Phytochemical and antitrypanosomal investigation of the fractions and compounds isolated from Artemisia elegantissima

Abstract

Context: Trypanosoma brucei brucei (T.b. brucei) infection causes death in cattle, while the current treatments have serious toxicity problems. However, natural products can be used to overcome the problems associated with parasitic diseases including T.b. brucei.

Objective: Artemisia elegantissima Pamp (Asteraceae) was evaluated phytochemically for its constituents and antitrypanosomal potential against T.b. brucei for the first time. Scopoletin isolated from A. elegantissima has shown better potential then the standard drug suramin, used against T.b. brucei.

Materials and methods: The ethanol extract of the aerial parts of A. elegantissima was fractionated by column and preparative thin-layer chromatography into six fractions (A–F) yielding 13 compounds, these were evaluated for their antitrypanosomal activity against T.b. brucei at different concentrations.

Results: Thirteen compounds were isolated from A. elegantissima: (Z)-p-hydroxy cinnamic acid, stigmasterol, β-sitosterol, betulinic acid, bis-dracunculin, dracunculin, scopoletin, apigenin, dihydroluteolin, scoparol, nepetin, bonanzin, and 3′,4′-dihydroxy bonanzin. The fractions D–F were found to be active at the concentration of 20 µg/ml and three compounds isolated from these fractions, scopoletin (MIC ≤0.19 µg/ml), 3′,4′-dihydroxy bonanzin (MIC = 6.25 µg/ml) and bonanzin (MIC = 20 µg/ml), were found to be highly active.

Discussion and conclusion: Artemisia elegantissima was phytochemically and biologically explored for its antitrypanosomal potential against T.b. brucei. The number and orientation of phenolic hydroxyl groups play an important role in the antitrypanosomal potential of coumarins and flavonoids. The compounds 3′,4′-dihydroxy bonanzin and scopoletin with low MIC values, hold potential for use as antitrypanosomal drug leads.

• 3′
• 4′-dihydroxy bonanzin
• A. elegantissima
• antitrypanosomal
• dihydroluteolin
• phytochemical
• scopoletin
• T.b. brucei

Introduction

Several infectious diseases including Trypanosomiasis affect the lives of millions of people and livestock and are a major cause of high morbidity and mortality in Africa, South America, and Asia (Barrett et al., 2007). The species Trypanosoma brucei brucei (T.b. brucei) causes serious ailments to both wild and domestic animals in Africa (Zweygarth & Kaminsky, 1989). If T.b. brucei infections are left untreated, it can be fatal and sometimes can cause an epidemic (Simarro et al., 2011).

The current treatment regimens (diminazine, homidium, isometamidium, pentamidine, suramin, eflornithine, and melarsoprol) are inadequate, toxic, and show propensity for the development of drug resistance. Suramin, used as a standard drug against T.b. brucei, has shown variable efficacy against the early acute stage of infection and failure in the chronic phase of infection as well as being associated with adverse effects of toxicity (Ene et al., 2009). The development of drug-resistant parasites is producing additional major problems (Koehn & Carter, 2005; Rollinger et al., 2008). These facts emphasize the urgent need for the design and discovery of novel, nontoxic, cheap, and easy-to-administer pharmacophores for the treatment of T.b. brucei infection. This goal can be achieved with natural products which for several decades have continued to provide the majority of drug leads in almost all disease conditions including trypanosomal infections (Ogungbe & Setzer, 2009; Ali et al., 2013).

In vitro studies of the various phenolic compounds and their structure-based design analysis have revealed that compounds possessing a number of hydrogen bond accepting and/or donating groups like flavonoids and coumarins show extensive interactions with trypanosomal glycolytic enzyme, trypanosomatid glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which shows significant differences from the analogous human enzyme (Aronov et al., 1999).

The plants of Artemisia are historically important and are frequently used in the treatment of various ailments including cancer, malaria, hepatitis, infections, and inflammation by bacteria, fungi, and viruses (How et al., 1982). The plants are also known for their antiparasitic biology and some vital drug leads have been reported from the genus Artemisia, notably artemisinin isolated from A. annua Linn (Asteraceae); a molecule that is used today as a drug against the multi-drug-resistant strain of Plasmodium falciparum malaria (Brown, 2010).

Some of the compounds from Artemisia have been established as potential insecticides and allelopathic drugs and so the genus could potentially provide a rich source of drugs against trypanosomiasis (Salem & Werbovetz, 2006b).

