2f96dc2527edb3b81e5cc499e45f9d9b pharmaceutics_a2021v13n8p1153.pdf 8ddc651c69c0b252afdc48e1008569b48df22ef3 pharmaceutics_a2021v13n8p1153.pdf 39a8e99c3c5aba858b34f374183f16815edbd91e682056f06849a86e66ff8349 pharmaceutics_a2021v13n8p1153.pdf Title: Purification and Identification of Novel Antioxidant Peptides Isolated from Geoffroea decorticans Seeds with Anticoagulant Activity Subject: Geoffroea decorticans is a xerophilous deciduous tree present in most arid forests of southern South America, which is commonly used in traditional medicine. The seeds of this tree have been previously investigated for their singular chemical composition, but their protein content has been poorly investigated. Herein, we report the isolation, purification, and characterization of a set of thermostable peptides derived from Geoffroea decorticans seeds (GdAPs) with strong antioxidant and anticoagulant activities. The most potent antioxidant peptides showed a half maximal inhibitory concentration (IC50) of 35.5 0.3 g/mL determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH). They also caused a dose-dependent prolongation of the aPTT clotting time with an IC50 value of ~82 g/mL. Interestingly, MALDI-TOF/MS analysis showed the presence of three major peptides with low molecular weights of 2257.199 Da, 2717.165 Da, and 5422.002 Da. The derived amino-acid sequence of GdAPs revealed their unique structural features, exhibiting homology with various proteins present in the genome of Arachis hypogaea. All in all, our data suggest a direct applicability of GdAPs for pharmaceutical purposes. Keywords: Geoffroea decorticans; antioxidant; functional compound; thermostable protein; reactive oxygen species; anticoagulant; bioactive compound Author: Juliana Cotabarren, Brenda Ozón, Santiago Claver, Javier Garcia-Pardo and Walter David Obregón Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Jul 27 13:38:09 2021 CEST ModDate: Tue Jul 27 14:16:01 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 12 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1620954 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- PMZFBW+VnURWPalladioL Type 1 Custom yes yes no 10 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 15 0 RQBNQK+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 XLTTWG+URWPalladioL-Bold Type 1 Custom yes yes no 24 0 BPCWTG+URWPalladioL-BoldItal Type 1 Custom yes yes no 28 0 FFFSRE+URWPalladioL-Roma Type 1 Custom yes yes no 32 0 KTWDOO+CMSY10 Type 1 Builtin yes yes no 36 0 SOZKOC+VnURWPalladioL-Bold Type 1 Custom yes yes no 40 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 49 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 77 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 81 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 85 0 CZKGAS+CMR10 Type 1 Builtin yes yes no 103 0 MHJPCL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 117 0 MHJPCN+PalatinoLinotype TrueType WinAnsi yes yes no 120 0 MHJPCP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 123 0 MHJPCL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 137 0 MHJPCN+PalatinoLinotype TrueType WinAnsi yes yes no 140 0 MHJPCP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 143 0 MHJPCL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 157 0 MHJPCN+PalatinoLinotype TrueType WinAnsi yes yes no 160 0 MHJPCP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 163 0 XIRVNA+EURB10 Type 1 Builtin yes yes no 170 0 MHJPCL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 181 0 MHJPCN+PalatinoLinotype TrueType WinAnsi yes yes no 184 0 MHJPCP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 187 0 MHJPCL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 201 0 MHJPCM+PalatinoLinotype CID TrueType Identity-H yes yes yes 204 0 MHJPCN+PalatinoLinotype TrueType WinAnsi yes yes no 210 0 MHJPCP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 213 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 03:26:36 CEST RepresentationInformation: pharmaceutics_a2021v13n8p1153.