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Studies in Natural Products Chemistry
Volume 58
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Studies in Natural
Products Chemistry
Volume 58
Edited by
Atta-ur-Rahman, FRS
International Center for Chemical and Biological Sciences
H.E.J. Research Institute of Chemistry
University of Karachi
Karachi, Pakistan
Elsevier
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Contents
Contributors xiii
Preface xvii
1. Plant Lectins: Bioactivities and Bioapplications 1

Ana Cristina Ribeiro, Ricardo Ferreira, and Regina Freitas
Plant Lectins: Introduction 2
Plant Lectins: Legume Lectins 7
The Carbohydrate-Recognition Domain 7
Oral Toxicity to Higher Animals 10
Allergenicity to Higher Animals 11
Plant Lectins: Their Role in Defense 14
Protection Against Insect, Fungi, Bacteria, Predation,
and Man 14
Acute Toxicity vs Chronic Toxicity 16
Effects of Lectins on the Diet 16
Biomedical Application of Lectins 17
In Cancer 17
Molecular Mechanisms of Apoptosis/Autophagy
Induced by Lectins 19
Ricin-B Family 19
Gallanthus nivalis Family 21
Leguminous Lectin Family 23
Others Lectins 26
Biomedical Application of Lectins in Diagnostic 27
FDA-Approved Tumor Biomarkers 27
In Neurodegenerative Diseases 30
Immunotherapy Passive in Neurodegenerative Disease 31
Innovation in Lectins Application in Therapeutic 31
Biotechnological Applications 31
Conclusions 34
References 37
Further Reading 42
v
vi Contents
2. The Role of NADP(H) Oxidase Inhibition and Its

Implications in Cardiovascular Disease Management
Using Natural Plant Products 43
Melissa Reid, Jason Spence, Magdalene Nwokocha,
Javier Palacios, and Chukwuemeka R. Nwokocha
ROS and Oxidative Stress 43
NADPH Oxidase 45
NADPH Oxidase, ROS, and Diseases 47
NADPH Oxidase and the Cardiovascular System 48
NADPH Oxidase Inhibitors 48
Insights on Therapeutic Targets Using Natural Products 51
References 56
3. The Current and Potential Therapeutic Uses

of Parthenolide 61
Snezana Agatonovic-Kustrin and David W. Morton
Introduction 61
Feverfew Plant, Active Constituents, and Its Medicinal Uses 62
Sesquiterpene Lactones, Chemical Structure, and Activities 64
Antiinflammatory Activity, Mechanism of Action,
and Clinical Trials 67
Cytotoxic and Anticancer Activity 71
Strategies for Overcoming the Limited Therapeutic Efficacy
Associated With the Low Solubility of Parthenolide 72
The Structure–Activity Relationship of Parthenolide and Parthenolide
Analogues 74
The Use of Parthenolide in Combination With Other Cancer
Drugs and Radiation Therapy 78
Migraine, 5-Hydroxytryptamine, and Antiserotonergic Activity 79
Parthenolide, Cocaine, and Inhibition of Neurotransmitter
Transporters 81
Structural Requirements for Activity 83
Conclusion 85
References 86
4. Caged Garcinia Xanthones: Synthetic Studies

and Pharmacophore Evaluation 93
Oraphin Chantarasriwong, Bashayer D. Althufairi,
Nicholas J. Checchia, and Emmanuel A. Theodorakis
Introduction 94
Chemical Classification of CGXs 94
Biogenesis and Biosynthesis of CGXs 97
Synthetic Planning Toward the CGXs 98
Representative Examples on the Synthesis of CGX-Containing
Natural Products 101
Contents vii
Synthesis of Forbesione and Structurally Related Natural Products 101

Synthesis of Desoxymorellin, Desoxygaudichaudione A,
Gambogin, Isomorellin, and Gaudichaudione A 103
Synthesis of Lateriflorone Derivatives 106
Representative Examples on the Synthesis of Designed
Caged Xanthones 107
Synthesis of Simplified Caged Xanthones 107
Synthesis of Cluvenone 109
Synthesis of MAD28 110
Structure/Function Studies of the CGX Pharmacophore 113
General Pharmacology and Toxicology of Gambogic Acid 113
Molecular Mechanism of Action of Gambogic Acid 113
Gambogic Acid: Derivatives and SAR Studies 116
Gambogic Acid: Conjugation and Formulation Studies 118
Cluvenone and MAD28: Mode-of-Action Studies 120
Concluding Remarks 123
Acknowledgments 123
References 125
5. Phytoestrogens as Potential Therapeutic Agents

for the Treatment of Anxiety and Affective Disorders 133
Juan F. Rodrı́guez-Landa, Abraham Puga-Olguı́n,
León J. Germán-Ponciano, Oscar J. Olmos-Vázquez,
and Blandina Bernal-Morales
Introduction 133
Phytoestrogens in Nature 134
Interactions Between Phytoestrogens and Estrogen Receptors:
Impact on Anxiety and Depressive Symptoms 137
Phytoestrogens in Anxiety- and Depressive-Like Behavior:
Animal Studies 138
Phytoestrogens in Anxiety and Mood Disorders: Human Studies 144
Phytoestrogens and Brain Function 149
Acknowledgments 155
References 155
6. Applications of Flavonoids, With an Emphasis on

Hesperidin, as Anticancer Prodrugs: Phytotherapy
as an Alternative to Chemotherapy 161
Danijela Stanisic, Amanda F. Costa, Guilherme Cruz,
Nelson Durán, and Ljubica Tasic
Introduction 162
Hesperidin 163
Antioxidant Activity of Hesperidin 165
Antiinflammatory Properties of Flavonoids 167
Antiproliferative Properties of Flavonoids 168
viii Contents
Bioavailability of the Flavonoids and Hesperidin 169

Introduction to Cancer Heterogeneity Mechanisms 170
Apoptosis 171
Hesperidin and Its Derivatives: Anticancer Mechanisms
and Activities 175
The Use of Hesperidin Against Colon Cancer 175
Gastric Cancer Cells 178
Skin Cancer 178
Tongue Cancer 180
Lung Cancer 181
Hepatic and Renal Cancers 182
Breast Cancer 183
Final Remarks 184
Clinical Research and Hesperidin 185
Toxicity 185
Drug Delivery Systems 191
Types of Nanocarriers for Flavonoid Delivery 192
Solid Lipid Nanoparticles and Nanostructured
Lipid Carriers 192
Liposomes 193
Dendrimers 193
Gold Nanoparticles 193
Superparamagnetic Iron Oxide Nanoparticles 194
Nanogels 194
Polymeric Nanoparticles 194
Routes for Bioflavonoid Administration 195
Transdermal Drug Delivery 195
Oral Administration 196
Inhalation 196
Intravenous 197
Hesperidin and Other Citrus Flavonoids in DDS 197
Conclusions 202
Acknowledgments 202
Appendix 203
References 207
7. Augmented Cell Signaling by Natural Polyphenols

and Flavonoids: Insights Into Cancer Cell
Remodeling and Prevention 213
Sugapriya Dhanasekaran and Ravindran Jaganathan
Introduction 213
Anticancer Properties of Polyphenols and Flavonoids 220
Antioxidant and Free Radical Quenching Effects 220
Targeting of Cell Signaling Pathway 222
Clinical Trials of Polyphenols and Flavonoids: Anticancer Activity 233
Clinical Trials of Curcumin: A Promising Anticancer Drug 234
Clinical Trials of Resveratrol: An Anticancer Agent 235
Contents ix
Clinical Trials of Tea ( )-Epigallocatechin-3-Gallate:

A Chemopreventive Drug 235
Clinical Trials of Genistein: An Anticancer Drug 236
Clinical Trials of Quercetin: An Anticancer Agent 236
Conclusions 237
Acknowledgments 238
Declaration of Interest 238
Financial Disclosures 238
References 240
8. Beneficial Effects of Anthocyanin From Natural

Products on Lifestyle-Related Diseases Through
Inhibition of Protease Activities 245
Takuya Yamane
Introduction 245
Beneficial Effects of Anthocyanins on Hypertension
and Cardiovascular Disease 250
Beneficial Effects of Anthocyanins on Type 2 Diabetes and Obesity 256
References 260
9. Quercetin: Prooxidant Effect and Apoptosis

in Cancer 265
Paola G. Mateus, Vanessa G. Wolf, Maiara S. Borges,
and Valdecir F. Ximenes
Molecular Oxygen, Spin Restriction, and Physiological
Generation of ROS 265
Molecular Oxygen, Autoxidation of Polyphenols,
and Generation of ROS 268
Polyphenols as Cocatalyst in Peroxidase-Mediated Oxidation 269
Chemistry of Polyphenols 270
Quercetin as a Source of ROS 271
Apoptosis 275
Apoptosis and Cancer 277
Oxidation Therapy and Induction of Apoptosis
as an Anticancer Strategy 278
Induction of Apoptosis in Cancer Cells by Quercetin:
Involvement of Prooxidant Activity 279
Leukemia Cells 279
Hepatocellular Carcinoma Cell Lines 281
Cervical and Breast Cancer Cell Lines 282
Gastric Carcinoma and Thyroid Cancer Cell Lines 283
Acknowledgments 284
References 285
x Contents
10. Bioactive Components of Saffron and Their

Pharmacological Properties 289
Azam Bolhassani
Introduction 289
General Description of Saffron 290
Anticancer Mechanisms of Saffron and Its Ingredients 291
Saffron 291
Crocetin 293
Crocin 294
Safranal 296
Other Pharmacological Effects of Saffron and Its Components 296
Toxicity of Saffron and Its Ingredients 306
References 308
11. Anthraquinones: An Overview 313

Gaspar Diaz-Muñoz, Izabel L. Miranda, Su
elen K. Sartori,
Daniele C. de Rezende, and Marisa A.N. Diaz
Introduction 313
Biosynthesis and Occurrence of Anthraquinones 315
Biosynthesis 315
Natural Anthraquinones 317
Methods of Extraction, Purification and Characterization
of Anthraquinones 319
Characterization 319
Chemical, Pharmacological and Technological Applications
of Anthraquinones 322
Relevant Chemical Syntheses of Anthraquinones 327
References 335
12. The Genus Hymenaea (Fabaceae): A Chemical

and Pharmacological Review 339
Fernanda G. da Silva Oliveira, Camila de Souza Araújo,
Larissa A. Rolim, Jos
e M. Barbosa-Filho,
and Jackson R.G. da Silva Almeida
Introduction 339
Ethnobotanic and Pharmacological Studies 340
Hymenaea courbaril L. 345
Hymenaea stigonocarpa Mart. ex Hayne 348
Hymenaea martiana Hayne 350
Hymenaea palustris Ducke 352
Hymenaea intermedia Ducke 352
The Chemistry of Hymenaea Species 352
Hymenaea courbaril L. 374
Contents xi
Hymenaea stigonocarpa Mart. ex Hayne 379

