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Antimicrobial Constituents of Artemisia afra Jacq. ex Willd. against Periodontal Pathogens

The phytochemical investigation of an ethanol extract of Artemisia afra led to the isolation of six known compounds, acacetin (1), 12α,4α-dihydroxybishopsolicepolide (2), scopoletin (3), α-amyrin (4), phytol (5), and a pentacyclic triterpenoid betulinic acid (6). The compounds were evaluated for antimicrobial activity against Gram positive (Actinomyces naeslundii, Actinomyces israelii, and Streptococcus mutans), Gram negative bacteria (Prevotella intermedia, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans previously known as Actinobacillus actinomycetemcomitans), and Candida albicans. The crude extract of A. afra inhibited the growth of all tested microbial species at concentration range of 1.6 mg/mL to 25 mg/mL. The compounds 1–6 also showed activity range at 1.0 mg/mL to 0.25 mg/mL. Three best compounds (scopoletin, betulinic acid, and acacetin) which showed good antimicrobial activity were selected for further studies. Cytotoxicity of extract and compounds was determined using the XTT cell proliferation kit. The antioxidant activity of the extract and compounds was done using the DPPH scavenging method. The extract showed good antioxidant activity with an IC50 value of 22.2 μg/mL. Scopoletin had a strong transformation of the DPPH radical into its reduced form, with an IC50 value of 1.24 μg/mL which was significant to that of vitamin C (1.22 μg/mL). Acacetin and betulinic acid exhibited a decreased scavenging activity with the IC50 of 2.39 and 2.42 μg/mL, respectively. The extract and compounds showed moderate toxicity on McCoy fibroblast cell line and scopoletin was relatively nontoxic with an IC50 value of 132.5 μg/mL. Acacetin and betulinic acid also showed a smooth trend of non-toxic effects with IC50 values of 35.44 and 30.96 μg/mL. The obtained results in this study confirm the use of A. afra in the treatment of microbial infections.


Introduction
Periodontal disease is a chronic, multifactorial disease of the tissue supporting teeth [1]. It is characterised by local infection and inflammation in teeth supporting tissue leading to connective tissue destruction and alveolar bone loss [2]. The etiology of periodontitis is the oral bacteria [3,4]. If left untreated, periodontitis can have medical consequences such as weight loss, chronic pain, sore or loss teeth, swollen gums, tooth decay, breakage of the maxillary or mandibular bones, and renal, coronary, and hepatic diseases [5][6][7]. The population of periodontal bacteria begins to increase as an anaerobic environment is produced due to oxygen scavenging activity of the early subgingival colonizers [4]. These bacterial populations lead to biofilm formation which consists of microcolonies, extracellular layers, fluid channels, and communication systems [8]. Biofilms may consist of more than 700 different microbial species which leave symbiotically and incubate each other [9]. The biofilm formation and associated disease can be prevented by daily tooth brushing and chemotherapeutic agents such as chlorhexidine (CHX), fluorides biguanide antiseptics, quaternary ammonium-antiseptics and phenol derivatives [10,11]. Although these chemotherapeutic agents are effective they can cause side effects, such as gastrointestinal irritation, tooth staining, and gum irritation [11].
Dental treatment usually is expensive and not so easily accessible, especially in developing countries; therefore humans have turned to the use of natural traditional remedies to prevent oral ailments [12][13][14][15]. Artemisia afra (African wormwood, family Asteraceae) is widely distributed along the eastern parts of Africa. It grows in thick, bushy areas, usually with tall stems up to 2 m high but sometimes as low as 0.6 m. A. afra is a common species in South Africa with a wide distribution from the Cederberg Mountains in the Cape, northwards to tropical East Africa and stretching as far north as Ethiopia [16,17]. Worldwide there are about 500 species of Artemisia, mainly from the northern hemisphere. Many of the other Artemisia species are aromatic perennials and are used medicinally [18]. In southern Africa it is used to treat coughs, colds, diabetes, malaria, sore throat, asthma, headache, dental care, gout and intestinal worms [19]. In vitro studies done have revealed that A. afra is a potential antidepressant, cardiovascular, spasmolytic effects, antioxidant, and antimycobacteria [18,20,21]. Furthermore, the extracts of this plant species have shown activity against Trypanosoma brucei brucei [22].
The rationale of this study was to determine the antimicrobial, antioxidant, and cytotoxicity of Artemisia afra and isolated compounds against oral microorganisms which are responsible for dental caries, gingivitis, and periodontitis.

Plant
Material. Artemisia afra was collected at the South African National Botanical Institute (SANBI), Pretoria. Voucher specimen was prepared and identified at the H. G. W. J. Schwelcherdt Herbarium (PRU), University of Pretoria.