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 11:58:49 CEST Size: 1620954 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 432 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Materials Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Chemicals Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Sample Collection Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Crude Extract Preparation and Determination of the Protein Content Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Purification of Antioxidant Peptides Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Initial Purification by Heat Treatment Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Size-Exclusion Chromatography Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: MALDI-TOF Mass Spectrometric Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Antioxidant Activity Evaluation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: DPPH Radical-Scavenging Activity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: ABTS Radical-Scavenging Activity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Anticoagulant Activity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Children: Item: Title: Isolation and Purification of the Antioxidant Peptides Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Identification and Antioxidant Properties of GdAPs Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: GdAPs Display Strong Anticoagulant Activiy against the Intrinsic Pathway Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Info: Title: Purification and Identification of Novel Antioxidant Peptides Isolated from Geoffroea decorticans Seeds with Anticoagulant Activity Author: Juliana Cotabarren, Brenda Ozón, Santiago Claver, Javier Garcia-Pardo and Walter David Obregón Subject: Geoffroea decorticans is a xerophilous deciduous tree present in most arid forests of southern South America, which is commonly used in traditional medicine. The seeds of this tree have been previously investigated for their singular chemical composition, but their protein content has been poorly investigated. Herein, we report the isolation, purification, and characterization of a set of thermostable peptides derived from Geoffroea decorticans seeds (GdAPs) with strong antioxidant and anticoagulant activities. The most potent antioxidant peptides showed a half maximal inhibitory concentration (IC50) of 35.5 0.3 g/mL determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH). They also caused a dose-dependent prolongation of the aPTT clotting time with an IC50 value of ~82 g/mL. Interestingly, MALDI-TOF/MS analysis showed the presence of three major peptides with low molecular weights of 2257.199 Da, 2717.165 Da, and 5422.002 Da. The derived amino-acid sequence of GdAPs revealed their unique structural features, exhibiting homology with various proteins present in the genome of Arachis hypogaea. All in all, our data suggest a direct applicability of GdAPs for pharmaceutical purposes. Keywords: Geoffroea decorticans; antioxidant; functional compound; thermostable protein; reactive oxygen species; anticoagulant; bioactive compound Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Jul 27 13:38:09 CEST 2021 ModDate: Tue Jul 27 14:16:01 CEST 2021 ID: 0x53076199189e8167a80cc26c64ef2ded, 0x53076199189e8167a80cc26c64ef2ded Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: FFFSRE+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 243 FontDescriptor: FontName: FFFSRE+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: RQBNQK+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 121 FontDescriptor: FontName: RQBNQK+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: KTWDOO+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: KTWDOO+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: CZKGAS+CMR10 FontSubset: true FirstChar: 40 LastChar: 61 FontDescriptor: FontName: CZKGAS+CMR10 Flags: Symbolic FontBBox: -40, -250, 1009, 750 FontFile: true Font: BaseFont: XLTTWG+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: XLTTWG+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: SOZKOC+VnURWPalladioL-Bold FontSubset: true FirstChar: 226 LastChar: 226 FontDescriptor: FontName: SOZKOC+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: PMZFBW+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 226 FontDescriptor: FontName: PMZFBW+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XIRVNA+EURB10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: XIRVNA+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true Font: BaseFont: BPCWTG+URWPalladioL-BoldItal FontSubset: true FirstChar: 71 LastChar: 116 FontDescriptor: FontName: BPCWTG+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: VVSYYB+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: VVSYYB+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 168, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 397, 641, 408, 654 Annotation: Subtype: Link Rect: 82, 358, 154, 367 Annotation: Subtype: Link Rect: 35, 346, 136, 355 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 541, 243, 548, 252 ActionDest: 217 Annotation: Subtype: Link Rect: 549, 243, 556, 252 ActionDest: 217 Annotation: Subtype: Link Rect: 208, 143, 215, 152 ActionDest: 217 Annotation: Subtype: Link Rect: 218, 143, 225, 152 ActionDest: 217 Annotation: Subtype: Link Rect: 136, 17, 312, 28 Annotation: Subtype: Link Rect: 383, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 446, 87, 465 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 528, 711, 535, 721 ActionDest: 217 Annotation: Subtype: Link Rect: 397, 686, 404, 695 ActionDest: 217 Annotation: Subtype: Link Rect: 308, 661, 315, 670 ActionDest: 217 Annotation: Subtype: Link Rect: 323, 649, 330, 658 ActionDest: 220 Annotation: Subtype: Link Rect: 331, 649, 343, 658 ActionDest: 220 Annotation: Subtype: Link Rect: 522, 624, 534, 633 ActionDest: 220 Annotation: Subtype: Link Rect: 184, 611, 196, 620 ActionDest: 220 Annotation: Subtype: Link Rect: 241, 611, 253, 620 ActionDest: 220 Annotation: Subtype: Link Rect: 321, 611, 333, 620 ActionDest: 220 Annotation: Subtype: Link Rect: 334, 611, 346, 620 ActionDest: 220 Annotation: Subtype: Link Rect: 459, 611, 471, 620 ActionDest: 220 Annotation: Subtype: Link Rect: 372, 586, 384, 595 ActionDest: 220 Annotation: Subtype: Link Rect: 387, 586, 399, 595 ActionDest: 220 Annotation: Subtype: Link Rect: 257, 561, 268, 570 ActionDest: 220 Annotation: Subtype: Link Rect: 400, 536, 412, 545 ActionDest: 220 Annotation: Subtype: Link Rect: 478, 523, 490, 532 ActionDest: 220 Annotation: Subtype: Link Rect: 543, 523, 555, 532 ActionDest: 220 Annotation: Subtype: Link Rect: 257, 511, 269, 520 ActionDest: 220 Annotation: Subtype: Link Rect: 204, 498, 216, 507 ActionDest: 220 Annotation: Subtype: Link Rect: 230, 473, 242, 482 ActionDest: 220 Annotation: Subtype: Link Rect: 245, 473, 257, 482 ActionDest: 220 Annotation: Subtype: Link Rect: 462, 435, 474, 444 ActionDest: 220 Annotation: Subtype: Link Rect: 475, 435, 487, 444 ActionDest: 220 Annotation: Subtype: Link Rect: 529, 372, 541, 382 ActionDest: 220 Annotation: Subtype: Link Rect: 477, 347, 488, 356 ActionDest: 220 Annotation: Subtype: Link Rect: 492, 347, 503, 356 ActionDest: 224 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 457, 546, 469, 555 ActionDest: 224 Annotation: Subtype: Link Rect: 505, 344, 517, 353 ActionDest: 224 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 530, 710, 542, 719 ActionDest: 224 Annotation: Subtype: Link Rect: 472, 594, 560, 606 Annotation: Subtype: Link Rect: 166, 581, 187, 594 Annotation: Subtype: Link Rect: 264, 495, 276, 504 ActionDest: 224 Annotation: Subtype: Link Rect: 253, 278, 