Hymenaea martiana Hayne 380
Hymenaea palustris Ducke 380
Hymenaea intermedia Ducke 380
Hymenaea oblongifolia Huber 381
Hymenaea parvifolia Huber 381
Hymenaea verrucosa Gaertn 381
Hymenaea mexicana Poinar & Brown 382
Hymenaea protera Poinar 382
Acknowledgments 383
References 383
13. Phenolic Acids From Plants: Extraction

and Application to Human Health 389
Samar Al Jitan, Saeed A. Alkhoori, and Lina F. Yousef
Introduction 389
Phenolic Acids in Plants 390
Extraction of Phenolic Acids From Plant Biomass 395
Soxhlet Extraction 395
Ultrasound Extraction 396
Microwave-Assisted Extraction 396
Supercritical Fluid Extraction 400
Accelerated Solvent Extraction 403
Ionic Liquids and Deep Eutectic Solvents 405
Comparison of Extraction Methods 405
Application of Phenolic Acids to Human Health 407
References 413
14. Cannabidiol in Refractory Epilepsy 419

Ashish Dhir
Introduction 419
Phytocannabinoids: Biosynthetic Pathway 421
Endocannabinoid System 422
Cannabidiol in Preclinical Animal Models of Epilepsy 424
Mechanism of Antiseizure Action of Cannabidiol 427
Cannabidiol in Human Cases of Epilepsy 432
References 435
Index 439
Contributors
Numbers in Parentheses indicate the pages on which the author’s contributions begin.
Snezana Agatonovic-Kustrin (61), School of Pharmacy, Monash University Malaysia,
Selangor Darul Ehsan, Malaysia
Samar Al Jitan (389), Khalifa University of Science and Technology, Abu Dhabi,
United Arab Emirates
Saeed A. Alkhoori (389), Khalifa University of Science and Technology, Abu Dhabi,
Bashayer D. Althufairi (93), School of Pharmacy, Kuwait University, Safat, Kuwait;
University of California, San Diego, La Jolla, CA, United States
José M. Barbosa-Filho (339), Postgraduate Program in Natural and Synthetic
Bioactive Products, Federal University of Paraı́ba, Joao Pessoa, Paraı́ba, Brazil
Blandina Bernal-Morales (133), Universidad Veracruzana, Xalapa, Mexico
Azam Bolhassani (289), Department of Hepatitis and AIDS, Pasteur Institute of Iran,
Tehran, Iran
Maiara S. Borges (265), UNESP—São Paulo State University, Bauru, SP, Brazil
Oraphin Chantarasriwong (93), Department of Chemistry, King Mongkut’s
University of Technology Thonburi, Bangkok, Thailand
Nicholas J. Checchia (93), University of California, San Diego, La Jolla, CA, United
States
Amanda F. Costa (161), Institute of Chemistry, University of Campinas (UNICAMP),
Campinas, Brazil
Guilherme Cruz (161), Institute of Chemistry, University of Campinas (UNICAMP),
Campinas, Brazil
Jackson R.G. da Silva Almeida (339), Center for Studies and Research of Medicinal
Plants, Federal University of the San Francisco Valley, Petrolina, Pernambuco,
Brazil
Fernanda G. da Silva Oliveira (339), Center for Studies and Research of Medicinal
Plants, Federal University of the San Francisco Valley, Petrolina, Pernambuco,
Brazil
Daniele C. de Rezende (313), Universidade Federal de Minas Gerais, Belo Horizonte,
Minas Gerais, Brazil
Camila de Souza Araújo (339), Center for Studies and Research of Medicinal Plants,
Federal University of the San Francisco Valley, Petrolina, Pernambuco, Brazil
xiii
xiv Contributors
Sugapriya Dhanasekaran (213), College of Applied Medical Sciences, Prince Sattam

Bin Abdulaziz University, Riyadh Province, Kingdom of Saudi Arabia
Ashish Dhir (419), Department of Neurology, School of Medicine, University of
California, Davis, Sacramento, CA, United States
Gaspar Diaz-Muñoz (313), Universidade Federal de Minas Gerais, Belo Horizonte,
Marisa A.N. Diaz (313), Universidade Federal de Viçosa, Vicosa, Minas Gerais,
Brazil
Nelson Durán (161), Institute of Chemistry, University of Campinas (UNICAMP);
NanoBioss—Institute of Chemistry, University of Campinas, Campinas; Nanome-
dicine Research Unit (Nanomed), Federal University of ABC (UFABC), Santo
André, Brazil
Ricardo Ferreira (1), Higher Institute of Agronomy, DRAT, Universidade de Lisboa,
Lisbon, Portugal
Regina Freitas (1), Higher Institute of Agronomy, DRAT, Universidade de Lisboa,
Lisbon, Portugal
León J. Germán-Ponciano (133), Universidad Veracruzana, Xalapa, Mexico
Ravindran Jaganathan (213), Universiti Kuala Lumpur Royal College of Medicine
Perak (UniKL-RCMP), Ipoh, Malaysia
Paola G. Mateus (265), UNESP—São Paulo State University, Bauru, SP, Brazil
Izabel L. Miranda (313), Universidade Federal de Minas Gerais, Belo Horizonte,
David W. Morton (61), School of Pharmacy and Applied Science, La Trobe Institute
for Molecular Sciences, La Trobe University, Bendigo, VIC, Australia
Chukwuemeka R. Nwokocha (43), University of the West Indies, Mona, Jamaica
Magdalene Nwokocha (43), University of the West Indies, Mona, Jamaica
Oscar J. Olmos-Vázquez (133), Universidad Veracruzana, Xalapa, Mexico
Javier Palacios (43), Universidad Arturo Prat, Iquique, Chile
Abraham Puga-Olguı́n (133), Universidad Veracruzana, Xalapa, Mexico
Melissa Reid (43), University of the West Indies, Mona, Jamaica
Ana Cristina Ribeiro (1), Faculty of Pharmacy, DCTB; Higher Institute of Agronomy,
DRAT, Universidade de Lisboa, Lisbon, Portugal
Juan F. Rodrı́guez-Landa (133), Universidad Veracruzana, Xalapa, Mexico
Larissa A. Rolim (339), Center for Studies and Research of Medicinal Plants, Federal
University of the San Francisco Valley, Petrolina, Pernambuco, Brazil
Suélen K. Sartori (313), Universidade Federal de Minas Gerais, Belo Horizonte,
Jason Spence (43), University of the West Indies, Mona, Jamaica
Danijela Stanisic (161), Institute of Chemistry, University of Campinas (UNICAMP),
Campinas, Brazil
Contributors xv
Ljubica Tasic (161), Institute of Chemistry, University of Campinas (UNICAMP);

NanoBioss—Institute of Chemistry, University of Campinas, Campinas, Brazil
Emmanuel A. Theodorakis (93), University of California, San Diego, La Jolla, CA,
United States
Vanessa G. Wolf (265), UNESP—São Paulo State University, Bauru, SP, Brazil
Valdecir F. Ximenes (265), UNESP—São Paulo State University, Bauru, SP, Brazil
Takuya Yamane (245), Osaka Prefecture University, Sakai, Japan
Lina F. Yousef (389), Khalifa University of Science and Technology, Abu Dhabi,
Preface
The 58th volume of Studies in Natural Products Chemistry comprises

14 chapters that cover a variety of components derived from natural products
and their effective use in different ailments. I hope that it will be received
with the same enthusiasm as previous volumes of this long-standing series.
In Chapter 1, Ribeiro reviews the potent biological activity of plant lectins.
They have a prominent role in plant protection. They are also effective in
diagnosis and treatment of various ailments. The role of lectins for developing
anticancer agents, both for preventive and for curative purposes, is discussed.
NADP(H) oxidase-derived reactive oxygen species plays a significant role in
the vascular system. In Chapter 2, Nwokocha et al. focus on the utilization
of natural plant products as novel therapeutic agents in the management
and treatment of cardiovascular diseases that are linked to oxidative stress.
In Chapter 3, Agatonovic-Kustrin and Morton provide an overview of the
role of parthenolide in the inhibition of cocaine-induced behaviors in an
animal model. They summarize the mechanisms of action of parthenolide
as a migraine prophylactic and therapeutic agent for cocaine addiction/
intoxication.
Theodorakis et al. in Chapter 4 present the different approaches to the syn-
thesis of caged Garcinia xanthone-derived natural products. They find impor-
tance in traditional Eastern medicine and have potential for the development
of effective anticancer agents. In Chapter 5, Rodrı́guez-Landa and colleagues
have contributed an interesting review on phytoestrogens for managing
anxiety and depressive symptoms.
The chemoprotective role of flavonoids (especially hesperidin) in carcino-
genesis has been discussed by Tasic et al., in Chapter 6. Similarly, in
Chapter 7, Dhanasekaran and Jaganathan have also focused on the pharmaco-
logical efficacy of flavonoids and polyphenols for the prevention of cancer.
Yamane in Chapter 8 presents an informative review on anthocyanins and
highlights their protective role as protease inhibitors on lifestyle-related
diseases.
Ximenes et al. in Chapter 9 discuss the tumoricidal effects of quercetin.
The authors describe it as a redox-active compound that acts via its anti- or
prooxidant action. The occurrence and incidence of depression are progres-
sing rapidly in almost every part of the world. In Chapter 10, Bolhassani
has discussed the current advancements in the pharmacological effectiveness
of saffron and its components in depression and other diseases.
xvii
xviii Preface
Diaz-Muñoz et al. in Chapter 11 have presented the different characteris-

