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  • Research article
  • Open Access
  • Open Peer Review

Screening of neuraminidase inhibitory activities of some medicinal plants traditionally used in Lingnan Chinese medicines

Contributed equally
BMC Complementary and Alternative MedicineBMC series – open, inclusive and trusted201818:102

https://doi.org/10.1186/s12906-018-2173-1

  • Received: 23 March 2017
  • Accepted: 15 March 2018
  • Published:
Open Peer Review reports

Abstract

Background

Neuraminidase (NA) is one of the key surface protein of the influenza virus, and has been established as a primary drug target for anti-influenza therapies. This study aimed to screen bioactive herbal extracts from some medicinal plants traditionally used in Lingnan Chinese Medicines by NA activity high-throughput screening assay.

Methods

One hundred ninety herbal extracts from 95 medicinal plants collected in Guangzhou were screened for their potential inhibitory activities against A (H1N1) influenza neuraminidase, and the most active extracts were further evaluated for their anti-influenza virus activities using virus-induced cytopathic effect (CPE).

Results

Among the tested 190 herbal extracts, 14 extracts inhibited significantly NA activity (IC50 < 40 μg/mL), and the extracts 15, which were obtained from Amomurn villosum Lour, Melaphis chinensis (Bell) Baker, Sanguisorba officinalis and Flos Caryophylli, showed potent inhibitory activity against NA with IC50 values ranging from 4.1 to 9.6 μg/mL. Moreover, the most bioactive extracts 15 were found to protect MDCK cells from A (H1N1) influenza virus infection with very low cytotoxicity to the host cells (EC50 values ranged from 1.8 to 14.1 μg/mL, CC50 values ranged from 97.0 to 779.2 μg/mL, SI values ranged from 14 to 438). In addition, quantitative RT-PCR analysis showed that the extracts 15 inhibited viral RNA synthesis in a dose-dependent manner.

Conclusion

We performed in vitro screening of anti-neuraminidase activities of herbal extracts from medicinal plants used in Lingnan Chinese Medicines, and the results indicate that some bioactive extracts are worth further studies to identify the bioactive components responsible for anti-influenza virus activities, to elucidate their modes of action and finally determine their clinical potentials.

Keywords

  • A (H1N1) influenza virus
  • Neuraminidase inhibitor
  • Anti-influenza agents
  • Medicinal plant
  • Lingnan Chinese medicines

Background

Influenza virus causes an acute contagious respiratory tract infection, which is a major contributor to morbidity and mortality among human population. Historically pandemic flu has caused widespread human deaths, most notably the 1918 “Spanish Flu” (A/H1N1) which killed 25–50 million people worldwide [1]. Novel swine-origin influenza A (H1N1 subtype) virus identified in Mexico in 2009 emerges to spread rapidly worldwide via human-human transmission [2] and led to at least 17,798 deaths in 214 countries. Therefore, pandemic influenza A viruses such as the H1N1subtype becomes a serious global public health problem, which calls for more agents of anti-influenza therapies as possible.

Neuraminidase (NA) is an antigenic glycoprotein on the surface of influenza virus, which takes charge of catalyzing the cleavage of neuraminic acid residues to facilitate the detachment from the host cell surface at the end of the viral replication cycle and suppresses their self-aggregation of the virions [3, 4]. NA plays a critical role for virus replication and spread in infected tissues during infection, and has been well established as a primary drug target for anti-influenza therapies [5, 6]. Some potent NA inhibitors, including oseltamivir, zanamivir, laninamivir and peramivir, have been designed and applied in clinical treatments [7, 8]. Unfortunately, resistance to these NA inhibitors has been extensively reported [911]. Therefore, there is a continuing need for developing novel NA inhibitors as anti-influenza agents. Medicinal plants may be a probable source for the discovery of natural NA inhibitors and might provide leads to develop the NA inhibitors [12].

In order to search for novel anti-influenza agents from natural resources, a library of 190 extracts of 95 medicinal plants traditionally used in Lingnan Chinese Medicines were screened for in vitro inhibitory activity against A (H1N1) influenza virus neuraminidase using high-throughput assay. The most active five extracts (15) were selected to further study their action upon the replication of influenza viruses using cytopathic effect (CPE) reduction assay and quantitative RT-PCR analysis. The results showed that these herbal extracts significantly inhibited the NA activity and the replication of influenza viruses, and exhibited very low cytotoxicity to the host cells.

Methods

Plant materials

Ninety nine medicinal plants traditionally used in Lingnan Chinese Medicines were collected in Guangzhou in 2009. The identity of the plants samples was verified by Dr. Guangtian Peng (Guangzhou University of Chinese Medicine). Voucher specimens of these materials were deposited for references in the Research Center of Medicinal Plants Resource Science and Engineering, Guangzhou University of Chinese Medicine. The samples were stored in the shade at room temperature and pulverized before use.

Standard extraction preparation

Dried powdered plants (100 g) were extracted with ethyl acetate (EtOAc, 250 mL × 3) and methanol (MeOH, 250 mL × 3) by ultrasound wave at 40 kHz and 400 W at 45 °C for 30 min, the filtrates were evaporated under vacuum at 45 °C to give the EtOAc and MeOH extracts, respectively. A total of 190 herbal extracts were obtained. A stock solution for each extract was prepared by dissolution to dimethyl sulfoxide (DMSO), 50 mg of each extract was suspended in 1 ml of DMSO ensuing stock concentration of 50 μg/μL. The solutions were filtered by using 0.22 μm filters, and stored at − 20 °C. The concentration of DMSO in test dilutions was restricted to no more than 0.5% (v/v) to minimize potential effects of the solvent on enzyme activity and cell growth.

