Phytotoxicity , cytotoxicity and antioxidant activity of the invasive shrub Austroeupatorium inulifolium ( Kunth ) R . M . King

Methanol and dichloromethane extracts of root, stem, leaves and flowers of invasive plant Austroeupatorium inulifolium were tested for cytotoxic, phytotoxic, antioxidant and antifungal activities. Significantly higher phytotoxicity was detected in methanol extracts of leaves and dichloromethane extracts of roots. This effect was most pronounced against amaranth seeds where the seed germination was reduced to 3.74 % with the addition of methanol extracts of leaves at 3000 mg/L. The dichloromethane extract of roots of A. inulifolium showed potent antifungal activity against Cladosporium cladosporioides. Cytotoxic activity was found in dichloromethane extract of roots (LD50 = 27.91 ± 8.55 mg/L), methanol extract of flowers (LD50 = 15.22±7.89 mg/L) and leaves (LD50 = 22.92±11.76 mg/L) against Artemiasalina (brine shrimp) larvae. The results also revealed significant antioxidant activity in methanol extract of leaves (IC50 =33.66± 0.03 mg/L) against the reference α-tocopherol (IC50 =10.02 ± 0.01 mg/L).


INTRODUCTION
Plants have co-evolved with their respective environments for millions of years. During this process, secondary metabolites played a critical role in plants' adaptations to insect and microbial attacks and climatic conditions. Plant metabolites with various bioactivities including phytotoxic and antioxidant activities allow plants to live in a state of equilibrium with the environment. However, some plant species use these bioactivities to transform themselves into noxious weeds. The indigenous flora of Sri Lanka comprises about 7,500 species. Of the 3,154 flowering plants, about 894 (28 %) species are endemic to the island, some carrying varying levels of biological activity (Wijesundara et al., 2012;Bandara et al., 1989a;Bandara et al., 1990a;Hewage et al., 1997;Hewage et al., 1998). Many bioactivities in plants have been used in medicinal applications (Banadara et al., 1989b;Bandaraet al., 1989c;Bandaraet al., 1990b;Williams et al., 2011). Plant ecologists were always puzzled by the fact that exotic plant species with low densities in their native ranges eventually produce large populations in their introduced ranges. The 'novel weapons hypothesis' proposed that these exotic plants possess novel biochemical compounds with powerful bioactivities (Callaway and Ridenour, 2004). Austroeupatorium inulifolium (Kunth) King and Robinson (Asteraceae) which is an aggressive invasive shrub native to South America has become a noxious invader in the up-country, wet zone of Sri Lanka, invading many natural and man-made ecosystems (Madawala et al., 2014). It is also listed as an 'agricultural and environmental weed in the Global Compendium of weeds (Randall, 2012). Even though it has been well identified as an invasive plant in many countries, it has been categorized as an invasive plant in Sri Lanka fairly recently. A. inulifolium has been invading the Cymbopogon-dominated grasslands in the Knuckles Conservation Area (KCA), displacing the grass. Once established, A. inulifolium can form mono-specific stands in their introduced range, influencing the growth and survival of the native flora (Haluwana and Madawala, 2013).
There have been many hypotheses to explain the mechanisms of exotic plant invasions into new landscapes and among them the 'novel weapon hypothesis' has attracted contrasting views since it was proposed in 2004 ( Blair et al., 2006;Duke et al., 2009). According to this hypothesis, some exotic species credit their success of spread due to the production of bioactive compounds that native species never encountered before (Callaway and Ridenour, 2004;Thorpe et al., 2009;Callaway and Aschehoug, 2000). Previous studies on other invasive species have confirmed the presence of bioactive compounds (Yan et al., 2010;Shao et al., 2010;Ens et al., 2009;Xie et al., 2010). These bioactive compounds can reach the soil environment through leaching, root exudation or litter decomposition. In the present study, we attempted to investigate the potential role of bioactive compounds in A. inulifolium by using bioassays with extracts of different parts of the shrub including stems, roots, flowers and leaves and to relate them to its invasive success. Apart from the isolation of nine new norlabdane derivatives with antimicrobial activity in A. inulifolium (Saito et al., 2011), no studies have been carried out to assess the bioactive profile of A. inulifolium with an aim to explain its invasive nature of the plant. We report herein, the bioactivities of A. inulifolium including cytotoxic, phytotoxic, antioxidant and antifungal properties.

MATERIALS AND METHODS
Austroeupatorium inulifolium, belongs to the family Asteraceae, is a shrub that can grow up to 1-5 m in height. It bears a creamy white fragrant inflorescence ( Figure 1). Leaves are opposite, spear-shaped and pubescent, abruptly narrowing to a wedge-shaped petiole. It is well known as an aggressive invader in many countries.

Extraction of potential bioactive compounds
Plant samples were collected from Riverston area in the Knuckles Forest Reserve (KFR) in the central of Sri Lanka. Plant parts (roots, stems, leaves and flowers) were separated and air-dried before grinding into a powder. The resulting powdered plant material (≈600 g) was subjected to sequential extraction with 5 L of CH2Cl2 and CH3OH separately after maceration for 24 hours (in cleaned and dried glass bottles) with constant shaking. The extract was then filtered and concentrated using a rotary evaporator at 30 °C to obtain the final yield of extract in paste form. The crude extract was then stored at 4 °C until further use. The CH2Cl2 and CH3OH crude extracts were subjected to cytotoxic, phytotoxic, antioxidant and antifungal bioassays.