tics and utilization of anthraquinones. They can be used as anticancer, antiin-
flammatory, diuretic, antiarthritic, antifungal, antibacterial, and antimalarial
agents. In Chapter 12, the pharmacological and chemical aspects related to
genus Hymenaea have been reviewed by Almeida et al. In Chapter 13, Yousef
et al. highlight different techniques for extracting phenolic acids. In the last
chapter, Dhir discusses the role of cannabidiol for the treatment of epilepsy.
I would like to express thanks to Miss Taqdees Malik for assistance in the
preparation of this volume. I am also grateful to Mr. Mahmood Alam for
providing editorial assistance.
Prof. Atta-ur-Rahman, FRS
Patron-in-Chief
International Centre for Chemical & Biological Sciences
University of Karachi
Karachi, Pakistan
Chapter 1
Plant Lectins: Bioactivities

and Bioapplications
Ana Cristina Ribeiro*,†,1, Ricardo Ferreira† and Regina Freitas†
*
Faculty of Pharmacy, DCTB, Universidade de Lisboa, Lisbon, Portugal
†
Higher Institute of Agronomy, DRAT, Universidade de Lisboa, Lisbon, Portugal
1
Corresponding author: e-mail: acribeiro@ff.ulisboa.pt
Chapter Outline
Plant Lectins: Introduction 1 Ricin-B Family 19
Plant Lectins: Legume Lectins 7 Gallanthus nivalis Family 21
The Carbohydrate-Recognition Leguminous Lectin Family 23
Domain 7 Others Lectins 26
Oral Toxicity to Higher Biomedical Application of
Animals 10 Lectins in Diagnostic 27
Allergenicity to Higher Animals 11 FDA-Approved Tumor
Plant Lectins: Their Role in Biomarkers 27
Defense 14 In Neurodegenerative
Protection Against Insect, Fungi, Diseases 30
Bacteria, Predation, and Man 14 Immunotherapy Passive
Acute Toxicity vs Chronic in Neurodegenerative
Toxicity 16 Disease 31
Effects of Lectins on the Diet 16 Innovation in Lectins Application
Biomedical Application of Lectins 17 in Therapeutic 31
In Cancer 17 Biotechnological Applications 31
Molecular Mechanisms of Conclusions 34
Apoptosis/Autophagy Induced References 37
by Lectins 19 Further Reading 42
Studies in Natural Products Chemistry, Vol. 58. https://doi.org/10.1016/B978-0-444-64056-7.00001-5

© 2018 Elsevier B.V. All rights reserved. 1
2 Studies in Natural Products Chemistry
PLANT LECTINS: INTRODUCTION

From bacteria to men, the plasma membrane of each cell contains glycopro-
teins and glycolipids with an array of oligosaccharide side chains projecting
outward. Such oligosaccharide structures have been considered the third code
of life and one for which no template is believed to exist. How cells synthe-
size their appropriate complement of oligosaccharide side chains remains a
challenging mystery of modern biology. If there is a code, a decoding system
is required. This role is, at least in part, fulfilled by lectins, a large and wide-
spread protein family, defined as glycan-specific (mono- or oligosaccharides
presented by glycoconjugates or polysaccharides) receptors, distinguished
from antibodies, from enzymes using carbohydrates as substrates, and from
transporters of free saccharides [1]. It is believed that a large number of lec-
tins remain to be discovered.
Despite the huge number of lectins already described and their ubiquitous
presence in living cells, their physiological roles are often poorly characterized
or even unknown. Many are known to be involved in cell–cell, cell–molecule,
and molecule–molecule recognition. Thus, for example, they participate in the
symbiotic and pathogenic interactions between eukaryotes on one hand and
microorganisms and viruses on the other, as well as in the fixation of cancer
cells released from primary tumors into the blood stream to initiate metastatic
processes.
Lectins display great potential in disease control. Thus, for example, a lec-
tin specific for a given oligosaccharide side chain may be fused to any metab-
olite, peptide, or protein and used to target specific cell tissues or organisms.
This is something nature “discovered” a long time ago, as may be exemplified
by ricin, an extremely toxic protein present in castor bean (Ricinus communis)
seeds. Ricin is composed of two disulfide-linked polypeptide chains: The
B chain, exhibiting galactose and N-acetylgalactosamine-specific lectin activ-
ity, binds to galactose-containing cell surface receptors, mediating attach-
ment, endocytosis, and intracellular trafficking of ricin in host cells; the
A chain irreversibly inactivates eukaryotic ribosomes by hydrolytic cleavage
of a conserved adenosine residue of the 28S rRNA, thus inhibiting protein
synthesis.
One other striking example is provided by Blad, a remarkable 20-kDa
polypeptide exhibiting lectin activity with a unique biosynthetic route [2–4].
This 173-amino acid residue polypeptide occurs naturally as part of a lectin,
the Blad-containing 210 kDa oligomer (BCO), in Lupinus cotyledons between
4 and 14 days after the onset of germination. The oligomer is synthesized by
limited proteolysis from b-conglutin, a vicilin, lectin, and the major seed stor-
age protein present in Lupinus albus seeds [5,6].
Blad was discovered in 1991, patented in 2005, and entered the US market
as a crop fungicide in 2015 under the trade name Fracture™. It was included
in the FRAC (fungicide resistance action committee) Code List© in 2016.
Plant Lectins: Bioactivities and Bioapplications Chapter 1 3
Blad constitutes a changing paradigm for human, animal, and plant patho-
gens in what toxicity and efficacy of fungicides are concerned. In addition to
a general shortage in efficient fungicides, most commercially available anti-
fungal compounds fall within two major groups: those with high efficiency,
which are highly toxic; those with a low-level toxicity, which exhibit low
efficacy. Low efficacy is typically associated to a high death rate in animals
or to crop yield losses above 40%–50%, both of which are unacceptable to
present day society. Except for fungi and certain bacterial species, Blad is
nontoxic (it is actually readily edible to man and animals) but exhibits effi-
ciencies which are equal or higher than those of the best chemical and toxic
fungicides available. The recent advances in the chemistry of additives have
allowed the development of several excellent and nontoxic formulations, sus-
ceptible to be used in all kinds of farming, which concede in some cases the
increment of Blad efficiency up to 100%.
The potential use of proteins (e.g., lectins) or protein derivatives in disease
control offers a number of advantages over secondary metabolites:
(i) While most peptides are considered within secondary metabolism, pro-
teins (especially those with a defensive role) were somewhat neglected
in what concerns plant defense. Unlike many bioactive secondary meta-
bolites, such as antioxidant polyphenols, which show a beneficial or
harmful effect in a dose-dependent manner, proteins do not usually
exhibit this differential effect as a function of concentration, from bene-
ficial to harmful, when their concentration increases.
(ii) When compared to secondary metabolites, defensive proteins often
exhibit multitarget action mechanisms, which may widen their action
spectra, may increment their efficacy, and/or may reduce the chances
of resistance development by the target organisms.
(iii) The advances in protein engineering and molecular biology allow the
buildup of nonnatural fusion molecules by combining peptides and
proteins with each other or with other molecules (e.g., secondary meta-
bolites), thus offering an almost unlimited array of new possibilities.
Different components of the chimeric molecule may target distinct
organisms, distinct targets in the same organism, or direct the molecule
to a specific cell type.
Lectins are extensively dispersed in nature and ample in plants. In plants, lec-
tins are established in diverse species of major taxonomical groups and most
of as seed storage proteins [6]. Lectins also originated in vegetative organs
like roots, leaves, rhizomes, bulbs, tubers, corms, stems, bark, flowers, fruits,
phloem sap, latex, and nectar [7]. Plant lectins have analogous biological
activities and chemical properties [8–10], they are distributed in various
families and, consequently, are ingested daily in substantial amounts by both
humans and animals (Table 1.1). One of the most nutritionally significant fea-
tures of plant lectins is their ability to survive digestion, permitting lectins to
TABLE 1.1 Plant Lectins Examples and Their Families

Plant Species Lectin Abbrev. Family
Abrus precatorius Abrin Abrin Leguminosae
Adenia digitata Modeccine Modeccine Passifloraceae
Allium sativum Garlic lectin ASA Amaryllidaceae
Amaranthus Amaranthin Amaranthin Amaranthaceae

caudatus
Arachis hypogaea Peanut agglutinin PNA Leguminosae
Artocarpus Jacalin Jacalin Moraceae

heterophylus
Canavalia Concanavalin A ConA Leguminosae

ensiformis
Cratylia argentea Cratylia lectin CAL Leguminosae
Croton tiglium Crotin Crotin Euphorbiaceae

Datura stramonium Datura stramonium DSL Solanaceae
lectin
Dolichos biflorus Dolichos biflorus DBA Leguminosae

agglutinin
Galanthus nivalis Galanthus nivalis GNA Amaryllidaceae

agglutinin
Glycine max Soybean agglutinin SBA Leguminosae
Griffonia Griffonia simplicifolia GSL Leguminosae

simplicifolia lectin
Lens culinaris Lentil agglutinin LCA Leguminosae
Lycopersicum Tomato lectin LEL Solanaceae

esculentum
Lupinus albus Blad-containing BCO Leguminosae

oligomer
Maackia amurensis Maackia amurensis MAA Leguminosae
agglutinin
Musa paradisiaca Banana lectin BanLec Musaceae
Phaseolus vulgaris Phytohemagglutinin PHA Leguminosae
Pisum sativum Pea agglutinin PSA Leguminosae

Ricinus communis Ricin Ricin Euphorbiaceae
Solanum Potato agglutinin STA Solanaceae
tuberosum
TABLE 1.1 Plant Lectins Examples and Their Families—Cont’d
Plant Species Lectin Abbrev. Family

