Antiviral and antiparasitic activities of clovamide : the major constituent of Dichrostachys cinerea ( L . ) Wight et Arn

Article history: Received on: 05/07/2017 Accepted on: 17/08/2017 Available online: 30/09/2017 The application of different chromatographic and spectroscopic techniques to the aqueous alcoholic leaves extract of Dichrostachys cinerea (L.) Wight et Arn. (Mimosaceae) led to the isolation and identification of ten phenolic compounds. Among them, apigenin-7-O-apiosyl (1→2) glucoside, chrysoeriol-7-O-apiosyl (1→2) glucoside and clovamide were isolated for the first time from the plant. The rest of the compounds are: flavonol glycosides (quercetin-3-O-rhamnopyranoside, quercetin-3-O-glucopyranoside, myricetin-3-Orhamnopyranoside and myricetin-3-O-glucopyranoside), and three aglycones (myricetin, apigenin and kaempferol). The crude extract of D. cinerea showed a significant antitrypanosomal and antiviral effects. Clovamide as a major constituent was investigated for its antiviral and antitrypanosomal activities. It showed a significant antiviral effect against H5N1 influenza A virus with inhibition rate (74%) and a momentous trypanocidal effect against Trypanosoma evansi with IC50 value of 3.27 μg/ml, compared with the standard drug; diminazene aceturate (IC50=0.72μg/ml). Therefore, clovamide is playing an important role in antitrypanosomal and antiviral activities for D. cinerea extract and it can be considered a new candidate for the treatment of these two infections.


INTRODUCTION
Dichrostachys cinerea (L.) Wight et Arn.(Mimosaceae) known as Sicklebush, Bell mimosa, Chinese lantern tree or Kalahari Christmas tree, is a semi-deciduous to deciduous fast growing tree, typically grows up to 7 meters in height.It characterized by strong alternate smooth spines (up to 8 cm long), dark grey-brown fractures on old branches and stems and bark on younger branches (Zeid et al., 2009).
D. cinerea is one of the very useful wild medicinal plants used in folk medicine across Africa and Asia (Aworet-Samseny et al., 2011).Its bark is used to prepare concoction for treatment of dysentery, headache and elephantiasis.The root infusions are used to treat gonorrhea coughs, syphilis and sore eye and also used as laxative, anthelmintic and strong diuretic.Leaves are good fodder for domesticated animals and seeds are edible (Neondo et al., 2012).Pharmacological studies on D. cinerea have shown antibacterial, antiviral, antilice, antiplasmodial, and antitrypanosomal effects (Aworet-Samseny et al., 2011;Vijayalakshmi et al., 2010;Atindehou et al., 2004).Phytochemical studies on D. cinerea revealed the isolation of various constituents, such as sterols, alkaloids, tannins, triterpenes, polyphenols, phenolic acids, flavonoids (Zeid et al., 2009) and cardiotonic heterosides (Tillement et al., 1977).
Trypanosoma evansi, an animal-pathogenic protozoan parasite, infects various big animals including buffaloes, camels, equines, cattle, goats, and sheep causing great economic losses in the infected areas, where thousands of animals die from T. evansi infections (Luckins 1988).Diminazene aceturate and suramin are the most commonly used drugs for the treatment of T. evansi infection.However, they cause severe side effects and the development of drug resistant trypanosomes has occurred in many regions (Elkhateeb et al. 2012).
Influenza, a respiratory disease caused by influenza A viruses, is still one of the major epidemics worldwide that are highly transmissible pathogens for humans and some animals (Droebner et al., 2007).H5N1 is one of the highly pathogenic avian IV strains.It is rarely infected humans and poses a severe threat because of its high pathogenicity (Pleschka et al. 2009).The development of influenza A viruses resistant to antiviral drugs draw attention to the need for other antiviral drugs.Although several antiviral compounds have been developed against influenza virus, they are of limited efficacy due to toxicity and/or the appearance of drug-resistant virus mutants (Droebner et al., 2007).
The crude extract of D. cinerea showed a remarkable antitrypanosomal and antiviral effects (Atindehou et al., 2004;Aworet-Samseny et al., 2011).Therefore, it was of a great importance to check the antitrypanosomal and antiviral activities of its major constituents which my lead to an alternative drug with least side effects and higher efficacy for the treatment of T. evansi and H5N1infections.

General
Mass spectra were measured on a JEOL JMS-AX500 spectrometer, Graduate School of Agriculture, Hokkaido University, Japan.NMR was recorded on a Bruker AM-500 FT-NMR spectrometer, Graduate School of Agriculture, Hokkaido University, Japan.UV spectrophotometer (Shimadzu UV-240/ National Research Center/Egypt)

Plant material
Fresh leaves of D. cinerea were collected from Wadi Halfa, South Egypt, in April 2013.The plant was authenticated by Prof. Dr. S. Kawashty, Department of Phytochemical and Plant Systematics, NRC.A voucher specimen was deposited in the Herbarium of NRC (CAIRC, Cairo, Egypt).