Cytotoxic Activity
Cytotoxic activity was determined using a brine shrimp lethality assay (McLaughlin, 1982). Brine shrimp eggs were added to fresh artificial sea water and allowed to hatch in a beaker which was aerated and illuminated using a 20 W bulb. The temperature was maintained at 27 °C. A series of concentrations (0. 75, 2, 7.5, 20, 75, 200, 750, 2000 mg/L) of the dichloromethane and methanol extracts of different plant parts were tested with 1day old brine shrimps (three replicates and 10 shrimps per experiment). After 24 hours, the number of surviving nauplii of brine shrimps was counted and LD50 value (concentration at 50 % survival) was calculated using probit analysis of MINITAB version 16. The known cyctotoxic lactone, (4 S)-4-methyl-2-(11-dodecynyl)-2 butenolide, from the genus Hortonia was used as the positive control (Ratnayake et al., 2001).

Antioxidant activity
Antioxidant activity was determined using 2, 2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay (Budzianowski and Budzianowska, 2006). The reduction of DPPH in the extract was measured using a concentration series ranging from 1 -500 mg/L at 517 nm using a UV-visible spectrophotometer (Shimadzu UV-1800) after 30 minutes against the CH3OH blank. The initial absorbance of DPPH and each test solution (without DPPH) were measured at 517 nm separately. α-Tocopherol (Vitamin E), concentration series ranging from 1 -500 mg/L was used as the positive control. Tests were carried out in triplicate for each concentration. Percentage antioxidant activity was calculated using absorbance readings using the equation, [A0 -At/At] x 100 where A0 is the initial absorbance value and At is the absorbance of the test sample after 30 min of adding DPPH. The % antioxidant activity was plotted against the concentration gradient for all three trials separately and trend lines were constructed for the linear range of the plot. The IC50 value was calculated for each trend line from which the average was taken.

Antifungal activity
Quantitative analysis of antifungal activity using the TLC bioassay method with the fungus, Cladosporium cladosporioides was carried out. A concentration series of the extracts (250, 750 and 2500 mg/L) of different plant parts was prepared and spotted on a TLC plate. A conidial suspension of C. cladosporioides was carefully sprayed using an atomizer onto the TLC plates. After 2 days of incubation, the presence of antifungal compounds was detected using inhibition zones. Antifungal active spots appeared white against the background of grey-green mycelia.

Yields of potential bioactive extracts
After extraction and concentration, crude yields of methanol and dichloromethane extracts of different plant parts of A. inulifolium were obtained (Table 1). In both extracts, the highest percentage yield was obtained from the leaves.

Phytotoxic activity
Germination index (GI) generally decreased with increasing concentrations of CH3OH and CH2Cl2 extracts of A. inulifolium (Table 2). However, CH3OH-leaf, CH2Cl2-root and CH2Cl2-stem extracts showed the most significant reductions in germination index with increasing concentrations (from 250 to 3000 mg/L). As an example, CH3OH-leaf extracts decreased the GI from about 68 % to 4 % in amaranth seeds, while CH2Cl2-root extracts reduced the GI of same seeds from 61 % to 8 %, when increase the concentration from 250 mg/L to 3000 mg/L. In contrast, all concentrations of CH2Cl2-flower extracts showed no influence on the germination of seeds tested in the assay. CH2Cl2-leaf extracts too showed no impact on the germination of maize seeds.
Both CH3OH and CH2Cl2 extracts of A. inulifolium roots and shoots negatively influenced the growth of both monocot and dicot seedlings tested, and this inhibition gradually increased with increasing concentrations (Figure 2). CH3OH extracts of A. inulifolium leaves negatively affect the growth of seedlings while CH2Cl2 extracts did not show clear inhibition apart from amaranth seedlings. Interestingly, CH3OH-leaf and CH3OH-flower extracts showed higher inhibition on the growth of dicot seedlings compared to that of monocots. In contrast to the effects of other extracts, CH2Cl2flower extracts stimulated the growth of dicot and monocot seedlings, showing highest stimulatory effect at 250 mg/L concentration, though the differences are not significant. Furthermore, the monocot seedlings were more stimulated than that of dicot seedlings (Figure 2).

Antioxidant activity of A. inulifolium
When comparing the antioxidant activity of CH3OH and CH2Cl2 extracts of A. inulifolium with α-tocopherol (IC50 =10.02 ± 0.01 mg/L), the CH3OH extract of leaves (IC50 =33.66± 0.03 mg/L) showed a significant antioxidant activity among the extracts at P<0.05.

Antifungal activity of A. inulifolium
Interestingly, the antifungal activity was only observed in CH2Cl2-root extracts. According to the results obtained, the average diameters of inhibition zones shown in the presence of the CH2Cl2 extract of Austroeupatorium roots were 16.0 and 24.6 mm at concentrations 750 and 2500 mg/L, respectively (Table 3).