Triticum aestivum Wheat germ WGA Gramineae
agglutinin
Ulex europaeus Ulex agglutinin UEA Leguminosae
Vicia faba Faba agglutinin VFA Leguminosae
Viscum album Mistletoe agglutinin VAA Santalaceae
Wisteria floribunda Wisteria lectin WFL Leguminosae
bind to membrane glycosyl groups of the cells lining the digestive tract. As a
result, a series of injurious local and systemic reactions are elicited conveying
this molecule as a group of antinutritive and/or toxic substances. Locally, they
can affect the turnover and loss of gut epithelial cells, wound the luminal
membranes of the epithelium, interfere with nutrient digestion and absorp-
tion, stimulate shifts in the bacterial flora, and modulate the immune state
of the digestive tract. Systemically, they can disrupt lipid, carbohydrate,
and protein metabolism, promote enlargement and/or atrophy of key internal
organs and tissues, and alter the hormonal and immunological status. In high
quantity, lectins can seriously threaten the growth and health of consuming
animals [11].
Lectin toxicity to animals was first described in 1888, when Stillmark
[12] published his work on the deleterious effects of a proteinaceous sub-
stance present in castor bean (R. communis). Shortly, similar toxic substances
were discovered in the seeds of Croton tiglium (crotin) and Abrus precator-
ius (abrin) and in the bark of Robinia pseudoacacia (robinia) [13]. After the
discovery of blood group lectin specificity [14,15], the interest increased and
from the realization that phytohemagglutinin (from Phaseolus vulgaris)
induced mitosis in mature, nondividing human lymphocytes [16], many other
lectins were identified and isolated. Some of these were found to be toxic or
antinutritional for man and animals.
Elevated concentration of lectin in foods, such as beans, cereal grains,
seeds, and nuts, may be injurious to human and animals. Leading to nutri-
tional deficiencies, gastrointestinal distress, immune allergic reactions, and
food poisoning. Lectins might also be causing agent of rigorous inflammation
and devastation of epithelial cells, edema, hyperemia, and hemorrhages in the
lymphatic tissues. Lectin when injected to animals produces local necrosis
and fatty changes can be detecting in the liver tissue, whereas hemorrhages
are observed in the stomach, the intestinal wall, and other organs [17].
Plant defense is the most likely function of plant lectins. While direct inter-
ference with viruses and microorganisms is rather exceptional, the deleterious
effects of plant lectins on predatory invertebrates and higher animals are evi-
dent. Considering the abundance of lectins in storage organs and their storage
protein-like behavior, it is highly probable that plants store part of their nitro-
gen reserve in the form of carbohydrate-binding proteins, which then can be
used as passive-defense proteins [7,8].
A number of plant lectins such as concanavalin A (ConA), wheat germ
agglutinin (WGA), PHA, and the lectin from R. pseudoacacia were found
to be toxic to mammalian cells both in vitro and in vivo. While the lectins like
ricin, abrin, and modeccine are toxic and move down through neuronal pro-
cesses to the cell body where they inactivate ribosomes resulting in neuronal
death. The defensive effect of ricin, ConA, and GSL against tumor growth in
experimental animals has been described. The shielding effect of ricin and
abrin in humans against tumor growth and in grouping with anticancer drugs
has also been illustrated [17].
Lectin-dependent cytotoxicity can be explained by killing of target cells
by cytotoxic T-lymphocytes (CTL) through the specific recognition by the
affected cells, mediated by lectin. The mitogenic lectins include no immune
specificity to both effected and target cells. They do not participate in the
intercellular recognition, but modify the surface of the target cells leading
to T-lymphocyte recognition. Lectins, from wheat germ and Griffonia simpli-
cifolia, hold the capacity to mediate carbohydrate explicit binding of mouse
macrophages and tumor cells and to induce killing of the tumor cells by the
macrophages. Lectin-mediated cytotoxicity may also take place in vivo, by
intraperitoneal injection of G. simplicifolia lectin extract [17].
Plant lectins, an exceptional group of proteins and glycoproteins with
potent biological activity, occur in foods like wheat, corn, tomato, peanut,
kidney bean, banana, pea, lentil, soybean, mushroom, rice, and potato [18].
However, due to their diversity, it has been difficult to achieve a general
agreement for their classification. One of the oldest classification of plant lec-
tins is based on their carbohydrate-binding specificities and presented a good
approach to catalogue and study them. This classification grouped lectins in
six families such as (1) mannose- and mannose/glucose-, (2) mannose/maltose-,
(3) Gal/GalNAc-, (4) N-acetylglucosamine (GlcNAc)/(GlcNAc)-, (5) fucose-,
and (6) sialic acid-biding lectins. However, structurally unrelated proteins could
also get included under these classes [13,19]. To overcome this, a system of
classifying lectins based on their three-dimensional structures was created
and six major groups were identified: alpha-D-mannose-specific monocot lec-
tins, agglutinins with a hevein domains, beta-prism plant lectins, beta-trefoil
lectins, cyanovirin-N homologs, and legume lectins [20].
Another classification is based on the overall domain architecture of plant
lectins, into four major groups: (1) merolectins, e.g., hevein, holding only one
CRD (carbohydrate-recognition domain) cannot agglutinate cells; (2) hololec-
tins, with of two or more identical or very homologous CRDs, can agglutinate
cells and/or precipitate glycoconjugates, e.g., Phaseolus vulgaris agglutinin

(PHA), specific for galactose; (3) superlectins, possessing of two or more
inequivalent CRDs to recognize structurally unrelated carbohydrates, e.g., the
lectin from tulip bulbs has two distinctly different CRDs that bind mannose
in one CRD and N-acetylgalactosamine (GalNAc) in another; and (4) chimero-
lectins, composed of one or more CRDs and an independent with biological
activity, e.g., type 2 ribosome-inactivating protein [19,21]. Most of the plant
lectins isolated and characterized to date are hololectins. Although not many
chimerolectins have been isolated and studied, the sequence analysis of com-
plete plant genomes reveals that they are very abundant in plants [20,22].
A seven-family classification based on sequence similarities and serologi-
cal relatedness was suggested by Van Damme and coauthors [23], which, after
taking into consideration sequence and structural homology, was updated to a
12-family classification: (1) Agaricus bisporus agglutinin homologs, (2) amar-
anthins, (3) class V chitinase homologs with lectin activity, (4) cyanovirin
family, (5) Euonymus europaeus agglutinin family, (6) Galanthus nivalis agglu-
tinin (GNA) family, (7) proteins with hevein domains, (8) jacalins, (9) proteins
with legume lectin domains, (10) lysine motif domain, (11) Nictaba family, and
(12) Ricin-B family [24].
PLANT LECTINS: LEGUME LECTINS

Legume lectins represent the largest and most thoroughly studied family of
these sugar-binding proteins. Over 100 members of this family are known,
almost all isolated from seeds of the plants [13,25,26]. Because they are
readily obtainable in purified form and exhibit an amazing variety of sugar
specificities, these lectins are eminently suitable for tackling the question
of how proteins bind carbohydrates.
Many lectins resist digestion, survive gut passage, and bind to gastroin-
testinal cells and/or enter the circulation intact to maintain full biological
activity. Several lectins have been found to possess anticancer properties
in vitro, in vivo, and in human case studies; they are used as therapeutic agents,
preferentially binding to cancer cell membranes or their receptors, causing
cytotoxicity, apoptosis, and inhibition of tumor growth [18]. In the recent years,
accumulating evidence suggests that plant lectins have become an attractive
research field due to their role in cell agglutination, toxicity, and antifungal, anti-
bacterial, antiviral, antiproliferative, and antitumor effects [27].
Proteins with legume lectin domains have been most widely studied
because of their presence in large quantities in the seeds. Proteins with legume
lectin domain family have been widely reported to have a number of links to
many pathological processes including cancer [28].
The Carbohydrate-Recognition Domain

Life as we know it is essentially dependent on biological codes. Best known are
the genetic and protein codes. However, knowledge about a third code has more
recently emerged, whose coding capacity is immensely greater than those of the
other two [29,30].
All cells, from bacteria to human cells, are surrounded by the plasma
membrane. Embedded in this complex structure are glycolipids and glyco-
proteins, which project outward their oligosaccharidic moieties, collectively
termed the exoglycome. A typical mammalian cell membrane may contain
several hundred oligosaccharides projecting outward into the external milieu.
This exo-, oligoglycocode plays a crucial role in cell–cell and cell–molecule
interactions, ranging from plant cell interactions with symbiotic and patho-
genic microbes to virus host recognition.
For any code to operate, one or more decoding systems are required.
Thus, while the individual oligosaccharides function as keys, discrete, mod-
ular CRDs present in lectins act as the corresponding decoding locks, i.e.,
they are responsible for recognition of each lectin carbohydrate target. In this
respect, Peumans and Van Damme [7] defined lectins as “all proteins posses-
sing at least one noncatalytic domain which binds reversibly to a specific
mono- or oligosaccharide.”
In similarity with secondary metabolites, the occurrence of many lectins
is often restricted to specific taxonomic groups. On the other hand, there is
one common feature among all lectins: by definition, they all possess at least
one CRD capable of binding carbohydrates nonimmunologically and in a sta-
ble manner. There are many different types of CRDs which seem to have
evolved independently. Nevertheless, they share a number of important fea-
tures [31,32]:
(i) CRDs correspond typically to a small portion of the lectin molecule;
(ii) A comparative analysis of CRD sequences and of the resulting three-
dimensional structures reveals the existence of several families, with con-
siderable homologies among the members of each family (Table 1.2);
(iii) Shallow cavities on protein surfaces are usually responsible for the low-
affinity binding sites for monosaccharides. Deeper depressions are typically
required for the higher-affinity binding sites for oligosaccharides, while the
highest affinities usually require oligomers of polypeptides exhibiting lectin
activities. In other words, the association of more than one CRD in a single
lectin allows for increased affinity toward oligosaccharides.
Several features have been selected to classify lectins in sequence and evolu-
tionary related multiple families, based on their sugar specificity, expression
patterns (inducible or constitutive expression), or the amino acid residue
sequence of the CRD. Thus, lectins from a specific family are characterized
by sharing a pattern of highly conserved residues.
Table 1.2 presents a proposed classification of lectins in families based on
structural and evolutionary relationships among CRDs [33]. Twelve families
have been successfully suggested for plant lectins [23], providing a much
more accurate method than the previous attempts to group plant lectins based
on sugar specificity.
TABLE 1.2 Lectin Plant Families

