Planta Med 2018; 84(09/10): 684-695
DOI: 10.1055/a-0590-5153
Biological and Pharmacological Activity
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Discovery of Bioactive Natural Products for the Treatment of Acute Respiratory Infections – An Integrated Approach[*]

Ulrike Grienke
1   Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Vienna, Austria
,
Christina E. Mair
1   Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Vienna, Austria
,
Johannes Kirchmair
2   Center for Bioinformatics, Department of Informatics, MIN Faculty, Universität Hamburg, Hamburg, Germany
,
Michaela Schmidtke
3   Section of Experimental Virology, Department of Medical Microbiology, Jena University Hospital, Jena, Germany
,
Judith M. Rollinger
1   Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, Vienna, Austria
› Author Affiliations
Further Information

Correspondence

Univ.-Prof. Mag. pharm. Dr. Judith M. Rollinger
Department of Pharmacognosy
Faculty of Life Sciences
University of Vienna
Althanstrasse 14
1090 Vienna
Austria   
Phone: + 43 14 27 75 52 55   
Fax: + 43 1 42 77 85 52 55   

Publication History

received 16 November 2017
revised 01 March 2018

accepted 07 March 2018

Publication Date:
19 March 2018 (online)

 

Abstract

In this work, an integrated approach for the identification of new antiviral agents from natural sources for the treatment of acute respiratory infections is presented. The approach comprises (i) the selection of starting material based on traditional knowledge, (ii) phenotypic screening of extracts for antiviral activity, and (iii) the implementation of in silico predictions to identify antiviral compounds and derive the molecular mechanism underlying their biological activity. A variety of starting materials from plants and fungi was selected for the production of 162 extracts. These extracts were tested in cytopathic effect inhibition assays against influenza virus A/Hong Kong/68 (HK/68), rhinovirus A2 (RV-A2), and coxsackie virus B3 (CV-B3). All extracts were also evaluated regarding their cytotoxicity. At an IC50 threshold of 50 µg/mL, 20, 11, and 14% of all tested extracts showed antiviral activity against HK/68, CV-B3, and RV-A2, respectively. Among all active extracts (n = 47), 68% showed antiviral activity against one of the investigated viruses, whereas 31% inhibited at least two viruses. Herein, we present a comprehensive dataset of probed extracts along with their antiviral activities and cytotoxicity. Application examples presented in this work illustrate the phytochemical workflow for the identification of antiviral natural compounds. We also discuss the challenges, pitfalls, and advantages of the integrated approach.


#

Abbreviations

HK/68: influenza virus A/Hong Kong/68 (H3N2)
ARI: acute respiratory infection
CC50 : 50% cytotoxic concentration
CPE: cytopathic effect
CV: coxsackie virus
CV-B3: coxsackie virus B3
EV: enterovirus
RV: rhinovirus
RV-A2: rhinovirus type A2
IAV: influenza A viruses
IBV: influenza B viruses
IV: influenza viruses
LLE: lead-like enhanced extract
MDCK: Madin-Darby canine kidney
NA: neuraminidase
NAI: neuraminidase inhibitor
RNA: ribonucleic acid
SI: selectivity index
 

Introduction

Acute respiratory infections (ARIs) affect the lives of millions of people each year. They are the leading cause of morbidity and mortality related to infectious diseases worldwide [1]. ARIs are typically caused by enteroviruses (EVs), e.g., coxsackie viruses (CVs) and the closely related rhinoviruses (RVs), or influenza A viruses (IAVs) and influenza B viruses (IBVs). Virus infections might also occur in combination with bacterial infections caused by Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, or Pseudomonas aeruginosa [2], [3], [4].

Neither vaccines nor antiviral drugs are available for the prevention or treatment of EV infections [5], [6]. For IAV and IBV infections, the gold standard for prevention is vaccination [7], [8]. Treatment options are limited to ion channel blockers (M2 inhibitors) and NAIs. Most circulating IAVs are resistant to approved M2 inhibitors (amantadine and rimantadine in particular) [9]. Also, the portfolio of NAIs is small, with oseltamivir and zanamivir being the only two NAIs approved in most countries. Further NAIs include laninamivir, approved in Japan, and peramivir, approved in Japan, China, South Korea, and the USA [10]. Japan was the first country to approve the stockpiling of favipiravir, an RNA polymerase inhibitor, for use during influenza pandemics in 2016 [11].

The risk of emerging NAI-resistant IAVs was demonstrated by the recent local [12], [13] and global [14] outbreaks of seasonal H1N1 IAV that acquired mutations compensating the fitness loss caused by the H275Y mutation [12].

The lack of anti-EV drugs, the limited efficacy of NAIs against IVs, resistance issues, and the limited availability of favipiravir demand for the identification of novel highly active anti-EV and anti-IV agents as leads for drug development. Natural products are a primary resource for the discovery and development of new antivirals [15]. For the identification of bioactive secondary metabolites from plant, fungal, microbial, or marine sources, a variety of approaches such as (i) the exploitation of chemotaxonomic or ethnopharmacological knowledge, (ii) extract screening followed by bioassay-guided isolation, and (iii) computational approaches have been developed and applied [16], [17]. Probing multicomponent mixtures and their constituents is generally approached by two different strategies: (1) phenotypic screening, where the identification of promising starting material is based on the constituentsʼ ability to trigger a desired biological response without knowledge of the underlying mode of action, and (2) target-based screening, where a specific working hypothesis serves as a starting point for the identification of bioactive compounds effective on a specific molecular target [18].

In this study, an integrated approach combining, in particular, knowledge from traditional medicine and phenotypic screening of extracts is presented. The objective was to identify antiviral natural products against three pathogens involved in ARIs: HK/68, RV-A2, and CV-B3. The application, scope, and limitations of this integrated approach are discussed and supported by a number of examples. Screening data of 162 extracts are presented to guide the selection of further promising candidates for the discovery of natural products targeting viral proteins.


#

Results and Discussion

Ethnopharmacological sources comprising the books “De materia medica” by Pedanios Dioscurides [19], “Naturalis historiae libri” by Plini the Elder [20], “Das große Buch der Heilpflanzen” and sources cited therein [21], and the published final report of an EU-Interreg-II project, “Volksmedizin in Tirol” [22] served as a starting point. The key words “cough”, “cold”, “catarrh”, “sore throat”, “fever”, “lung disease”, “pneumonia”, “flu”, “bronchitis”, and “pleuritis” were used for searching in these four sources to select plant and fungal species with a traditional background for the treatment of symptoms related to ARIs.

The final selection comprised 141 diverse plant and fungal species belonging to 66 different families, with Asteraceae (10%), Lamiaceae (10%), Apiaceae (6%), and Fabaceae (4%) being the most prominent ones ([Table 1]). For some of the selected species, individual extracts were produced from different organs, resulting in a total of 162 extracts.

Table 1 Antiviral activity and cytotoxicity of extracts. Their mean IC50 values against IV A/Hong Kong/68 (HK/68), coxsackie virus (CV-B3), and rhinovirus (RV-A2) in the CPE inhibition assay (MDCK cells for HK/68; HeLa cells for CV-B3 and RV-A2) as well as their mean CC50 values are presented (n = 3).

Code

Species

Family

Organ

Extract type

HK/68

CV-B3

RV-A2

CC50 [µg/mL] in

IC50 [µg/mL] of CPE

Selectivity index [CC50/IC50]

IC50 [µg/mL] of CPE

Selectivity index [CC50/IC50]

IC50 [µg/mL] of CPE

Selectivity index [CC50/IC50]

MDCK cells

HeLa cells

*No IC50 could be determined due to toxicity. The last noncytotoxic test concentration with an antiviral effect is reported instead. Abbreviations: n. a. = not active, n. d. = not determined, D = dichloromethane, M = methanol, LLE = lead-like enhanced extracts [23], [24], H2O = water, E = ethanol

1

Abutilon theophrasti Medik.

Malvaceae

seeds

LLE

n. a.

n. a.

n. a.

> 100

> 100

2

Aegle marmelos (L.) Corrêa

Rutaceae

fruit

LLE

n. a.

n. d.

n. d.

> 100

n. d.

3

Allium sativum var. sativum L.

Amaryllidaceae

bulb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

4

Anchieta pyrifolia (Mart.) G. Don

Violaceae

herb

LLE

n. a.

n. a.

n. a.

> 100

> 100

5

Andographis paniculata (Burm. F.) Nees

Acanthaceae

herb

LLE

79

> 1.3

n. a.

n. a.

> 100

64

6

Angelica sinensis (Oliv.) Diels

Apiaceae

root

LLE

n. a.

n. a.

n. a.

> 100

> 100

7

Annona squamosa L.

Annonaceae

seeds

LLE

n. a.

n. a.

n. a.

n. d.

6.3

8

Aquilegia vulgaris L.

Ranunculaceae

herb

LLE

n. a.

n. d.

n. d.

