitative or
quantitative difference in the metabolite profile of the 2 different treatments of the calli (1 mg lÀ1 or 2 mg lÀ1 of Kinetin and
2,4D), results not shown.
PCA of E. lysistemon MeJA elicited and control suspension
culture observed in negative ionization mode
Cell culture was further subjected to MeJA treatment to determine its impact on reprogramming of secondary metabolites as
revealed via UPLC-MS analysis. To assess for changes in
metabolite composition in response to elicitation as monitored
via UPLC-MS traces of the different callus samples harvested
at 0, 12 and 2 h post MeJA elicitation (Suppl. Fig. 2), principal


Erythrina lysistemon cell culture metabolomics

685

Table 1 Metabolites identified in E. lysistemon L. cell suspension methanol extract using UPLC–PDA–MS/MS in negative/positive
ionization modes.
Peak

rt (s)

UV (nm) Metabolite

Class

1

122

285

Phenolic acid 447.1139[M À H]À

2
3
4

161
198
209

294, 325
294, 326
287, 312

5
6
7
8
9
10
11
12

13
14
15
16
17
18
19
20
21
22
23
24
25
26
27

220
237
238
238
245
252
262
263
277
278
289
292
350
350

357
369
380
395
408
485
485
507
510

294,
282
272,
280
270,
325
nd
nd
282,
280,
282,
272,
262,
282,
232,
280,
284
280
280,
280,

230,
nd
270,

28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50

531
531

537
552
562
567
596
601
621
630
642
655
659
665
678
680
689
691
714
721
748
762
779

287, 330
287. 320
287, 311
286, 323
287,322
282
287, 304
285, 340

282
281
280
283
272
286
281, 287
287,322
285,
280
280
280
280
nd
282

51
52
53

785
806
838

287,330
nd
nd

325
340

332

286
308
286
319
308
286
285
335

335
310
287
307

Dihydroxybenzoic acid
pentosylhexoside
N-Caffeoylaspartic acid
N-p-Coumaroylaspartic acid
N-(Hydroxycinnamoyl)
tyraminehexoside
N-Feruloylaspartic acid
Erysotrine
Apigeninpentosyl hexoside
DOPA methyl ether
Dihydroxyflavone hexoside
Erythrartine/11-Methoxyerysodine
Apigeninhexosylmalonate
Erysotramidine

Demethylmedicarpin hexosylmalonate
Diacetoxy benzoic acid
Demethylmedicarpin hexosylmalonate
N-Cinnamoyl-Aspartic acid
Dihydroxyisoflavone
Demethylmedicarpin
Erythribyssin B
Eryvarin D
Unknown isoflavone
Unknown isoflavone
Apigenin
Unknown isoflavone
Vogelin A
Oleanolic acid trihexoside
5-Deoxyglyasperin
F/5-Deoxylicoisoflavanone
Unknown isoflavanone
Licoisoflavanone/Ficuisoflavone
Isoneorautenol
Dihydroisoneorautenol
Eryvarin I/Erypoegin B
Erythrabissin I
5-Deoxylicoisoflavanone
Erysubin A
Unknown
Unknown
Erystagallin B
Unknown isoflavone
Unknown
Unknown triterpene

Sandwicensin
Dimethoxyisoflavone
Lysisteisoflavone
Hydroxy-9,11-octadecadienoic acid
Unknown
40 -O-Methylalpinumisoflavone
Erylysin A
Unknown sterol
Hydroxy-9,11-octadecadienoic
acid isomer
Unknown
Unk307.2/878
299.1/419
527.2/922
528.8/559
508.3/769
283.3/936
281.2/877
295.2/828
295.1/542
398.2/444
398.2/937
415.2/878
447.2/683
391.2/826
391/258
449.1/223
257.2/919
277.1/309
780.5/551

670.4/919
718.4/877
351.2/444
348.2/877
349.2/877
351.1/505
353.1/438
187.1/309
209.1/309
163/259
617.3/361
634.4/571
627.4/877
651.4/877
650.4/877
583.5/877
603.4/919
635.4/856
635.4/882
863.4/524
797.5/524
880.7/919
911.5/524
925.5/565
885.7/877
993.5/527
994.5/527
928.5/527
969.5/603
973.5/510

971.5/527
990.5/509
974.5/510
945.5/509
958.5/425
957.5/509
956.5/426
947.6/919
324.2/919
535.5/919
293.2/562
471.3/826
278.2/684
369.1/381
276.2/639
271.2/919
669.4/919
737.4/920
717.4/877
211.1/877
171.1/878
235.2/638
586.5/936
602.4/919
879.7/919
889.7/936
924.5/565
957.5/426
955.5/426
337.1/441

338.1/587
315.2/704
309.2/643
281.2/936
568.4/769
397.2/444
379.2/683
431.2/684
361.2/639
633.4/571
649.4/877
347.2/877
796.5/524
923.5/565
884.7/877
927.5/527
939.5/498
989.5/509
941.5/545
323.2/919
317.2/683
297.1/555
812.7/919
883.7/877
940.5/498
327.2/476
409.2/857
275.2/639
633.4/861
795.5/524

601.4/919
614.4/683
944.5/509
330.2/444
410.2/857
254.1/351
585.5/936
285/353
364.2/684
582.5/877
811.7/919
926.5/527
988.5/509
967.5/561
324.1/555
534.5/919
256.2/919
280.2/877
940.5/545
295.2/782
277.2/684
613.4/683
987.5/509
925.5/527
329.2/444329.2/478
581.5/877
283/447
939.5/545
943.5/509
295.2/695

533.5/919
363.2/684
293.2/713
296.2/683
279.2/877
253/351
255.2/919
942.5/509

0.0

-0.4

(B)

337.1/684

161/259

-0.2

PC2

0.2

0.4

PC1 (92%)

322.1/542

323.1/555

353.1/534
337.1/587

Erythrabissin I

321.1/542

Isoneorautenol

Hydroxy-octadecadienoic acid
295.2/683
271.2/816

-0.2

0.0

-0.2

-0.4

-0.6

PC1
Fig. 2 UPLC-qTOF-negative ionization MS (m/z 100–1000) principal component analyses of E. lysistemon unelicited cell culture (), cell
cultures treated with 1.0 mM MeJA at 0 h (D), 12 h (D) and 24 h (+) (n = 3). The metabolome clusters are located at the distinct positions
in two-dimensional space prescribed by two vectors of principal component 1 (PC1 = 92%) and principal component 2 (PC2 = 7%). (A)
Score Plot of PC1 vs PC2 scores. (B) Loading plot for PC1 and PC2 contributing to mass peaks and their assignments, with each

metabolite denoted by its mass/rt (s) pair.


Erythrina lysistemon cell culture metabolomics

687

12 h

0

PC2 (6%)

2000

(A)

0h
-2000

24 h

-50000

0

5000

0.0

-0.2

-0.1

PC2

0.1

0.2

PC1 (93%)
439.4/841

425.3/891
713.5/919

283.1/683

Dimethoxy isoflavone

Unknown sterol

425.4/859
379.3/886
274.3/479
403.2/758
439.4/853
337.1/510
476.2/211
361.1/683

562.2/238
393.1/398
377.3/825
565.2/244
440.4/841
503.1/290
426.3/891
426.4/859
163/398
407.4/859
671.4/741
377.1/510
671.4/781
495.3/747
393.4/841
321.1/337
445.2/683
479.4/852
631.4/741
459.3/894
335.1/555
380.3/886
284.3/938
519.1/263
522.4/705
393.3/638
284.1/683
358.2/292
177.1/221
191.1/398

440.4/853
339.3/637
302.3/548
299.1/230
404.2/758
205.2/859
322.1/337
593.3/919
271.1/263
437.3/671
411.4/936
299.2/509
437.3/742
226.1/240353.3/865
340.2/302
563.2/238
935.7/841
246.2/401
275.3/479
672.4/741
479.4/840
339.3/886
217.2/859
885.8/859
631.4/781
457.2/799
179/683
437.3/781
393.4/884
455.1/204

504.1/290
313.3/852
282.1/241
547.1/244
494.2/212
566.2/244
338.2/510
621.3/810
290.3/487
177.1/178
378.3/825
519.1/325
477.2/211
335.2/877
587.1/244
265.3/886
672.4/781
659.3/904
377.3/807
495.1/568
353.1/398
886.8/859
460.3/894
411.4/883
408.4/859
632.4/741
373.7/742
541.2/337
351.2/826
394.1/398