Neuraminidase, virus and cells

The human influenza virus strains A/PR/8/34 (H1N1) was kindly provided by China Centers for Disease Control, and was used as the source of NA; Madin-Darby canine kidney (MDCK) and A549 cell lines were obtained from the National Center for Pharmaceutical Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences. Madin-Darby canine kidney (MDCK) cells were grown in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere. MDCK cells were used for virus infection, and were washed with PBS buffer before infection. 2′-(4-methylunbelliferyl)-α-D-acetyl-neuraminic acid (MUNANA), 2-(N-Morpholino)-ethanesulfonic acid (MES) and 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma. DMEM, FBS, and 0.25% trypsin-EDTA were purchased from Gibco. Ribavirin with purity more than 98%, and zanamivir with purity more than 98% were purchased from Sigma (Lot#020 M4003) and Full Land international trade company in Shanghai of China (Lot#091209-005LY), respectively. They were used as references in NA and CPE inhibition assays.

In vitro screening of plant extracts for NA activity

Inhibition of influenza virus NA activity was determined by a standard fluorimetric method [13, 14] using4-methylumbelliferyl-α-D-N-acetyl-neuraminate (MUNANA) (Sigma) as substrate, in 96-well microplates. The reaction mixture containing the extracts or compounds, and NA enzyme in MES buffer (32.5 mM) and calcium chloride (4 mM, pH 6.5) was incubated for 60 min. After incubation, the reaction was terminated by adding NaOH (34 mM). Fluorescence intensity (M) was quantified with excitation wavelength at 360 nm and emission wavelength at 450 nm. Percentage inhibition was calculated relative to a blank reaction mixture (solvent control) containing virus NA and solvent (% Inhibition = [1-(Mextract/Mcontrol)] × 100). The 50% inhibitory concentration (IC50) was defined as the concentration of NA inhibitor necessary to reduce NA activity by 50% relative to a blank reaction mixture. IC50 values displayed represent the mean of three individual determinations each performed in triplicate assays. Zanamivir (Sigma) was used as the reference compound.

Cytotoxicity assay

The cytotoxicity of medicinal plant extracts was determined with the MTT (Sigma) method as described previously [15]. Briefly, different concentrations of the extracts and compounds were added to each well of a 96-well culture plate containing a confluent cell monolayer in triplicate, blank medium was used as the control. After incubation at 37 °C in an atmosphere of 5% CO2 for 72 h, 12 μL of MTT solution (5 mg/ml in phosphate buffered saline) was added to each well. The plate was further incubated at 37 °C for 3 h to allow formation of formazan product. After removing the medium, 100 μL of DMSO was added to dissolve the formazan crystals. After 15 min, the contents of the wells were homogenized on a microplate shaker. The optical densities (OD) were then determined by measuring absorbance with a microplate spectrophotometer at a wavelength of 540 nm and a reference wavelength of 620 nm. The median cytotoxic concentration (CC50) was calculated as the concentration of the constituent that reduced the viable cells to 50% of the untreated control. The maximal non-cytotoxic concentration (MNCC) was defined as the maximal concentration of the sample that did not exert a cytotoxic effect and resulted in more than 90% viable cells.

CPE reduction assay

The anti-viral activity of the extracts was measured by a virus-induced cytopathic effect (CPE) reduction assay as described previously [14, 16]. Briefly, 100 μL of virus suspension of 200 tissue culture infective dose (TCID50/mL) was added to each well of a 96-well culture plate containing confluent a MDCK cells monolayer. After incubation at 37 °C for 2 h, the virus solution was removed, and 100 μL of serial dilutions of the extracts and ribavirin were added to each well of the 96-well culture plates, using the maximal non-cytotoxic concentration (MNCC) as the highest concentration. The plates were incubated at 37 °C in a humidified 5% CO2 atmosphere for 48 h, and then the CPE was assessed. The virus-induced CPE was scored as follows: 0 = no CPE, 1 = 0–25% CPE, 2 = 25%–50% CPE, 3 = 50%–75% CPE, and 4 = 75%–100% CPE. Apart from test group, there were control group (treated with FBS-free medium instead of extracts and virus) and model group (treated with FBS-free medium and virus instead of extracts and virus). The CPE inhibition ratios were calculated using the equation: CPE inhibition % = 100 -[(ODtest-ODcontrol) *100/ (ODmodel- ODcontrol)]. The ODtest, ODmodel, and ODcontrol mean the optical density of test group, model group, and control group, respectively. At least three independent experiments with three parallel experiments were performed to determine the mean and SD value.

Measurement of viral RNA synthesis by quantitative and reverse transcription PCR (qPCR)

A549 cells were grown in RPMI1640 to about 90% confluence and were infected with influenza virus A/PR/8/34 (H1N1) influenza virus at 100 TCID50, followed by administration of test extracts for 5 h. To determine the expression level of hemagglutinin (HA) gene mRNA of influenza virus, cells were harvested and the total RNA was extracted by TRIzol (Invitrogen) according to the manufacture’s instruction. The primer sequences which were designed by Primer-BLAST from NCBI for quantitative real-time PCR of influenza virus were 5’-CCTGCTCGAAGACAGCCACAACG-3′ (sense) and 5’-TTCCCAAGAGCCATCCGGCGA-3′ (antisense). The GAPDH were used as internal control of cellular RNAs, with primer sequence of 5′- TGCTCCGAAGGGTGGCCCTTA-3′(sense) and 5′- TGCGTGTTTCCAGAGCCGTGC-3′(antisense). The total RNA was reverse transcribed into cDNA using the TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). The cDNA was used as template for real-time PCR conducted by SsoFast EvaGreen PCR 2 × master mix (Bio-Rad) using CFX 96 Realtime PCR system (Bio Rad location) according to the manufacture’s protocol. The data was analyzed using the mode for normalised expression (2-ΔΔCq).