> 100

n. d.

9

Arctostaphylos uva-ursi (L.) Spreng.

Ericaceae

leaves

LLE

n. a.

n. a.

n. a.

n. d.

82

10

Arisaema sp.

Araceae

rhizome

LLE

68

> 1.5

n. a.

32*

> 100

68

11

Artemisia absinthum L.

Asteraceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

129

12

Artemisia anomala S. Moore

Asteraceae

herb

LLE

n. a.

n. a.

n. a.

> 100

> 100

13

Artemisia argyi Levl. & Vant.

Asteraceae

leaves

LLE

24

2.5

n. a.

32*

60

67

14

Artemisia vulgaris L.

Asteraceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

15

Aster tataricus L. f.

Asteraceae

root

LLE

n. a.

n. a.

n. a.

> 100

> 100

16

Azadirachta indica A. Juss.

Meliaceae

fruit

LLE

n. a.

n. d.

n. d.

84

n. d.

17

Boswellia serrata Roxb. ex Colebr.

Burseraceae

resin

D

9.0

2.4

n. d.

n. a.

21

16

18

Buddleja officinalis Maxim.

Loganiaceae

flowers

LLE

n. a.

n. a.

n. a.

> 100

81

19a

Burkea africana Hook.

Fabaceae

bark

D

24

3

32*

29

2.1

71

60

19b

Burkea africana Hook.

Fabaceae

bark

M

5.6

11

n. a.

n. a.

63

16

20a

Burkea africana Hook.

Fabaceae

heartwood

D

22

> 4.5

n. a.

n. a.

> 100

> 100

20b

Burkea africana Hook.

Fabaceae

heartwood

M

48

> 2.1

32*

n. a.

> 100

74

21

Calamintha menthifolia L.

Lamiaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

22

Calendula officinalis L.

Asteraceae

flowers

LLE

n. a.

n. d.

n. d.

> 100

n. d.

23

Capsella bursa-pastoris (L.) Medik.

Brassicaceae

herb

LLE

44

2.9

n. a.

n. a.

126

> 200

24

Carlina acaulis L.

Asteraceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

145

25

Carlina acaulis L.

Asteraceae

root

LLE

n. a.

n. a.

n. a.

n. d.

> 200

26

Carum carvi L.

Apiaceae

fruit

LLE

n. a.

n. a.

n. a.

n. d.

> 200

27

Castanea sativa Mill.

Fagaceae

leaves

LLE

n. a.

46

1.7

n. a.

n. d.

80

28a

Centaurea ragusina L.

Asteraceae

leaves

LLE

2.7

2.4

n. a.

n. a.

6.4

9.0

28b

Centaurea ragusina L.

Asteraceae

leaves

H2O

n. a.

n. d.

n. d.

62

n. d.

28c

Centaurea ragusina L.

Asteraceae

leaves

E

n. a.

n. d.

n. d.

2.3

n. d.

29

Cetraria islandica (L.) Ach.

Parmeliaceae

thallus

LLE

44

3.3

50*

50*

148

91

30

Chenopodium ambrosioides (L.) Mosyakin & Clemants

Amaranthaceae

leaves

LLE

75

> 1.3

n. a.

n. a.

> 100

> 100

31

Chrysanthemum indicum L.

Asteraceae

flowers

LLE

n. a.

n. a.

n. a.

> 100

> 100

32

Cinnamomum mairei H. Lév.

Lauraceae

bark

LLE

n. a.

n. a.

n. a.

> 100

> 100

33

Cordia curassavica (Jacq.) Roem. & Schult.

Boraginaceae

leaves

LLE

30

2.0

n. a.

n. a.

60

31

34

Cynanchum paniculatum (Bunge) Kitag. ex H.Hara

Apocynaceae

root

LLE

n. a.

n. a.

n. a.

4.7

> 100

35

Cynanchum stauntonii (Decne.) Schltr. ex H.Lév.

Apocynaceae

root

LLE

9.1

2.1

n. a.

n. a.

19

> 100

36

Cynomorium songaricum Rupr.

Cynomoriaceae

herb

LLE

n. a.

n. a.

n. a.

> 100

> 100

37

Daucus carota L.

Apiaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

38

Drynaria fortunei (Kunze) J. Sm.

Polypodiaceae

rhizome

LLE

n. a.

n. a.

n. a.

> 100

> 100

39

Elettaria cardamomum (L.) Maton

Zingiberaceae

fruit

LLE

n. a.

n. d.

n. d.

> 100

n. d.

40

Epimedium sagittatum (Sieb. & Zucc.) Maxim.

Berberidaceae

herb

LLE

86

> 1.2

n. a.

n. a.

> 100

> 100

41

Equisetum arvense L.

Equisetaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

42

Equisetum hiemale L.

Equisetaceae

herb

LLE

n. a.

n. a.

n. a.

> 100

> 100

43

Euphrasia officinalis ssp. rostkoviana Hayne (L.)

Orobanchaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

44

Evodia rutaecarpa (Juss.) Benth.

Rutaceae

fruit

LLE

n. a.

1.0*

n. a.

48

0.8

45

Fagopyrum esculentum Moench.

Polygonaceae

seeds

LLE

n. a.

n. a.

n. a.

> 200

> 100

46

Foeniculum vulgare L.

Apiaceae

fruit

LLE

n. a.

n. a.

n. a.

n. d.

152

47

Fomes fomentarius J. J. Kickx. (strain 19)

Polyporaceae

fruit body

E

n. a.

n. a.

n. a.

> 100

> 100

48

Fomitopsis pinicola Karst. (strain 10)

Fomitopsidaceae

fruit body

E

n. a.

n. a.

n. a.

18

16

49

Forsythia suspensa (Thunb.) Vahl

Oleaceae

fruit

LLE

22

3.5

32*

n. a.

76

40

50

Fraxinus sp.

Oleaceae

bark

LLE

91

> 1.1

n. a.

91

> 1.1

> 100

> 100

51

Galeopsis tetrahit L.

Lamiaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

52

Ganoderma applanatum (Pers.) Pat. (strain 12)

Ganodermataceae

fruit body

E

n. a.

n. a.

10*

18

16

53

Ganoderma lucidum Karst.

Ganodermataceae

fruit body

LLE

37

2.5

n. a.

n. a.

94

54

54

Ganoderma sinense Zhao, Xu & Zang

Ganodermataceae

fruit body

LLE

n. a.

n. a.

n. a.

> 100

> 100

55

Ganoderma tsugae Murill.

Ganodermataceae

fruit body

E

n. a.

n. a.

28

2.0

48

55

56

Gardenia jasminoides Ellis.

Rubiaceae

fruit

LLE

n. a.

n. a.

n. a.

> 100

> 100

57

Glechoma hederacea L.

Lamiaceae

herb

LLE

n. a.

n. a.

> 50

n. d.

109

58

Gleditsia sinensis Lam.

Fabaceae

fruit

LLE

n. a.

n. a.

n. a.

66

29

59

Gloeophyllum odoratum Imazeki (strain 23)

Gloeophyllaceae

fruit body

E

13

> 7.7

36

2.2

19

4.1

> 100

77

60

Gloeophyllum odoratum Imazeki (strain 28)

Gloeophyllaceae

fruit body

E

9.4

> 11

16

2.5

27

1.4

> 100

39

61

Gloeophyllum odoratum Imazeki (strain 54)

Gloeophyllaceae

fruit body

E

15

> 6.8

31

> 3.3

16

> 6.3

> 100

> 100

62

Glycyrrhiza glabra L.

Fabaceae

root

LLE

n. a.

n. a.

47

> 4.3

n. d.

> 200

63

Hedera helix L.

Araliaceae

leaves

LLE

n. a.

n. a.

n. a.

n. d.

> 200

64

Helianthus annuus L.

Asteraceae

flowers

LLE

n. a.

n. a.

n. a.

n. d.

> 200

65

Hepatica nobilis Schreb.

Ranunculaceae

leaves

LLE

n. a.

n. a.

n. a.

n. d.

> 200

66

Hericium erinaceus (Bull.) Pers.

Hericiaceae

fruit body

LLE

n. a.

n. a.

n. a.

> 100

> 100

67

Imperata cylindrica var. major (Nees) C. E. Hubb

Poaceae

root

LLE

n. a.

n. a.

n. a.

> 100

> 100

68

Inonotus obliquus (Ach. ex Pers.) Pilát

Hymenochaetaceae

fruit body

LLE

31

4.7

n. a.

n. a.

147

52

69

Ischnoderma benzoinum Karst. (strain 38)

Fomitopsidaceae

fruit body

E

n. a.

n. a.

32*

68

71

70

Kaempferia galanga L.

Zingiberaceae

rhizome

LLE

n. a.

n. d.

n. d.

> 100

n. d.

71

Lactuca sativa L.

Asteraceae

herb

LLE

n. a.

n. d.

n. d.

> 100

n. d.