191.2/860
203.1/510
936.7/841
322.1/306
429.2/826
419.3/895
362.1/683
495.4/671
577.3/261
895.7/841
913.7/841
580.2/238
461.2/683
226.1/292
314.2/264
943.5/350
342.3/669
480.3/698
335.1/683
218.2/305
457.4/841
441.4/841
302.1/683
179.2/841
379.3/937
378.1/510
480.4/852
496.4/747
332.1/221
339.2/683

295.7/841
391.2/592
581.3/810
259.2/841
595.2/435
637.3/904
443.4/859
281.1/316
281.1/287
368.4/686
366.3/645
278.2/842
541.3/761
375.1/554
477.2/193
304.2/877
337.1/434
629.4/730
296.3/479
405.2/683
203.1/670
896.7/841
907.8/859
446.2/683
359.1/670
223.1/398
594.3/919
425.4/909
290/382
973.5/434

177.1/741
525.1/290
520.1/263
541.2/308
495.3/878
495.3/778
339.3/810
308.3/797
627.2/347
391.2/652
619.3/724
203.2/859
351.1/684
625.2/436
214.1/398
455.2/730
458.2/799
409.1/398
632.4/781
302.1/244
271.1/283
271.1/216
177.1/781
330.3/613
523.4/705
438.3/671
433.1/204
433.1/216
555.2/321
373.7/781

374.2/741
541.1/263
491.2/229
577.8/261
335.1/336
867.8/859
306.2/747
571.2/300
514.2/390
337.3/779
163/318
573.2/435
311.1/683
325.1/435
394.4/841
353.1/319
393.1/511
625.2/466
585.2/466
455.4/741
727.5/758
507.2/449
427.3/891
971.5/390
181.1/234
687.5/935
330.1/683
355.2/413
438.3/781
382.7/741

555.3/765
341.2/302
340.3/637
374.3/837
541.2/287
376.3/894
533.1/298
170/683
572.3/387
480.4/840
495.2/212
335.1/317
335.1/398
492.2/209
333.2/877
337.1/374
337.1/466
164/398
310.1/221
195.1/741
627.3/678
189.2/859
615.4/899
394.3/638
914.7/841
935.7/852
908.8/859
579.3/723
207.1/683
321.2/724

320.3/554
445.2/828
455.4/781
451.4/883
455.4/671
457.1/242
457.1/196
247.2/887
358/382
429.2/841
272/258
699.3/350
501.3/852
230.3/481
506.2/434
261.2/841
417.2/798
263.2/825
649.4/741
643.2/413
271.1/325
265.2/178
673.4/741
520.1/325
522.4/691
374.2/781
660.3/904
375.1/398
376.3/808
541.2/764

548.2/244
377.1/671
341.1/381
544.3/705
306.2/671
481.2/895
573.2/308
478.2/211
481.3/776
757.2/348
868.8/860
573.2/348
311.1/244
496.4/671
465.4/909
338.2/670
127/205
325.1/347
567.2/244
496.1/569
337.1/424
337.3/825
163/171
304.3/866
351.2/417
403.2/826
625.3/636
353.1/308
627.2/316
191/258

611.2/401
615.4/873
581.3/387
581.8/387
937.7/841
215.2/860
887.8/859
588.1/244
322.1/400
323.1/397
447.2/350
359.2/292
245.2/841
257.2/853
357.3/886
699.8/350
412.4/883
231.2/860
273.1/197
285.1/298
313.3/766
313.1/209
949.5/526
269.1/435
271.1/310
671.4/817
177.2/742
182.6/684
178.1/221
329.2/750

525.4/841
326.2/191
370.4/734
277.7/852
438.3/742
437.2/350
383.2/741
382.7/781
542.2/337
549.2/453
555.2/367
541.2/466
378.3/862
381.3/886
375.1/670
376.3/886
377.1/683
535.1/291
563.3/558
339.3/621
304.7/841
306.3/938
474.3/558
877.7/841
564.2/238
465.4/859
337.3/919
309.1/558
337.3/723
771.2/381

325.1/380
337.1/221
481.3/698
481.1/460
337.1/448
161/682
309.2/825
200/258
351.2/455
195.1/781
189.1/826
394.4/884
617.4/558
189.1/374
622.3/810
623.1/261
190.5/683
395.3/675
353.1/552
943.5/387
581.2/238
207.2/859
899.8/859
582.3/810
206.1/510
206.1/670
588.4/696
207.6/566
318/382
453.3/698

444.4/859
462.2/683
456.1/205
462.3/698
247.2/671
247.1/193
255.2/841
257.2/859
255.1/258
275.2/417
411.4/841
407.2/826
225.1/193
733.7/558
233.2/841
407.1/486
498.2/449
506.7/434
272.1/325
427.2/815
411.4/903
223.1/554
502.3/796
502.3/698
272.1/263
596.2/435
423.1/683
658.3/260
638.3/904
641.2/349

441.3/860
439.4/449
287.2/840
285.3/938
287.1/178
289.7/705
292/382
301.2/936
974.5/434
971.5/456
972.5/390
265.3/873
266.3/886
188.1/176
673.4/781
180.2/841
179.5/683
328.1/325
330.1/413
521.1/263
328.2/278
354.3/865
354.1/398
369.4/686
369.3/752
363.1/683
437.3/896
282.1/238
665.2/233
343.3/669

381.3/939
344.2/262
557.2/394
549.2/421
542.3/761
671.2/318
549.7/453
541.2/361
340.3/887
349.1/683
379.3/825
175/454
171/683
305.2/935
772.5/756
572.8/387
576.2/352
515.4/841
485.2/366
483.4/738
512.2/325
564.3/396
786.6/936
815.5/755
483.2/308
339.3/779
782.6/919
817.2/336
307.1/193
463.4/916

573.2/274
493.1/193
309.3/797
311.1/220
493.3/817
325.1/318
811.5/435
311.1/278
801.2/404
578.2/233
337.1/333
479.2/189
574.2/435
478.3/649
337.1/530
163/486
336.2/825
162/683
336.1/683
337.1/602
123/684
337.1/551
352.2/826
203.1/477
615.3/400
351.3/867
405.2/814
192.1/398
617.3/663
191.2/909

395.3/887
395.4/840
397.4/859
627.2/379
391.1/431
617.3/766
620.3/723
197.1/825
203.1/452
393.1/487
393.1/670
611.2/385
205.2/852
206.1/245
897.7/841
578.3/261
878.7/841
204.2/860
205.1/398
203.2/904
203.2/884
579.2/181
584.1/244
321.2/704
320.1/244
321.1/394
320/382
323.2/826
458.4/841
445.2/690

445.2/734
456.4/671
456.2/344
445.1/244
447.3/886
247.2/401
247.2/852
254.1/229
258.1/209
256.1/225
261/280
253.1/683
360/382
245.2/825
243.1/734
430.2/826
498.4/840
419.1/827
497.4/861
593.3/406
273.1/404
505.1/290
407.4/910
421.3/841
502.3/852
423.4/916
733.4/558
601.2/189
507.1/229
499.2/447

703.4/692
727.4/650
411.1/398
261.2/638
497.4/893
749.2/381
261.2/772
272.1/244
739/558
423.3/825
650.4/742
653.3/340
643.3/810
641.2/389
642.4/676
633.4/741
439.4/742
444.2/212
443.1/224
439.4/466
443.2/815
288.3/514
286.7/840
289.2/841
283.2/859
283.1/237
302.1/466
302.6/683
957.5/452
973.5/358

264.1/278
271.1/470
266/244
188/684
281.1/456
280.7/705
185.1/444
177.2/859
183.5/220
675.3/766
182/258
328.1/352
526.1/290
522.1/290
525.2/381
330.6/683
520.3/654
357.1/287
357.3/692
356.2/413
353.3/772
355.1/403
365.7/741
367.3/912
369.2/918
369.1/683
364.3/611
366.1/221
363.1/522
279.2/762

437.2/587
437.2/615
434.1/216
431.2/787
347.1/258
344.5/258
350.3/867
541.1/325
546.3/558
554.3/558
343.2/724
343.2/936
554.8/558
348.1/212
666.2/233
546.3/575
173.1/919
377.1/437
343.2/877
551.4/575
166.1/209
168.1/826
543.1/278
373.2/637
341.3/887
993.5/390
995.5/434
532.2/575
542.1/325
541.7/467

542.2/287
344.3/713
555.1/298
170.5/683
557.1/291
341.1/306
529.1/244
534.1/298
542.1/263
349.2/772
346.2/278
663.3/747
542.2/764
378.1/683
345.1/683
537.2/435
539.2/448
545.3/705
669.3/724
539.1/484
341.1/365
340.2/683
771.5/756
563.8/558
564.3/558
573.3/387
137.1/841
517.2/598
477.3/896
493.3/701