Statistical analysis

Statistical analysis was performed using the Student’s unpaired t-test. The results were presented as mean ± S.D. (n = 3). *p < 0.05 and **p < 0.001 indicate a statistically significant difference as compared to the untreated control.

Results

NA has been validated as one of the most important targets to screen the drugs of anti-influenza virus. We first examined the ability of 190 organic extracts from 95 medicinal plants to inhibit NA activity by in vitro screening assay. Zanamivir was used as a positive control, its IC50 value to NA inhibition was 0.05 μg/mL. 14 extracts were found to effectively inhibit the NA activity at the concentration of 40 μg/mL. Among them, 5 extracts exhibited potent inhibition of NA activity, 9 extracts exhibited moderate NA inhibitory activity with IC50 values ranged from 4.1 to 37.3 μg/mL. The bioactive extracts and their NA inhibition activity were summarized in Table 1. The highest activity was demonstrated by MeOH extracts of Melaphis chinensis (1) and Amomurn villosum Lour (2) with IC50 = 4.1 and 4.9 μg/mL, respectively. Significant activity with IC50 = 5.0–10 μg/mL was also shown by MeOH extract of Sanguisorba officinalis (3), EtOAc extract of Melaphis chinensis (4) and MeOH extract of Flos Caryophylli (5). While other plant extracts (614) showed a moderate inhibitory activity on NA with the IC50 values ranging from 20.3 to 37.3 μg/mL. These results demonstrated that these plant extracts possessed significant inhibitory activities against influenza virus NA and the most active extracts 15 were then selected to further study their effects on the replication of influenza virus.
Table 1

Inhibitory activities of Chinese herbs extract on A(H1N1) influenza virus neuraminidase

No.

Positive control and Botanical name

Botanical part

Extract

Inhibition (%)a

IC50b

Voucher No.

Zanamivir

99.8

0.05

1

Melaphis chinensis (Bell)Baker

cecidium

MeOH

103.6

4.1

MCB091101

2

Amomurn villosum Lour.

fruit

MeOH

92.2

4.9

CG20080829

3

Sanguisorba officinalis L.

root

MeOH

100.8

5.1

SOL091101

4

Melaphis chinensis (Bell)Baker

cecidium

EtOAc

99.3

5.3

MCB091101

5

Flos Caryophylli

flowers

MeOH

94.1

9.1

SA091101

6

Areca catechu Linn

fruit

MeOH

85.1

19.3

ACL091101

7

Artemisia capillaries Thunb

whole plant

MeOH

91.3

19.4

ACT091101

8

Terminalia chebula Retz

fruit

EtOAc

78.4

20.3

TCR091101

9

Duchesnea indica (Andr.) Focke

whole plant

EtOAc

69.1

23.3

DIF091101

10

Terminalia chebula Retz.

fruit

MeOH

68

24.3

TCR091101

10

Murraya exotica L.

stem and leaves

MeOH

65.7

28.9

MEL091101

11

Geranium carolinianum L.

whole plant

MeOH

64.8

28.9

GCL091101

12

Polygonum cuspidatum

rhizome

EtOAc

63.9

29.8

PC091101

13

Saposhnikovia divaricata (Turez.) Schischk.

root

EtOAc

53.1

37.3

SDS091101

14

Callicarpa formosana Rolfe

fruit

MeOH

47.9

NTd

CFR091103

15

Gardenia jasminoides Ellis

fruit

MeOH

46.6

NT

GJE091101

16

Duchesnea indica (Andr.) Focke

whole plant

EtOAc

46.1

NT

DIF091101

17

Rosa laevigata Michx.

stem and leaves

EtOAc

45.8

NT

RLM091103

18

Euphorbia humifusa Willd. ex Schlecht.

whole plant

MeOH

43.9

NT

EHW091101

19

Litchi chinensis Sonn.

seed

EtOAc

43.9

NT

LCS091101

20

Punica granatum L.

fruit peel

MeOH

43.4

NT

PGL091101

21

Scutellaria baicalensis Georgi

root

EtOAc

41.3

NT

SBG091101

22

Amomum villosum Lour.

fruit

EtOAc

40.5

NT

CG20080829

23

Geranium carolinianum L.

whole plant

EtOAc

40.1

NT

GCL091101

24

Isatis indigotica Fort

stem and leaves

EtOAc

40.1

NT

IIF091103

25

Onosma gmelinii Ledeb

root

EtOAc

40

NT

OGL091101

26

Houttuynia cordata Thunb

whole plant

EtOAc

38.5

NT

HCT091101

27

Altingia chinensis (Champ.) Oliver ex Hance

stem and leaves

EtOAc

37.3

NT

ACO091103

28

Pogostemon cablin (Blanco) Bent.

whole plant

EtOAc

36.7

NT

PCB091101

29

Polygonum cuspidatum

rhizome

MeOH

36.1

NT

PC091101

30

Punica granatum L.

fruit peel

EtOAc

35.5

NT

PGL091101

31

Rosa laevigata Michx.

