In vitro anti-influenza virus activity of a cardiotonic glycoside from Adenium obesum (Forssk.)

Article Outline

• Abstract
• Introduction
• Materials and methods
  • General experimental procedures
  • Plant material
  • Preparation of the crude plant extracts
  • Fractionation of the methanolic extract of Adenium obesum
  • Isolation of compound A
  • Compound A (oleandrigenin-β-d-glucosyl-(1→4)-β-d-digitalose)
  • Cells and viruses (Nagai et al., 1992)
  • In vitro anti-influenza virus activity and cytotoxicity of samples (Nagai et al. 1992, 1995a,b)
• Results and discussion
• References
• Copyright

Abstract 

Methanolic extracts of six Saudi plants were screened for their in vitro antiviral activity using influenza virus A/PR/8/34 (H1N1) and MDCK cells in an MTT assay. The results indicated that the extracts of Adeniumobesum and Tephorosianubica possessed antiviral activity (99.3 and 93.3% inhibition at the concentration of 10μg/ml, respectively). Based on these results A. obesum was selected for further study by applying bioactivity-guided fractionation to isolate its antiviral principle. The antiviral principle was isolated from the chloroform fraction through solvent fractionation, combined open liquid chromatography and HPLC. The isolated active compound A was identified as oleandrigenin-β-d-glucosyl (1→4)-β-d-digitalose, on the basis of its spectral analysis (MS, 1D and 2D NMR). The isolated glycoside showed reduction of virus titre by 69.3% inhibition at concentration of 1μg/ml (IC₅₀=0.86μg/ml).

Keywords: Adenium obesum, Tephorosia nubica, Oleandrogenin-glucosyldigitaloside, Antiviral, Influenza virus A/PR/8/34 (H1N1), MDCK cells, MTT assay

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Introduction 

The influenza virus is a highly infective that causes acute respiratory diseases. In more serious cases, influenza causes pneumonia, which can be particularly fatal in young children, immunocompromised patients, elderly people, and patients with cardiopulmonary diseases. In addition, influenza spreads around the world in seasonal epidemics, and killing numerous people in pandemic years (Hein et al. 2004). Zanamivir and oseltamivir have been used for the treatment and prophylaxis of influenza viruses (De Clercq 2004), however their utilities have been limited by their side effects and the emergence of resistant viral strains (Ryan et al., 1995, Hayden and Hay, 1992).

The development of safe, effective, and inexpensive antiviral drugs is among top global priorities in drug development, as many virus diseases are not yet curable. The new approach in antiviral chemotherapy is seeking for new antiviral agents from plant origin (Jassim and Naji 2003). Large numbers of extracts and pure compounds have been tested and showed selective antiviral activity (Vanden Berghe et al. 1986).

The flora of Saudi Arabia is rich in wild plants which have been used by the local communities for the treatment of a large number of ailments. Therefore, plants of Saudi flora still need more investigation to detect their importance in the discovery of new source for new molecules that can be used in treatment of different ailments following their traditional uses.

The aim of the present study is to identify the anti-influenza viral ingredients in the methanolic extracts of some Saudi plants against influenza virus A/PR/8/34 (H1N1).

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Materials and methods 

General experimental procedures 

Optical rotations were measured with a DIP-360 automatic polarimeter (JASCO Co., Tokyo). IR spectra were taken out on a JASCO FTIR-230 IR spectrometer. Mass spectra were measured on JEOL JMS DX-700 Mstation (high resolution mass spectrometry) or JEOL JMS-AX505HA (low resolution mass spectrometry). FAB-MS was conducted with thioglycerol–glycerol (1:1 by vol.) as matrix, acceleration voltage (8kV), emitter current (3mA), gun High Voltage (6kV), gas (Xe). NMR spectra were measured on Varian Unity-400 using C₅D₅N. HPLC was performed on Shimadzu LC-20AT pump equipped with Shimadzu SPD-6A spectrophotometric detector, Pegasil ODS column (4.6I.D.×250mm, Senshu Scientific Co., Tokyo, Japan) and X-Bridge™ Prep ODS column (10I.D.×250mm, Waters Co., Milford, MA, USA). The samples were eluted at a flow rate of 4ml/min, and detected at 210nm.