Although the essential oil and extract of most of the Artemisia species has been tested for antibacterial, antifungal, insecticidal, and antitrypanosomal activities, there has not been any report on the antitrypanosomal activities of the compounds obtained from Artemisia spp. Therefore, this paper investigated whether the compounds obtained from A. elegantissima Pamp could yield antitrypanosomal activities. The ethno-pharmacological and chemotaxonomic importance of the genus Artemisia against infectious diseases prompted us to undertake antitrypanosomal studies on A. elegantissima as so far this plant is phytochemically and pharmacologically uninvestigated.

Materials and methods

Plant material

The aerial parts (stems and leaves) of A. elegantissima Pamp were collected from Pirchanasi (Muzaffarabad, Kashmir, India) in August 2009. The plant was identified by Dr. Tanveer Akhtar, Department of Botany, University of Azad Jammu and Kashmir, Muzaffarabad, India. A voucher specimen (Bot. 20021 (PUP)) was deposited at the Herbarium of the Botany Department, University of Peshawar, KPK, Pakistan.

General

Fractionation of the crude extract and isolation of the compounds was achieved using silica gel 60 (0.063–0.200 mm; Merck, Darmstadt, Germany) for column chromatography (CC) and silica gel 60 F₂₅₄ on aluminum sheets (0.2 mm thickness, Merck, Darmstadt, Germany) for preparative thin layer chromatography (Prep. TLC). Sephadex LH20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) were also used for the purification of the compounds. TLC of separated compounds was visualized under UV light (254/365 nm), spraying with ceric sulfate solution (dissolving 0.13 g of ceric sulfate in 5 ml of sulfuric acid and diluted up to 50 mL) followed by heating. Melting points (m.p.) were determined using a Stuart digital melting point apparatus (SMP 10; Staffordshire, UK). The compounds were characterized using NMR instruments (Bruker AVANCE DRX 500 and 400 MHz, Karlsruhe, Germany) with deutrated solvents. The mass spectra (EIMS) were recorded on a JEOL-MS Route (Middletown, NY) through direct insertion probe. Optical rotation was measured with a Perkin–Elmer 243B automatic polarimeter (PerkinElmer Informatics, Waltham, MA) (MeOH at 25 °C). All chemicals used were from Sigma Aldrich (Dorset, UK).

Extraction and isolation

Air-dried and ground samples of A. elegantissima (3.0 kg) were extracted three times by maceration with 10 L of EtOH in a closed container each time for 48 h. The extract concentrated in vaccuo, yielded a dark greenish mass (195 g) which was subjected to CC eluted successively with n-hexane:CH₂Cl₂ (7:2, 7:3, 7:4, 1:1, 3:7, 1:4, and 0:1) and CH₂Cl₂:MeOH (9:1, 1:1, 1:3, and 0:1) yielding 11 major fractions which on TLC resulted in six major fractions A–F. Fraction A was subjected to CC eluted with n-hexane:EtOAc (9.5:0.5, 7:3) yielded compounds 1–3. Fraction B on CC eluted with n-hexane:EtOAc (4.2:0.8) yielded compound 4. Fraction C on CC eluted with n-hexane:EtOAc (5:2) yielded compound 5. Fraction D on CC eluting with n-hexane:EtOAc (5:2) yielded a mixture of compounds 6 and 7, which were further purified by preparative TLC developed with n-hexane:EtOAc (5:2) (R_f 0.56 and 0.47 for compounds 6 and 7, respectively). Fraction E on preparative TLC developed with n-hexane:EtOAc (1:1) yielded compounds 8–10 (R_f 0.85, 0.97, and 0.54, respectively). Fraction F on preparative TLC developed with n-hexane:EtOAc (4:6) yielded compounds 11–13 (R_f 0.77, 0.62 and 0.48, respectively). Compounds 12 and 13 were further purified on a Sephadex LH-20 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden).

Antitrypanosomal assay

The method was followed as detailed in Clark et al. (2012). Unless otherwise stated, chemicals and solvents were of reagent or anhydrous grade and used as obtained from commercial sources without further purification. The T.b. brucei bloodstream form strain 427 isolated from sheep in Uganda were cultured in the HMI-9 medium containing 20% (v/v) heat-activated fetal calf serum (FCS), in a 5% CO₂ atmosphere at 37 °C.

Minimum inhibitory concentrations (MICs µg/ml) for the fractions and compounds were carried out in duplicate. The assay was conducted in 96-well microplates. Stock solutions (10 mg/ml) of the test agents were prepared in dimethylsulfoxide (DMSO) and then diluted to a concentration of 1 mg/ml using HMI-9 medium. From the stock solution, various concentrations, i.e., 20, 10, and 5 µg/ml of the sample were prepared.