72

Laetiporus sulphureus (Bull.) Murrill (strain 43)

Fomitopsidaceae

fruit body

E

n. a.

n. a.

n. a.

> 100

62

73

Lantana camara L.

Verbenaceae

leaves

LLE

n. a.

32*

n. a.

66

25

74

Lepidium apetalum Willd.

Apiaceae

seeds

LLE

n. a.

32*

n. a.

> 100

88

75

Liquidambar orientalis Mill.

Hamamelidaceae

resin

LLE

47

2.0

n. a.

13*

93

31

76

Lonicera japonica Thunb.

Caprifoliaceae

twigs

LLE

n. a.

n. a.

n. a.

> 100

> 100

77

Lophaterum gracile Brongn.

Poaceae

herb

LLE

n. a.

n. a.

n. a.

> 100

> 100

78

Lycopodium clavatum L.

Lycopodiaceae

herb

LLE

n. a.

n. a.

50*

n. d.

78

79

Lycopodium clavatum L.

Lycopodiaceae

spores

LLE

n. a.

n. a.

n. a.

> 200

122

80

Lycopus lucidus var. hirtus Regel.

Lamiaceae

herb

LLE

76

> 1.3

32*

n. a.

> 100

96

81

Lygodium japonicum (Thunb.) Sw.

Lygodiaceae

spores

LLE

41

> 2.4

n. a.

n. a.

> 100

> 100

82

Magnolia sp.

Magnoliaceae

flowers

LLE

n. a.

n. a.

n. a.

> 100

40

83

Matricaria chamomilla L.

Asteraceae

flowers

LLE

n. a.

n. a.

n. a.

n. d.

> 200

84

Melissa officinalis L.

Lamiaceae

leaves

LLE

n. a.

n. a.

n. a.

n. d.

84

85

Morus alba L.

Moraceae

root bark

M

30

> 3.4

n. d.

n. d.

> 100

n. d.

86

Nelumbo nucifera Gaertn.

Nelumbonaceae

leaves

LLE

n. a.

n. a.

n. a.

n. d.

75

87

Nelumbo nucifera Gaertn.

Nelumbonaceae

root

LLE

48

1.5

22

1.9

13*

74

42

88

Nelumbo nucifera Gaertn.

Nelumbonaceae

seeds

LLE

n. a.

n. a.

n. a.

n. d.

> 200

89

Origanum vulgare L.

Lamiaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

90

Papaver rhoeas L.

Papaveraceae

flowers

LLE

n. a.

n. a.

n. a.

n. d.

> 200

91

Papaver somniferum L.

Papaveraceae

seeds

LLE

n. a.

n. a.

n. a.

n. d.

> 200

92

Peucedanum ostruthium (L.) Koch

Apiaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

93

Peucedanum ostruthium (L.) Koch

Apiaceae

root

LLE

48

1.2

n. a.

n. a.

66

45

94

Pharbitis sp.

Convolvulaceae

seeds

LLE

32

1.2

n. a.

n. a.

39

20

95

Phellinus robustus (L.) Quel. (strain 25)

Hymenochaetaceae

fruit body

LLE

92

> 1.2

n. a.

n. a.

> 100

88

96

Pimenta dioica (L.) Merr.

Myrtaceae

fruit

LLE

n. a.

n. d.

n. d.

> 100

n. d.

97

Pimpinella anisum L.

Apiaceae

fruit

LLE

n. a.

n. a.

n. a.

n. d.

> 200

98

Pimpinella major (L.) Huds.

Apiaceae

root

LLE

n. a.

n. a.

n. a.

n. d.

155

99

Pinguicula vulgaris L.

Lentibulariaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

100

Piper nigrum L.

Piperaceae

fruit

LLE

n. a.

11

1.5

n. a.

n. d.

17

101

Piptoporus betulinus Karst. (strain 29)

Fomitopsidaceae

fruit body

E

10*

10*

8.3

2.3

54

22

102

Piptoporus betulinus Karst. (strain 39)

Fomitopsidaceae

fruit body

E

10*

10*

9.9

3.8

40

38

103

Plantago lanceolata L.

Plantaginaceae

leaves

LLE

n. a.

n. a.

n. a.

n. d.

> 200

104

Polygala senega L.

Polygalaceae

root

LLE

n. a.

n. a.

n. a.

n. d.

147

105

Polygala sp.

Polygalaceae

root

LLE

n. a.

n. a.

n. a.

> 100

> 100

106

Polygala vulgaris L.

Polygalaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

107

Polygonum aviculare L.

Polygonaceae

herb

LLE

n. a.

n. d.

n. d.

> 100

n. d.

108

Polypodium vulgare L.

Polypodiaceae

root

LLE

n. a.

n. a.

n. a.

n. d.

> 200

109

Potentilla anserinae L.

Rosaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

110

Potentilla aurea L.

Rosaceae

herb

LLE

n. a.

n. d.

n. d.

> 100

n. d.

111

Prunella grandiflora D. Torre & Sarnth.

Lamiaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

112

Pterocarpus santalinus L. f.

Fabaceae

wood

LLE

12

4.6

n. d.

n. d.

54

n. d.

113

Pyrrosia sp.

Polypodiaceae

leaves

LLE

n. a.

n. a.

n. a.

> 100

> 100

114

Ribes nigrum L.

Grossulariaceae

leaves

LLE

n. a.

> 50

n. a.

n. d.

89

115

Ribes nigrum L.

Grossulariaceae

fruit

LLE

n. a.

n. a.

n. a.

n. d.

> 200

116

Rosa canina L.

Rosaceae

fruit

LLE

n. a.

n. a.

n. a.

n. d.

> 200

117

Rosmarinus officinalis L.

Lamiaceae

leaves

LLE

n. a.

8.0

4.0

n. a.

n. d.

32

118

Rubus fruticosus L.

Rosaceae

leaves

LLE

n. a.

n. a.

n. a.

n. d.

> 200

119

Rubus fruticosus L.

Rosaceae

root

LLE

n. a.

n. a.

n. a.

n. d.

> 200

120

Ruta graveolens L.

Rutaceae

herb

LLE

8.5

< 0.7

3.5

3.5

n. a.

< 6.3

12

121

Salvia glutinosa L.

Lamiaceae

herb

LLE

n. a.

50*

25*

n. d.

127

122

Sambucus nigra L.

Adoxaceae

flowers

LLE

n. a.

n. a.

n. a.

n. d.

> 200

123

Sambucus nigra L.

Adoxaceae

fruit

LLE

n. a.

n. a.

n. a.

n. d.

> 200

124

Saussurea costus (Falc.) Lipsch.

Asteraceae

root

LLE

n. a.

n. d.

n. d.

56

n. d.

125a

Sclerocarya birrea (A. Rich.) Hochst.

Anacardiaceae

bark

D

13

> 7.9

n. a.

n. a.

> 100

46

125b

Sclerocarya birrea (A. Rich.) Hochst.

Anacardiaceae

bark

M

3.4

> 29

n. a.

n. a.

> 100

> 100

125c

Sclerocarya birrea (A. Rich.) Hochst.

Anacardiaceae

bark

E

3.9

> 26

n. d.

n. d.

> 100

n. d.

126a

Sclerocarya birrea (A. Rich.) Hochst.

Anacardiaceae

heartwood

D

38

> 2.6

n. a.

n. a.

> 100

96

126b

Sclerocarya birrea (A. Rich.) Hochst.

Anacardiaceae

heartwood

M

n. a.

n. a.

n. a.

> 100

> 100

127

Scrophularia nodosa L.

Scrophulariaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

128

Scrophularia nodosa L.

Scrophulariaceae

root

LLE

n. a.

n. a.

n. a.

n. d.

> 200

129

Scutellaria barbata D. Don

Lamiaceae

herb

LLE

n. a.

n. a.

n. a.

> 100

> 100

130

Sida cordifolia L.

Malvaceae

herb

LLE

n. a.

n. d.

n. d.

> 100

n. d.

131

Sinomenium acutum (Thunb.) Rehd. & Wils.

Menispermaceae

twigs

LLE

n. a.

n. a.

n. a.

> 100

> 100

132

Solanum dulcamara L.

Solanaceae

twigs

LLE

n. a.

n. a.

50

> 4

n. d.

> 200

133

Solanum paniculatum L.

Solanaceae

leaves

LLE

n. a.

n. a.

n. a.

77

51

134

Solanum pseudoquina A. St.-Hil.

Solanaceae

leaves

LLE

81

> 1.2

n. a.

n. a.

> 100

87

135

Solanum torvum Sw.

Solanaceae

leaves

LLE

n. a.

n. a.

n. a.

> 100

> 100

136

Sophora flavescens Ait.

Fabaceae

root

LLE

n. a.

32*

n. a.

> 100

60

137

Stachys officinalis L.

Lamiaceae

herb

LLE

n. a.

n. a.

50

> 4

n. d.