Preparation of Extracts.
Fresh plant material was soaked in 96% ethanol and homogenized into fine mesh. The extract was then filtered through the Whatman No.1 filter paper. The filtrates were evaporated to dryness in a (BUCHI) Rotavapor under reduced pressure of 40 • C.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Microbicidal Concentration (MMC).
The microdilution technique using 96-well microplates [23] was used to obtain the MIC and MMC values of the crude extract against microorganisms under study. The extract was serially diluted in the 96-well plate with 48 hours old microorganisms (5 × 10 6 CFU/mL) grown at 37 • C and the final concentration of extract and positive control (CHX) ranged from 25.0 mg/mL to 0.8 mg/mL. Microbial growth was indicated by adding 40 μL of (0.2 mg/mL) piodonitrotetrazolium violet (INT) (Sigma-Aldrich, South Africa) to microplate wells and incubated at 37 • C for 48 hours. MIC was defined as the lowest concentration that inhibited the colour change of INT. The MMC was determined by adding 50 μL of the suspensions from the wells, which did not show any growth after incubation during MIC assays, to 150 μL of fresh broth. These suspensions were reincubated at 37 • C for 48 hours. The MMC was determined as the lowest concentration of extract which inhibited 100% growth of microorganisms [24].

Antioxidant
Activity. The free radical scavenging activity was measured using 1, 1 diphenyl-2-picryl-hydraxyl (DPPH) assay [25] with slight modifications. The ethanol extract of A. afra and Vitamin C (positive control) 1000 μg/mL (20 μL) was added in the first three wells of a 96-well plate containing 200 μL of distilled water to make up final concentration of 100 μg/mL and the remaining wells were filled with 110 μL of distilled water. The first raws containing the extract/compounds were serially diluted to wells which contain 110 μL of distilled water, and later, 90 μL of methanolic solution of DPPH (90 mM) was added to all the wells. The final concentrations of the extract/compounds ranged from 100 to 0.8 μg/mL. The plates were incubated at 37 • C for 30 min and the absorbance was measured at 517 nm using the ELISA plate reader. The percent radical scavenging activity by A. afra was determined by comparison with ethanol (blank). The inhibition ratio was calculated as follows: % DPPH radical-scavenging = (AC-AS)/AC × 100, where AC is absorbance of the control solution (containing only DPPH solution), and AS is the absorbance of the sample in DPPH solution. The percentage of DPPH radicalscavenging was plotted against the plant extract/compounds concentrations (μg/mL) to determine the concentration of extract/compound required to scavenge DPPH by 50% (EC 50 ).  (1 × 10 5 mL) was seeded onto a microtiter plate and incubated for 24 h to allow the cells to attach to the bottom of the plate. Dilution series were made of the extract and compound and the various concentrations (400 to 3.1 μg/mL) were added to the microtitre plate and incubated for 48 h. The XTT reagents were added to a final concentration of 0.3 mg/mL and the cells were incubated for 1-2 hours. The positive drug control (Zearalenone) at concentrations range of (10 μg/mL to 0.6 μg/mL) was included in the assay. After incubation the absorbance of the colour was spectrophotometrically quantified using an ELISA plate reader, which measured the optical density at 490 nm with a reference wavelength of 690 nm. The assay was carried out in triplicate.

Statistical Analysis.
Statistical analysis was conveyed as means ± SD using GraphPad Prism 4.0 with a significant difference of (P < 0.05).

Isolation and Identification of Compounds
The antibacterial compounds present in A. afra extract were determined by the direct bioautography method of chromatograms using S. mutans [27]. The extract was spotted onto a TLC plate and developed using hexane: ethyl acetate at different ratios (1 : 1, 3 : 7, and 7 : 3). The plates were thoroughly dried and then the chromatograms were sprayed with a dense culture of S. mutans, incubated overnight at 37 • C. The plates were further sprayed with 0.2 mg/mL of p-iodonitrotetrazolium (INT) (Sigma). Clear zones of inhibition indicated compounds which inhibited bacterial growth. The isolation of compounds was performed using column chromatography. Fractionation was preceded by using silica gel 60 (70-230 mesh) and Sephadex LH 20. Thin layer chromatography (TLC) was performed on aluminum sheets coated with silica gel 60 F 254 (Merck) and UV light was used to detect compounds. TLC plates were further sprayed with vanillin/sulphuric acid reagent. H 1 -NMR and 13 C-NMR spectra were obtained by using Nuclear magnetic resonance (NMR) Germin 200 AT 199, 50 Hz, respectively. The Isolation of A. afra was started with 200 g of ethanol extract on to a 100 mm diameter column. The column was filled with 2 kg silica gel, eluted with a mixture of hexane: ethyl acetate of increasing polarity (100 : 0 to 0 : 100). Forty fractions were obtained and combined to make up 12 (I-XII) main fractions according to similarities of compounds as determined by TLC plate. A Sephadex column was conducted on the fraction X using 100% MeOH and it yielded 25 subfractions which were combined in to three subfractions (L, M, N). Fraction N was fractionated using MeOH and yielded a pure compound 1. Fraction M was isolated using a gradient of solvents DCM: Hex, 9 : 1 increasing polarity to 3% to obtain a florescent blue compound 3.  Fraction VII was chromatographed using DCM: MeOH (95 : 5), and 20 fractions were obtained and combined to 3 subfractions. Fraction G was fractionated using DCM: MeOH, increasing polarity on a Sephadex column and two fractions were obtained of which one was a pure compound 2, the second fraction was again fractionated using DCM: MeOH (95 : 5) and compound 6 was obtained. Compound 5 was isolated using Hex: EtoAc, at a ratio of 9 : 1, using silica gel column. A succession of a blue-coloured compound was observed on a TLC plate after application of vanillin/sulphuric acid. Subfraction H from fraction VII was chromatographed using a sephadex column with ethanol as its mobile phase and it yielded a white precipitate of a pure compound 4.