Typical Saccharide Predicted
Lectin Family Ligands Localization
Plant Agaricus bisporus GlcNAc/GalNAc, Nucleus, cytosol
lectin family galactose
Amaranthin family GalNAc Nucleus, cytosol
Chitinase-related High-mannose Vacuole, membrane
agglutinin family N-glycans bound
Cyanovirin family Mannose Nucleus
Euonymus Galactosides, high- Nucleus, cytosol
europaeus lectin mannose N-glycans
family
Galanthus nivalis Mannose Vacuole, nucleus,
lectin family cytosol, or membrane
bound
Hevein family Chitin Vacuole
Jacalin family Mannose- and Nucleus, cytosol,
galactose-specific vacuole
subgroup
Legume family Mannose Vacuole, nucleus,
cytosol, or membrane
bound
LysM family Chitin, peptidoglycan Vacuole, nucleus,
cytosol, or membrane
bound
Nicotiana (GlcNAc)n, high- Nucleus, cytosol
tabacum lectin mannose and
family complex N-glycans
Ricin-B family Gal/GalNAc, Vacuole, nucleus,
sialylated Gal/ cytosol
GalNAc
Table extracted from K. Schutter, E.J.M. Van Damme, Molecules 20 (2015) 9029–9053.
Three further points deserve a special mention: (i) Few lectin families
have been found so far containing both animal and plant lectins, namely, cal-
nexin and calreticulin and the R-type lectins (e.g., ricin). One other example is
provided by pufflectin, a lectin from the skin mucus of the pufferfish (Fugu
rubripes), which does not show amino acid sequence homology with any
known animal lectin but shares homology with lectins from monocotyledon-
ous plants [34]. (ii) The CRD-based lectin classification should be extended
to include the fungal, bacteria, and virus lectins. (iii) There is no correlation
between the sugar specificity-based and the CRD-based classifications of lec-
tins. Thus, lectins from CRD-based distinct families, which are structurally
unrelated, may recognize the same carbohydrates [35], whereas different car-
bohydrate structures may be recognized by similar CRDs [33].
Oral Toxicity to Higher Animals

Lectins are found in a wide range of vegetables and some fruits [36], and
though not all lectins are toxic, many lectins present toxicity at different levels.
In humans, they have caused extensive damage, including mass food poisoning
with raw or undercooked kidney beans [37,38] and hemolytic anemia and jaun-
dice from Mexican fava beans [39]. Plant lectins that are not efficiently
degraded by digestive enzymes and that have an affinity for the surface of
gut epithelial cells, such as those existing in the Leguminosae family, can be
poisonous [11]. Acute symptoms following ingestion include nausea, vomiting,
and diarrhea. Long-term consumption in rodent models is characterized by
increased cell turnover, gut hyperplasia, and weight loss. Table 1.3 illustrates
LD50 values found for some lectin ingestion.
Epithelial cell necrosis and even zones of complete epithelial cell denu-
dation areas are seen in biopsies of the stomach and intestine of mammals
[40] and insects [41] fed with plant lectins. However, when cells are treated
with lectins in vitro, even at very high doses, necrosis is not witnessed,
although many other responses have been detected, including vacuole forma-
tion [42], inhibition of exocytosis [43], and mitogenesis [44]. These effects
are not found, or are decreased, when the meals include heated beans. Lec-
tins can, also, be a source of leptin resistance, which may translate into dis-
eases, particularly it could be responsible for obesity in humans who have
high levels of leptin [21].
It has been extensively confirmed that plant lectins are effective
biological agents against insect attack [45]. In fact, insect-resistant transgenic
plants produced by expression of lectin genes are currently a reality [46].
TABLE 1.3 Examples of Animal Lectin Toxicity by Ingestion
Lectin LD50 (mg/kg Body Weight) Animal Reference

Abrin 0.02 Mouse [173]
Ricin 0.001 Mouse [173]
PHA 50 Rat [174]
SBA 50 Rat [175]
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CHAPTER IX
THE AFTERNOON CALL
“W hich way?” asked Ned as, a few minutes later, they went
through the gate.
“Let’s go down to the river and along the road and see all the
booful automobiles,” said Laurie.
“It’s not my idea of a pleasant walk,” returned Ned, “to get entirely
covered with dust and then run over!”
“We’re not going to walk,” announced Polly. “Anyway, not yet.
We’re going this way.” She and Mae turned toward School Park.
“Not going to walk?” exclaimed Laurie. “What are we going to do?
Polly, don’t tell me you’ve gone and bought an automobile!”
“We’re going calling,” said Polly.
“What!” protested Ned. “Calling, did you say? Not much, Polly! At
least, I’m not.”
“Now, Ned Turner—” began Polly.
“Oh, never mind him,” broke in Laurie. “I’ll go calling with you,
Polly. I just love to go calling. Have you any one specially in mind?
Or shall we just take them as they come?”
They were crossing the street now diagonally, Polly and Mae in
the lead. Laurie was smoothing his hair and settling his tie
smirkingly. Ned looked rebellious. “Who are we going to call on?” he
demanded dejectedly.
“You’ll know soon enough,” laughed Polly. And he did, for the next
instant she had pushed open a little gate between the lilac hedges
and was leading the way up the short path to Miss Comfort’s door.
“Gee!” murmured Laurie. But he and Ned followed obediently and
stared questioningly at Polly while somewhere at the rear of the little
house, a bell jangled in response to her tug at the brown crockery
knob. “What’s the big idea?” whispered Laurie to Mae, who was
nearest. But Mae only shook her head. And then, with such
promptitude as to suggest to Ned that he had not just imagined that
face at the front window, the door opened, and Miss Comfort was
giving them welcome. There were introductions in the small hall,
during which Ned trod on Laurie’s foot and Laurie pushed Mae into
an umbrella-stand which had once been a length of drain-pipe and
which now bore a faded design of cat-o’-nine-tails and swallows; and
then, somehow, they were all seated in the front parlor, Laurie, who
had neglected in the confusion to leave his cap in the hall, trying to
stuff it into a side pocket.
The room was not over-furnished. There was a walnut sofa
covered with faded green rep across one corner, a marble-topped
walnut table between the two front windows, a bookcase midway of
the inner wall, a number of straight chairs placed formally along the
sides of the room, and an easy-chair at each window. There were
also two foot-stools covered with crewel work, one of which Ned
narrowly escaped, and a brightly hued Brussels carpet. A fireplace,
surmounted by a white marble shelf, was blankly, inexorably closed
by a glossy black sheet of iron. Two gilt candelabra adorned the
ends of the mantel, and a black marble clock, whose stumpy hands
had stopped at twelve minutes to nine on some long-past day, stood
squarely in the center. There was a purple and green square of
embroidery on the table and a few books of unexciting appearance.
Everything was spotlessly clean, immaculately neat, depressingly
orderly.
Polly and Mae, as usual, crowded into one of the easy-chairs, and
Miss Comfort sat erectly in the other. Miss Comfort proved to be
small and rather thin, with lightish hair that wasn’t brown and wasn’t
white. She had small, delicate features and dark eyes that remained
very bright and clear. Miss Comfort might be nearly seventy, as Polly
had stated, but there was something youthful in her pleasant face,
her quick movements, and her thin, soft voice. Laurie was receiving
these impressions when that thin, soft voice pronounced his name
and he discovered that his hostess had turned from the girls and was
looking toward him, her head pushed forward a little as if, despite
their brightness, her eyes were not as serviceable as they had been.
“Mr. Laurie,” Miss Comfort was saying, “I want to thank you for
your interest in my affairs. I do think it was extremely kind of you to
send that telegram to my brother-in-law. Although I am convinced
that nothing will come of it, I assure you that I appreciate your
helpfulness.”
It was rather a precise and formal little speech, and it is probable
that Miss Comfort had prepared it in advance of the occasion. It left
Laurie surprised and sputtering.
“But—but—why, that’s all right—if you mean—”
Polly came to his rescue: “It was mama who told, Laurie. She
really didn’t mean to, but if you knew her as well as I do you’d know
that she simply can’t keep a secret, no matter how hard she tried.”
“Oh,” said Laurie. “Well, you don’t need to thank me—us a bit,
Miss Comfort. I—we were mighty glad to do anything we could, and
we wish there was more we might do. I guess Polly’s told you that
that—er—that your brother-in-law hasn’t answered yet.”
Miss Comfort nodded. “Yes, and I’m not surprised. Mr. Goupil is a
very busy man, I suppose, and I dare say he hasn’t time to—to look
after all matters himself.”
“Well, if you ask me—” began Laurie indignantly.
“But she hasn’t,” interrupted Ned warningly. “I guess what Laurie
was going to say, Miss Comfort, is that he—that is, we—both of—
neither of us—” Laurie was smiling enjoyably—“can understand how
your brother-in-law could act so—so—”
“Rotten,” supplied the irrepressible Laurie.
“I know,” replied Miss Comfort. “Perhaps I can explain a little. You
might say that Mr. Goupil and I are strangers. Yes, that is scarcely an
exaggeration. My sister Amanda met him in New Jersey fourteen
years ago when she was teaching school there. Amanda was much
younger than I and—and impulsive. I knew nothing about Mr. Goupil
until she wrote to me from Chicago saying that she was married and
on her way west with her husband. I was dreadfully surprised, as you
can well understand, for Amanda was—” Miss Comfort hesitated,
coughed and continued—“was almost fifty years of age, and I had
never thought of her becoming married. In my surprise, I fear that my
letter to her was not—well, quite as sympathetic as it should have
been. I suppose I showed her that I was a little bit hurt because she
had not confided in me earlier. That was most unfortunate, because
it led to a—a misunderstanding. I tried very hard to atone, but she
never forgave me, and after two years she stopped answering my
letters.” Miss Comfort was silent a moment, gazing down at the thin
hands folded in her lap. “I fear,” she went on at length, “Amanda
gathered the impression that I didn’t approve of her husband. Well, I
don’t suppose I did. I mean that I didn’t approve of him for her. You
see, he was younger than Amanda by several years, and then he
was a foreigner.”
“A foreigner!” exclaimed Polly. “Why, I didn’t know that, Miss
Comfort.”
Miss Comfort nodded. “Yes, he was a Frenchman, Polly. Of course
there are undoubtedly many most estimable French gentlemen, but it
did seem to me that if Amanda had to marry she might have found a
man of her own race.” Miss Comfort sighed and then she laughed
apologetically. “I don’t know why I’m telling you all this. Oh, yes, I
was trying to explain about Mr. Goupil, wasn’t I? Well, you see, after
Amanda was married I never saw either her or her husband. They
lived in Chicago a year or so and then moved further west, and after
that I lost all trace of them until I received word lately of Amanda’s
death. After that came this letter from the lawyer about the house.
Maybe, you see, Mr. Goupil doesn’t feel very kindly toward me, and if
he doesn’t I don’t suppose I should blame him one bit.”
“This house belongs to him now?” asked Laurie.
“Yes. My mother left a will that gave everything to Amanda, but
allowed me the use of this place until Amanda’s death. Of course
mother never meant it the way she wrote it. She just got a little mixed
up, and as she didn’t employ a lawyer to do it for her, why, it stood
just as she wrote it. I’ve often wondered,” added Miss Comfort,
wrinkling her forehead, “what she did mean. I suppose she meant
me to live here until my death, and not Amanda’s.”
“I’ll bet you could break a will like that,” declared Laurie eagerly.
“So Mr. Whipple told me,” responded Miss Comfort. “He was the
lawyer. He’s dead now. But I didn’t like to do it. It seemed kind of—of
disrespectful to mother. Besides, I never had any suspicion that I
would outlast poor Amanda.”
In the ensuing silence Polly and Mae gazed sympathetically at
Miss Comfort, who, smoothing the old black dress over her knees,
appeared lost in her thoughts. Finally:
“Well,” began Laurie. Then he stopped, cleared his throat, and
said: “Look here, Miss Comfort, I’d like to ask you— It may sound
cheeky— Well, what I mean is, haven’t you—that is, are you—”
Laurie’s cheeks reddened as he floundered on. “Haven’t you any—
any means at all? Maybe it’s none of my business—”
“No, Mr. Laurie, I haven’t,” replied Miss Comfort quietly. “There
wasn’t ever much money after my father died, and mother’s will left
what there was to Amanda. That was just as it should have been, for
as long as I had this house I was quite all right.” She smiled gently.
“But, land sakes, I don’t want you young folks to trouble your heads
about me and my affairs. Troubles aren’t for the young, Mr. Laurie.”
“That’s all right,” was the dogged response, “but—but something—
somebody— It doesn’t seem right for you to have to go to—to that
place!”
“Why, I don’t know,” said Miss Comfort thoughtfully. “I guess lots of
perfectly respectable folks have gone to the poor-farm. I dare say
there’s no disgrace. And they do say that the—the institution is
conducted very nicely. No doubt I’ll be quite comfortable there. And
—and it isn’t as though I’d have to stay very long.”
“Oh,” exclaimed Ned relievedly, “then you expect to—” But Polly
interrupted him.
“Now, Miss Comfort,” cried Polly indignantly, “don’t you talk like
that! Why, goodness gracious, you aren’t old at all! The—the idea!”
“I should say not!” said Mae warmly. “The idea!”
Miss Comfort chuckled softly. “Well, I ain’t helpless yet, I know,
Polly, but I’m—” she coughed daintily— “I’m getting along in years,
my dear.”
“Seems to me,” exploded Laurie, “there ought to be some place in
this town where you could go. Wouldn’t you a whole lot rather live in
a—a—” he had started to say “barn,” but changed it to—“a—a shed
than go to that poor-farm place?”
“Why, yes, I don’t know but what I would,” said Miss Comfort, “as
long as it had a roof and I could go on with my work. But I’m afraid I
couldn’t even pay the rent for a shed, Mr. Laurie. Now I ain’t going to
let you talk a minute longer about me. Why, I’m just ashamed of
myself!” She arose quickly and crossed to the door with short, firm
steps. “Will you excuse me a minute?” she asked.
When she had gone the four visitors looked at each other silently.
Finally, “Rotten shame, I call it,” muttered Laurie. Ned nodded
agreement. Polly, whose gaze was fixed on Laurie expectantly, said
suddenly: “Laurie, if you have anything in mind I think you’d ought to
tell her. It might make her feel more comfortable.”
“Anything in mind?” echoed Laurie. “I haven’t. At least, only—”
Miss Comfort’s return with a dish of cake stopped him.
A little later they were outside again, walking silently away from
the little white house with the brown shutters. When they were at last
out of sight of the front windows Polly turned eagerly toward Laurie.
“What were you going to say?” she demanded. “You have thought
of some plan, haven’t you?”
Laurie hesitated, frowning thoughtfully. “Not much of a one,” he
answered. “I guess it doesn’t amount to anything. Only—well, now
look here, doesn’t it seem that there ought to be some place
somewhere in this town that would do for her? It wouldn’t have to be
much, would it? Maybe just a sort of shed that could be fixed up and
made comfortable? Or a nice stable that has rooms above it. You
know some stables have quarters for the coachman or chauffeur or
gardener. Maybe—”
“Why, I think it’s a perfectly stunning idea!” cried Polly. “No one
thought of that!”
“But she’d have to pay rent just the same, wouldn’t she?” asked
Ned dubiously. “Some rent, anyhow? And she said—”
“If we explained about her,” said Polly, “I’m sure no one would
think of asking rent for just a stable attic—” Laurie’s chuckles
interrupted. “Well, whatever you call it. Loft, isn’t it? Anyhow, perhaps
just a—a nominal rent would be all they’d ask.”
“Why don’t we look right now and see if we can’t find something?”
asked Mae excitedly.
“Why don’t we?” cried Polly eagerly.
“Just what I was about to propose,” said Laurie a bit patronizingly,
“when Ned butted in. Let’s start in and do the old burg systematically.
Which way shall we go first?”
Dusk had settled over Orstead when the four, footsore and weary,
returned to the shop. Their quest had been fruitless.
CHAPTER X
THE COACH MAKES A PROMISE
“T urner,” said Coach Mulford, taking the vacant place on the