> 200

138

Stachys sylvatica L.

Lamiaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

139

Stemona sp.

Stemonaceae

root

LLE

n. a.

n. a.

n. a.

> 100

> 100

140

Styrax calamitus L.

Styracaceae

resin

LLE

50

1.5

n. a.

13*

74

59

141

Syzygium aromaticum (L.) Merr. & L. M.Perry

Myrtaceae

flowers

LLE

73

> 1.4

n. d.

n. d.

> 100

n. d.

142

Terminalia chebula Retz.

Combretaceae

fruit

LLE

14

2.0

n. a.

n. a.

28

33

143

Teucrium chamaedrys L.

Lamiaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

144

Thymus pulegioides L.

Lamiaceae

herb

LLE

n. a.

n. a.

50

3.0

n. d.

152

145

Tilia cordata Mill.

Malvaceae

flowers

LLE

n. a.

n. a.

n. a.

n. d.

> 200

146

Trametes gibbosa (Pers.) Fr. (strain 52)

Polyporaceae

fruit body

E

n. a.

n. a.

n. a.

> 100

> 100

147

Tussilago farfara L.

Asteraceae

leaves

LLE

n. a.

n. a.

n. a.

n. d.

> 200

148

Vaccinium vitis-ideae L.

Ericaceae

leaves

LLE

n. a.

n. a.

n. a.

n. d.

> 200

149

Valeriana officinalis L.

Valerianaceae

root

LLE

n. a.

n. a.

n. a.

n. d.

> 200

150

Verbascum densiflorum Bertol.

Scrophulariaceae

flowers

LLE

n. a.

n. a.

n. a.

n. d.

> 200

151

Verbena officinalis L.

Verbenaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

152

Veronica officinalis L.

Plantaginaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

153

Viola odorata L.

Violaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

154

Viola tricolor L.

Violaceae

herb

LLE

n. a.

n. a.

n. a.

n. d.

> 200

155

Viscum coloratum (Komar.) Nakai

Loranthaceae

herb

LLE

n. a.

n. a.

n. a.

> 100

> 100

A recently reported protocol for the preparation of LLEs [23], [24] was adapted for the production of small-scale extracts of the acquired natural materials. Additionally, 15 plant extracts (produced either by extraction with dichloromethane, methanol, ethanol, or water) were included in the extract screening ([Table 1]).

The ability of extracts to inhibit the virus-induced CPE was evaluated with a phenotypic assay described previously [25], [26]. In this work, multicomponent mixtures were screened in a cellular model to test whether the contained compounds were able to protect the cells from the respective virus. The observed effects served as a basis for further experiments to determine the bioactive compounds and their biological targets. All 162 extracts were tested for their anti-influenza activity against HK/68 in MDCK cells. The majority of the extracts (88%) were also tested against CV-B3 and RV-A2 in HeLa cells ([Table 1] and [Fig. 1 a]). Forty-seven extracts (29%) were active (i.e., having IC50 values no higher than 50 µg/mL) against at least one of the three viruses. Of these extracts, 33 (20%), 16 (11%), and 20 (14%) extracts were active against HK/68, CV-B3, and RV-A2, respectively. IC50 values were below 30 µg/mL for 21 (13%), 8 (6%), and 14 (10%) extracts, respectively. Sixty-eight percent showed antiviral activity (defined as IC50 values no higher than 50 µg/mL) against one of the investigated viruses, whereas 15 extracts (31%) inhibited at least 2 viruses. Cytotoxicity was determined for all samples as a prerequisite for the estimation of selectivity of antiviral activity. The SI of each antiviral active extract was calculated to evaluate the specificity of antiviral activity ([Table 1]). Raw data for the determination of IC50 and CC50 values (and respective CI) of the most active extracts are given in Table 2S, Supporting Information.

Two-dimensional plots of activity and cytotoxicity data allowed for the prioritization and targeted selection of starting materials for further phytochemical and pharmacological investigations ([Fig. 1]). In particular, the extracts were classified into four different categories based on their measured antiviral activity and cytotoxicity ([Fig. 1 a]).

Zoom Image
Fig. 1a Activity of extracts against HK/68, CV-B3, and RV-A2 versus their cytotoxicity (CV-B3 and RV-A2 in HeLa cells; HK/68 in MDCK cells). Candidates with high antiviral activity (IC50 ≤ 50 µg/mL) and low cytotoxicity (CC50 ≥ 50 µg/mL) are top left (green quadrangle). General activity (IC50 ≤ 50 µg/mL) and cytotoxicity frontiers (CC50 ≥ 50 µg/mL) are indicated by bold, red lines. All inactive extracts were set to an IC50 = 200 µg/mL in order to be able to display them in the graphic. Accordingly, CC50 values above 100 or 200 µg/mL were set to 200 µg/mL. b Enlarged section of a revealing the identity of respective extracts (see [Table 1]). Extracts where it was not possible to determine an IC50 value due to interference with cytotoxicity are marked with “x”.

Category A extracts (i.e., extracts located in quadrant A of [Fig. 1 a]) had distinct antiviral activity (IC50 ≤ 50 µg/mL) and no or low cytotoxicity (CC50 ≥ 50 µg/mL), giving an SI > 5. These extracts were considered to be most promising for further processing. Category B and D extracts (located in the respective quadrants of [Fig. 1 a]) showed weak or no antiviral activity against the tested viruses (IC50 ≥ 50 µg/mL) and were therefore not investigated further. Category C extracts (located in quadrant C of [Fig. 1 a]) were active (IC50 ≤ 50 µg/mL), but also cytotoxic (CC50 ≤ 50 µg/mL). These extracts are potentially worthwhile investigating because the observed cytotoxicity may be mediated by components other than those responsible for the extractʼs antiviral activity, and those components could potentially be separated. However, cytotoxic compounds may be able to mimic biological activity and cause false-positive assay readouts (measurements potentially affected by this type of assay interference are indicated with an asterisk in [Table 1]). The probability of false-positive outcomes resulting from cytotoxicity is a function of the ratio of CC50 and IC50, denoted as the SI. The higher the SI, the lower is the risk of false-positive results. Accordingly, extracts with SI values greater than 2 are candidates worthwhile investigating further. An enlarged depiction of this area of interest (quadrant A and the upper part of quadrant C) is shown in [Fig. 1 b].

In the following paragraphs, we report on the most relevant findings obtained from the extract screening and show how the integrated approach can help to overcome some of the pitfalls in the discovery of antiviral natural compounds.

The majority of extracts was prepared according to the protocol for the preparation of LLE [23], [24]. In addition, some of the already available extracts were produced without the application of defatting or tannin depletion procedures. These include the extracts from the bark of Burkea africana (no. 19a and 19b) and Sclerocarya birrea (no. 125a, 125b, and 125c), which showed potent anti-influenza activity without a significant level of cytotoxicity (Table 2S, Supporting Information). These plant materials as well as the fruits of Terminalia chebula (no. 142) are known to contain high amounts of tannins. Tannins tend to build nonselective protein complexes [27], [28], [29] and were previously shown to prevent virus adsorption to the host cells [30], [31], [32]. The focus of our project was to identify novel antiviral compounds active against viral targets other than those involved in adsorption. Accordingly, following the specific antiviral activity in category A, extracts from the bark of B. africana and S. birrea, and the fruits of T. chebula were generated on a larger scale for phytochemical investigations. For further evaluation of the antiviral activity of these extracts, a polyamide column was employed to separate the extracts into fractions free of polyphenols, fractions with low molecular weight polyphenols, and fractions with polymerized polyphenols (e.g., tannins).

In the case of S. birrea and T. chebula, the in vitro results indicated a strong influence of tannins on the antiviral activity. While the tannin-depleted fractions were not or only weakly active in the CPE assay, an antiviral effect was observed for the fractions with polyphenols present. Therefore, the phytochemical investigation of these two plant materials was discontinued.

As recently published, the situation was different in the case of B. africana, where tannin-free fractions showed distinct activities against influenza HK/68, with IC50 values of about 3 µg/mL [33]. Accordingly, polyphenol-rich fractions were neglected, whereas the tannin-free fractions were investigated further (Fig. 2S, Supporting Information). Eight novel triterpene saponins from lupane and oleanane types were identified as the bioactive principles (Fig. 2S, Supporting Information). In the CPE assay, the most active compounds showed IC50 values between 0.05 µM and 0.27 µM against HK/68 and the 2009 pandemic H1N1 strain A/Jena/8178/09 [33].

To explore the limitations of this integrated approach, the extracts of Piper nigrum fruits (no. 100) and Ruta graveolens herbs (no. 120) were selected for further analysis. These extracts out of category C exerted significant antiviral activities, but also problematic levels of cytotoxicity (SI between 1 and 2). The aim here was to determine whether the antiviral activity and cytotoxicity are mediated by different components and whether those could be separated.