496.3/878
305.3/877
757.7/387
307.7/841
473.3/791
478.2/193
485.2/574
485.2/396
489.2/302
514.7/390
483.4/859
496.4/778
758.2/348
335.2/920
337.2/885
561.4/789
785.6/918
510.2/210
564.8/396
787.2/362
804.2/291
811.5/453
514.2/456
338.1/466
765.2/330
510.1/244
466.4/909
475.2/698
491.2/274
511.3/642

337.3/699
339.1/554
495.4/920
479.2/799
763.2/329
334.1/368
304/258
306.3/710
337.1/584
397.2/757
352.2/416
200.5/258
296.2/841
198.2/204
192.2/853
621.3/779
621.4/935
198.1/886
353.1/258
192.5/221
617.3/642
199.6/683
352.1/683
615.3/919
395.4/873
198.1/683
626.2/466
627.1/570
391.2/606
391.3/665

393.2/674
393.1/445
351.1/695
215.2/841
582.3/387
915.7/841
923.5/461
218.2/859
207.1/887
903.4/564
217.2/909
589.2/221
589.3/332
204.1/510
207.1/431
579.3/733
590.2/408
590.2/399
584.6/244
215.1/510
217.1/520
219.2/859
581.2/229
589.1/244
586.2/466
217.1/183
215.1/566
589.2/587
589.2/558
591.2/584

322.2/724
322.3/802
319.2/825
317.1/683
321.1/671
323.1/347
318.1/171
456.4/742
452.4/883
459.2/826
461.4/782
455.4/434
446.2/827
447.2/683
461.1/214
457.1/302
451.2/274
457.2/778
455.1/216
249.2/852
256.1/290
357.3/762
248.1/178
246.1/292
257.3/843
250.1/448
247.1/176
254.1/445
257.1/245
360.1/670

257.1/265
358.1/683
359.1/586
360/281
429.3/792
427.4/865
427.3/857
431.1/221
243.2/841
598.2/352
232.1/229
233.2/671
407.4/396
409.4/671
712.5/918
277.2/475
412.4/841
241.2/840
503.4/841
505.9/841
600.4/841
413.3/582
233.2/859
506.4/840
417.1/224
593.8/406
595.4/935
273.1/242
749.2/322
509.1/445

507.1/494
507.2/359
425.3/672
226.6/510
229.2/859
223.1/212
426.4/909
595.3/919
701.4/643
735.6/887
275.2/860
603.1/244
509.1/647
411.1/466
507.1/445
406.2/683
425.3/785
263/258
499.1/244
595.2/655
239.1/213
699.4/605
424.1/684
273/258
409.1/541
734/558
412.4/936
237/189
715.3/683
641.4/635

638.3/461
649.4/781
635.3/461
653.8/340
655.2/379
656.3/340
659.2/330
657.2/316
641.3/723
647.3/725
657.2/721
441.4/853
442.4/840
443.4/909
443.1/282
443.1/297
283.3/935
287.1/395
289.1/265
283.3/886
285.1/255
285.1/473
285.1/683
314.2/249
313.1/289
313.2/790
311.6/683
299.2/860
302.6/244
950.5/526

944.5/386
957.5/359
973.5/390
271.2/859
263.5/258
269.7/852
271/258
264.2/825
269.1/887
271.1/356
265.1/684
269.1/413
187.1/671
280.1/186
281.1/181
279.2/842
279.7/859
281/258
683.4/759
684.4/759
177.2/936
687.4/741
179.1/522
683.5/865
679.2/272
674.4/742
679.2/305
687.2/233
695.2/294
685.2/356

691.2/400
675.2/719
677.2/309
677.4/669
679.2/683
673.3/683
184.1/691
677.5/825
184/258
187/258
330.2/257
522.6/289
524.1/482
329.2/278
329.2/209
523.1/525
326.1/435
524.4/705
521.3/655
355.3/865
354.1/220
357.1/526
357.1/361
357.3/920
367.2/417
371.2/443
361.1/257
365.3/842
368.3/886
361.1/315

367.1/599
372.2/558
364.1/554
367.1/535
369.1/427
279.1/176
438.2/587
431.2/692
433.1/325
435.1/292
437.2/461
433.1/283
436.7/682
283.1/554
547.5/935
548.5/936
549.2/558
349.2/510
546.8/558
551.3/511
546.8/460
557.3/558
558.3/559
557.8/558
526.4/841
987.5/508
377.1/587
382.3/348
390.3/836
344.1/318

345.1/379
667.2/233
549.7/421
527.1/290
663.4/390
543.1/264
544.3/635
546.8/575
556.3/766
555.8/558
340.3/810
341.1/527
529.1/494
979.5/449
999.5/509
376.8/886
378.1/670
379.1/510
383.2/384
383.1/684
344.2/280
349.1/534
349.2/825
549.1/333
553.2/408
663.2/294
536.1/291
540.3/706
540.2/448
389.2/855

349.1/555
543.1/327
375.1/588
527.1/303
340.1/554
531.4/824
377.1/224
378.1/631
377.1/318
380.2/683
555.2/342
340.3/873
537.1/452
539.3/706
374.8/825
998/526
379.1/683
349.1/709
173/279
177/454
533/919
374.3/825
669.3/785
377.1/631
527.2/683
347.1/175
469.3/858
309.1/526
467.4/693
306.7/671

575.3/386
563.2/243
103.1/292
123.1/841
463.2/936
786.6/919
573.8/387
466.3/526
467.1/526
474.8/508
161.1/522
337.1/258
759.6/919
767.5/936
517.3/527
564.3/386
307.1/229
575.8/387
135.1/936
484.4/738
487.2/318
492.2/229
517.4/790
337.6/258
331.3/866
335.3/642
562.7/558
753.5/918
757.4/387
829.2/291

121.1/878
564.8/559
569.4/575
571.2/599
790.2/290
789.2/291
788.2/291
805.2/290
575.1/325
576.3/387
577.2/352
135.1/877
466.3/574
468.2/510
473.4/698
149.1/877
151.5/292
161.5/264
484.4/819
491.2/324
491.3/766
494.4/840
514.7/456
515.2/358
337.3/666
482.1/461
497.1/568
766.2/330
767.4/904
772.2/381

335.3/663
304.3/590
306.1/435
309.1/395
827.4/399
125.5/265
511.1/244
510.6/244
568.2/244
575.2/335
479.3/649
161.1/825
493.2/225
337.1/718
760.6/935
777.2/424
496.4/814
511.3/656
566.4/696
569.1/495
335.1/409
137.1/826
469.1/254
473.1/683
473.2/799
477.3/856
478.3/829
336.7/825
518.3/614
338.3/825

337.3/636
325.1/487
477.3/829
563.2/621
307.2/842
575.2/497
495.4/814
305/289
482.3/758
135/258
151/224
337.1/606
336/382
156/220
150/733
163/467
332.2/238
325.1/409
337.1/574
305.3/866
631.4/793
353.1/424
195.1/936
295.2/751
615.8/401
199.1/566
352.1/233
189.2/884
191.5/258
195.2/671

350.8/865
351.1/292
294.1/244
294.1/291
296.7/841
391.2/864
394.1/511
400.3/607
401.2/221
404.2/826
297.3/478
618.3/400
622.3/779
191.1/189
353.1/658
188.5/683
611.3/766
615.2/616
615.2/419
294.1/225
295.1/683
391.3/671
392.2/606
393.1/380
393.1/631
397.3/841
403.2/732
192.1/176
616.4/873
619.3/700

626.2/436
200.2/635
296.1/278
295.1/552
392.3/652
395.4/936
397.1/682
619.2/716
621.2/602
198.5/566
352.1/566
391.2/787
199/258
351.1/366
617.2/546
190/292
191/279
202/258
394/382
392/381
199/221
393.1/683
203.2/741
923.5/558
927.5/527
935.7/936
580.3/379
924.5/558
925.5/387
928.5/527

931.5/526
936.7/936
207.2/903
207.6/683
907.5/558
904.4/564
942.5/498
221.1/183
579.2/190
579.3/345
926.5/387
591.1/325
204.1/670
205.5/382
206.1/825
207/258
206.6/510
208.1/566
919.4/510
919.4/564
923.5/581
924.5/581
579.2/220
219.1/286
580.8/405
587.1/539
585.2/243
589.4/696
591.2/688
579.2/522