stem and leaves

MeOH

34.4

NT

RLM091103

32

Dianella ensifolia (Linn.) Redouté

fruit

EtOAc

31.5

NT

DER091103

33

Elsholtzia ciliata (Thunb.) Hyland.

whole plant

MeOH

31.3

NT

ECH091101

34

Atractylodes Lancea (Thunb) DC.

root

EtOAc

30.4

NT

ALD091101

35

Cynanchum otophyllum Schneid.

root

EtOAc

29.3

NT

COS091101

36

Homalocladium platycladum (F. Muell.) Bailey

whole plant

MeOH

29.1

NT

HPB091101

37

Cinnamomum cassia Presl

branch

MeOH

28.9

NT

CCP091101

38

Elsholtzia ciliata (Thunb.) Hyland.

whole plant

EtOAc

28.1

NT

ECP091101

39

Sarcandra glabra (Thunb.) Nakai

stem and leaves

EtOAc

26.8

NT

SGN091103

40

Altingia chinensis (Champ.) Oliver ex Hance

stem and leaves

MeOH

25.8

NT

ACO091103

41

Litchi chinensis Sonn.

seed

MeOH

25.5

NT

LCS091101

42

Phellodendron chinense Schneid

bark

EtOAC

25.4

NT

PCS091101

43

Euphorbia humifusa Willd. ex Schlecht.

whole plant

EtOAc

23.6

NT

EHW091101

44

Glycyrrhiza uralensis Fisch.

rhizome

EtOAc

23.1

NT

GUF091101

45

Woodwardia japonica (L. f.) Sm.

rhizome

MeOH

23

NT

WJS091101

46

Ardisia japonica (Thunb) Blume

whole plant

MeOH

22.7

NT

AJB091101

47

Cinnamomum cassia Presl

branch

EtOAc

22.7

NT

CCP091101

48

Equisetum hyemale L.

whole plant

EtOAc

22.1

NT

EHL091101

49

Fraxinus rhynchophylla Hance

bark

EtOAc

22.1

NT

FRH091101

50

Ardisia japonica (Thunb.) Blume

whole plant

EtOAc

21.7

NT

AJB091101

51

Andrographis paniculata (Burm. f.) Nees

whole plant

EtOAc

20.8

NT

APN091101

52

Punica granatum Linn.

stem

EtOAc

20.2

NT

AGL091103

53

Syzygium aromaticum

flowers

EtOAc

19.5

NT

SA091101

54

Artemisia capillaris Thunb.

whole plant

EtOAc

19.2

NT

ACT091101

55

Nepeta cataria L.

whole plant

MeOH

18.9

NT

NCL091101

56

Lonicera japonica Thunb.

flowers

MeOH

18

NT

AJT091101

57

Woodwardia japonica (L. f.) Sm.

rhizome

EtOAc

17.9

NT

WJS091101

58

Nepeta cataria L.

whole plant

EtOAc

17.4

NT

NCL091101

59

Dendranthema indicum (L.) Des Moul.

flowers

EtOAc

16.5

NT

DID091101

60

Senecio scandens Buch. -Ham. ex D. Don

whole plant

MeOH

16.3

NT

SSB091101

61

Onosma gmelinii Ledeb

root

MeOH

15.9

NT

OGL091101

62

Evodia rutaecarpa (Juss.) Benth.

fruit

MeOH

15.5

NT

ERB091101

63

Ligusticum chuanxiong Hort.

root

MeOH

15.5

NT

LCH091101

64

Atractylodes Lancea (Thunb.) DC.

root

MeOH

15.2

NT

ALD091101

65

Punica granatum L.

leaves

MeOH

15

NT

PGL091101

66

Artemisia indices Willd.

leaves

MeOH

14.8

NT

AIW091101

67

Serissa japonica (Thunb.) Thunb.

stem and leaves

EtOAc

14.8

NT

SJT091101

68

Prunella vulgaris L.

whole plant

MeOH

14.1

NT

PVL091101

69

Dicliptera chinensis (L.) Juss.

whole plant

MeOH

14

NT

DCJ091101

70

Glycyrrhiza uralensis Fisch.

rhizome

MeOH

13.7

NT

GUF091101

71

Platycladus orientalis (L.) Franco

leaves

EtOAc

13.4

NT

POF091101

72

Angelica dahurica (Fisch. ex Hoffm.) Benth.

root

MeOH

13.3

NT

ADB091101

73

Sarcandra glabra (Thunb.) Nakai

stem and leaves

MeOH

13.3

NT

SGN091101

74

Cynanchum otophyllum Schneid.

root

MeOH

13

NT

COS091101

75

Clerodendrum fortunatum Linn.

stem and leaves

EtOAc

12.5

NT

CFL091101

76

Scutellaria baicalensis Georgi

root

MeOH

12.2

NT

SBG091101

77

Sophora flavescens Alt.

root

MeOH

11.6

NT

SFA091101

78

Paris verticillata M.Bieb.

rhizome

EtOAc

11.4

NT

PVM091101

79

Semiaquilegia adoxoides (DC.) Makino

whole plant

EtOAc

11.4

NT

SAM091101

80

Magnolia liliflora Desr.

flowers

EtOAc

11.3

NT

MLD091101

81

Albizia julibrissin Durazz.

flowers

MeOH

NAc

NT

AJD091101

82

Albizia julibrissin Durazz.

flowers

EtOAc

NA

NT

AJD091101

83

Andrographis paniculata (Burm. f.) Nees

whole plant

MeOH

NA

NT

APN091101

84

Angelica dahurica (Fisch. ex Hoffm.) Benth.