The extract of R. graveolens (no. 120; Figs. 4S and 5S, Supporting Information) obtained IC50 values of 8.5 and 3.5 µg/mL in the phenotypic assay with HK/68 and CV-B3, respectively. Due to cytotoxicity (CC50 = 12 µg/mL in HeLa cells and below 6.3 µg/mL in MDCK cells), no exact IC50 for RV-A2 could be determined but was estimated by visual evaluation to be in the range of 12 to 25 µg/mL. Sixteen metabolites isolated from aerial parts of R. graveolens were evaluated in a previous study [34]. These are the alkaloids S-ribalinine, arborinine, isoplatydesmine, (−)-edulinine, and norgraveoline, the coumarins 7-methoxycoumarin, 6,7,8-trimethoxycoumarin, daphnoretin methyl ether, rutamarin, isoimperatorine, psoralen, bergapten, and 8-methoxy psoralene, and the phenyl propionic acid derivatives methyl 3-hydroxy-3-(4-hydroxy-3,5-dimethoxyphenyl) propanoate, methyl 3-(6-hydroxy-7-methoxy-benzofuran-5-yl) propanoate, and methyl 3-(4-hydroxy-3,5-dimethoxyphenyl)oxirane-2-carboxylate). In that study, the potential targets of these metabolites were predicted with a pharmacophore-based in silico approach. For one out of five metabolites predicted as inhibitors of the RV coat protein (i.e., arborine), an inhibitory activity was detected by an experiment against the capsid protein of RV-A2 (IC50 = 3.2 µM) [34]. Also 6,7,8-trimethoxycoumarin (not picked up by the in silico approach) showed activity against the capsid protein of RV-A2, with an IC50 of 12 µM. The CC50 values of both of these compounds were greater than 50 µM. This case demonstrates the successful isolation of noncytotoxic, antiviral constituents from a category C extract. In the present study, the 16 metabolites previously isolated from R. graveolens were further assayed for their CPE inhibition on HK/68 and CV-B3. The furanocoumarin rutamarin protected cells from a HK/68- and CV-B3-induced CPE with IC50 values of 2.7 µM and 5.1 µM, respectively ([Table 2], [Fig. 2]). All other constituents were inactive (data not shown). Since rutamarin is one of the main constituents identified for this extract [34], its cytotoxic and antiviral activity might reflect that of the whole extract. However, an effect of the high level of cytotoxicity of rutamarin (CC50 in MDCK cells: 4.7 µM; CC50 in HeLa cells: 4.6 µM) on the observed anti-influenza and anti-CV-B3 activity cannot be excluded.

Table 2 Antiviral activity of selected pure compounds. Their mean IC50 values against IV A/Hong Kong/68 (HK/68), coxsackie virus (CV-B3), and rhinovirus (RV-A2) in the CPE inhibition assay (MDCK cells for HK/68; HeLa cells for CV-B3 and RV-A2) as well as their mean CC50 values are presented (n = 3).

Name

Inhibition of virus-induced cytopathic effect

Cytotoxicity

HK/68

CV-B3

RV-A2

CC50 (CI95) [µM] in

IC50 (CI95) [µM] in MDCK cells

Selectivity index [CC50/IC50]

IC50 (CI95) [µM] in HeLa cells

Selectivity index [CC50/IC50]

IC50 (CI95) [µM] of CPE in HeLa cells

Selectivity index [CC50/IC50]

MDCK cells

HeLa cells

*Tested only once; **tested only twice; n. a. = not active; n. d. = not determined

Rutamarin

2.7 (2.4 – 3.2)**

1.7

5.1 (3.6 – 7.1)**

0.9

n. d.

4.7*

4.6*

Piperine

n. d.

n. a.

41 (15 – 79)

2.2

n. d.

88 (77 – 87)

Feruperine

n. d.

n. a.

n. a.

n. d.

> 100

Piperylin

n. d.

51 (32 – 75)

> 1.9

79 (36 – 139)

> 1.3

n. d.

> 100

1-[(2E,4E,8E)-9-(1,3-Benzodioxol-5-yl)-1-oxo-2,4,8-nonatrienyl]-pyrrolidine

n. d.

61 (44 – 79)

> 1.6

n. a.

n. d.

> 100

N-trans-feruloyl-piperidine

n. d.

n. a.

n. a.

n. d.

> 100

Piperoleine A

n. d.

22 (18 – 25)

1.2

n. a.

n. d.

25 (18 – 34)

Dehydropipernonaline

n. d.

24 (14 – 35)

1.4

n. a.

n. d.

34 (24 – 44)

Pipernonaline

n. d.

~ 32

0.7

n. a.

n. d.

21 (13 – 31)

Chabamide

n. d.

9.1 (7.1 – 11)

1.2

n. a.

n. d.

11 (8.6 – 14)

Pipertipine

n. d.

n. a.

n. a.

n. d.

21 (16 – 27)

Ganoderol B

17 (13 – 31)

> 5.9

n. a.

65 (39 – 93)

> 1.5

> 100

> 100

The extract of P. nigrum fruits (no. 100; Figs. 6S and 7S, Supporting Information) revealed an antiviral activity against CV-B3 (IC50 = 11 µg/mL). However, due to significant cytotoxicity observed with HeLa cells (CC50 = 16.8 µg/mL), the obtained antiviral activity data are questionable. For a more detailed analysis, ten piperamides that have been extracted as part of a previous study [35] [i.e., piperine, feruperine, piperylin, 9-(1,3-benzodioxol-5-yl)-1-(1-pyrrolidinyl) 2E,4E,8E-nonatrien-1-one, N-trans-feruloyl-piperidine, piperoleine A, dehydropipernonaline, pipernonaline, chabamide, and pipertipine] were scrutinized for their CV-B3 inhibiting CPE ([Fig. 2]). The most active compounds were chabamide (IC50 = 9.1 µM; CC50 = 11 µM), piperoleine A (IC50 = 22 µM; CC50 = 25 µM), and dehydropipernonaline (IC50 = 24 µM; CC50 = 34 µM) (see [Table 2]). However, in this case, attempts to separate the antiviral and cytotoxic constituents were not successful.

Phenotypic assays can provide valuable information for the prioritization of extracts for phytochemical processing and pharmacological analysis. There is a clear added value in integrating computational methods into this screening setup. In silico methods can, e.g., guide the identification of the mode of action or mechanism of toxicity. They can also identify the most promising constituents of extracts for isolation and testing on a target of interest, e.g., for influenza NA [36], [37], [38]. In a previously performed computational study, we identified secondary metabolites present in the fruit body extract of Ganoderma lucidum Karst. (no. 53) that likely exhibit activity on anti-influenza and anti-RV targets [39]. A database of 279 known constituents of the fungus (mostly lanostane-type triterpenes) was compiled and screened with a pharmacophore-based approach for activity against a selection of viral targets. As one outcome of this study, ganoderol B ([Fig. 2]) was identified as a potential inhibitor of the RV coat protein [39], and was therefore selected here for experimental testing on HK/68 and RV-A2. In the phenotypic CPE assay, previously isolated ganoderol B [40] showed moderate activity against HK/68 and RV-A2 (IC50 = 17 µM and 65 µM, respectively; [Table 2]), even though the crude extract (no. 53; Figs. 8S and 9S, Supporting Information) did not show any activity against RV-A2 ([Table 1]).

With the integrated strategy for the identification of bioactive compounds from extracts that we present in this work, several of the shortcomings of extract screening (e.g., false-positive assay readouts caused by interference or cytotoxicity provoked by multicomponent mixtures) can be leveraged. In particular, the combination of ethnopharmacological knowledge with effective, phenotypic screening technologies can accelerate and improve the prioritization of promising extracts as starting materials. Furthermore, the integration of computational methods can contribute valuable insights on the mode of action and mechanism of toxicity of individual constituents and provide guidance to in vitro analyses.

The data on extracts and their biological and toxicological profiles reported in this work shall serve as a starting point for future investigations. Moreover, the generated extract library can serve as a valuable platform for the scientific community. This collection of well-defined LLEs is now available for further studies including biological tests, analytical studies, or a combination of both.

Besides promising extracts with no or low cytotoxicity (i.e., category A extracts), extracts from the upper part of quadrant C in [Fig. 1 b] (i.e., category C extracts that exhibited antiviral activity but also some degree of cytotoxicity) may also be worthwhile probing for bioactive ingredients, while bearing in mind that the observed cytotoxicity may interfere with the phenotypic assay.

Zoom Image
Fig. 2 Chemical structures of the tested pure compounds.

#

Materials and Methods

Natural materials

Some of the plant and mushroom materials were collected in Tyrol/Austria and identified by J. M. Rollinger or U. Peintner (Institute of Microbiology, University of Innsbruck, Austria). Further plant material was purchased from commercial providers, and some materials/extracts were obtained from collaboration partners (Table 1S, Supporting Information). Voucher specimens are deposited in the Herbarium of the Department of Pharmacognosy, University of Vienna, Austria, unless otherwise stated in Table 1S, Supporting Information.