588.2/387
590.2/587
591.2/520
215.1/356
592.2/612
205.1/683
321.2/802
323.1/526
317.2/877
318.1/258
320.6/244
322.1/683
318.2/826
320.2/867
321.1/684
453.2/587
457.4/635
460.2/563
459.2/770
460.7/563
461.1/225
461.2/566
445.1/254
449.3/936
451.4/903
453.2/277
458.1/243
457.4/610
454.3/698
455.1/245

455.4/762
447.1/578
460.3/649
453.3/785
457.4/650
455.4/856
456.1/216
257.1/325
260.2/841
360.2/692
247.7/671
251.1/176
255.5/258
257.1/290
257.2/865
358.3/886
359.2/789
250.2/917
253.1/523
256.1/435
257.1/516
248.1/189
246.2/824
257.1/303
358.6/683
248.2/867
250.2/931
251.1/198
249/258
253/279

603.3/707
728.5/758
497.4/840
239.2/865
409.4/741
275.2/454
601.4/526
507.7/449
419.8/755
508.4/919
507.1/366
407.4/575
407.4/409
423.4/387
409.3/782
731.2/316
734.4/558
275.2/558
738.7/558
243.2/884
244.1/734
235.2/909
235.2/852
240.2/502
503.9/841
508.2/389
509.4/841
428.2/815
410.4/671
233.1/363

504.4/842
420.3/895
425.4/396
411.4/636
262.1/292
593.2/225
405.2/468
417.1/279
417.1/290
418.1/224
423.4/351
594.2/353
594.3/407
701.5/825
704.4/692
713.3/833
272.1/408
596.3/406
272.3/572
734.7/558
739.3/558
276.3/479
277.1/244
602.4/527
603.1/291
605.4/787
745.5/575
751.5/921
429.1/686
235.2/859

508.1/494
427.4/778
407.1/511
407.1/498
411.1/684
413.4/909
413.4/883
415.2/692
506.1/290
500.3/649
411.4/807
422.2/312
423.2/571
427.3/883
226.6/659
499.1/435
593.1/263
695.4/878
695.2/748
697.2/765
696.2/294
417.1/266
418.1/244
424.1/297
272.5/244
727.4/669
728.4/650
729.3/684
736.6/887
277.2/826

277.1/494
602.2/190
609.2/250
609.2/419
750.2/381
602.4/574
413.1/683
239.1/303
412.2/705
409.1/321
227.1/683
262.2/761
501.2/358
501.2/412
417.1/244
272.1/309
597.4/826
607.2/626
609.2/387
605.2/622
409.1/390
425.1/577
734.3/683
745.2/566
408.3/917
221.2/931
263.1/409
716.3/683
603.2/403
699.3/683

733.3/683
605.2/570
240/292
228/190
261/308
262/382
272/382
274/382
409.1/684
407.2/683
638.8/461
642.4/636
635.8/461
654.3/340
636.3/461
637.3/809
656.8/340
645.3/348
633.5/936
647.3/852
659.2/766
645.3/750
644.2/412
643.4/676
635.2/683
635.3/723
641.2/466
644.4/733
657.1/220
643.2/785

643.2/684
645.3/771
439.4/563
439.4/671
441.8/754
440.4/449
441.4/917
443.2/731
443.2/750
439.4/651
441.2/843
441.1/265
283.3/669
288.1/264
289.3/514
287.1/353
291.5/445
313.3/865
315.1/435
311.6/244
313.3/736
299.1/399
298.3/936
301.1/212
300.2/707
299.1/362
299.1/316
301.1/307
299.1/683
299.1/402

301.2/860
302.6/466
303.1/244
303.2/826
303.1/683
302/292
954.5/508
951.5/526
947.5/564
946.5/527
947.5/526
959.5/442
972.5/456
945.5/581
947/526
264/258
264.1/292
271.1/408
265.1/292
265.5/258
268.2/401
263.2/705
266.2/852
269.1/319
269.1/772
270.1/435
269.1/825
265.3/867
187.2/884
188/587

280.5/258
281.2/683
281.1/244
281.2/705
281.2/825
281.1/416
281.2/713
280.3/803
279.7/655
280/258
281.1/437
279.2/826
685.4/759
181.2/936
177.2/671
680.2/272
681.2/253
177.5/350
685.2/785
178.1/178
179.2/878
679.3/650
685.5/877
688.5/935
694.2/313
693.3/683
184.2/252
673.2/399
186.1/201
674.3/683

684.2/685
681.2/587
185.1/289
672.4/825
678.2/566
183.5/541
683.2/684
183/258
677.2/566
180/683
328.2/935
525.2/581
329.2/936
331.2/919
525.1/526
327.3/827
519.1/291
521.1/325
523.4/905
329.1/326
330.1/554
331.1/554
519.3/614
326.1/257
523.1/626
331.1/243
331.1/684
326.1/409
523.1/482
523.2/717

328.1/566
521.1/463
521.1/438
523.1/598
523.1/439
355.2/559
353.4/621
354.3/772
355.3/644
353.2/936
355.1/437
355.1/587
354.1/552
355.6/566
355.1/605
355.1/372
357.1/534
357/350
365.1/566
365.2/461
369.2/865
369.3/693
363.1/504
365.7/461
367.1/566
361.1/671
362.3/931
366.2/554
369.1/464
369.1/281

369.3/894
367.2/842
367.2/724
363.1/586
366.3/825
367.1/587
371.1/709
362.1/422
369.2/826
367.2/435
278.1/292
278.2/638
278.1/448
279.1/733
279.1/387
278.2/851
434.2/856
438.3/896
433.1/432
433.6/292
437.3/536
439.1/266
433.1/414
433.2/671
437.3/521
438.1/554
438.3/830
437.3/829
437.7/682
282.5/463

529.5/936
346.3/351
349.2/445
375.2/443
530.5/936
541.1/526
343.2/802
350.3/783
997.5/527
549.1/292
549.8/558
348.1/258
545.1/463
546.3/461
547.3/558
526.8/547
982.5/508
555.3/558
533.7/516
384.2/877
385.3/792
388.2/443
342.3/935
345.1/258
548.4/919
550.1/292
552.1/292
340.2/565
349.3/827
527.2/419

169.5/292
669.2/304
558.8/559
559.1/325
340.3/780
528.2/559
530.2/239
532.1/350
984.5/572
983.5/572
983.5/508
988.6/537
993.6/918
176.5/244
533.7/564
537.3/766
538.4/942
541.1/290
541.1/565
377.1/466
380.1/566
381.1/378
381.1/733
382.3/886
383.1/646
384.1/221
389.1/692
389.1/492
545.2/218
549.2/244

552.4/575
553.2/385
554.4/886
554.9/887
375.1/583
375.3/837
375.3/824
376.2/825
342.1/613
341.3/939
345.6/683
346.2/242
347.1/772
350.1/683
350.1/709
165.5/350
174.5/350
665.2/289
547.1/315
559.3/877
530.1/466
531.3/793
176.1/686
535.1/216
537.1/281
541.1/462
667.2/821
385.1/566
348.1/243
543.2/683

543.4/829
346.1/225
350.1/199
171.6/683
663.3/716
664.2/588
670.3/724
555.2/435
529.3/856
530.2/555
531.1/412
531.1/522
532.1/390
671.3/785
377.1/606
379.1/554
346.6/566
557.5/826
661.3/743
663.2/588
532.2/557
667.2/690
667.2/683
670.3/785
537.3/661
164/683
541/258
667.2/717
348/221
172/221

171/362
661.2/610
378.1/577
169/566
527.7/683
347.2/826
376.1/683
492.3/526
492.8/526
485.2/526
339.1/526
483.4/819
485.4/861
337.2/596
482.2/497
306.3/935
493.3/526
483.4/782
495.4/896
758.6/936
752.5/918
470.3/858
474.8/558
153.1/936
164/258
310.1/352
517.7/599
517.8/581
338.1/291
338.3/581

486.2/526
495.3/537
759.5/873
761.5/558
511.7/571
337.2/587
563.3/810
496.4/896
763.5/508
779.5/544
509.8/547
332.2/501
803.5/550
466.8/526
467.4/870
468.4/693
139.5/258
471.4/701
311.1/467
491.2/387
494.3/786
494.3/700
513.4/671
517.8/527
338.2/587
495.3/643
497.2/390
762.5/558
766.5/818
766.5/526

767.5/508
335.3/772
304.1/264
563.5/877
462.3/535
481.1/527
481.3/249
752.2/261
755.2/333
828.2/291
105.1/264
109.1/919
121.1/936
119.1/878
509.8/599
324.2/881
332.3/519
308.1/292
567.1/291
567.4/558
797.5/387
799.5/523
804.5/550
812.2/292
817.4/558
820.1/291
819.5/387
133.1/878
334.1/220
334.3/459

