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Erschienen in: BMC Complementary Medicine and Therapies 1/2018

Open Access 01.12.2018 | Research article

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

verfasst von: Jiawei Liu, Mian Zu, Kaotan Chen, Li Gao, Huan Min, Weiling Zhuo, Weiwen Chen, Ailin Liu

Erschienen in: BMC Complementary Medicine and Therapies | Ausgabe 1/2018

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.
Abkürzungen
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.

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.

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.

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.
Not applicable.
Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Metadaten
Titel
Screening of neuraminidase inhibitory activities of some medicinal plants traditionally used in Lingnan Chinese medicines
verfasst von
Jiawei Liu
Mian Zu
Kaotan Chen
Li Gao
Huan Min
Weiling Zhuo
Weiwen Chen
Ailin Liu
Publikationsdatum
01.12.2018
Verlag
BioMed Central
Erschienen in
BMC Complementary Medicine and Therapies / Ausgabe 1/2018
Elektronische ISSN: 2662-7671
DOI
https://doi.org/10.1186/s12906-018-2173-1

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