root

EtOAc

NA

NT

ADB091101

85

Arctium lappa L.

seed

MeOH

NA

NT

ALL091101

86

Arctium lappa L.

seed

EtOAc

NA

NT

ALL091101

87

Areca catechu Linn

fruit

EtOAc

NA

NT

ACL091101

88

Artemisia argyi Levl. et Van.

leaves

MeOH

NA

NT

AAL091101

89

Artemisia argyi Levl. et Van.

leaves

EtOAc

NA

NT

AAL091101

90

Artemisia carvifolia Buch. -Ham. ex Roxb.

whole plant

EtOAc

NA

NT

ACB091101

91

Artemisia carvifolia Buch. -Ham. ex Roxb.

whole plant

MeOH

NA

NT

ACB091101

92

Artemisia indices Willd.

leaves

EtOAc

NA

NT

AIW091103

93

Bidens pilosa Linn.

whole plant

EtOAc

NA

NT

BPL091103

94

Bidens pilosa Linn.

whole plant

MeOH

NA

NT

BPL091103

95

Bupleurum tenue Buch-Ham. ex D. Don

root

EtOAc

NA

NT

BTB091101

96

Bupleurum tenue Buch-Ham. ex D. Don

root

MeOH

NA

NT

BTB091101

97

Callicarpa formosana Rolfe

fruit

EtOAc

NA

NT

CFR091103

98

Clerodendrum fortunatum Linn.

stem and leaves

MeOH

NA

NT

CFL091103

99

Clinopodium megalanthum

seed

EtOAc

NA

NT

CMC091101

100

Clinopodium megalanthum

seed

MeOH

NA

NT

CMC091101

101

Crataegus pinnatifida Bge.

fruit

MeOH

NA

NT

CPB091101

102

Crataegus pinnatifida Bge.

fruit

EtOAc

NA

NT

CPB091101

103

Dendranthema indicum (L.) Des Moul.

flowers

MeOH

NA

NT

DID091101

104

Dendranthema morifolium (Ramat.) Tzvel.

flowers

EtOAc

NA

NT

DMT091101

105

Dendranthema morifolium (Ramat.) Tzvel.

flowers

MeOH

NA

NT

DMT091101

106

Dianella ensifolia (Linn.) Redouté

fruit

MeOH

NA

NT

DER091103

107

Dicliptera chinensis (L.) Juss.

whole plant

EtOAc

NA

NT

DCJ091103

108

Duchesnea indica (Andr.) Focke

whole plant

MeOH

NA

NT

DIF091103

109

Epaltes australis Less.

whole plant

EtOAc

NA

NT

EAL091101

110

Epaltes australis Less.

whole plant

MeOH

NA

NT

EAL091101

111

Equisetum hyemale L.

whole plant

MeOH

NA

NT

EHL091101

112

Euchresta japonica Hook. f. ex Regel

root

EtOAc

NA

NT

EJH091101

113

Euchresta japonica Hook. f. ex Regel

root

MeOH

NA

NT

EJH091101

114

Eupatorium catarium Veldkamp

whole plant

MeOH

NA

NT

ECV091103

115

Eupatorium catarium Veldkamp

whole plant

EtOAc

NA

NT

ECV091103

116

Eupatorium fortunei Turcz.

whole plant

EtOAc

NA

NT

EFT091101

117

Eupatorium fortunei Turcz.

whole plant

MeOH

NA

NT

EFT091101

118

Eupolyphaga seu Steleophaga

insect

EtOAc

NA

NT

ESS091101

119

Eupolyphaga seu Steleophaga

insect

MeOH

NA

NT

ESS091101

120

Evodia rutaecarpa (Juss.) Benth.

fruit

EtOAc

NA

NT

ERB091101

121

Ficus hirta Vahl

leaves

MeOH

NA

NT

FHV091101

122

Ficus hirta Vahl

leaves

EtOAc

NA

NT

FHV091101

123

Forsythia suspensa (Thunb.) Vahl

fruit

MeOH

NA

NT

FSV091101

124

Forsythia suspensa (Thunb.) Vahl

fruit

EtOAc

NA

NT

FSV091101

125

Fraxinus rhynchophylla Hance

bark

MeOH

NA

NT

FRH091101

126

Gardenia jasminoides Ellis

fruit

EtOAc

NA

NT

GJE091101

127

Homalocladium platycladum (F. Muell.) Bailey

whole plant

EtOAc

NA

NT

HPB091103

128

Homalomena occulta (Lour.) Schot

rhizome

MeOH

NA

NT

HOS091101

129

Homalomena occulta (Lour.) Schot

rhizome

EtOAc

NA

NT

HOS091101

130

Houttuynia cordata Thunb

whole plant

MeOH

NA

NT

HCT091101

131

Ilex cornuta Lindl

stem

MeOH

NA

NT

ICL091103

132

Ilex cornuta Lindl

stem

EtOAc

NA

NT

ICL091103

133

Inula japonica Thunb.

flowers

MeOH

NA

NT

IJT091101

134

Inula japonica Thunb.

flowers

EtOAc

NA

NT

IJT091101

135

Isatis indigotica Fort

stem and leaves

MeOH

NA

NT

IIF091103

136

Ligusticum chuanxiong Hort.

root

EtOAc

NA

NT

LCH091101

137

Lobelia chinensis Lour.

whole plant

MeOH

NA

NT

LCH091101

138

Lobelia chinensis Lour.

whole plant

EtOAc

NA

NT

LCL091101

139

Lonicera confusa (Sweet) DC.

stem and leaves

MeOH

NA

NT

LCD091103

140

Lonicera confusa (Sweet) DC.

stem and leaves

EtOAc

NA

NT

LCD091103

141

Lonicera japonica Thunb.

flowers

EtOAc

NA

NT

LJT091101

142

Lonicera japonica Thunb.

stem and branch

MeOH

NA

NT

LJT091101

143

Lonicera japonica Thunb.

stem and branch

EtOAc

NA

NT

LJT091101

144

Lycium chinense Mill.

root bark

MeOH

NA

NT

LCM091101

145

Lycium chinense Mill.