Results and Discussion
The TLC profile gave a clear antibacterial activity of the extract and guide to isolate ideal compounds. In all solvent systems tested on TLC, both polar and nonpolar bends demonstrated activity by inhibiting the growth of S. mutans. Isolation from A. afra extract yielded six compounds (Figure 1), one known flavone Acacetin (1), sequiterpe-ne12α,4α-dihydroxybishopsolicepolide (2), a diterpene Phytol (5) [28,29], and two pentacyclic triterpenes (4). A thorough revision of literature indicated that the data for pentacyclic triterpene matched with those of α-Amyrin, a widely spread triterpene in nature [30,31]. The presence of the signal at 3.0 of H-17 confirms the structure of Betulinic acid (6). We have compound (3) of which the forgoing data is identical with the known compound Scopoletin. All these compounds were characterized by their 1 H-NMR and 13 C-NMR spectra. The strong antimicrobial activity demonstrated by the ethanol extract of A. afra has provided us with more evidence that needed further chemical investigation of bioactive compounds present. The extract of A. afra showed good inhibitory effects against all Gram positive bacteria. It can be noted that the MIC and MMC values varied from 1.6 to 25.0 mg/mL among Gram positive bacteria. C. albicans which is a thick grower fungus was inhibited at a concentration of 6.3 mg/mL. However, the MMC value insignificant as compared to the results of the MIC. The A. actinomycetemcomitans was the resistant Gram negative bacteria as compared to all other tested bacteria ( Table 1). The lowest MIC and MMC value of isolated compounds was recorded at 0.25 mg/mL of compounds (6), (3), and (1) against A. israelii and A. naeslundii. Of all microorganisms, A. actinomycetemcomitans and C. albicans showed to be resistant against all compounds tested ( Table 1).

Evidence-Based Complementary and Alternative Medicine
The antioxidant activity of three selected compounds (scopoletin, acacetin and betulinic acid) revealed that they are effective antioxidant agents ( Figure 2). Scopoletin had a strong transformation of the DPPH radical into its reduced form, with an IC 50 value of 1.24 μg/mL which was in close range to that of vitamin C (1.22 μg/mL). Acacetin and Betulinic acid exhibited a slightly low DPPH scavenging activity with the IC 50 of 2.39 and 2.42 μg/mL, respectively.
The cytotoxicity effects of ethanol crude extract of A. afra and three selected compound on the growth of Fibroblast cells are shown in Figure 3 and Table 2. The extract as a mixture of different components showed to be nontoxic on lower concentrations of 6.12 and 3.06 μg/mL, with the cell viability of 120%. However, toxic effects were apparent at higher concentration range of 12.50 to 400 μg/mL, with the cell viability of 60 to 20%. Acacetin and betulinic acid also showed a smooth trend of nontoxic effects at lower concentrations and toxic at higher concentrations with IC 50 values of 35.44 and 30.96 μg/mL respectively. The effect of acacetin on lung cancer (A549) cell proliferation was observed to have a dose-dependent manner with an IC 50 value of 9.46 μM [32]. Apoptotic activity of betulinic acid against murine melanoma B16 cell line was reported and it was discovered that betulinic acid induces apoptotic effects with an IC 50 of 22.5 μg/mL [33]. Reports postulates that triterpenes with a carboxyl group at c-28 shows more cytotoxic activity against cancer cell lines [34][35][36] and induce apoptosis [37]. Unexpectedly, one out of three compounds tested, scopoletin was relatively nontoxic with an IC 50 value of 132.5 μg/mL. Evidence-Based Complementary and Alternative Medicine