571.3/559
575.3/671
575.8/592
577.3/461
468.4/869
473.2/649
473.4/537
476.3/852
477.2/684
478.3/896
145.1/559
472.4/701
471.3/786
147.1/877
150.1/826
151.1/841
160.5/292
483.3/758
515.3/886
517.2/279
517.4/936
338.2/877
338.2/885
497.2/427
759.5/938
335.2/552
304.3/522
560.1/932
561.3/723
463.2/615

756.4/330
763.2/374
786.2/485
855.2/244
481.1/290
109.1/878
117.1/919
117.1/686
509.9/840
511.1/446
510.4/848
324.3/766
566.5/921
567.4/916
571.2/408
463.3/824
795.3/566
802.6/828
333.1/613
333.2/639
333.2/685
573.1/290
574.2/419
575.1/290
575.2/660
464.2/937
478.3/856
466.8/574
467.4/839
468.7/510

470.2/502
473.4/778
474.2/750
475.2/826
469.3/772
339.2/472
336.1/535
492.2/675
513.2/685
515.3/738
563.2/567
764.2/329
123.1/826
324.1/622
335.2/826
561.2/504
763.2/485
333.1/744
306.3/867
569.1/518
333.2/238
335.1/725
335.1/612
137.1/566
467.2/675
469.2/553
495.2/564
338.1/584
339.2/485
481.4/798

303.2/803
515.2/342
337.6/566
91.1/258
324.2/826
333.1/221
473.2/785
339.1/358
338.1/659
758/387
763/386
101/259
510.4/723
308/258
145/221
119/200
338.1/551
617.4/761
194.1/444
352.3/936
352.2/455
191.2/936
395.3/749
618.4/558
624.4/757
199.2/877
352.1/724
394.3/721
189.1/176
189.1/592

189.1/491
191.1/814
192.1/734
196.1/772
201.2/728
202.2/402
612.2/376
614.4/855
351.1/613
295.1/436
295.1/176
298.2/840
393.1/258
392.3/671
393.4/903
395.3/698
396.3/698
396.3/887
401.1/643
401.3/859
405.1/691
405.2/548
617.4/581
617.4/593
620.5/773
625.4/936
197.2/877
391.1/455
395.1/599
193.1/789

194.1/566
201.2/884
203.1/710
611.1/557
611.1/587
612.2/435
613.2/520
613.4/912
351.1/717
351.1/749
295.2/686
298.1/597
392.1/431
392.3/823
396.1/510
396.2/566
396.4/873
617.4/826
624.4/826
627.2/466
625.4/757
195.1/253
352.1/750
395.1/510
395.1/399
193.5/216
611.2/435
612.2/384
297.1/826
392.1/566

392.1/684
393.2/857
393.2/825
623.4/826
196.5/566
197.1/566
394.1/445
405.1/566
401.1/684
194.1/866
393.1/581
395.1/566
193/258
395.1/683
396.2/442
201/289
394.1/683
396.2/450
190/380
203.1/683
190/200
196/631
196/534
296.1/171
297/605
926.5/508
217.1/936
213.2/936
208.5/258
205.2/936

941.5/498
207.2/877
207.5/258
215.2/884
217.1/877
579.2/233
580.3/733
925.5/558
219.2/877
580.2/190
581.1/292
583.2/580
582.8/387
583.4/551
584.3/387
584.5/935
585.4/558
587.1/291
586.4/559
209.1/554
211.2/877
213.1/409
207.1/422
208/258
216.6/566
897.5/510
220.1/587
221.1/707
579.1/297
579.2/706

580.3/405
218.6/587
218.6/659
218.5/541
581.4/919
582.1/291
589.2/441
590.2/821
590.2/419
593.1/325
583.3/387
585.3/797
209.2/826
210.6/587
204.1/683
587.2/609
581.2/528
588.2/360
592.4/880
215.1/660
206.6/566
215.1/279
591.2/663
592.2/744
592.2/785
592.2/520
219.6/541
587.2/360
591.2/492
590.2/558

210/258
591.2/785
211/258
208/201
591.2/612
321.2/842
320.2/751
317.3/581
321.2/877
321.2/936
322.2/704
317.2/264
317.3/570
320.2/877
321.1/292
318.1/734
320.3/826
323.2/860
323.1/654
319.1/541
317.2/826
318.1/566
323.1/485
318/292
319/262
462.3/461
454.2/587
455.2/856
459.1/290
459.3/642

459.4/715
451.2/650
452.4/903
456.4/896
459.1/490
461.2/650
449.2/788
445.2/658
447.1/255
455.4/920
459.4/691
462.2/614
445.1/504
452.2/368
247.2/935
248.2/935
256.3/843
245.2/724
247.2/860
245.2/704
259.2/671
360.3/635
248.2/734
253.1/732
254.6/446
255.1/394
255.2/883
257.1/277
260.1/292
359.1/529

359.1/510
248.2/887
246.1/514
251.2/340
259.1/224
358.2/605
254.1/683
255.1/683
358/281
251/258
247.2/866
227.1/240
426.4/526
233.1/547
408.4/526
413.3/936
503.2/527
233.1/581
423.4/558
597.3/581
737.4/897
240.1/261
410.4/741
503.3/796
225.2/671
500.7/545
711.4/836
597.8/581
747.5/461
244.3/517

245.1/567
509.1/464
429.3/895
429.4/880
430.4/880
235.2/680
240.2/866
413.3/596
413.3/894
415.2/308
413.9/919
503.3/698
505.2/559
421.3/865
422.4/919
425.4/575
407.4/599
223.1/228
225.2/877
263.1/274
263.1/308
695.5/918
699.2/281
408.4/547
423.4/852
595.2/224
699.5/936
700.4/444
703.2/392
424.4/558

272.1/283
731.5/558
273.5/258
599.2/352
739.7/558
599.4/558
601.4/575
743.4/558
746.5/575
750.2/344
429.1/504
429.1/643
413.2/787
234.6/605
237.2/826
238.2/397
427.4/904
410.1/553
411.1/615
412.4/903
413.4/826
416.2/691
415.8/682
231.2/883
409.1/510
409.2/222
410.1/566
420.2/683
426.1/577
228.2/740

223.1/683
223.2/295
225.1/733
261.2/760
263.1/436
501.2/585
699.3/792
417.1/189
417.1/562
595.4/907
700.4/605
701.1/272
715.4/897
717.1/293
596.4/826
275.1/244
598.2/573
599.2/522
743.4/537
744.4/536
747.2/414
748.2/359
604.5/936
604.2/404
413.1/587
235.6/584
239.1/269
407.1/566
410.1/684
410.1/415

500.3/710
411.3/545
425.4/817
223.2/826
714.3/566
597.2/435
732.3/683
599.2/655
411.1/442
425.1/684
427.1/566
406.1/198
409.3/931
227.6/587
713.3/566
595.4/826
700.3/683
717.3/683
717.2/586
731.3/683
275.2/826
602.2/518
606.2/570
607.2/417
509.1/587
500.2/785
409.2/526
411.1/450
226.2/432
409.1/338

601.2/470
609.2/550
407.1/540
409/258
600.2/524
429.1/566
237/290
410/258
228/244
262/279
272.1/216
245/216
499.2/750
274/291
507.3/856
499.2/785
632.5/936
648.2/304
657.4/521
657.4/570
646.2/541
643.2/558
634.5/866
634.4/826
641.2/220
641.2/644
655.5/825
658.2/721
645.3/821
644.2/684

633.3/720
633.5/919
640.3/630
646.3/744
648.3/785
643.2/750
647.3/785
658.2/683
443.4/937
439.4/545
439.4/636
440.4/563
441.1/301
441.4/558
444.4/874
441.3/824
441.2/653
441.1/684
441.1/245
444.1/281
440.2/683
444/258
284.1/566
284.2/461
284.2/559
284.7/558
287.1/526
291.5/463
290.3/515
291.2/710

287.1/290
292.1/522
290.2/704
290.3/501
286.1/361
292.1/200
291/445
293/309
314.3/865
313.1/609
316.2/840
311.3/514
312.1/186
312.1/566
314.1/587
315.1/238
313/279
300.2/860
299.1/522
302.2/826
300.2/749
299.1/416
300.1/547
300.1/631
301.2/826
303.2/733
948.5/508
948.5/564
964.5/508
971.5/602