DISCUSSION
When consider the extraction of potentials of bioactive compounds in A. inulifolium, the CH3OH extracts recorded a higher yield than that of CH2Cl2extracts. This may be due to the higher content of polar compounds in A. inulifolium compared to that of non-polar compounds. Cytotoxic results suggested that A. inulifolium roots, flowers and leaves contain cytotoxic compounds which can be useful as antiproliferative and antitumor activities, as well as pesticidal and other bioactive agents. There is evidence to support that natural cytotoxic compounds are good contenders for anticancer drugs (Rahman et al., 2001). In addition, these cytotoxic compounds also play an important role in these plants to minimize pest attacks compared to co-existing natives, determining their successful establishment in their introduced ranges (McGraw and Eloff, 2008).
The effects of bioactive compounds on seed germination have been tested using germination index (GI) in previous studies (Chiapuso et al., 1997). GI is considered as a sensitive indicator of phytotoxic effects (Ahmed and Wardle, 1994).
Results showed that germination of tested species were affected by all extracts which either delayed or reduced germination of all test seeds. However, a significantly higher phytotoxic effect was detected in CH3OH-leaf and CH2Cl2-root extracts of A. inulifolium (P<0.001). The CH3OH-leaf extract at 3000 mg/L showed the most pronounced impact against amaranth seeds with GI of 3.74 %. The analysis showed that CH2Cl2-flower extract had no significant effect on the GI (P < 0.05) of both monocot and dicots, although it has declined the seed germination. The degree of effect on germination increased with the increasing concentrations of extracts and this was evident in extracts of all plant parts of A. inulifolium. The delayed seed germination can have important biological implications, as this can negatively affect the establishment of seedlings of cooccurring natives (Chaves et al., 2001), and thereby increasing the chances of competing for resources with neighbouring species (Xingxinag et al., 2009).
However, the effect was more pronounced in CH3OH extracts of A. inulifolium, where polar compounds are commonly found. Methanolic extracts of some plant species were known to contain phenolics and other toxic substances (Belicova et al., 2001;Al Harun et al., 2015). These phenolics may inhibit the germination process through their interference in the indole acetic acid metabolism, or synthesis of protein and Some bioactivities of Austroeupatorium inulifolium 97 iron uptake by plants (Blum, 1998;Castro et al., 1984).
Phytotoxic results showed that the root and shoot development of germinating test seeds are highly inhibited by the CH2Cl2 extracts of root and stem, and by all extracts of CH3OH. In contrast, slight stimulatory effects on the root and shoot growth of monocot seedlings was shown by CH2Cl2 extracts of leaves and flowers. The shoot growth appeared less sensitive to A. inulifolium extracts than the radicle, which eventually grows into roots. The phytotoxic compounds are highly active against meristmatic tissue in growing roots. This perhaps is the reason for differing responses of root and shoot to A. inulifolium extracts. Phytotoxicity has been suggested as the key strategy for the impressive success of many invasive plants that has been dominated in their invaded plant communities (Ridenour and Callaway, 2001).
In some instnaces, there were slight stimulatory effects on root and/or shoot by different extracts of A. inulifolium. According to results, inhibition or stimulation of root and shoot and the germination index varied with plant extract, concentration and seed type. The stimulatory bioactive compounds can be used to develop eco-friendly, cheap and effective Green Growth Promoters (Oudhia et al., 1998). Phytotoxic compounds inhibit germination and seedling growth probably by affecting cell division and elongation, processes that are very important at early stages, or by interfering with enzymes involved in the mobilization of nutrients necessary for germination (Batlang and Shush, 2007).
Antioxidant results revealed that the MeOH extract of leaves contains compounds with antioxidant activity. Generally, plant polyphenolic compounds are responsible for antioxidant activity. In addition, they also act as iron chelators. The latter property may be important in an invasive plant like A. inulifolium where it contributes to altering the soil bacterial flora in their growing habitats (Morel et al., 1993).
In the antifungal assay, larger inhibition zones were observed with CH2Cl2 extracts, suggesting that the solvent has the potential to extract many active antifungal compounds. Chromene, an antifungal compound, found in roots of the invasive plant Eupatorium riparium which also belongs to the same genus as A. inulifolium (Bandara et al., 1992).
Furthermore, the antifungal, phytotoxic and antioxidant activities were also tested in essential oils extracted from roots and inflorescence of Eupatorium adenophorum (Ahluwalia et al., 2014), further supporting this genus's worldwide distribution.

CONCLUSIONS
The results suggest that A. inulifolium contained phytotoxic, cytotoxic and antifungal activities. The presence of phytochemicals in A. inulifolium may suppress the growth of other plants in the habitat they invade. Cytotoxic compounds can kill pests thereby minimizing pest attacks. Antifungal compounds can reduce fungal attacks on the plant too. These characteristics can favour invasive species to invade and establish in their introduced ranges successfully. Therefore, the survival and spread of A. inulifolium may have been favored by the presence of these phytotoxic compounds where they can inhibit the growth of co-habiting plants. These factors may have contributed to the aggressive nature of A. inulifolium.