bench beside Laurie and laying a hand on his knees, “Turner,
they tell me you’re grooming a dark horse.”
“Sir?” Laurie looked blank. Pinky’s smile told him that there was a
joke somewhere about, but the phrase was a new one to him and he
didn’t get the coach’s meaning. Mr. Mulford laughed.
“They tell me that you’re training a new pitcher for us,” he
explained. “How about it?”
Laurie reddened a bit. He wasn’t surprised that the coach knew
about it, for his crazy boast and his daily work-outs with Kewpie were
known all over school and he was being joked unmercifully. Those
morning sessions now were being attended by something of a
gallery of interested spectators who were generous with suggestions
and applause. But it occurred to him now that Coach Mulford must
think him rather a fool.
“I—well, I’m sort of helping Kewpie Proudtree,” he answered
haltingly. “He wants to learn to pitch, Mr. Mulford.”
“I see.” The coach evidently didn’t disapprove of the proceeding.
Laurie gathered that from his tones. “I see. How’s he getting on?”
Laurie shook his head. “Not very well,” he said frankly.
“Sorry to hear that,” was the grave reply. “Still, there’s quite a while
yet, and I dare say we’ll manage to get along with Beedle and the
others until your man’s ready.” Mr. Mulford slapped Laurie’s knee
again and again laughed. Laurie laughed, too, but it wasn’t a whole-
hearted laugh. Aware of the coach’s amused regard, he felt slightly
resentful. After a moment he said offhandedly:
“I reckon he’ll be ready for the Farview game, sir.”
“Think so? Fine!” Mr. Mulford chuckled as he arose. “Well, let me
know when he is ready, Turner.”
“If I do will you give him a trial?” asked Laurie quickly.
“What?” Mr. Mulford paused in his departure and looked back.
“Give him a trial? Why, I don’t know, Turner,” he continued slowly,
“but I might.”
“You—you wouldn’t care to make that a promise, would you, sir?”
asked Laurie. Pinky’s round, red face smiled back as, after a
perceptible pause, he nodded.
“Yes, I’ll make it a promise, Turner,” he agreed. “But, mind you,
you mustn’t ask me to waste my time. If your Great Unknown gets so
he can really pitch, you let me know, and I’ll look him over. But no
duds, Turner!”
When, just before supper that evening, Laurie jubilantly repeated
the conversation to Kewpie, Kewpie was all swelled up over that title
of Great Unknown until Ned dryly remarked that most Great
Unknowns never amounted to a hill of beans. Even that pessimistic
utterance failed to dispel all of Kewpie’s pleasure, however.
“That’s all right,” he said. “But some of them make good, don’t
they? Well, here’s one of ’em. You ask Nod if I didn’t pitch some
mighty nice curves this morning.”
“Yeah,” agreed Laurie glumly, “they curved all right, but you
mustn’t think that a batter’s going to step out of his box to hit your
balls, Kewpie. Batters aren’t that accommodating!”
“Gosh,” complained Kewpie, “you don’t give a fellow credit when
he deserves it. If you think it’s any fun going through that stunt every
morning—”
“Who started it?” demanded Laurie.
“Well, that’s all right, but—”
“You’ll get a nice long rest pretty soon,” said Ned soothingly.
“Spring recess’ll be along in less than two weeks, old son.”
Kewpie made no reply for a moment. Then, “Well,” he began
hesitantly, “I was thinking, Nid, that maybe I ought—oughtn’t—
oughtn’t to go home at recess.”
“Not go home! For goodness’ sake, why?”
“Well, I’d lose a whole week, wouldn’t I? You and Laurie will be
here, won’t you?”
“Yes,” replied Ned, with a notable lack of enthusiasm. He and
Laurie weren’t at all keen on remaining at school during the spring
vacation, but it lasted only eight days, and as the journey to
California occupied four, why, as Laurie put it, “they’d meet
themselves coming back!”
“Sure,” continued Kewpie. “Well, I ought to stay, too, I guess, and
get a lot of practice in. Don’t you think so, Nod?”
“Why, I don’t know.” Laurie looked startled. The prospect of seven
long days with nothing to do but to catch Kewpie’s drops and curves
seemed decidedly lacking in attraction. There were moments when
Laurie’s determination wavered, and this was one of them. “I
suppose it would be a mighty good idea, though,” he added listlessly.
Ned’s mouth trembled in a smile.
“Absolutely corking, Kewpie,” he declared. “Of course, you ought
to stay. But what about your folks? Won’t they expect you home?”
Kewpie nodded. “But I wrote yesterday and told them that maybe I
wouldn’t be able to.”
“I’d like to have seen that letter,” chuckled Ned.
Kewpie grinned. “I just told them that I might have to stay here on
account of baseball practice,” he explained innocently.
“Of course,” agreed Ned gravely. “Well, you and Laurie can have a
fine old time during recess. No recitations to bother you or anything.”
“O Death, where is thy sting?” murmured Laurie.
“There is, though,” observed Ned, throwing his legs over the side
of the Morris chair and eyeing Laurie quizzically, “just one
complication that occurs to me. I’ve heard talk of the baseball team
taking a Southern trip during recess. In that case, Laurie, I suppose
you’d go along.”
“Honest?” exclaimed Kewpie anxiously. “I didn’t know that!”
“Nor any one else,” said Laurie, frowning. “Don’t you know yet
when Ned’s joshing?”
“Oh,” breathed Kewpie with immense relief. “I thought maybe—”
“A swell chance I’d have of going with the team if it did go,” said
Laurie. “I can’t play ball. I didn’t make a hit this afternoon. Couldn’t
even see the old pill! Guess I’ll quit and go in for—for soccer or
rowing.”
“Yes, rowing would be nice for you,” said Ned. “You’re so big and
strong! It’s a wonder to me they haven’t grabbed you for the boat
before this!”
“I’ll bet I could row as well as you, you old bluffer!”
“There goes the bell!” yelped Kewpie. “Gosh, I didn’t know it was
so late! S’long!” He collided with a chair and rushed out.
A week passed, a week of ideal weather. The days were mildly
warm and spring-like, and Polly’s possible snow didn’t develop. It
showered occasionally, usually at night, and never enough to
interfere with baseball practice. Tennis came into its own again, and
Bob Starling was torn between the desire to remain at home and
speed the making of the court behind the big house and the longing
to go over to the school field and engage in combat with his ancient
rivals. The crews were on the river daily. The education of Kewpie
Proudtree as a baseball pitcher continued. Laurie regained his
batting eye in a measure and talked no more of abandoning the
diamond for the courts or the four-oared shells. Ned borrowed three
golf-clubs from as many different acquaintances, bought a fourth,
and accompanied Joe Stevenson, captain of last autumn’s football’s
eleven, around the links. Mr. Goupil, of Sioux City, Iowa, continued to
emulate the Sphinx, and Miss Comfort was temporarily installed in
one of the up-stairs rooms at the Widow Deane’s, Polly sleeping in
the room below.
This arrangement had come about as the result of an eleventh-
hour hitch in the program that was to have placed Miss Comfort in
the poor-farm, down the river about two miles. It turned out that
gaining admission to that institution was not such a simple matter as
one might suppose. There was a great deal of red tape to be untied,
and the untying of it occupied the energies of several of Orstead’s
influential citizens. There was no doubt that eventually Miss Comfort
would reach that haven, but meanwhile there ensued a delay that
might last a week—a fortnight—even longer. Bob Starling’s father,
instigated by his sister, who, since the death of Bob’s mother, had
kept house for them, offered very generous assistance of money.
Other individuals had sought to aid, as, too, had the congregation of
the little church that Miss Comfort attended. But all such offers had
been gratefully and firmly declined. Hospitality the little old lady
would have accepted, but charity in the form of money was, to her
mind, something quite different and most repugnant. So, until the last
knot in the mass of red tape had been untied, she was to remain as
Mrs. Deane’s guest, an arrangement that brought as much pleasure
to the Widow and Polly as it did to Miss Comfort.
Even Polly had now accepted the inevitable. That first search for a
modest habitation for the exile had been discouragingly
unsuccessful, as had a second and more half-hearted one, and the
four sympathetic young folks had finally agreed that the situation was
beyond them. If Polly was a wee bit disappointed in Laurie because
of his failure to find a solution of the problem—and I think she was—
she doubtless recognized the injustice of that emotion and
concealed it. Laurie, once satisfied that everything had been done
that could be done, philosophically banished the matter from his
mind. Of course, he was just as sorry as ever for Miss Comfort, but
that didn’t keep him from giving his full attention to matters of more
personal interest, such as trying to beat Elk Thurston out for the
position of first substitute catcher, and striving, sometimes
hopelessly, to make Kewpie into a pitcher. It is always so much
easier to view another’s misfortunes with philosophy than one’s own.
Hillman’s played two games during the week preceding the spring
vacation and won one of them. The second, with Lincolndale High
School, went to ten innings at 7 to 7 and was then called to allow the
visitors to catch a train. Laurie, to his oddly mingled relief and
disgust, saw action in neither of the contests. Elk Thurston took the
place of Cas Bennett, the regular catcher, for the last two innings in
the first encounter, but in the second game Cas worked through to
the end. Laurie had to acknowledge that Elk did pretty well that
Wednesday as a catcher—better, probably, than he could have
done. Laurie’s modesty, though, did not keep him from telling himself
that, while he might have performed less skillfully behind the plate
than Elk had, he was mighty sure he could have done better at the
bat. The Orstead High School pitcher, the third since the beginning
of the game, had nothing on the ball, was, in fact, scarcely more of a
twirler than Kewpie Proudtree, and yet Elk had swung ingloriously at
the first three offerings and had failed to so much as tickle one of
them. “Bet you,” thought Laurie, “I’d have fouled one, anyhow!”
The Lincolndale game was on Friday, and the next day vacation
began. By noon the school was pretty well depopulated, although
there remained a scattering of unfortunate fellows who, like Ned and
Laurie, lived too far from Orstead to allow of a home visit, or who
could not afford the trip. Kewpie had reached a compromise with his
parents. He was to go home and remain until Tuesday morning.
Then he was to return to school and the demands of baseball. Ned
was cynical after Kewpie’s departure.
“Bet you we won’t see Kewpie again until a week from to-morrow,”
he said to Laurie.
Laurie shook his head. “I don’t know,” he replied, “but I have a
hunch that he will be back Tuesday. Kewpie’s taking this pretty
seriously, Ned, and he’s really trying mighty hard. Sometimes I think
that if only he wasn’t so outrageously like a dumpling he could do
something at it!”
CHAPTER XI
ON LITTLE CROW
M ae Ferrand was not on hand the next afternoon when the twins
and Bob Starling reached the Widow Deane’s. Mae, Polly
informed them, had gone to Poughkeepsie to spend Sunday with her
grandmother. They decided to go down to the river for their walk this
afternoon, and were soon descending Walnut Street. At the station
they crossed the tracks, passed the freight-shed, and went
southward beside the river, blue and sparkling in the spring sunlight.
Then they had to return again to the tracks and cross a bridge that
spanned a narrow inlet. The inlet connected the river with a shallow
stretch of marsh and water known as the Basin which lay between
the tracks and the big rock-quarry. The quarry was slowly but very
surely removing the hill called Little Crow, and the face of the quarry
was fully eighty feet in height from the boulder-strewn base to the
tree-topped summit. It was here that stone was being obtained for
the work on which Mr. Starling’s company was engaged. Spur-tracks
ran from the railroad to the base of the high cliff, about two hundred
yards distant, and from the railroad again to the stone-walled dock
wherein the quarry company loaded to lighters for water
transportation. The Basin was a favorite place for skating in winter,
and Ned reminded the others of several episodes of three months
back.
“Remember the time Elk Thurston tried to get ashore over there by
the rushes?” asked Ned. “Every time he put his foot down the ice
broke and let him through.”
“And he got angrier and angrier,” laughed Polly, “and tried to hurry
and—”
“Fell flat,” chuckled Laurie. “They told him the ice wouldn’t hold
him over there, but he always knows a little more than any one else.
And, look, there’s the old Pequot Queen over there yet. It’s a wonder
some one doesn’t take her away or break her up or something.”
“Nobody knows who she belongs to, I heard,” said Bob. “The old
ferry company went bust three or four years back, and the quarry
company can’t touch her because she isn’t theirs. I heard they had a
bill for dockage as long as my arm against the Queen, though.”
“Still, that’s the quarry dock she’s in,” said Ned, “and she must be
in the way there. I don’t see why they don’t push her out and let her
float down the river.”
“She’d be a menace to navigation,” replied Bob knowingly. “The
law would get them if they tried that.”
“Sort of like a fellow driving an automobile into your front yard and
leaving it there and going off,” laughed Laurie. “You couldn’t put it out
into the street because that would be against traffic rules and you
couldn’t take possession of it—”
“You could send it to a garage, though,” said Bob.
“Yes, and pay the garage bills!”
“The quarry folks could see that it got on fire accidently,” said Ned.
“It would only burn to the water-edge. The hull would be just as
much in the way as the whole thing,” objected Bob.
“I hope they’ll let it stay just where it is,” said Polly. “I’m sure it
comes in very handy when we come here skating. Remember that
perfectly ferocious day just after Christmas, Laurie, when we were all
nearly frozen and you made a fire in the—the fireplace—”
“Fireplace!” echoed Ned. “That’s corking!”
“Well, the—the—why, I don’t see why it isn’t a fireplace, Smarty.
It’s the place you build the fire, isn’t it?”
“Boiler,” said Bob.
“Well, anyway, it just about saved my feet from freezing right off,”
declared Polly. “And we had a lot of fun on the boat, and I hope no
one will do anything to it at all!”
“Guess you needn’t worry,” said Laurie. “Looks as if she’d stay
right here and rot to pieces. Guess she’s got a good start already.”
Their homeward way led them through the woods and around the
slope of Little Crow Hill, at first by an old wood-road and then by
devious trails through the now leafless forest. That was the nearer
way, but there was a longer, more arduous, and far more attractive
route that took them to the summit of Little Crow and laid the world at
their feet; for from above the face of the quarry they could look for
miles and miles up and down the broad river and across it and
westward to the rising foot-hills of the mountains. Since to-day was
as clear as a whistle and the air held that crisp quality that makes
exertion a pleasure, Bob’s suggestion that they go up to the top of
the hill was accepted with enthusiasm by Ned and Laurie. Polly,
glancing solicitously at her dress, hesitated. But she was, in the
boys’ parlance, “a good sport,” and she didn’t want to spoil their fun.
So after a brief moment she, too, agreed, although with less
enthusiasm, and they turned northward from the wood-road and
ascended, for a time almost parallel to the railroad, a narrow path
where the branches clutched mischievously at Polly’s skirt and
proved that she had had cause for indecision.
Laurie led, with Polly next. For a while the going was not hard, but
then outcropping boulders set the path to twisting and winding, and
soon they were helping themselves upward by branches and setting
their feet carefully in the moist tangles of root and moss. It was half-
way up a more than usually severe stretch, when every muscle was
tense, that Laurie suddenly stopped short, turned about and
exclaimed “Say!” in such an unexpected and explosive burst of
sound that Polly, thrown from her balance by her attempt to avoid
collision with Laurie, and startled out of her wits, fell back against
Ned. Only Bob’s prompt support from the rear saved the situation.
The three glared at the offender in outrage.
“Say,” exclaimed Ned, “what do you want to do? Break all our
necks? What’s the matter with you, anyway, stopping like that and
shouting like a crazy man?”
Laurie stared back for an instant as though he neither saw Ned
nor heard him. Then his gaze fell and he turned away. “Sorry,” he
muttered.
“But—but what was it?” gasped Polly. “Did you see a snake or—or
something?”
Laurie shook his head and began to climb again. “I just thought of
something,” he said.
“Well, for the love of lime-drops!” scolded his brother. “Don’t think
any more until we get to the top, you poor prune!”
They went on, but it wasn’t difficult to perceive that Laurie wasn’t
obeying Ned’s injunction. If he had been he wouldn’t have stumbled
over everything in his course and he wouldn’t have missed the path
above the big fern-clad rock near the summit and gone wandering off
into the brush all by himself until called back by the others. Ned
observed him pityingly as he sheepishly rejoined them.
“We’ll have to hold you when we get to the top,” said Ned
crushingly. “If we don’t you’ll probably walk right over the edge! What
in the world’s got into you?”
“Nothing,” answered Laurie, an absent expression possessing his
features again. “What are you stopping here for?”
“Well, there is something,” said Ned accusingly, “and I know what
it is. You’ve got some crazy idea in your bean.” He turned to Polly.
“He’s always like that when he thinks he’s discovered something big,
like perpetual motion or—or how to make a million dollars. We’ll
have to watch him until he recovers, or he will do himself harm. You
go first, Bob, and I’ll keep an eye on him.”
The rest of the climb was accomplished without further incident,
and they at last emerged in a small cleared space at the top of the
hill. I don’t mean cleared in the sense of free from rubbish, for
occasional picnic-parties had offended against nature as they have a
way of doing, and the scanty grass was littered with paper and
empty cracker-boxes and an occasional bottle or tin. Ned viewed the
scene disgustedly.
“Funny what human hogs some folks are,” he growled, kicking an
empty olive-bottle over the edge of the cliff. He paused until, after an
appreciable interval, the distant tinkling sound of breaking glass met
his ears. “It’s enough to make you sick. Folks who can’t stand a
speck of dust on their automobile will get out and eat their lunch and
leave the place looking like a pigsty. Ought to be brought back and
made to eat every scrap of the mess they leave behind them.”
“Right-o,” agreed Bob, “but I don’t believe these folks were
automobilists, Ned. It’s a long way up here from the road.”
“Doesn’t matter,” said Ned, “whether they came in a car or walked;
they’re hogs just the same.”
“Well, let’s sit down and get our breaths,” said Polly, suiting action
to words. “That’s a perfectly frightful climb, isn’t it. I don’t think I tore
my dress, though.” She was making inspection and looked vastly
relieved as no damage showed.
“Better luck going down,” said Bob cheerfully, and Polly made a
face at him as he sprawled beside Ned. Laurie had not joined them
on the grass, but instead was lounging toward the edge of the cliff,
his hands in his pockets.
“Laurie, please don’t go so close,” called Polly from a dozen feet
away. “It makes me feel sort of squirmy.”
Perhaps Laurie didn’t hear her. He was very near the edge now,
close by a pine that leaned outward at an angle, its roots clinging to
the thin crust of earth that hid the rock beneath. Ned glanced toward
him, and an expression of disapproval came to his face.
“He thinks he’s smart,” he said contemptuously. “He’s always liked
to walk on roofs and act silly goat that way.” He raised his voice.
“Laurie!”
Laurie gave a start. “Yes?” he answered. Then—well, then
everything happened all at once and with incredible speed. They
saw Laurie grasp suddenly at the leaning tree, saw him miss it, saw
one foot disappear over the edge in a tiny cloud of brown dust, and
then, in almost the same instant, Laurie just wasn’t there!
CHAPTER XII
ON THE QUARRY SHELF
T here was an instant of incredulous horror on the cliff top. Then