944.5/544
970.5/508
973.5/508
972.5/602
967.5/537
955/508
956/508
265.3/936
266.3/935
269.2/842
269.3/559
265.1/237
267.3/936
269.1/337
271.5/258
270.1/886
263.6/409
266.2/432
270.1/381
270.1/413
270.1/683
266.3/867
268.1/566
269.1/409
269/258
187.1/826
187.5/566
279.2/752
279.2/877
281.1/683

280.2/826
183.1/749
672.4/817
683.5/920
185.1/703
185.2/936
181.2/877
678.5/935
177.5/244
687.5/921
694.5/849
685.2/668
183.5/554
177.5/200
671.4/825
186/258
691.3/683
526.2/547
326.3/827
329.2/536
526.3/490
525.7/581
329.2/554
327.3/602
519.4/918
520.2/537
325.3/590
327.2/895
325.2/768
521.2/868

521.2/888
523.1/507
327.1/605
330.3/890
525.2/366
327.6/566
353.2/519
355.2/683
355.2/631
356.1/318
357.1/254
354.1/605
369.2/703
367.1/221
373.1/724
367.1/689
373.1/412
361.1/521
363.1/622
371.1/435
363.1/483
369.1/520
278.2/703
278.2/444
279/258
437.2/236
432.2/626
439.1/566
437.2/682
281.5/258

283/258
282/494
283.1/446
282.3/867
176.1/444
661.5/759
559.1/526
381.3/618
373.2/936
344/258
349.3/887
526.3/527
975.5/508
981.5/508
173.1/683
533.2/516
345.2/936
549.5/935
553.1/547
167.1/936
341.3/553
987.5/537
997.5/508
531.1/350
535.8/520
537.3/490
381.3/897
383.3/755
386.1/333
545.2/271

377.1/408
169.1/211
547.1/292
557.5/919
560.1/526
559.3/852
341.2/826
341.2/782
993.5/560
661.2/328
532.5/919
534.2/490
541.1/396
553.1/599
544.5/877
559.1/289
559.1/452
559.1/932
374.1/566
529.1/467
531.1/477
671.3/685
538.2/280
666.4/764
545.1/246
173.1/826
374.1/522
175.5/554
541.1/442
377.3/878

551.1/598
168.5/200
668.2/683
341.3/758
659.2/719
663.3/668
379.1/402
526.4/767
528.2/683
375.1/532
165/258
378.1/534
172/291
542.3/655
172/682
346.1/566
670.3/750
537.1/572
669.2/541
551.7/566
172/200
340.1/422
164/171
379.1/566
661.3/655
377.1/577
491.7/508
474.3/508
490.7/545
332.2/250

483.3/544
305.1/527
820.5/522
131.5/258
795.5/544
149.1/936
163.1/936
310.3/514
336.1/258
336.6/258
482.7/497
765.5/526
768.5/936
495.4/937
517.2/547
561.3/937
858.7/919
857.7/919
109.1/936
303.3/590
566.1/292
784.6/935
799.5/508
334.1/292
466.3/874
137.5/258
140.5/258
484.4/782
515.1/279
337.1/408

309.2/794
482.4/775
495.3/825
519.1/244
563.1/442
481.3/643
763.4/877
103.1/919
566.4/842
567.3/395
783.5/508
788.4/903
807.7/919
820.2/358
132.5/258
307.3/938
571.3/355
574.3/387
575.3/592
577.1/290
578.1/290
468.4/904
139.1/936
311.1/330
485.1/841
493.3/786
338.1/605
338.2/789
338.3/779
511.4/820

335.2/695
560.2/201
117.1/720
325.1/573
334.2/566
574.4/689
465.3/832
468.3/802
147.5/258
163.1/877
310.2/825
337.1/500
486.2/389
487.2/408
517.1/435
309.2/772
497.2/621
785.2/485
128/258
510.2/409
510.1/522
149.1/684
339.1/676
109.1/826
575.2/601
578.2/655
473.1/223
473.2/750
495.2/541
577.2/522

469.2/503
763.2/566
576.2/574
477.3/675
146/258
162/258
123/279
105/280
134/258
156/350
123/521
565.2/587
575.2/548
149/513
311.1/366
147/383
147/683
578.2/597
338.2/683
163/566
476.3/675
575.2/573
351.3/936
295.2/443
194.1/477
195.2/936
351.1/724
627.1/547
615.4/544
615.4/497

621.4/877
198.2/306
193.2/877
614.4/835
294.7/671
294.2/919
295.2/602
391.2/901
396.3/749
628.4/877
203.1/605
189.1/720
201.2/919
613.4/835
294.2/720
294.2/868
393.1/595
395.1/554
621.2/630
623.3/734
629.3/394
189.1/415
199.2/789
352.1/694
352.1/785
353.1/633
191.5/587
192.1/554
614.4/880
294.2/889

297.1/733
632.2/614
189.1/622
401.3/939
297.6/554
621.2/750
623.2/459
353.1/343
353.1/505
189.1/550
201/258
394.1/566
631.2/614
353.1/566
297.1/658
394.1/469
201/418
617.2/569
629.2/599
198/587
353.1/469
391.1/683
393.1/566
209.2/936
211.1/308
929.5/506
927.5/508
215.2/919
930.7/877
929.7/877

219.2/919
580.5/878
591.1/290
206.1/691
215.1/547
215.6/789
215.3/508
217.1/826
219.2/662
590.4/860
211.1/387
216.6/587
219.6/587
589.2/821
579.2/571
587.2/587
591.4/880
206.1/683
219/418
219/258
581/877
215.1/533
587.2/387
319.2/936
317.2/919
322.1/264
318.1/626
321.1/668
322.3/877
322.1/416

323.1/379
320/292
321.1/495
323.1/622
323.1/534
455.4/698
457.4/497
461.2/877
453.2/816
457.4/893
455.1/283
455.4/537
455.2/462
455.4/830
445.1/566
460.1/213
449.2/675
248.1/239
248.2/852
247.1/763
249.1/579
257.1/411
259.1/212
260.1/264
360.1/573
247.1/533
360.1/659
257.2/762
251.1/566
253.1/566

247/258
407.4/526
501.8/508
427.3/444
407.4/547
502.2/508
601.5/919
427.7/523
504.3/537
699.4/444
743.4/903
243.2/874
509.3/598
428.4/861
504.8/537
504.8/560
507.2/509
507.7/426
229.2/828
498.2/545
426.4/547
408.2/281
419.2/509
227.2/936
701.5/936
424.4/508
734.2/333
276.1/292
600.4/508
606.5/508

751.4/868
244.3/503
237.2/919
239.2/764
427.4/527
415.2/865
503.2/575
505.3/537
422.4/841
411.2/223
227.1/302
229.2/883
261.2/515
722.2/281
721.2/281
273.2/828
600.4/877
601.4/547
609.3/898
751.2/261
237.1/882
237.1/858
242.2/308
508.1/445
428.3/419
231.1/925
507.1/566
507.3/821
500.2/387
406.8/682

421.3/884
422.9/919
427.1/344
227.1/308
229.1/221
224.1/485
225.1/545
408.4/574
417.1/432
425.1/510
701.5/919
705.5/877
597.2/628
276.2/826
601.2/420
602.2/626
610.2/440
747.2/359
750.5/922
602.5/877
243.2/826
429.1/522
430.1/566
234.1/803
407.2/385
229.1/316
499.2/334
699.2/472
701.5/877
600.2/597

601.3/734
607.2/430
431.1/587
504.3/758
407.2/605
261.2/826
502.2/338
243.1/356
410.1/366
730.3/566
594.2/509
595.2/546
749.3/566
509.2/409
223/258
225/258
409.1/566
274/258
732.3/566
274/350
598.3/363
748.3/566
223/605
501.2/338
593.2/509
731.3/566
408.2/683
229.1/683
593.2/571
224.1/534

641.4/760
641.4/728
634.4/544
647.5/919
641.4/921
652.4/838
654.4/733
643.3/779
647.2/466
645.2/419
647.3/683
646.3/683
645.3/644
645.3/683
657.2/683
440.3/877
442.4/508
440.1/566
441.1/587
291.2/502
289.3/501
287.2/874
283.3/575
283.3/553
291.1/619
288.1/292
291.2/667
292.1/733
287.1/248
289.2/658

289.2/826
285.1/522
291.1/598
285.1/294
291/463
283.3/866
315.2/841
315.2/919
313.1/683
313.1/587
314.3/675
314.1/238
301.2/765
300.1/534
299.2/707
301.1/484
299.1/566
302.2/803
299.1/631
962.5/508
963.5/508
947.5/508
955.5/508
959.5/425
968.5/508
957.5/426
965.5/544
957.5/508
964/508
269.1/176