Root bark

EtOAc

NA

NT

LCM091101

146

Magnolia liliflora Desr.

flowers

MeOH

NA

NT

MLD091101

147

Melia azedarach L.

bark

EtOAc

NA

NT

MAL091103

148

Melia azedarach L.

bark

MeOH

NA

NT

MAL091103

149

Murraya exotica L.

stem and leaves

EtOAc

NA

NT

MEL091103

150

Mussaenda pubescens Ait. f.

stem and leaves

EtOAc

NA

NT

MPA091103

151

Mussaenda pubescens Ait. f.

stem and leaves

MeOH

NA

NT

MPA091103

152

Paris verticillata M.Bieb.

rhizome

MeOH

NA

NT

PVM091101

153

Perilla frutescens (L.) Britt.

flowers

EtOAc

NA

NT

PFB091103

154

Perilla frutescens (L.) Britt.

flowers

MeOH

NA

NT

PFB091103

155

Peucedanum praeruptorum Dunn

root

EtOAc

NA

NT

PPD091101

156

Peucedanum praeruptorum Dunn

root

MeOH

NA

NT

PPD091101

157

Phellodendron chinense Schneid

bark

MeOH

NA

NT

PCS091101

158

Phytolacca acinosa Roxb.

root

EtOAc

NA

NT

PAR091101

159

Phytolacca acinosa Roxb.

root

MeOH

NA

NT

PAR091101

160

Pinellia ternata (Thunb.) Breit.

stem

MeOH

NA

NT

PTB091101

161

Pinellia ternata (Thunb.) Breit.

stem

EtOAc

NA

NT

PTB091101

162

Platycladus orientalis (L.) Franco

leaves

MeOH

NA

NT

POF091101

163

Pogostemon cablin (Blanco) Bent.

whole plant

MeOH

NA

NT

PCB091101

164

Prunella vulgaris L.

whole plant

EtOAc

NA

NT

PVL091101

165

Punica granatum L.

leaves

EtOAc

NA

NT

PGL091103

166

Punica granatum Linn.

stem

MeOH

NA

NT

PGL091103

167

Sanguisorba officinalis L.

root

EtOAc

NA

NT

SOL091101

168

Saposhnikovia divaricata (Trucz.) Schischk.

root

MeOH

NA

NT

SDS091101

169

Scaphium wallichii Shott & Endl.

seed

MeOH

NA

NT

SWS091101

170

Scaphium wallichii Shott & Endl.

seed

EtOAc

NA

NT

SWS091101

171

Semiaquilegia adoxoides (DC.) Makino

whole plant

MeOH

NA

NT

SAM091101

172

Senecio scandens Buch-Ham. ex D. Don

whole plant

EtOAc

NA

NT

SSB091101

173

Serissa japonica (Thunb.) Thunb.

stem and leaves

MeOH

NA

NT

SJT091103

174

Sophora flavescens Alt.

root

EtOAc

NA

NT

SFA091101

175

Stemona japonica (Bl.) Miq.

root

MeOH

NA

NT

SJM091101

176

Stemona japonica (Bl.) Miq.

root

EtOAc

NA

NT

SJM091101

177

Strobilanthes cusia (Ness) W. Ktze.

stem and leaves

MeOH

NA

NT

SCW091101

178

Strobilanthes cusia (Ness) W. Ktze.

stem and leaves

EtOAc

NA

NT

SCW091101

179

Thlaspi arvense L.

whole plant

MeOH

NA

NT

TAL091103

180

Thlaspi arvense L.

whole plant

EtOAc

NA

NT

TAL091103

181

Turczaninovia fastigiata (Fisch.) DC.

flowers

MeOH

NA

NT

TFD091101

182

Turczaninovia fastigiata (Fisch.) DC.

flowers

EtOAc

NA

NT

TFD091101

183

Vitex trifolia L.

stem and leaves

EtOAc

NA

NT

VTL091103

184

Vitex trifolia L.

stem and leaves

MeOH

NA

NT

VTL091103

185

Wikstroemia indica (Linn.) C. A. Mey.

whole plant

MeOH

NA

NT

WIC091103

186

Wikstroemia indica (Linn.) C. A. Mey.

whole plant

EtOAc

NA

NT

WIC091103

187

Xanthium sibiricum Patrin ex Widder

fruit

EtOAc

NA

NT

XSP091103

188

Xanthium sibiricum Patrin ex Widder

fruit

MeOH

NA

NT

XSP091103

189

Zanthoxylum nitidum (Roxb.) DC.

root

MeOH

NA

NT

ZND091101

190

Zanthoxylum nitidum (Roxb.) DC.

root

EtOAc

NA

NT

ZND091101

aPercentage inhibition was calculated relative to a blank group containing virus NA but no inhibitors, final concentration at 40 μg/mL; bIC50 values represent the concentration that caused 50% NA enzyme activity loss, the average of at least three independent assays, IC50 values are in μg/mL. c: not active; d: not test