265, 287 ActionDest: 224 Annotation: Subtype: Link Rect: 198, 74, 210, 83 ActionDest: 224 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 193, 556, 441, 568 Annotation: Subtype: Link Rect: 362, 442, 369, 454 ActionDest: 107 Annotation: Subtype: Link Rect: 487, 332, 494, 341 ActionDest: 217 Annotation: Subtype: Link Rect: 495, 332, 502, 341 ActionDest: 217 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 196, 694, 203, 706 ActionDest: 107 Annotation: Subtype: Link Rect: 297, 644, 304, 656 ActionDest: 127 Annotation: Subtype: Link Rect: 359, 619, 366, 631 ActionDest: 127 Annotation: Subtype: Link Rect: 363, 609, 375, 618 ActionDest: 224 Annotation: Subtype: Link Rect: 378, 609, 390, 618 ActionDest: 224 Annotation: Subtype: Link Rect: 439, 521, 451, 530 ActionDest: 224 Annotation: Subtype: Link Rect: 373, 245, 380, 257 ActionDest: 94 Annotation: Subtype: Link Rect: 533, 245, 540, 257 ActionDest: 147 Annotation: Subtype: Link Rect: 549, 220, 556, 232 ActionDest: 147 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 200, 694, 207, 706 ActionDest: 147 Annotation: Subtype: Link Rect: 361, 352, 373, 361 ActionDest: 224 Annotation: Subtype: Link Rect: 385, 301, 397, 311 ActionDest: 224 Annotation: Subtype: Link Rect: 418, 276, 430, 285 ActionDest: 224 Annotation: Subtype: Link Rect: 367, 201, 379, 210 ActionDest: 224 Annotation: Subtype: Link Rect: 382, 201, 394, 210 ActionDest: 224 Annotation: Subtype: Link Rect: 197, 136, 204, 148 ActionDest: 167 Annotation: Subtype: Link Rect: 390, 123, 397, 135 ActionDest: 167 Annotation: Subtype: Link Rect: 468, 113, 480, 122 ActionDest: 220 Annotation: Subtype: Link Rect: 483, 113, 495, 122 ActionDest: 220 Annotation: Subtype: Link Rect: 434, 73, 441, 85 ActionDest: 167 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 327, 400, 339, 409 ActionDest: 224 Annotation: Subtype: Link Rect: 218, 349, 230, 358 ActionDest: 224 Annotation: Subtype: Link Rect: 230, 349, 242, 358 ActionDest: 224 Annotation: Subtype: Link Rect: 287, 312, 299, 321 ActionDest: 224 Annotation: Subtype: Link Rect: 300, 312, 312, 321 ActionDest: 224 Annotation: Subtype: Link Rect: 267, 274, 279, 283 ActionDest: 220 Annotation: Subtype: Link Rect: 312, 274, 324, 283 ActionDest: 224 Annotation: Subtype: Link Rect: 418, 274, 430, 283 ActionDest: 224 Annotation: Subtype: Link Rect: 477, 274, 489, 283 ActionDest: 220 Annotation: Subtype: Link Rect: 544, 274, 556, 283 ActionDest: 220 Annotation: Subtype: Link Rect: 195, 260, 202, 271 ActionDest: 167 Annotation: Subtype: Link Rect: 520, 193, 531, 201 ActionDest: 220 Annotation: Subtype: Link Rect: 520, 182, 531, 190 ActionDest: 224 Annotation: Subtype: Link Rect: 520, 171, 531, 179 ActionDest: 224 Annotation: Subtype: Link Rect: 520, 160, 531, 168 ActionDest: 224 Annotation: Subtype: Link Rect: 520, 149, 531, 157 ActionDest: 220 Annotation: Subtype: Link Rect: 520, 138, 531, 146 ActionDest: 220 Annotation: Subtype: Link Rect: 520, 121, 531, 130 ActionDest: 224 Annotation: Subtype: Link Rect: 520, 105, 531, 113 ActionDest: 224 Annotation: Subtype: Link Rect: 520, 94, 531, 102 ActionDest: 220 Annotation: Subtype: Link Rect: 520, 83, 531, 91 ActionDest: 220 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 374, 707, 386, 716 ActionDest: 224 Annotation: Subtype: Link Rect: 278, 619, 290, 628 ActionDest: 224 Annotation: Subtype: Link Rect: 324, 566, 331, 578 ActionDest: 191 Annotation: Subtype: Link Rect: 364, 491, 371, 503 ActionDest: 191 Annotation: Subtype: Link Rect: 307, 443, 319, 452 ActionDest: 224 Annotation: Subtype: Link Rect: 486, 418, 498, 427 ActionDest: 224 Annotation: Subtype: Link Rect: 239, 405, 251, 414 ActionDest: 224 Annotation: Subtype: Link Rect: 354, 405, 366, 414 ActionDest: 224 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 352, 722, 364, 731 ActionDest: 224 Annotation: Subtype: Link Rect: 465, 594, 472, 606 ActionDest: 167 Annotation: Subtype: Link Rect: 