269.2/680
266.3/590
267.2/293
269.1/215
267.1/339
267.1/263
271.2/482
269.1/510
269.1/684
267.1/461
267.7/681
267/258
279.5/258
280.2/919
694.5/918
677.5/936
686.5/937
685.5/937
185.5/245
178/566
180/541
327.2/935
331.2/460
521.4/919
327.1/627
326.1/616
523.2/338
521.1/607
519.3/681
353.3/877

356.1/484
356.1/566
356.3/920
362.3/585
363.1/415
367.1/520
369.1/764
362.1/554
371.1/380
373.1/522
436.3/674
282/463
381.3/571
387.2/443
543.8/490
387.2/477
176.1/477
342.2/683
543.1/526
544.3/490
991.5/560
530.1/463
551.1/292
345.2/797
550.5/918
546.4/763
341.2/877
531.2/938
992.5/560
175.1/838

540.4/920
342.3/792
543.5/877
531.1/489
537.4/844
379.1/362
544.2/218
173.1/788
539.1/462
377.1/484
661.2/582
537.1/484
343.1/535
346.3/765
347.1/541
375.1/605
165/350
344.1/588
341.1/483
341.1/587
551.2/566
171/541
669.3/750
340.1/510
377.1/525
484.3/508
492.3/508
798.5/523
819.5/523
492.8/508

309.2/365
484.8/508
513.3/537
514.2/602
324.3/936
571.2/547
473.8/544
517.3/581
335.2/515
132.1/292
148.5/258
309.2/919
761.5/896
335.3/836
511.2/571
784.6/919
787.5/835
819.2/358
577.4/841
471.4/920
339.1/669
513.8/537
495.4/834
305.2/840
562.5/877
124.5/292
809.7/919
809.4/902
133.5/292
479.3/515

467.4/904
161.1/878
515.2/777
123.1/919
307.1/802
337.1/748
485.2/427
575.2/628
163.5/554
311.1/566
337.1/317
307.1/361
573.2/419
338.1/573
134/265
577.2/655
110/258
132/258
156/292
478.3/681
144.1/183
335.1/535
336.1/566
339.2/566
123/554
149/734
631.5/936
351.2/543
192.5/258
194.1/703

627.4/877
199.2/935
195.1/444
293.2/782
295.2/703
399.4/934
192/258
351.2/877
351.3/852
189.2/877
613.4/855
350.3/790
395.1/547
395.1/261
401.3/764
629.4/877
188.2/789
610.3/882
298.2/791
631.4/808
618.4/683
625.3/868
395.1/411
613.4/880
297.1/580
617.4/653
352.1/541
615.2/571
197/587
297.1/541

393.1/554
391.1/566
353.1/487
616.4/653
925.5/508
943.5/544
213.1/443
580.4/938
584.4/840
583.1/560
213.2/841
205.1/734
580.4/910
589.4/860
579.2/626
592.2/584
211.6/541
207.1/587
591.2/744
208.6/541
219/289
205/566
319.2/743
318.2/816
319.2/877
318.2/638
323.1/684
317.2/686
321.1/554
322.1/535

321.1/416
318/200
322.1/588
461.2/614
257.1/354
251.2/749
255.1/435
258.1/449
256.1/244
257.1/825
258/292
359.1/659
359.1/551
425.4/526
500.2/544
508.8/425
499.7/508
509.3/547
421.3/772
423.4/581
733.2/333
599.4/508
502.7/508
277.2/887
275.2/668
275.2/619
241.2/919
423.4/775
601.5/877
605.5/935

749.2/344
425.3/445
427.1/280
704.5/877
608.3/868
609.2/440
509.1/561
409.1/468
425.1/566
225.1/587
226.1/764
605.4/919
412.1/344
424.1/566
607.2/651
509.1/522
234/219
703.5/877
720.3/557
608.2/599
699.2/586
276.7/680
408.3/931
719.3/557
409.3/565
423.1/566
498.4/681
409.1/366
607.2/599
647.2/304

658.5/752
645.5/877
632.4/808
633.4/826
646.3/717
645.3/717
440.1/244
440.8/877
441.4/877
284.3/935
288.3/501
293.2/476
285.3/590
288.2/842
288.6/554
290.7/768
293/350
316.3/570
316.2/264
316.2/789
317.1/616
317.1/607
313.3/675
299.2/645
301.3/553
303.2/919
299.1/379
299.1/547
963/508
944.5/526

264.6/258
269.2/789
264.1/237
269.1/442
187.2/919
281.1/362
281.1/200
185.1/477
680.5/920
689.5/919
679.3/558
331.2/502
327.2/877
522.4/905
325.3/895
330.1/243
369.2/444
361.2/803
365.1/560
369.2/477
371.2/877
363.1/554
437.2/921
433.1/273
282.2/250
348.3/827
343.2/829
533.2/563
527.2/559
539.4/920

165.1/877
547.1/289
557.2/768
175.1/877
659.4/921
553.4/912
379.1/437
547.4/767
483.8/508
573.4/762
482.3/544
332.2/763
473.3/545
490.2/545
339.2/703
482.8/545
565.1/292
782.5/508
787.4/903
514.9/841
337.2/936
339.3/531
463.3/860
479.4/884
333.2/541
333.2/649
161.1/707
309.2/731
575.2/481
487.4/826

119/258
107/258
339.1/387
126/292
307.1/422
156/200
337.1/659
295.1/291
618.4/508
351.2/703
352.2/476
298.1/263
393.3/703
293.2/649
617.5/936
612.4/860
200.1/554
393.3/691
617.4/683
199.1/541
351.1/566
193.1/566
395.2/442
209.5/258
588.2/440
583.3/863
579.4/911
206.1/566
581.1/200
321.3/551

318.3/531
323.2/773
324.1/389
321.1/588
459.1/212
255.1/290
259.3/919
257.2/837
361.1/541
361.1/422
272.5/258
597.4/762
277.2/443
499.2/508
425.4/547
427.2/523
507.2/426
504.3/560
261.2/683
245.1/308
603.5/936
235.1/877
410.4/860
409.3/826
605.3/928
241.2/772
426.2/803
221.2/877
597.3/355
594.2/419

239.1/686
425.1/541
406.2/385
409.1/554
409.1/415
599.2/597
409.1/409
411.1/566
409.3/553
729.3/566
728.8/566
747.3/566
728.3/566
637.4/683
648.3/750
441.4/508
291.2/877
291.1/788
284.1/587
285.1/361
315.2/766
315.2/619
317.1/733
299.3/530
303.1/200
299.1/534
967.5/508
964.5/544
266.1/264
179.1/703

679.5/919
689.5/935
181/554
329.1/308
327.1/566
353.2/489
355.1/318
362.1/587
278.2/712
282.3/574
283.1/566
534.7/527
986.5/537
377.1/555
539.2/768
539.2/788
173.1/765
172.5/554
174/350
376.3/877
341.1/468
531.2/557
377.1/534
343.1/588
375.1/683
491.2/508
797.5/523
781.5/508
798.5/508
337.2/877

484.4/903
491.2/559
480.4/536
336.2/607
510.1/489
565.4/843
465.3/920
339.1/441
465.3/731
154/554
463.3/683
336/200
296.2/816
351.1/560
201.2/841
353.1/391
400.3/812
297.1/554
351.1/541
393.1/469
209.2/683
580.4/919
319.2/762
319.2/782
321.2/683
321.1/535
454.3/675
246.2/877
257.1/877
359.1/573

499.2/497
421.3/765
499.7/497
423.4/508
427.3/419
605.4/508
508.1/463
599.4/877
242.3/581
277.2/855
509.1/412
715.4/919
608.3/888
241.1/566
409.4/860
273.2/279
601.2/626
245.1/514
503.7/541
633.4/544
636.4/508
632.4/683
442.3/755
442.3/778
443.4/874
286.1/292
283.2/249
293.2/919
283.1/485
315.2/250

316.1/554
963.5/544
946.5/508
271.1/317
264.2/877
271.1/554
268.1/541
280.2/683
281.1/382
688.5/920
187/566
326.2/877
326.2/845
354.2/443
355.1/566
356.1/534
278.1/350
279.1/484
543.3/490
529.1/463
345.2/804
347.1/788
350.3/877
535.4/937
376.3/772
483.3/508
308.2/365
485.4/765
565.3/762
324.3/590