The plate was arranged by adding DMSO as a negative control in column 1, suramin as a positive control in column 12, and the samples to be tested in columns 2–11. The trypanosomes were diluted to 3 × 10⁴ microbes/ml and added in suspension (100 µl) to each well of the assay plate. Alamar Blue® (Trek Diagnostic Systems, Inc., Cleveland, OH) was added to all wells of the plate to give a final concentration of 10% (v/v) and a total well volume of 200 µl. The microplates were incubated at 37 °C in a 5% CO₂ humidified atmosphere for 24 h. The level of florescence was measured using a Perkin Elmer Victor 2 microplate reader in fluorescence mode (excitation 560 nm, emission 590 nm). The percentage (%) of the DMSO control values were recorded as % D control.

Results

Compounds isolated

The aerial parts of A. elegantissima yielded 13 compounds. The structures of the compounds were determined using spectroscopic techniques (1D and 2D NMR, UV, IR, and MS) and the data were compared with those reported in the literature. The compounds isolated were: (Z)-p-hydroxycinnamic acid (1) (Shimoji et al., 2002), stigmasterol (2) (Kamboj et al., 2011), β-sitosterol (3) (Kamboj et al., 2011), betulinic acid (4) (Krasutsky, 2006), bis-dracunculin (5) (Wu et al., 2001) dracunculin (6) (Hofer et al., 1986), scopoletin (7) (Murray, 1997), dihydroluteolin (8) (Owen et al., 2003), apigenin (9) (Sandhar et al., 2011), scoparol (10) (Wollenweber & Dietz, 1981), nepetin (11) (Carlo et al., 1999), bonanzin (12) (Tang et al., 2000), and 3′,4′-dihydroxy bonanzin (13) (Colegate & Molyneux, 1993). Structures of compounds 1–13 are given in Figure 1.

Figure 1. Structures of compounds 1–13.

Antitrypanosomal activities of the fractions obtained from the aerial parts of A. elegantissima

The six fractions obtained were tested against T.b. brucei. In this preliminary screening, the fractions whose values of % D control were less than 5 were considered to be active. Fractions A, B, and C were found to be inactive at all the three concentrations: 5, 10, and 20 µg/ml. Fractions D and E were found to be active at the concentrations of 10 and 20 µg/ml, showed −6.7, −2.4% D control and −10.5, −6.0% D control, respectively; while they were found to be inactive at the concentrations of 5 µg/ml. Fraction F was found to be active at all the three concentrations: 5, 10, and 20 µg/ml, showed −3.5, −5.7, and 2.1% D control, respectively (Table 1).

Table 1. Preliminary antitrypanosomal activities of the fractions obtained from the aerial parts of A. elegantissima.

		T.b. brucei^a % of D control
S. no	Fractions	20 µg/ml	10 µg/ml	5 µg/ml
1	A	22.9	82.5	91.5
2	B	7.4	1.8	11.4
3	C	5.7	0.3	2.4
4	D	−6.7	−10.5	81.4
5	E	−6.0	−2.4	86.3
6	F	−3.5	−5.7	2.1

D, DMSO.

^aT.b. brucei, Trypanosoma brucei brucei.

Three of the 13 isolated compounds (7, 12, and 13) were found to be active. Scopoletin (7) was found to be the most active compound (MIC ≤0.19 µg/ml) and the flavonoids; bonanzin (12) (MIC = 20 µg/ml), and 3′,4′-dihydroxy bonanzin (13) (MIC = 6.25 µg/ml) also showed good activities. The (MIC µg/ml) values were only calculated for the highly active compounds (7, 12, and 13).

Discussion

From the antitrypanosomal study of the 6 fractions (A–F) and 13 compounds (1–13), phenolic compounds (coumarins and flavonoids) were the only ones active against T.b. brucei. By comparing the structures and activities of coumarins (5–7) and flavonoids (8–13) (Table 2 and Figure 1), it was deduced that the molecular size, the number of active hydroxyl groups, and the substitution pattern of the hydroxyl group on coumarin and flavonoids skeleton played an important role in their antiparasitic potential against T.b. brucei.

Table 2. Preliminary antitrypanosomal activities of the compounds isolated from the aerial parts of A. elegantissima.