#

Generation of small-scale extracts

Combined dichloromethane and methanol extracts of plant and fungal species were prepared as described recently [23], [24]. Briefly, ground-dried material was defatted with n-hexane. The dried, defatted material was then extracted successively with dichloromethane and methanol, whereby the two resulting extracts were combined. Finally, tannin depletion via polyamide gel was carried out in order to remove compounds likely to cause assay interference.

In a few cases, the materials were not defatted and the dichloromethane (D) and methanol (M) extract were kept separately. Moreover, ethanol (E) or aqueous (H2O) extracts were also generated for a small number of natural materials.


#

Cell culture and viruses

H3N2 IV strain HK/68 (strain collection of the Department of Virology and Antiviral Therapy, Jena, Germany), CV-B3 (CV-B3 Nancy; Institute of Poliomyelitis and Virus Encephalitides, Moscow, Russia), and RV-A2 (Medical University of Vienna, Vienna, Austria) were used in antiviral studies. IVs were propagated in MDCK cells (Friedrich-Loeffler Institute, Riems, Germany) in serum-free Eagleʼs minimum essential medium, 2 µg/mL trypsin, and 1.2 mM bicarbonate [41]. CV-B3 and RV-A2 were grown and tested in Eagleʼs minimal essential medium supplemented with 2% neonatal calf serum (PAA, Cölbe, Germany). Cells were proved to be free of mycoplasma contamination before using. Titers of virus stocks were determined according to Reed and Muench [42] in MDCK cells and HeLa cells, respectively.


#

Determination of cytotoxicity and cytopathic effect inhibition

The CC50 is defined as the concentration of a sample (in our case extract or pure compound) where the viability of treated cells in comparison to untreated control cells (mean viability of six controls is set to 100%) is reduced by half [25]. The IC50 is the concentration of a virus inhibitor (in our case extract or pure compound) where the response (grade of cell destruction, i.e., CPE caused by the virus) is reduced by half [25]. The IC50 was determined on 2-day-old confluent MDCK cell monolayers (for HK/68) and on 1-day-old and 2-day-old confluent HeLa Ohio cells for RV-A2 and CV-B3, respectively [43]. The cells were grown in 96-well plates as described previously (maximum tested concentration: 100 or 200 µg/mL for extracts and 100 µM for compounds) [25]. The maximum applied solvent concentration was 0.5% (v/v). Cytotoxicity was analyzed 72 h after adding the extracts. CPE inhibition was measured 48 h after infection for HK/68 and CV-B3, and 72 h after infection for RV-A2. A multiplicity of infection of 0.003, 0.001 – 0.002, and 0.02 TCID50/cell of HK/68, CV-B3, and RV-A2, respectively, resulted in a complete CPE at this time point. CC50 and IC50 values were calculated from mean dose-response curves of at least three independent experiments. Linear regression using Microsoft Excel was therefore used in the linear scaled dose-dependent sample concentrations (in µg/mL) ([Tables 1] and 2S, Fig. 1S, Supporting Information).

These CC50 and IC50 values were used (i) to easily evaluate the specificity of antiviral activity after calculating the SI (CC50/IC50) and (ii) for categorization of activity. Then, the mean CC50 and IC50 values and confidence intervals of most active extracts (SI > 5) as well as some antiviral but also cytotoxic examples (SI < 5) were calculated. Oseltamivir, guanidine hydrochloride, and pleconaril served as positive controls for HK/68, CV-B3, and RV-A2, respectively.


#
#
#

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

The authors thank B. Jahn (Department of Virology and Antiviral Therapy, Jena University Hospital, Germany) for technical assistance, and A. Kaserer and C. Draschl (Institute of Pharmacy/Pharmacognosy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Austria) for extract preparation. This work was supported by the Austrian Science Fund (FWF: P24587) and the European Social Fund (ESF & TMWAT Project 2011 FGR 0137).

* Dedicated to “Women in Natural Products Science”.