766.4/903
561.5/877
464.4/566
467.3/802
333.2/566
164/554
148/200
628.3/394
191.1/554
455.3/675
250.2/484
501.3/508
508.2/425
277.2/646
277.2/638
221.2/919
425.1/423
607.3/868
507.2/389
411.1/374
595.2/569
411.1/344
597.1/200
443.4/832
302.2/683
326.2/554
354.2/477
356.3/811
282.3/553
281.3/877

340.3/553
553.4/863
544.2/271
529.2/240
341.3/792
348.1/554
546.4/768
342.1/541
333.2/803
137/258
758.6/919
576.5/919
485.2/389
337.1/356
325.1/541
617.4/508
403.1/424
192.6/789
611.4/860
297.2/791
395.2/450
942.5/545
207.1/468
216.1/587
324.1/419
237.1/733
288.1/554
271.2/765
183.1/232
353.2/506

355.1/484
431.2/626
283.1/587
985.5/537
347.3/918
171.1/192
797.5/508
465.3/874
469.4/885
510.4/925
467.3/789
577.2/597
518.3/681
351.2/476
587.2/440
319.3/553
455.4/896
257.1/449
256.1/350
509.3/765
507.1/463
275.3/919
609.3/882
291.1/928
293.2/868
312.3/811
525.4/767
433.2/856
553.4/764
534.2/527

529.4/764
535.5/919
668.2/541
512.8/537
196/566
209/258
579.4/919
579.4/937
317.2/638
361.1/554
499.7/545
225.1/788
596.3/355
291.1/733
302.2/919
265/258
325.2/802
439.1/244
557.5/683
379.1/383
378.1/566
765.4/903
337.2/789
191.1/686
353.1/605
208.1/541
227.2/816
593.2/419
640.4/761
293.2/888

293.2/720
300.3/553
966.5/508
279.2/919
280.1/200
185/258
281.3/551
348.3/842
572.2/419
337.1/683
615.5/936
941.5/544
214.3/507
319.2/751
245.2/877
635.4/508
443.3/811
303.2/877
302.1/733
269.1/275
269.1/381
535.3/527
552.4/863
335.2/595
478.3/515
163/554
318.2/712
320.3/550
323.1/389
607.3/888

599.2/616
315.2/789
302.3/590
355.3/827
347.3/853
512.3/537
483.4/903
335.2/607
574.5/921
338.2/566
191.2/852
631.4/683
596.4/762
509.1/489
647.3/750
441.3/754
285/446
349.2/877
497.3/682
339.1/510
318.2/703
319.2/695
639.4/761
441.3/764
Oleanolic acid
341.2/683
346.1/541
325.2/845
575.5/919
499.2/544

425.2/802
284.3/590
965.5/508
339.1/422
255.1/244
265/262
187.2/789
331.2/763
331.2/829
317.2/646
361.1/587
163/258
282.1/566
340.1/587
317.2/816
311.3/827
263.2/877
460.3/531
277.2/712
667.2/541
298.3/530
500.8/508
509.4/925
325.2/825
277.1/350
338.3/531
469.4/866
945.5/508
240.2/919
552.4/763

346.2/869
534.5/919
131.1/292
399.3/812
317.3/531
441.3/778
301.2/683
524.4/767
353.2/443
528.2/240
225.1/764
315.2/669
323.1/419
267.1/541
172/554
500.3/508
302.1/200
347.1/554
295.2/816
258.3/919
355.3/797
463.3/777
320.2/683
464.3/765
325.1/554
353.2/477
420.3/765
311.3/811
270.1/554
508.3/765

279.2/683
280.3/550
571.2/419
317.2/722
301.1/733 355.1/534
377.1/566
147/200
355.3/811
325.2/877
496.3/681
318.3/553
trihexoside 595.4/762
376.3/763
324.1/541
255.1/350
301.2/919
944.5/508
347.3/842
337.1/566
345.1/541
317.2/712
317.2/703
339.1/587
551.4/764375.3/772
345.2/869
316.3/531
239.2/919
507.3/765
943.5/508
419.3/765

319.2/683
463.3/765
257.3/919

Unknown pterocrapan
281.1/566

Dihydroisoneorautenol

269.1/554

-0.2

0.0

(B)

323.1/541

-0.2

-0.4

PC1
Fig. 3 UPLC-qTOF-positive ionization MS (m/z 100–1000) principal component analyses of E. lysistemon unelicited cell culture samples
(), cell cultures treated with 1.0 mM MeJA at 0 h (o), 12 h (D) and 24 h (+) (n = 3). The metabolome clusters are located at the distinct
positions in two-dimensional space prescribed by two vectors of principal component 1 (PC1 = 93%) and principal component 2
(PC2 = 6%). (A) Score Plot of PC1 versus PC2 scores. (B) Loading plot for PC1 and PC2 contributing to mass peaks and their
assignments, with each metabolite denoted by its mass/rt (s) pair.

E. lysistemon plant

E. lysistemon callus

E. Lysistemon MeJA
elicited cell culture

O

Callus iniƟaƟon
R
H3C
H3C

O

MeJA
H 3C

O
CH3
H3C

O

1

CH3

O

Isoneorautenol
CH3

Erythrabissin
N

O

CH
H3

CH3

H3C

HO

O

H3C

OH
O

OH

R

OH

CH3
CH3

O

HO

Pterocarpans

H 3C

CH
H3

Oleanolic acid

O

R=CH2, R1=OCH3 Erythristemine
Erysotramidine
R=CO, R1=H

Triterpenes

Alkaloids

Fig. 4 Diagram showing major secondary metabolite pathways with represented major structures that are up regulated in E. lysistemon
plant, cell culture and in response to MeJA elicitation.

688

M.A. Farag et al.

Table 2 Quantification of metabolites identified in E. lysistemon cell suspension at 0, 12 and 24 h post 1.0 mM MeJA
elicitation using MS detection in positivea and negativeb
ionization modes. Values are expressed in lg gÀ1 as average
for 3 biological replicates.
Metabolites (lg gÀ1)
a

Erysotrine
Erysotramidinea
ErylysinAb
Sandwicensinb
Oleanolic acidb
ErythrabissinIb
Isoneorautenolb

Christoph Bo¨ttcher, Leibniz Institute of Plant Biochemistry,
Germany, for assistance with the UPLC-MS. We are grateful
to Dr. Tilo Lu¨bcken, University of Dresden, Germany, for
providing R scripts for UPLC-MS data analysis.
Appendix A. Supplementary material

E. lysistemon cell suspension
0h

12 h

24 h

2.8 ± 0.9
10.8 ± 3.8
36.0 ± 8.2
10.7 ± 2.9
406 ± 32.1
1268 ± 85
2217 ± 89

3.4 ± 2.1
8.9 ± 2.5
22.2 ± 5.4
3.4 ± 0.5
4907 ± 133
268 ± 59
473 ± 16

0.6 ± 0.5
6.0 ± 1.4
12.7 ± 2.6
2.2 ± 0.5
4838 ± 237
243 ± 18
564 ± 47

elicitation will help affirm induction hypothesis derived via
metabolite profiling. It should be noted that oleanolic acid
tri-hexoside conjugate was not detected by visual examination

of unelicited cell culture chromatograms, suggesting that coupling of metabolomics for analysis of elicited samples presents
a powerful methodology for identification of novel metabolites. Quantification of the major differential metabolites in elicited cell culture is presented in Table 2.
Conclusions
This study provides the first report on E. lysistemon cell suspension culture metabolite fingerprint via UPLC-MS. A metabolomic approach was used to investigate secondary
metabolites viz. alkaloids, flavonoids and triterpenes and their
reprogramming in response to MeJA elicitation. The results
confirm MeJA elicitation effect on terpenoid accumulation
and extend our knowledge base concerning secondary
metabolism in other legume species [27]. Comparative metabolic profiling of E. lysistemon cell suspension culture and in
response to elicitation using MeJA, revealed an activation in
sterol/triterpenes formation, see model depicted in Fig. 4.
The effect of other elicitors on secondary metabolites accumulation in Erythrina cell culture could also provide more holistic
insight into elicitation effect within that genus and how it can
reprogram its different secondary metabolite pathways.
Conflict of Interest
The authors declare that they have no conflict of interest.
Compliance with Ethics Requirements
This article does not contain any studies with human or animal
subjects.

Acknowledgments
Dr. Mohamed A. Farag acknowledges the funding received by
Science and Technology Development Fund STDF, Egypt
(grant number 12594), and the support of the Alexander von
Humboldt Foundation, Germany. We also thank Dr.

Supplementary data associated with this article can be found,
in the online version, at />07.002.
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