To validate whether these extracts 15 that exhibited NA inhibitory activity could protect host cells from influenza virus A (H1N1) infections, the CPE reduction assay was carried out in MDCK cells. The human influenza virus A/PR/8/34 (H1N1) strain was used to infect MDCK cells. Cells were incubated in the presence or absence of the extracts 1–5, after 48 h of incubation, their CPE reduction activity on virus multiplication was then examined. As shown in Table 2, the extracts 1–5 could protect MDCK cells from the infection of influenza virus A (H1N1), exhibited a drastic reduction of influenza virus-induced CPE. The EC50 values of the extracts 15 ranged from 1.8 to 14.1 μg/mL, similar to the results obtained in NA assays. Among the five extracts, the MeOH extract (2) from the fruits of Amomurn villosum had excellent CPE activity with very low EC50 values of 1.8 μg/mL, this is comparable to that of the positive compound ribavirin (3.2 μg/mL). The viability of MDCK cells incubated in the presence or absence of the extracts was evaluated by MTT assay, the CC50 values of the extracts 15 was found to be from 97.0 to 779.2 μg/mL, suggesting that the extracts protected significantly host cells from influenza virus infection and did not exhibit considerable cytotoxicity against MDCK cells. The maximal non-cytotoxic concentration (MNCC) of the extracts 1–5 were found to be from 30 to 300 μg/mL in MDCK cells. Their therapeutic selective index (SI) in MDCK cells ranged from 14 to 438, and among of them, the SI value of A. villosum was highest on basis of its low cytotoxicity and its high CPE effect. These data demonstrated that the extracts 1–5 protected MDCK host cells from viral damage with very low toxicity. Thus, in agreement with that these extracts inhibited NA activities, the extracts 15 reduced host cell damage caused by the influenza virus A (H1N1) infection.
Table 2

Inhibitory activity of Chinese herbs extracts (15) on A(H1N1) influenza virus by CPE assay

Sample No.

EC50a

CC50b

MNCCc

SId

1

7.7

184.3

30

24

2

1.8

779.2

300

438

3

8.1

478.4

100

59

4

7.2

97.0

30

14

5

14.1

744.3

300

53

Ribavirin

3.2

> 100

e

> 31

Zanamivir

> 90.4

> 1506.0

> 301.2

17

aEC50: Effective concentration required to protect 50% of cells; b CC50: Median (50%) cytotoxic concentration in MDCK cells; c MNCC: Maximal non-cytotoxic concentration in MDCK cells, values in μg/mL; d SI:Selectivity index, CC50/EC50.e: not test

To further examine whether the protective effect of the extracts15 is related with the inhibition of influenza viral replication, total RNA was extracted and subjected to quantitative reverse-transcription PCR in the A/H1N1 virus-infected A549 cells. Our results showed that treatment with the extracts 15 for 5 h resulted in a substantial reduction in viral RNA expression level in a dose-dependent manner (Fig. 1). All extracts 15 at the high concentration (30 μg/mL) had significant inhibitory effects on viral RNA expression as compared with untreated control, even more powerful than ribavirin (Fig. 1). The extracts 25 at medium concentration (10 μg/mL) also demonstrated significant inhibitory effects on viral RNA synthesis. Interestingly, the extracts 3 and 4 at low concentration of 3 μg/mL still significantly inhibited RNA synthesis of influenza viruses. These data indicate that the extracts 15 could inhibit significantly the replication of influenza viruses in cultures by RT-PCR analysis, which validated their anti-influenza viral activity obtained by CPE reduction assay.
Fig. 1
Fig. 1

Dose-dependent inhibitory effect of the extracts 15 on viral RNA synthesis. A549 cells were infected with 100 TCID50 influenza H1N1 viruses and treated with different concentrations of the extracts 15 (3, 10 or 30 μg/mL) and the DMSO (0.03%) for 5 h. The total RNA was extracted and followed by qPCR analysis. To quantify the changes in gene expression, the 2-ΔΔC(q) method was used to calculate relative changes which were normalized to the GAPDH gene and the untreated control (model group, which was set to 1). Value calculated as Mean ± SD of three independent tests, with * p < 0.05 and **p < 0.001, respectively

Discussion

In the course of our screening of NA inhibitors for influenza virus A (H1N1), a total of 190 extracts of 95 medicinal plants traditionally used in Lingnan Chinese Medicines were submitted to in vitro screening for their NA inhibitory activities. Among of them, the organic extracts 15, obtained from Melaphis chinensis, Amomurn villosum, Sanguisorba officinalis and Flos Caryophylli, were found to significantly inhibit the NA activity (IC50 < 10 μg/mL, Table 1) and the replication of influenza virus in a dose-dependent manner (Fig. 1), and exhibited very low cytotoxicity to the host cells with the high selective index (SI) values ranging 14 to 438 (Table 2). Therefore, these Chinese herb extracts might contain bioactive components responsible for anti-influenza virus activity at non-toxic concentration and they could be a promising source of natural NA inhibitors.

It was demonstrated previously that the aqueous extracts of barks, leaves and galls of Melaphis chinensis have anti-influenza virus activity and some compounds such as gallotannins isolated from M. chinensis are responsible for the anti-influenza virus effect [17]. The presence of such compounds in our EtOAc and MeOH extracts of galls of M. chinensis may explain the biological activities seen in our screenings.