424, 520, 560, 531 Annotation: Subtype: Link Rect: 166, 508, 310, 519 Annotation: Subtype: Link Rect: 59, 237, 97, 247 Annotation: Subtype: Link Rect: 59, 202, 97, 213 Annotation: Subtype: Link Rect: 103, 202, 139, 213 Annotation: Subtype: Link Rect: 161, 179, 199, 190 Annotation: Subtype: Link Rect: 415, 156, 453, 167 Annotation: Subtype: Link Rect: 462, 121, 499, 132 Annotation: Subtype: Link Rect: 59, 99, 97, 109 Annotation: Subtype: Link Rect: 103, 99, 139, 109 Annotation: Subtype: Link Rect: 520, 87, 557, 97 Annotation: Subtype: Link Rect: 59, 76, 95, 86 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 311, 722, 349, 732 Annotation: Subtype: Link Rect: 355, 722, 391, 732 Annotation: Subtype: Link Rect: 231, 698, 430, 709 Annotation: Subtype: Link Rect: 338, 675, 376, 686 Annotation: Subtype: Link Rect: 382, 675, 418, 686 Annotation: Subtype: Link Rect: 389, 652, 427, 663 Annotation: Subtype: Link Rect: 433, 652, 469, 663 Annotation: Subtype: Link Rect: 514, 629, 552, 640 Annotation: Subtype: Link Rect: 520, 606, 557, 617 Annotation: Subtype: Link Rect: 415, 583, 453, 594 Annotation: Subtype: Link Rect: 459, 583, 495, 594 Annotation: Subtype: Link Rect: 186, 549, 223, 559 Annotation: Subtype: Link Rect: 187, 515, 224, 525 Annotation: Subtype: Link Rect: 330, 491, 367, 502 Annotation: Subtype: Link Rect: 344, 468, 382, 479 Annotation: Subtype: Link Rect: 241, 446, 279, 456 Annotation: Subtype: Link Rect: 285, 446, 321, 456 Annotation: Subtype: Link Rect: 485, 422, 521, 433 Annotation: Subtype: Link Rect: 266, 399, 303, 410 Annotation: Subtype: Link Rect: 427, 376, 464, 387 Annotation: Subtype: Link Rect: 331, 353, 369, 364 Annotation: Subtype: Link Rect: 375, 353, 411, 364 Annotation: Subtype: Link Rect: 313, 330, 350, 341 Annotation: Subtype: Link Rect: 240, 307, 278, 318 Annotation: Subtype: Link Rect: 350, 284, 388, 295 Annotation: Subtype: Link Rect: 239, 261, 277, 272 Annotation: Subtype: Link Rect: 356, 238, 394, 249 Annotation: Subtype: Link Rect: 400, 238, 436, 249 Annotation: Subtype: Link Rect: 383, 215, 421, 226 Annotation: Subtype: Link Rect: 131, 181, 169, 191 Annotation: Subtype: Link Rect: 308, 157, 346, 168 Annotation: Subtype: Link Rect: 248, 134, 286, 145 Annotation: Subtype: Link Rect: 357, 100, 394, 111 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 362, 709, 399, 720 Annotation: Subtype: Link Rect: 453, 686, 490, 697 Annotation: Subtype: Link Rect: 277, 663, 314, 674 Annotation: Subtype: Link Rect: 59, 641, 97, 651 Annotation: Subtype: Link Rect: 376, 617, 414, 628 Annotation: Subtype: Link Rect: 122, 595, 159, 605 Annotation: Subtype: Link Rect: 416, 571, 453, 582 Annotation: Subtype: Link Rect: 460, 571, 496, 582 Annotation: Subtype: Link Rect: 59, 538, 97, 548 Annotation: Subtype: Link Rect: 103, 538, 139, 548 Annotation: Subtype: Link Rect: 170, 515, 208, 525 Annotation: Subtype: Link Rect: 342, 502, 379, 513 Annotation: Subtype: Link Rect: 171, 446, 208, 456 Annotation: Subtype: Link Rect: 250, 423, 288, 433 Annotation: Subtype: Link Rect: 185, 400, 223, 410 Annotation: Subtype: Link Rect: 278, 376, 315, 387 Annotation: Subtype: Link Rect: 510, 353, 548, 364 Annotation: Subtype: Link Rect: 315, 330, 352, 341 Annotation: Subtype: Link Rect: 232, 307, 270, 318 Annotation: Subtype: Link Rect: 293, 261, 331, 272 Annotation: Subtype: Link Rect: 161, 238, 199, 249 Annotation: Subtype: Link Rect: 271, 215, 308, 226 Annotation: Subtype: Link Rect: 370, 203, 560, 214 Annotation: Subtype: Link Rect: 56, 192, 208, 203 Annotation: Subtype: Link Rect: 424, 169, 461, 180 Annotation: Subtype: Link Rect: 520, 157, 557, 168 Annotation: Subtype: Link Rect: 361, 123, 398, 134 Annotation: Subtype: Link Rect: 446, 100, 483, 111 Annotation: Subtype: Link Rect: 283, 77, 320, 88 Checksum: d939e586 Type: CRC32 Checksum: 2f96dc2527edb3b81e5cc499e45f9d9b Type: MD5 Checksum: 8ddc651c69c0b252afdc48e1008569b48df22ef3 Type: SHA-1