Plant material 

The plants were collected from the western region of Saudi Arabia between March and May 2006. Plants under investigation were identified by staff members of Department of Biology, Faculty of Science, King Abdulaziz University, Saudi Arabia. Voucher specimens were deposited at the Herbarium of the Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia.

Preparation of the crude plant extracts 

Air-dried and powdered plant materials (aerial parts, 30g each) were refluxed with methanol (3×100ml). The solvent was removed under reduced pressure and the methanolic extracts were kept at 4°C.

Fractionation of the methanolic extract of Adenium obesum 

The dried powder of aerial parts of A. obesum (320g) was extracted with methanol (2l) using Ultra-turrax T25 homogenizer (Janke and Kunkel, IKA Labortechnik, Stauten, Germany) at a room temperature. The methanolic extract was evaporated to dryness to give (63.8g, 19.9%). The crude methanolic extract (30g) was dissolved with 50% MeOH (150ml) and extracted with n-hexane (3×200ml). The MeOH extract was concentrated and then the aqueous layer was partitioned successively with AcOEt (3×200ml) CHCl₃ (3×200ml) and n-BuOH (3×200ml). The n-hexane extract was designated as fraction A (8.4g), AcOEt extract as fraction B (16.1g), CHCl₃ extract as fraction C (0.3g), n-BuOH extract as fraction D (6.45g), and aqueous extract as fraction E (11.0g).

Isolation of compound A 

Fraction C (0.1g) was further fractionated by open silica gel column chromatography [Silica gel 60N, (20mm I.D.×130mm), Kanto Chemicals Co. Inc.] using CHCl₃–MeOH (5:1, 1.5l). Eluates were fractionated into 14ml per fraction to obtain 81 fractions. By comparison with TLC patterns of these fractions, 13 fractions (Fr. EC1 to EC12) were finally obtained. The column was washed with MeOH (200ml) to obtain EC13. The most potent anti-influenza virus active fraction (EC8, 31.66mg) was further purified by HPLC on X-Bridge ODS column (10mmI.D.×150mm, Waters Assoc.) with CH₃CN–H₂O (20:80) at a flow rate of 4.8ml/min. Compound A (4.51mg) was obtained as the peak eluted at a retention time of 26.96min.

Compound A (oleandrigenin-β-d-glucosyl-(1→4)-β-d-digitalose) 

Pale yellow amorphous powder [α]_D^23.4 −19.7 (CO.1625, MeOH); IR (KBr) υ_max 1739, 1624; UV λ_max nm (logɛ): 214 (4.0); LR-FABMS (glycerol–thioglycerol, positive mode): m/z 777 (M+Na)⁺, HR-FABMS (glycerol–thioglycerol, positive) Found 777.366 (Calcd. for C₃₈H₅₈O₁₅Na: 777.3673). ¹H NMR: δ ppm [400MHz, pyridine-d₅] δ_H 1.47 (2H, m, H-1), 1.67, 1.94 (H, m, H-2a,b), 4.33 (1H, d, J=2, H-3), 1.69, 1.83 (1H, m, H-4a,b), 1.96 (1H, brs, H-5), 1.21 (2× 2H, m, H-6, H-7), 1.74 (1H, m, H-8), 1.70 (1H, m, H-9), 2.10 (2H, m, H-11), 1.21, 1.45 (2H, m, H-12a,b), 2.04 (1H, dd, J=15, 2.1, H-15a), 2.80 (1H, dd, J=15, 9.2, H-15b), 5.69 (1H, ddd, J=9.2, 9.2, 2.1, H-16), 3.39 (1H, d, J=9.2, H-17), 1.06 (3H, s, H-18), 5.25 (1H, dd, J=18.4, 16, H-21a), 5.44 (1H, dd, J=18.4, 1.6, H-21b), 6.35 (1H, t, J=1.7, H-22), 1.85 (3H, s, OCOCH₃), 5.77 (1H, brs, –OH), 4.69 (1H, d, J=8.4, H-1′), 4.45 (1H, dd, J=8.4, 9.7, H-2′), 3.55 (1H, dd, J=9.7, 2.6, H-3′), 4.34 (1H, d, J=2.6, H-4′), 3.73 (1H, ddd, J=6.6,6.6,6.6, H-5′), 1.59 (3H, d, J=6.6, H-6′), 3.66 (3H, s, OCH₃), 5.16 (1H, d, J=7.5, H-1″), 3.99 (1H, dd, J=7.5, 8.8, H-2″), 4.25 (1H, dd, J=8.8, 8.8, H-3″), 4.2 (1H, dd, J=8.8, 8.8, H-4″), 3.99 (1H, brt, J=8.8, H-5″), 4.39 (1H, dd, J=11.9, 5.8, H-6″), 4.58 (1H, brd, J=11.9, H-6″). ¹³C NMR: δ ppm [100MHz, pyridine-d₅] δ_C 30.7 (C-1), 27.1 (C-2), 74.5 (C-3), 30.5 (C-4), 36.6 (C-5), 30.1 (C-6), 21.2 (C-7), 42.0 (C-8), 35.9 (C-9), 35.4 (C-10), 21.7 (C-11), 39.0 (C-12), 50.6 (C-13), 83.5 (C-14), 41.3 (C-15), 75.0 (C-16), 56.9 (C-17), 16.4 (C-18), 23.7 (C-19), 169.9 (C-20), 76.3 (C-21), 121.7 (C-22), 174.3 (C-23), 170.3 (OCOCH₃), 20.8 (OCOCH₃₎, 103.5 (C-1′), 71.5 (C-2′), 85.7 (C-3′), 76.7 (C-4′), 70.6 (C-5′), 17.8 (C-6′), 59.0 (OCH₃), 105.6 (C-1″), 76.2 (C-2″), 78.4 (C-3″), 71.9 (C-4″), 78.7 (C-5″), 63.2 (C-6″).