Supporting Information

  • References

  • 1 World Health Organization. The top 10 causes of death. Available at. http://www.who.int/mediacentre/factsheets/fs310/en Accessed September 29, 2017
  • 2 Jacobs SE, Lamson DM, St George K, Walsh TJ. Human rhinoviruses. Clin Microbiol Rev 2013; 26: 135-162
  • 3 McCullers JA. The co-pathogenesis of influenza viruses with bacteria in the lung. Nat Rev Microbiol 2014; 12: 252-262
  • 4 Melvin JA, Bomberger JM. Compromised defenses: exploitation of epithelial responses during viral-bacterial co-infection of the respiratory tract. PLoS Pathog 2016; 12: e1005797
  • 5 Bauer L, Lyoo H, van der Schaar HM, Strating JR, van Kuppeveld FJ. Direct-acting antivirals and host-targeting strategies to combat enterovirus infections. Curr Opin Virol 2017; 24: 1-8
  • 6 Rollinger JM, Schmidtke M. The human rhinovirus: human-pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery. Med Res Rev 2011; 31: 42-92
  • 7 Millner VS, Eichold 2nd BH, Franks RD, Johnson GD. Influenza vaccination acceptance and refusal rates among health care personnel. South Med J 2010; 103: 993-998
  • 8 Nguyen T, Henningsen KH, Brehaut JC, Hoe E, Wilson K. Acceptance of a pandemic influenza vaccine: a systematic review of surveys of the general public. Infect Drug Resist 2011; 4: 197-207
  • 9 World Health Organization. Facts sheet on seasonal influenza. Available at. http://www.who.int/mediacentre/factsheets/fs211/en Accessed November 30, 2016
  • 10 Hurt AC, Besselaar TG, Daniels RS, Ermetal B, Fry A, Gubareva L, Huang W, Lackenby A, Lee RT, Lo J, Maurer-Stroh S, Nguyen HT, Pereyaslov D, Rebelo-de-Andrade H, Siqueira MM, Takashita E, Tashiro M, Tilmanis D, Wang D, Zhang W, Meijer A. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2014–2015. Antiviral Res 2016; 132: 178-185
  • 11 Furuta Y, Komeno T, Nakamura T. Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase. Proc Jpn Acad Ser B Phys Biol Sci 2017; 93: 449-463
  • 12 Hurt AC, Hardie K, Wilson NJ, Deng YM, Osbourn M, Leang SK, Lee RT, Iannello P, Gehrig N, Shaw R, Wark P, Caldwell N, Givney RC, Xue L, Maurer-Stroh S, Dwyer DE, Wang B, Smith DW, Levy A, Booy R, Dixit R, Merritt T, Kelso A, Dalton C, Durrheim D, Barr IG. Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia. J Infect Dis 2012; 206: 148-157
  • 13 Takashita E, Kiso M, Fujisaki S, Yokoyama M, Nakamura K, Shirakura M, Sato H, Odagiri T, Kawaoka Y, Tashiro M. Characterization of a large cluster of influenza A(H1N1)pdm09 viruses cross-resistant to oseltamivir and peramivir during the 2013–2014 influenza season in Japan. Antimicrob Agents Chemother 2015; 59: 2607-2617
  • 14 Hurt AC, Ernest J, Deng YM, Iannello P, Besselaar TG, Birch C, Buchy P, Chittaganpitch M, Chiu SC, Dwyer D, Guigon A, Harrower B, Kei IP, Kok T, Lin C, McPhie K, Mohd A, Olveda R, Panayotou T, Rawlinson W, Scott L, Smith D, DʼSouza H, Komadina N, Shaw R, Kelso A, Barr IG. Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa. Antiviral Res 2009; 83: 90-93
  • 15 Newman DJ, Cragg GM. Natural products as sources of new drugs from 1981 to 2014. J Nat Prod 2016; 79: 629-661
  • 16 Rollinger JM, Langer T, Stuppner H. Strategies for efficient lead structure discovery from natural products. Curr Med Chem 2006; 13: 1491-1507
  • 17 Chen Y, de Bruyn Kops C, Kirchmair J. Data resources for the computer-guided discovery of bioactive natural products. J Chem Inf Model 2017; 57: 2099-2111
  • 18 Swinney DC, Anthony J. How were new medicines discovered?. Nat Rev Drug Discov 2011; 10: 507-519
  • 19 Berendes J. Des Pedanios Dioskurides aus Anazarbos Arzneimittellehre in fünf Büchern: reprint. Vaduz, Lichtenstein: Sändig Reprints Verlag; 1997
  • 20 König R, Hopp J, Glöckner W, Winkler G. Plinius Secundus d. Ä. Naturkunde, Books XX–XVII. München, Zürich: Artemis Publisher; 1985
  • 21 Pahlow M. Das große Buch der Heilpflanzen. München: Gräfe und Unzer; 1993
  • 22 Ausserer O. Volksmedizin in Tirol. Tisens, Italy: EU Interregnum-II-Project; 2001
  • 23 Camp D, Davis RA, Campitelli M, Ebdon J, Quinn RJ. Drug-like properties: guiding principles for the design of natural product libraries. J Nat Prod 2012; 75: 72-81
  • 24 Kratz JM, Mair CE, Oettl SK, Saxena P, Scheel O, Schuster D, Hering S, Rollinger JM. hERG channel blocking ipecac alkaloids identified by combined in silico – in vitro screening. Planta Med 2016; 82: 1009-1015
  • 25 Schmidtke M, Schnittler U, Jahn B, Dahse H, Stelzner A. A rapid assay for evaluation of antiviral activity against coxsackie virus B3, influenza virus A, and herpes simplex virus type 1. J Virol Methods 2001; 95: 133-143
  • 26 Makarov VA, Riabova OB, Granik VG, Wutzler P, Schmidtke M. Novel [(biphenyloxy)propyl]isoxazole derivatives for inhibition of human rhinovirus 2 and coxsackievirus B3 replication. J Antimicrob Chemother 2005; 55: 483-488
  • 27 Jones WP, Kinghorn DA. Vegetable Tannins. In: Sarker SD, Latif Z, Gray AI. eds. Natural Products Isolation. Totowa, New Jersey: Humana Press Inc.; 2006: 338
  • 28 Kinghorn AD, Pan L, Fletcher JN, Chai H. The relevance of higher plants in lead compound discovery programs. J Nat Prod 2011; 74: 1539-1555
  • 29 Spencer CM, Cai Y, Martin R, Gaffney SH, Goulding PN, Magnolato D, Lilley TH, Haslam E. Polyphenol complexation – some thoughts and observations. Phytochemistry 1988; 27: 2397-2409
  • 30 Yang ZF, Bai LP, Huang WB, Li XZ, Zhao SS, Zhong NS, Jiang ZH. Comparison of in vitro antiviral activity of tea polyphenols against influenza A and B viruses and structure-activity relationship analysis. Fitoterapia 2014; 93: 47-53
  • 31 Bahramsoltani R, Sodagari HR, Farzaei MH, Abdolghaffari AH, Gooshe M, Rezaei N. The preventive and therapeutic potential of natural polyphenols on influenza. Expert Rev Anti Infect Ther 2016; 14: 57-80
  • 32 Theisen LL, Erdelmeier CA, Spoden GA, Boukhallouk F, Sausy A, Florin L, Muller CP. Tannins from Hamamelis virginiana bark extract: characterization and improvement of the antiviral efficacy against influenza A virus and human papillomavirus. PLoS One 2014; 9: e88062
  • 33 Mair CE, Grienke U, Wilhelm A, Urban E, Zehl M, Schmidtke M, Rollinger JM. Anti-influenza triterpene saponins from the bark of Burkea africana . J Nat Prod 2018; DOI: 10.1021/acs.jnatprod.7b00774.
  • 34 Rollinger JM, Schuster D, Danzl B, Schwaiger S, Markt P, Schmidtke M, Gertsch J, Raduner S, Wolber G, Langer T, Stuppner H. In silico target fishing for rationalized ligand discovery exemplified on constituents of Ruta graveolens . Planta Med 2009; 75: 195-204
  • 35 Mair CE, Liu R, Atanasov AG, Wimmer L, Nemetz-Fiedler D, Sider N, Heiss EH, Mihovilovic MD, Dirsch VM, Rollinger JM. Piperine congeners as inhibitors of vascular smooth muscle cell proliferation. Planta Med 2015; 81: 1065-1074
  • 36 Grienke U, Schmidtke M, Kirchmair J, Pfarr K, Wutzler P, Durrwald R, Wolber G, Liedl KR, Stuppner H, Rollinger JM. Antiviral potential and molecular insight into neuraminidase inhibiting diarylheptanoids from Alpinia katsumadai . J Med Chem 2010; 53: 778-786
  • 37 Grienke U, Braun H, Seidel N, Kirchmair J, Richter M, Krumbholz A, von Grafenstein S, Liedl KR, Schmidtke M, Rollinger JM. Computer-guided approach to access the anti-influenza activity of licorice constituents. J Nat Prod 2014; 77: 563-570
  • 38 Grienke U, Richter M, Walther E, Hoffmann A, Kirchmair J, Makarov V, Nietzsche S, Schmidtke M, Rollinger JM. Discovery of prenylated flavonoids with dual activity against influenza virus and Streptococcus pneumoniae . Sci Rep 2016; 6: 27156
  • 39 Grienke U, Kaserer T, Pfluger F, Mair CE, Langer T, Schuster D, Rollinger JM. Accessing biological actions of Ganoderma secondary metabolites by in silico profiling. Phytochemistry 2015; 114: 114-124
  • 40 Grienke U, Mihaly-Bison J, Schuster D, Afonyushkin T, Binder M, Guan SH, Cheng CR, Wolber G, Stuppner H, Guo DA, Bochkov VN, Rollinger JM. Pharmacophore-based discovery of FXR-agonists. Part II: identification of bioactive triterpenes from Ganoderma lucidum . Bioorg Med Chem 2011; 19: 6779-6791
  • 41 Bauer K, Richter M, Wutzler P, Schmidtke M. Different neuraminidase inhibitor susceptibilities of human H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany from 2001 to 2005/2006. Antiviral Res 2009; 82: 34-41
  • 42 Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 1938; 27: 493-497
  • 43 Makarov VA, Braun H, Richter M, Riabova OB, Kirchmair J, Kazakova ES, Seidel N, Wutzler P, Schmidtke M. Pyrazolopyrimidines: potent inhibitors targeting the capsid of rhino- and enteroviruses. ChemMedChem 2015; 10: 1629-1634

Correspondence

Univ.-Prof. Mag. pharm. Dr. Judith M. Rollinger
Department of Pharmacognosy
Faculty of Life Sciences
University of Vienna
Althanstrasse 14
1090 Vienna
Austria   
Phone: + 43 14 27 75 52 55   
Fax: + 43 1 42 77 85 52 55   