Flos Caryophylli also known as cloves, is considered acrid, warm and aromatic in Traditional Chinese Medicines for the treatment of stomachache, diarrhea and dental pain [18]. It was reported that the hot water extract of Flos Caryophylli have been shown to have anti-herpes virus, anti-hepatitis C virus and anti-cytomegalovirus activities in vitro and in vivo, and compounds such as ellagitannin and eugeniin were identified as the bioactive components with anti-virus properties [19]. In the present study, the MeOH extract of Flos Caryophylli showed IC50 value of 9.1 μg/mL towards NA and EC50 value of 14.1 μg/mL against influenza virus. In our latest phytochemical study on the MeOH extract of Flos Caryophylli [14], a bioassay-guided isolation led to identification of ten flavonoids, seven tannins and two chromones as NA inhibitors with IC50 values ranging from 8.4 to 94.1 μM. These polyphenolic constituents were found to protect MDCK cells from A(H1N1) influenza infections (EC50 = 1.5–84.7 μM) with very low cytotoxicity to the host cells (CC50 = 374.3–1266.9 μM)), with selective index (SI) ranging from 7 to 297 [14].

The roots of S. officinalis (Rosaceae) are well-known Chinese herbs officially listed in the Chinese Pharmacopeia and have been used for the treatment of bleeding, diarrhea and burns. Early chemical studies showed that S. officinalis synthesize a variety of secondary metabolites, particularly polyphenols, triterpenoids, saponins and flavonoids with specific biological activities such as anti-asthmatic, anti-bacterial, anti-cancer and anti-inflammation [2025]. A variety of flavonoids, saponins and polyphenols isolated from medicinal plant have been studied extensively and exhibited anti-influenza activities [12]. The MeOH extract of S. officinalis showed strong activities towards NA (IC50: 5.1 μg/mL) and against influenza virus (EC50: 8.1 μg/mL). The anti-influenza activity may be due to the presence of flavonoids and polyphenols in the MeOH fraction.

The fruits of A. villosum (Zingiberaceae) were consumed widely as popular cooking spices in East Asian countries and have been traditionally used as a medicine to treat various digestive disorders [26]. The volatile oils of the fruits of A. villosum were shown to be the major components and suggested to be responsible for the different biological activities such as analgesic, anti-oxidation and anti-inflammation [27]. In this study, the MeOH extract of the fruits of A. villosumwas shown to significantly inhibit NA activities (IC50: 4.9 μg/mL) and protect the host cells from CPE damage (EC50: 1.8 μg/mL) without cytotoxicity, and its therapeutic selective index (SI) is 439 in MDCK cell culture.

In this study, we limit our study on EtOAc and MeOH extracts of medical plants since bioassay-guided isolation of neuraminidase inhibitors in aqueous extracts remains a challenging task for us. However, this may decrease the risk of false-positive results in the enzyme-based screening caused by some interfering components present within aqueous extracts. Future study will try to improve the screening methods on aqueous extracts that may also contain active components with anti-neuraminidase activity.

Conclusion

We carried out the in vitro screening of anti-neuraminidase activity of 190 herbal extracts from 95 medicinal plants traditionally used in Lingnan Chinese Medicines. Among the tested extracts, 5 extracts, obtained from Amomurn villosum, Melaphis chinensis, Sanguisorba officinalis and Flos Caryophylli, showed potent NA inhibitory activity. Comprehensive literature survey revealed that no study has been reported on the effects of the organic extracts of A. villosum and S. officinalis on anti-influenza virus activities and small-molecule NA inhibitors from these extracts have not been chemically identified yet. Further studies are underway to isolate bioactive components of these extracts by bioassay-guided fractionation, and to explore their antiviral mechanisms and finally determine their clinical potentials.

Abbreviations

CPE: 

Cytopathic effect

HA: 

Haemagglutinin

HHDP: 

Hexahydroxydiphenoyl

MDCK: 

Madin-Darby canine kidney

MNCC: 

Maximal non-cytotoxic concentration

MTT: 

3-[4,5-dimethyl-thiazol-2-yl]-2,5- diphenyl tetrazolium bromide

MUNANA: 

methylumbelliferyl-α-D-N-acetylneuraminate

NA: 

Neuraminidase

SI: 

Selectivity index.

Declarations

Acknowledgements

The authors would like to acknowledge all the fellows in Research Center of Medicinal Plants Resource Science and Engineering, Guangzhou University of Chinese Medicine for their great support and encouragement. We also thanks for their assistance in collecting medicinal plants from Jinxing Qiu, Guozheng He and Honghui Huang.

Funding

This work was supported by China National Natural Science Foundation Grant (No.81373432), Guangzhou Science and Technology Program Grant (No. 2014 J4100118) to JL, National Great Science and Technology Major Projects (2012ZX09301002-2013HXW-11) and Beijing Natural Science Foundation Grant (No. 7152103) to AL.

Availability of data and materials

The data sets used and /or analysed during the current study available from the corresponding authors on reasonable request.

Authors’ contributions

JL and AL conceived and designed the study. JL, KC and HM collected the herbs and prepared the herbal extracts. MZ, LG, WZ and AL carried out herbal screening and anti-influenza virus studies. JL, MZ and AL analyzed data. JL wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Ministry of Education Key Laboratory of Chinese Medicinal Resource from Lingnan, Research Center of Medicinal Plants Resource Science and Engineering, Guangzhou University of Chinese Medicine, 232 Waihuandong Road, Higher Education Mega Center, Guangzhou, 510006, People’s Republic of China
(2)
Beijing Key Laboratory of Drug Target Research and Drug Screening, Institute of Material Medica, Chinese Academy Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Xicheng District, Beijing, 100050, People’s Republic of China

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Copyright

© The Author(s). 2018

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