Cells and viruses (Nagai et al., 1992) 

Madin-Darby canine kidney (MDCK) cells were grown, and maintained as reported previously. Mouse-adapted influenza virus A/PR/8/34 (H1N1), which was maintained in Kitasato University (Tokyo, Japan), was grown in the allantoic cavity of 10-day-old embryonated hen eggs. The supernatant of allantoic fluid at 1000×g for 20min was stored at −80°C until use.

In vitro anti-influenza virus activity and cytotoxicity of samples (Nagai et al., 1992, Nagai et al., 1995a, Nagai et al., 1995b) 

Mouse-adapted influenza virus A/PR/8/34 (H1N1) was used in the in vitro antiviral assay as reported previously. 10% MeOH solution of samples (20μl/well, final concentration of the sample; 10μg/ml) or only 10% MeOH (control) was added to MDCK cells with or without the virus absorption, and cultured for 3 days at 37°C. The virus titre in the cultured medium was determined by measurement of influenza virus sialidase activity according to the procedure of Nagai et al. (1995a). Survival rate of MDCK cells was assessed using in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by viable cells (Hudson 1990) in accordance with the procedure of Nagai et al., 1995a, Nagai et al., 1995b.

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Results and discussion 

In this study, six Saudi medicinal plants were tested for their in vitro antiviral activity against influenza A/PR/8/34 (H1N1) virus strain. The methanolic extracts of the collected plants were tested at 10μg/ml. The results of standard drug and that of tested plants are presented in Table 1.

Table 1. In vitro anti-influenza virus activity of methanolic extracts of aerial parts of some Saudi plants.

No.	Plant name	Anti-influenza virus activity		Cytotoxicity against MDCK cell (%)b
		Virus titre (% of control)	Viable MDCK cell (%)a
1	Cucumis prophetarum L.	84.0	3.3	7.5
2	Euphorbia ammak (Latex)	12.0	0.4	99.4
3	Marrubium vulgare	84.2	9.1	0
4	Stachys sp. aff. Schimperi Vatke	79.7	3.8	0
5	Tephrosia nubica (Boiss.) Bak.	6.7	61.6	20.4
6	Adenium obesum (Forssk.) Roem & Schult	0.7	57.0	49.2
Ref	Zanamivir® (1μg/ml)	0.3	96.7	4.1
	Control (water)	100	0	0

From the results in Table 1, Euphorbia ammak, Tephrosia nubica and Adenium obesum potently reduced influenza virus titre, which were evaluated as viral sialidase activity. MDCK cell viability potently increased when either MeOH extract of T. nubica or A. obesum was added, however, the viability was low even by addition of the extracts of E. ammak and its cytotoxicity was very high. From the result of the present screening, it is expected that T. nubica or A. obesum contains anti-influenza virus ingredients. Subsequently, the methanolic extract of A. obesum was chosen in this study to isolate the active antiviral compound.