Polly’s smothered gasp broke the silence, and the two boys were
on their feet. Short of the edge, Ned faltered for a moment, sick and
trembling, and it was Bob who crouched on hands and knees and
looked first down the steeply sloping face of rock. Beside him the
earth was still trickling where Laurie’s unwary foot had broken off an
overhanging crust.
For a second Bob’s gaze, fearfully searching the rocky débris far
below, saw nothing. Then came a sharp cry of relief from Ned, who
had now dropped beside him, and at the same moment Bob’s gaze,
retraveling the face of rock, fell on Laurie.
About thirty feet below them he was, his feet set on a shelf
scarcely four inches wide, his right hand stretched high and its
fingers hooked over a still narrower ledge, his left hand flung
outward, its palm pressed against the smooth surface. His head
leaned against the raised shoulder, his forehead close to the rock.
Viewed from below the quarry face looked perpendicular, as, indeed,
it was farther around where the height was less, but here there was
a perceptible slope, slight but sufficient to have saved Laurie from a
headlong plunge to the strewn fragments at the base. His cap was
gone and the miniature landslide had powdered his head and
shoulders with red dust.
“Laurie!” called Ned tremulously.
For a space there was no answer. Then Laurie’s voice reached
them, weak and muffled. “Yeah?”
He didn’t raise his face.
“Are you hurt?” asked Bob anxiously.
“No, not—yet.” He stopped and then added, “Scraped a bit.”
“Can you hold on until we—we—” Ned stopped because he
couldn’t think just then what it was they could do.
“I reckon so,” answered Laurie. “Is there ... anything near my left
hand ... I can reach, Ned?”
“No. Wait. Yes, there’s a sort of edge about six inches higher. Can
you reach it? Further up. Nearer you now. That’s it!” Laurie’s
questing fingers had found the spot. It wasn’t much of a hold, only a
bit of rough rock projecting an inch or so from the smooth face. Ned
was suddenly aware that Polly was crouched beside him, crying
nervously. He tried hard to think clearly. After a moment he said:
“Laurie, we’re going for a rope. It will take some time, but—but it’s
the only thing I can think of. Can you hold on until we get back?”
“I’ll stick,” was the grim answer. His voice was clearer now and
steadier. “How far down am I?”
“About thirty feet.” Ned stumbled to his feet. “No use both of us
going, Bob,” he said hurriedly. “You stay. And Polly. I guess I can find
rope at the quarry.” He was off then, running down the path. Bob
dropped to his knees again beside Polly. Polly was speaking, trying
to make her voice steady and confident.
“It won’t be long, Laurie,” she called. “Be—be brave and—”
“Hello, Polly,” answered Laurie from below, a faint reminder of his
old insouciance in his voice. “Nice fix, eh?”
“Yes, but don’t worry, and—you’d better not talk.”
“Guess I’d rather,” answered Laurie. “Sort of keeps me from
thinking about—things.” After a moment he continued. “Position’s
sort of cramped, Polly. Bob there, or did he go, too?”
“No, I’m here,” answered Bob. “I’ve been thinking—”
“Don’t do it,” said Laurie. “I tried it, and now look at me! Wish my
legs wouldn’t tremble. How wide’s the thing I’m standing on, Bob?”
“Three inches. Maybe four. What I was—”