S. no.	Compounds	T.b. brucei % of D control 20 µg/ml	T.b. brucei % of D control 10 µg/ml	T.b. brucei MIC µg/ml
1	p-Hydroxy cinnamic acid (1)	93.2	99.7	NT^a
2	β-Sitosterol (2)	101.5	105.0	NT^a
3	Stigmasterol (3)	101.5	105.0	NT^a
4	Betulinic acid (4)	99.1	104.3	NT^a
5	bis-Dracunculin (5)	85.3	87.2	NT^a
6	Dracunculin (6)	89.0	106.2	NT^a
7	Scopoletin (7)	−0.4	−0.4	<0.19
8	Dihydroluteolin (8)	93.4	98.0	NT^a
9	Apigenin (9)	44.2	91.3	NT^a
10	Nepetin (10)	39.6	87.2	NT^a
11	Scopolin (11)	37.2	85.5	NT^a
12	Bonanzin (12)	−0.5	98.6	20
13	3′,4′-Dihydroxy bonanzin (13)	−0.8	−1.2	6.25

D, DMSO.

^aNT, not tested.

The prominent activity of the coumarin, scopoletin (7) (MIC ≤0.19 µg/ml), showed that the phenolics with small molecular size and active hydroxyl group mediated better antitrypanosomal activity. Among the flavonoids, the activity of hexahydroxy flavonols, bonanzin (12) (MIC = 20 µg/ml) and 3′,4′-dihydroxy bonanzin (13) (MIC = 6.25 µg/ml), showed that the flavonols with a greater number of active hydroxyl groups mediated high activity and methylation of hydroxyl groups had a negative effect. It has also been previously reported that the flavonoids potential against T.b. brucei and human cancer cell lines was closely linked to the number of phenolic hydroxyl groups and their structures and methylation was always associated with decreasing the activity and selectivity (Mamdelieva et al., 2011).

In the literature, in vitro antiprotozoal activities of scopoletin (IC₅₀ ≥30, 31, and 90 µg/ml, respectively) have been reported against Leishmania donovani, T.b. rodesiense, and T. cruzi, but has not been tested against T.b. brucei. Our result has proved the efficacy of scopoletin against trypanosomal infections. Several coumarin compounds have showed antitrypanosomal (GAPDH inhibition) and anti-HIV (HIV protease inhibition) activities (Oketch-Rabah et al., 1997). Furthermore, several inhibitors of the enzyme HIV integrase were structurally based on natural coumarins (Aronov et al., 1999; Zhao et al., 1997).

Among the flavonoids (8–13), the flavones (8–10) were found to be less active than the flavonols (11–13). Also amongst the flavonoids (8–13), the substitution of the hydroxy group with a methoxy group decreased the antitrypanosomal activity of the flavonoids at least two-fold.

According to the literature, all members of the flavanone subclass have some potential to inhibit the growth of African trypanosomes. The flavonoid activity against T.b. brucei was affected by the position of the ring “B” oxygenation as the flavanone; pinocembrin, and the flavones; chrysin showed adequate but different values of 50% inhibitory concentration (IC₅₀ = 11 and 14 µM, respectively) against the T.b. brucei blood stream forms. Furthermore, chrysin showed about a 12-fold selectivity for the parasites compared to mild cytotoxicity in human cancer cell lines (IC₅₀ = 124 µM) (Tasdemir et al., 2006). Acetylation of flavonoids reduced their antitrypanosomal potential which has been reported in the case of pinocembrin after acetylation (IC₅₀ ≥30 µM). In another study against T. cruzi trypomastigotes, the flavonoids, 3-methylquercetin, 3,6-dimethylquercetagetin, and 7,3-dimethylluteolin, were found to be less active (IC₅₀ = 128, 138, and 145 µg/ml, respectively) (Taleb-Contini et al., 2004). The isoflavone, calycosin, showed selective toxicity against T.b. brucei bloodstream forms (IC₅₀ = 12.7 µM) compared to L. donovani amastigotes and mammalian Vero cells (IC₅₀ = 100 and 159 µM, respectively) (Salem & Werbovetz, 2006a).

Similarly, the flavonoids, 7,8-dihydroxyflavone (IC₅₀ = 1.7, 0.068, and 6.6 µg/ml), and quercetin derivatives (IC₅₀ = 1.0, 8.3, and 3.0 µg/ml) were reported to be as potent and effective antiprotozoal agents against L. donovani, T.b. rodeseinse, and T. cruzi, respectively (Vieira et al., 2001).

Conclusion

Our results demonstrate the potential to help in directing the rational design of 3′,4′-dihydroxy bonanzin (flavonoid) and scopoletin (coumarin), as other similar secondary metabolites or synthetic analogues as potent and effective antiprotozoal agents against T.b. brucei.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Acknowledgements

This project was supported by the Higher Education Commission, Pakistan under the Indigenous 500 fellowship program (Project # 106-1074-Ps-062).