A. obesum is known as desert rose (Dimmit and Hanson 1991), and it has been reported the presence of several cardiotonic glycosides in addition to anthocyanidin glycosides and steroids (Yamauchi and Abe 1990).

The CHCl₃ fraction (Fr.C) of the methanolic extract of A. obesum revealed the highest antiviral activity with a lower cytotoxicity at a concentration of 5μg/ml (Table 2). Accordingly, Fr. C was further purified on a silica gel column following activity-guided fractionation, and compound A (Fig. 1) was only isolated as the anti-influenza virus active substance.

Table 2. In vitro anti-influenza virus activity of fractions (Frs. A-E) from the methanolic extract of A. obesum.

Test extract	Conc. (μg/ml)	Anti-influenza virus activity		Cytotoxicity against MDCK cell (%)b
		Virus titre (% of control)	Viable MDCK cell (%)a
Control	–	100.0	6.0–7.8	0
Hexane (Fr A)	1	121.1	6.5	10.2
	5	96.7	8.1	9.2
	10	95.5	16.1	8.8
	25	80.2	10.3	10.3
	50	79.6	16.3	18.0
AcOEt (Fr B)	1	91.1	5.6	17.2
	5	1.3	26.5	79.2
	10	0.3	0.9	99.2
	25	0.3	0.5	99.5
	50	0.2	0.4	99.4
CHCl3 (Fr C)	1	119.0	7.8	21.4
	5	4.6	54.1	40.6
	10	0.3	2.3	97.6
	25	0.1	0.5	99.4
	50	0.4	0.4	99.5
BuOH (Fr D)	1	129.1	2.9	5.0
	5	90.1	6.1	5.4
	10	82.2	28.2	0
	25	14.2	68.1	28.7
	50	0.2	4.4	95.4
H2O (Fr E)	1	119.1	5.0	6.1
	5	127.5	7.6	4.9
	10	140.6	5.8	6.4
	25	106.9	3.5	5.9
	50	112.3	5.2	8.7
Zanamivir	1	0.1–0.2	92.4–96.0	5.5–6.8

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• Fig. 1. 

  Characteristic HMBC correlation of compound A.

Because compound A had a considerable cytotoxicity against MDCK cells at concentrations of more than 2.5μg/ml (CC₅₀=1.53μg/ml) (data not shown), anti-influenza virus activity was tested at concentrations between 0.1 and 2.5μg/ml (Fig. 2). Compound A reduced influenza virus titre in culture medium in a dose-dependent manner between 0.1 and 1.5μg/ml (IC₅₀=0.86μg/ml, Fig. 2) improving the survival of MDCK cells. However, this effect was not observed more than 50% of survival rate of MDCK cells which was improved by compound A, and the IC₅₀ of the compound A could not be calculated from the point of view of MDCK cells survival (Fig. 2).

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• Fig. 2. 

  Anti-influenza virus activity and cytotoxicity on MDCK cells of compound A.

Compound A gave (M+Na)⁺ at m/z 777, and the molecular formula was determined to be C₃₈H₅₈O₁₅. IR spectrum of compound A revealed the presence of characteristic butenyllactone ring of cardiac glycosides (1739 and 1624cm⁻¹). Compound A was subjected to spectral analysis using ¹H NMR, ¹³C NMR, DEPT, TOCOSY1D as well as 2D NMR spectral techniques (HMQC, ¹H–¹H COSY, HMBC) and HR-MS. HMQC and HMBC spectra proposed that compound A was a glycoside of an oleandrigenin skeleton (Yamauchi and Abe, 1990) as an aglycon with an acetyl ester group at C-16 (Fig. 1). The ¹H and ¹³C NMR spectra showed the presence of two anomeric proton and carbon signals, which indicated the presence of two sugar moieties. The sugar moieties were identified as d-glucose linked 1→4 to d-digitalose (Fig. 1) as confirmed from HMBC spectrum and TOCOSY. From the results of spectral data, compound A was identified as oleandrigenin-β-d-glucosyl(1→4)-β-d-digitalose (Bai et al. 2010).

Although compound A has been previously isolated from the roots and aerial parts of A. obesum, this is the first report on the anti-influenza virus activity for this compound. Further studies will be required to determine structure-activity relationship among cardiotonic glycosides and to evaluate therapeutic value of this kind of natural products for influenza infection.

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