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Studies in natural products chemistry.

Volume 58. Bioactive natural products

Copyright © 2018 Elsevier B.V. All rights reserved.

No part of this publication may be reproduced or transmitted in any form or by any

For information on all Elsevier publications

Publisher: Susan Dennis

1. Plant Lectins: Bioactivities and Bioapplications 1

2. The Role of NADP(H) Oxidase Inhibition and Its

3. The Current and Potential Therapeutic Uses

4. Caged Garcinia Xanthones: Synthetic Studies

Synthesis of Forbesione and Structurally Related Natural Products 101

5. Phytoestrogens as Potential Therapeutic Agents

6. Applications of Flavonoids, With an Emphasis on

Bioavailability of the Flavonoids and Hesperidin 169

7. Augmented Cell Signaling by Natural Polyphenols

Clinical Trials of Tea ( )-Epigallocatechin-3-Gallate:

8. Beneficial Effects of Anthocyanin From Natural

9. Quercetin: Prooxidant Effect and Apoptosis

10. Bioactive Components of Saffron and Their

11. Anthraquinones: An Overview 313

12. The Genus Hymenaea (Fabaceae): A Chemical

Hymenaea stigonocarpa Mart. ex Hayne 379

13. Phenolic Acids From Plants: Extraction

14. Cannabidiol in Refractory Epilepsy 419

Sugapriya Dhanasekaran (213), College of Applied Medical Sciences, Prince Sattam

Ljubica Tasic (161), Institute of Chemistry, University of Campinas (UNICAMP);

The 58th volume of Studies in Natural Products Chemistry comprises

Diaz-Muñoz et al. in Chapter 11 have presented the different characteris-

Plant Lectins: Bioactivities

Studies in Natural Products Chemistry, Vol. 58. https://doi.org/10.1016/B978-0-444-64056-7.00001-5

PLANT LECTINS: INTRODUCTION

TABLE 1.1 Plant Lectins Examples and Their Families

Amaranthus Amaranthin Amaranthin Amaranthaceae

Artocarpus Jacalin Jacalin Moraceae

Canavalia Concanavalin A ConA Leguminosae

Croton tiglium Crotin Crotin Euphorbiaceae

Dolichos biflorus Dolichos biflorus DBA Leguminosae

Galanthus nivalis Galanthus nivalis GNA Amaryllidaceae

Griffonia Griffonia simplicifolia GSL Leguminosae

Lycopersicum Tomato lectin LEL Solanaceae

Lupinus albus Blad-containing BCO Leguminosae

Pisum sativum Pea agglutinin PSA Leguminosae

TABLE 1.1 Plant Lectins Examples and Their Families—Cont’d

Plant Species Lectin Abbrev. Family

cells and/or precipitate glycoconjugates, e.g., Phaseolus vulgaris agglutinin

PLANT LECTINS: LEGUME LECTINS

The Carbohydrate-Recognition Domain

TABLE 1.2 Lectin Plant Families

Oral Toxicity to Higher Animals

TABLE 1.3 Examples of Animal Lectin Toxicity by Ingestion

Lectin LD50 (mg/kg Body Weight) Animal Reference

“T urner,” said Coach Mulford, taking the vacant place on the

T here was an instant of incredulous horror on the cliff top. Then

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