  • References

  • 1 World Health Organization. The top 10 causes of death. Available at. http://www.who.int/mediacentre/factsheets/fs310/en Accessed September 29, 2017
  • 2 Jacobs SE, Lamson DM, St George K, Walsh TJ. Human rhinoviruses. Clin Microbiol Rev 2013; 26: 135-162
  • 3 McCullers JA. The co-pathogenesis of influenza viruses with bacteria in the lung. Nat Rev Microbiol 2014; 12: 252-262
  • 4 Melvin JA, Bomberger JM. Compromised defenses: exploitation of epithelial responses during viral-bacterial co-infection of the respiratory tract. PLoS Pathog 2016; 12: e1005797
  • 5 Bauer L, Lyoo H, van der Schaar HM, Strating JR, van Kuppeveld FJ. Direct-acting antivirals and host-targeting strategies to combat enterovirus infections. Curr Opin Virol 2017; 24: 1-8
  • 6 Rollinger JM, Schmidtke M. The human rhinovirus: human-pathological impact, mechanisms of antirhinoviral agents, and strategies for their discovery. Med Res Rev 2011; 31: 42-92
  • 7 Millner VS, Eichold 2nd BH, Franks RD, Johnson GD. Influenza vaccination acceptance and refusal rates among health care personnel. South Med J 2010; 103: 993-998
  • 8 Nguyen T, Henningsen KH, Brehaut JC, Hoe E, Wilson K. Acceptance of a pandemic influenza vaccine: a systematic review of surveys of the general public. Infect Drug Resist 2011; 4: 197-207
  • 9 World Health Organization. Facts sheet on seasonal influenza. Available at. http://www.who.int/mediacentre/factsheets/fs211/en Accessed November 30, 2016
  • 10 Hurt AC, Besselaar TG, Daniels RS, Ermetal B, Fry A, Gubareva L, Huang W, Lackenby A, Lee RT, Lo J, Maurer-Stroh S, Nguyen HT, Pereyaslov D, Rebelo-de-Andrade H, Siqueira MM, Takashita E, Tashiro M, Tilmanis D, Wang D, Zhang W, Meijer A. Global update on the susceptibility of human influenza viruses to neuraminidase inhibitors, 2014–2015. Antiviral Res 2016; 132: 178-185
  • 11 Furuta Y, Komeno T, Nakamura T. Favipiravir (T-705), a broad spectrum inhibitor of viral RNA polymerase. Proc Jpn Acad Ser B Phys Biol Sci 2017; 93: 449-463
  • 12 Hurt AC, Hardie K, Wilson NJ, Deng YM, Osbourn M, Leang SK, Lee RT, Iannello P, Gehrig N, Shaw R, Wark P, Caldwell N, Givney RC, Xue L, Maurer-Stroh S, Dwyer DE, Wang B, Smith DW, Levy A, Booy R, Dixit R, Merritt T, Kelso A, Dalton C, Durrheim D, Barr IG. Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia. J Infect Dis 2012; 206: 148-157
  • 13 Takashita E, Kiso M, Fujisaki S, Yokoyama M, Nakamura K, Shirakura M, Sato H, Odagiri T, Kawaoka Y, Tashiro M. Characterization of a large cluster of influenza A(H1N1)pdm09 viruses cross-resistant to oseltamivir and peramivir during the 2013–2014 influenza season in Japan. Antimicrob Agents Chemother 2015; 59: 2607-2617
  • 14 Hurt AC, Ernest J, Deng YM, Iannello P, Besselaar TG, Birch C, Buchy P, Chittaganpitch M, Chiu SC, Dwyer D, Guigon A, Harrower B, Kei IP, Kok T, Lin C, McPhie K, Mohd A, Olveda R, Panayotou T, Rawlinson W, Scott L, Smith D, DʼSouza H, Komadina N, Shaw R, Kelso A, Barr IG. Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa. Antiviral Res 2009; 83: 90-93
  • 15 Newman DJ, Cragg GM. Natural products as sources of new drugs from 1981 to 2014. J Nat Prod 2016; 79: 629-661
  • 16 Rollinger JM, Langer T, Stuppner H. Strategies for efficient lead structure discovery from natural products. Curr Med Chem 2006; 13: 1491-1507
  • 17 Chen Y, de Bruyn Kops C, Kirchmair J. Data resources for the computer-guided discovery of bioactive natural products. J Chem Inf Model 2017; 57: 2099-2111
  • 18 Swinney DC, Anthony J. How were new medicines discovered?. Nat Rev Drug Discov 2011; 10: 507-519
  • 19 Berendes J. Des Pedanios Dioskurides aus Anazarbos Arzneimittellehre in fünf Büchern: reprint. Vaduz, Lichtenstein: Sändig Reprints Verlag; 1997
  • 20 König R, Hopp J, Glöckner W, Winkler G. Plinius Secundus d. Ä. Naturkunde, Books XX–XVII. München, Zürich: Artemis Publisher; 1985
  • 21 Pahlow M. Das große Buch der Heilpflanzen. München: Gräfe und Unzer; 1993
  • 22 Ausserer O. Volksmedizin in Tirol. Tisens, Italy: EU Interregnum-II-Project; 2001
  • 23 Camp D, Davis RA, Campitelli M, Ebdon J, Quinn RJ. Drug-like properties: guiding principles for the design of natural product libraries. J Nat Prod 2012; 75: 72-81
  • 24 Kratz JM, Mair CE, Oettl SK, Saxena P, Scheel O, Schuster D, Hering S, Rollinger JM. hERG channel blocking ipecac alkaloids identified by combined in silico – in vitro screening. Planta Med 2016; 82: 1009-1015
  • 25 Schmidtke M, Schnittler U, Jahn B, Dahse H, Stelzner A. A rapid assay for evaluation of antiviral activity against coxsackie virus B3, influenza virus A, and herpes simplex virus type 1. J Virol Methods 2001; 95: 133-143
  • 26 Makarov VA, Riabova OB, Granik VG, Wutzler P, Schmidtke M. Novel [(biphenyloxy)propyl]isoxazole derivatives for inhibition of human rhinovirus 2 and coxsackievirus B3 replication. J Antimicrob Chemother 2005; 55: 483-488
  • 27 Jones WP, Kinghorn DA. Vegetable Tannins. In: Sarker SD, Latif Z, Gray AI. eds. Natural Products Isolation. Totowa, New Jersey: Humana Press Inc.; 2006: 338
  • 28 Kinghorn AD, Pan L, Fletcher JN, Chai H. The relevance of higher plants in lead compound discovery programs. J Nat Prod 2011; 74: 1539-1555
  • 29 Spencer CM, Cai Y, Martin R, Gaffney SH, Goulding PN, Magnolato D, Lilley TH, Haslam E. Polyphenol complexation – some thoughts and observations. Phytochemistry 1988; 27: 2397-2409
  • 30 Yang ZF, Bai LP, Huang WB, Li XZ, Zhao SS, Zhong NS, Jiang ZH. Comparison of in vitro antiviral activity of tea polyphenols against influenza A and B viruses and structure-activity relationship analysis. Fitoterapia 2014; 93: 47-53
  • 31 Bahramsoltani R, Sodagari HR, Farzaei MH, Abdolghaffari AH, Gooshe M, Rezaei N. The preventive and therapeutic potential of natural polyphenols on influenza. Expert Rev Anti Infect Ther 2016; 14: 57-80
  • 32 Theisen LL, Erdelmeier CA, Spoden GA, Boukhallouk F, Sausy A, Florin L, Muller CP. Tannins from Hamamelis virginiana bark extract: characterization and improvement of the antiviral efficacy against influenza A virus and human papillomavirus. PLoS One 2014; 9: e88062
  • 33 Mair CE, Grienke U, Wilhelm A, Urban E, Zehl M, Schmidtke M, Rollinger JM. Anti-influenza triterpene saponins from the bark of Burkea africana . J Nat Prod 2018; DOI: 10.1021/acs.jnatprod.7b00774.
  • 34 Rollinger JM, Schuster D, Danzl B, Schwaiger S, Markt P, Schmidtke M, Gertsch J, Raduner S, Wolber G, Langer T, Stuppner H. In silico target fishing for rationalized ligand discovery exemplified on constituents of Ruta graveolens . Planta Med 2009; 75: 195-204
  • 35 Mair CE, Liu R, Atanasov AG, Wimmer L, Nemetz-Fiedler D, Sider N, Heiss EH, Mihovilovic MD, Dirsch VM, Rollinger JM. Piperine congeners as inhibitors of vascular smooth muscle cell proliferation. Planta Med 2015; 81: 1065-1074
  • 36 Grienke U, Schmidtke M, Kirchmair J, Pfarr K, Wutzler P, Durrwald R, Wolber G, Liedl KR, Stuppner H, Rollinger JM. Antiviral potential and molecular insight into neuraminidase inhibiting diarylheptanoids from Alpinia katsumadai . J Med Chem 2010; 53: 778-786
  • 37 Grienke U, Braun H, Seidel N, Kirchmair J, Richter M, Krumbholz A, von Grafenstein S, Liedl KR, Schmidtke M, Rollinger JM. Computer-guided approach to access the anti-influenza activity of licorice constituents. J Nat Prod 2014; 77: 563-570
  • 38 Grienke U, Richter M, Walther E, Hoffmann A, Kirchmair J, Makarov V, Nietzsche S, Schmidtke M, Rollinger JM. Discovery of prenylated flavonoids with dual activity against influenza virus and Streptococcus pneumoniae . Sci Rep 2016; 6: 27156
  • 39 Grienke U, Kaserer T, Pfluger F, Mair CE, Langer T, Schuster D, Rollinger JM. Accessing biological actions of Ganoderma secondary metabolites by in silico profiling. Phytochemistry 2015; 114: 114-124
  • 40 Grienke U, Mihaly-Bison J, Schuster D, Afonyushkin T, Binder M, Guan SH, Cheng CR, Wolber G, Stuppner H, Guo DA, Bochkov VN, Rollinger JM. Pharmacophore-based discovery of FXR-agonists. Part II: identification of bioactive triterpenes from Ganoderma lucidum . Bioorg Med Chem 2011; 19: 6779-6791
  • 41 Bauer K, Richter M, Wutzler P, Schmidtke M. Different neuraminidase inhibitor susceptibilities of human H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany from 2001 to 2005/2006. Antiviral Res 2009; 82: 34-41
  • 42 Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 1938; 27: 493-497
  • 43 Makarov VA, Braun H, Richter M, Riabova OB, Kirchmair J, Kazakova ES, Seidel N, Wutzler P, Schmidtke M. Pyrazolopyrimidines: potent inhibitors targeting the capsid of rhino- and enteroviruses. ChemMedChem 2015; 10: 1629-1634

Zoom Image
Fig. 1a Activity of extracts against HK/68, CV-B3, and RV-A2 versus their cytotoxicity (CV-B3 and RV-A2 in HeLa cells; HK/68 in MDCK cells). Candidates with high antiviral activity (IC50 ≤ 50 µg/mL) and low cytotoxicity (CC50 ≥ 50 µg/mL) are top left (green quadrangle). General activity (IC50 ≤ 50 µg/mL) and cytotoxicity frontiers (CC50 ≥ 50 µg/mL) are indicated by bold, red lines. All inactive extracts were set to an IC50 = 200 µg/mL in order to be able to display them in the graphic. Accordingly, CC50 values above 100 or 200 µg/mL were set to 200 µg/mL. b Enlarged section of a revealing the identity of respective extracts (see [Table 1]). Extracts where it was not possible to determine an IC50 value due to interference with cytotoxicity are marked with “x”.
Zoom Image
Fig. 2 Chemical structures of the tested pure compounds.