Antiviral activity
Madin-Darby Canine Kidney (MDCK) cells were maintained in the Center of Scientific Excellence for influenza viruses at the National Research Centre/Cairo/Egypt.The cells were propagated till confluence in multi-well plates.The highly pathogenic avian influenza (HPAI) virus A/Chicken/Egypt/M7217B/2013 (H5N1) used in the current study was isolated from the infected chickens in Egypt in 2013 and characterized at immunologic and molecular levels.MTT assay was used to determine the LC 50 of the extract and clovamide according to the reported method (Mosmann et al., 1983).Briefly, the cells were cultured in 96 well-plates and incubated for 24 hrs at 37 ºC in 5% CO 2 .Then, they were treated with triplicates of the tested compounds.One day later, the supernatant was discarded and monolayers of cells were washed three times with sterile phosphate buffer saline.MTT solution was added to each well and incubated for 4 hrs at 37 °C.The formed formazan crystals in each well were dissolved in acidified isopropanol (200 µl).Absorbance of formazan solutions was measured at λmax 540 nm with 620 nm as a reference wavelength using a multi-well plate reader.The percentage of cytotoxicity compared to the untreated cells was determined as following: % of cytotoxicity = [(Absorbance of cell without treatment -Absorbance of cell with treatment)/ Absorbance of cell without treatment] x 100.The plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (TC 50 ).Plaque reduction assay was used to determine the antiviral activity for both of the extract and clovamide at safe concentrations (Hayden et al., 1980).

Antitrypanosomal activity
Trypanosoma evansi (H3 strain, isolated from deer in Thailand) were provided by Dr. Onuma, Graduate School of Veterinary Medicine, Hokkaido University, Japan.Trypomastigote forms of the parasite T. evansi were maintained in HMI-9 medium supplemented with 20% heat-inactivated horse serum (Sigma-Aldrich/Germany).In vitro antitrypanosomal test was performed to determine the 50% inhibitory concentration (IC 50 ) on parasite growth for clovamide (Bawm et al., 2008).Briefly, tests were performed in a 96-well microtiter plate using clovamide and standard trypanocidal drug, diminazene aceturate (Sigma-Aldrich/Germany).Both of the standard and the tested compound were dissolved in dimethyl sulfoxide (DMSO).Three-fold serial dilutions were prepared in HMI-9 medium.Trypomastigotes of T. evansi were incubated in each well in the presence of three-fold serial dilutions of tested compound.The plates were incubated at 37 ºC in 5% CO2 atmosphere for 72 h and the number of motile parasites was counted using hemocytometer
Compounds (1-3) were isolated for the first time from the plant.Compound 3, the major phenolic constituents of D. cinerea was elucidated and full characterized by 1D and 2D NMR (H-H COSY, HMQC and HMBC).The 1 H NMR spectrum showed two pairs of doublets at δ 6.41 and 7.17 ppm exhibiting a coupling constant of 15.7 Hz assigned to H-7 and H-8, respectively, which indicate two olefinic protons and their stereochemistry in trans.
The 1H NMR spectrum also showed two doublets of doublets at δ 2.89 (1H, dd, J=18.5, 5.0 Hz) and 2.71 (1H, dd, J=22.5, 8.5 Hz), due to the position in α of the asymmetric carbon (C7′), which indicates the presence of one methylene unit.H-8' appeared as multiplet at 4.37 ppm due to the presence of methyne group.

Antiviral activity
The cytotoxicity of the extract and clovamide LC 50 were found to be (30 and 68 μg/ml), respectively.Both showed a remarkable virus inhibition activity against influenza A virus (H5N1) infection up to 73% for the extract and 74% for clovamide at concentration (20 µg/µl).The pure compound clovamide showed higher inhibition rate than the extract at the used concentration.

Antitrypanosomal activity
Clovamide was also tested in vitro against T. evansi and showed a strong potential for antitrypanosomal activity with (IC 50 =3.27μg/ml) which compared well with the standard trypanocidal drug diminazene aceturate (IC 50 =0.72 μg/ml).Although diminazene aceturate still show a stronger antitrypanosomal activity against T. evansi than clovamide, its fatal side-effects (Peregrine et al., 1993) together with the comparatively low toxicity of clovamide determined by the relatively high LC 50 (68 μg/μl) nominate the later as a promising safe compound for further in vivo investigation.

CONCLUSION
Ten phenolic compounds were identified from D. cinerea, among them; compounds 1-3 were isolated for the first time from the studied taxa.To the best of our knowledge, this is the first report for the antiviral and antitrypanosomal activities of clovamide, which showed remarkable activity against H5N1 influenza A virus and a strong trypanocidal activity against T. evansi nominating it as a relatively safe promising antitrypanosomal compound for further investigation.

Figure 1 :
Figure 1: Chemical Structures of the isolated compounds from Dichrostachys cinerea