Potent bioactivities of the endemic Annonaceae heighten its dire conservation status

Sri Lanka records 17 endemic species of the Annonaceae family. Worldwide, the Annonaceae are known to possess compounds with pharmaceutically important properties such as anticancer and insecticidal actions. In an attempt to investigate the mosquito larvicidal and antioxidant activities of the endemic Annonaceae plants, twelve plant species were collected. Of the 71 extracts investigated from various plant parts, five plants showed significant larvicidal activity with the CH 2 Cl 2 extracts of the leaves of G. hookeri and G. gardneri (LC 50 at 48 h = 0.4 and 0.3 ppm, respectively) exhibiting potency compared to the known larvicide (4S)-4methyl-2-(11-dodecynyl)-2-butenolide (LC 50 =0.3 ppm). Compared to the percent radical scavenging activity of the standard dl-α tocopherol (55.84), the MeOH extracts of the stem of A. hortensis (56.30), the leaves of U. semecarpifolia (57.33), and the seeds of X. nigricans (62.06) showed very promising activity. Significantly, it is recorded that two of the Sri Lankan endemic Annonaceae plants are extinct (P. moonii and A. hortensis) and the rest, except for U. sphenocarpa, are critically endangered, endangered, vulnerable or near threatened. These grim statistics highlight not only the urgency and the importance of biodiversity conservation of the endemic Annonaceae of Sri Lanka but also of investigating the plants for new phamacophores.


INTRODUCTION
In an estimated number of the 270,000 species of vascular flora, 12 % are threatened (Walter & Gillett, 1998) and these plants are scattered in 369 families found in 200 countries. Because of the lack of information due to, either poor taxonomic knowledge or gaps in field work, the number could be much higher. When the sample assessed is the population within a species, or when there is genetic erosion among species, the situation becomes grimmer (Harper & Hawksworth, 1994;Gaston, 1996). In Sri Lanka, of the 3314 listed flowering plants, 455 are threatened with about 70 plants having become extinct by 1997 (Walter & Gillett, 1998).
The World Conservation Monitoring Center has designated Sri Lanka as a 'hotspot' in terms of rich biodiversity and threats faced (Caldecott et al., 1994). About 25 % of the flowering plant species are endemic to the island (Gunatilleke & Gunatilleke, 1990). The relict rainforest plant taxa of Gondwana-Deccan ancestry are now found only in some isolated forest pockets in Penninsular India and Southwestern Sri Lanka. The Deccan flora evolved in isolation in the late Cretaceous and early Tertiary periods, during the drifting of the Indian plate (Jayasekara, 1997). It has been hypothesized that biotic impoverishment, prior absence followed by colonization and later speciation has led to the tremendous endemic diversity in the island (Biswas, 2008). Except for a few reports (Bandara et al., 1989;Hewage et al., 1997;Hewage et al., 1998), there has not been a large-scale systematic search for bioactive agents from Sri Lankan flora so far. Although endemic plants such as Salacia reticulata var. diandra (Celastraceae) have underscored the potential of Sri Lankan plants (Gunatilaka et al., 1993;Dhanabalasingham et al., 1996;Yoshikawa et al., 1997), in general the bioactivity potential of Sri Lankan endemics remains relatively unknown. As such, there is an urgent need to investigate their therapeutic potential before they disappear forever.
The Annonaceae are woody trees, shrubs and vines comprising about 130 genera and 2,300 species worldwide. Considering its large size, it is chemically one of the least known of the tropical plant families (Leboeuf et al.,1982). However, plants of the Annonaceae have received increased phytochemical and pharmacological attention in recent years; this is mostly due to the discovery of Annonaceous acetogenins, a class of natural products with a variety of biological activities (Cave et al., 1997;Kojima & Tanaka, 2009;Liaw et al., 2010). In Sri Lanka, Annonaceae is centered in the lowland rainforests (Dassanayake & Fosberg, 1980). They extend to the dry and lower montane zones, but are absent from elevations above 1500 m. Due to extensive deforestation in the humid regions, many species have become rare. In Sri Lanka, 17 (Dassanayake & Fosberg, 1980), although many have not been collected for decades. It is in this backdrop, that the antioxidant and mosquito larvicidal activities of the extracts of 12 Annonacea belonging to 8 genera are reported in the present investigation. However, five endemic Annonaceae species could not be collected during plant collection between 2004 to 2010, which may have disappeared from their known habitats.

METHODS AND MATERIALS
Endemic Annonaceae plants were collected from Central Sri Lanka during 2004 -2010 (Table 1) and identified and deposited at the National Herbarium of Royal Botanic Gardens, Peradeniya.
Air dried, ground plant materials (100 -500 g) were extracted sequentially for 24 h at room temperature with CH 2 Cl 2 followed by MeOH (500 -1500 mL each) by using a bottle shaker. The combined extracts were concentrated in vacuo at 35 o C to obtain the respective crude extracts.
Radical scavenging activity (antioxidant activity) of plant extracts against stable DPPH radicals was determined spectrophotometrically by the slightly modified methods of Miliauskas et al. (2004) and Yen and Duh (1994). dl-α tocopherol was used as the positive control. All the determinations were performed in 3 replicates and averaged.

RESULTS AND DISCUSSION
Out of the 71 extracts studied, 25 extracts showed toxicity against A. aegypti . The extracts of G. gardneri and G. hookeri demonstrated exceptionally high larvicidal activity while A. hortensis, E. acuminata, X. championii, U. sphenocarpa and U. semecarpifolia showed significant activity ( Table 1). The most active was the dichloromethane leaf extracts of G. hookeri and G. gardneri (LC 50 at 48 h = 0.4 and 0.3 ppm, respectively). Significantly, larvicidal potency of extracts of G. hookeri and G. gardneri were comparable with the potent larvicide (4S)-4-methyl-2-(11-dodecynyl)-2butenolide (Ratnayake et al., 2001). Although no reports of either bioactivity or use in traditional medicine exists for these two plants in Sri Lanka, plants of the genus Goniothalamus are known for the presence of cytotoxic acetogenins and styryl-lactones (Leboeuf et al.,1982). However, of the large majority of the 166 Goniothalamus plants known globally, only 22 species have so far been investigated (Wiart, 2007).
Several extracts exhibited potent percent antioxidant activity. For example, MeOH extract of the stem of A. hortensis (56.30), the leaves of U. semecarpifolia (57.33), and the seeds of X. nigricans (62.06) showed higher radical scavenging activity compared to the standard dl-α-tocopherol (55.84). In recent times, free radicals have been implicated in inflammation processes, cardiovascular disease, rheumatoid arthritis, neurodegenerative disease, and the ageing process (Hollman & Katan, 1999). X. nigricans, X. parvifolia and X. championii, of which the latter two are non-endemic, are rich in isoquinoline alkaloids where some have shown potent antioxidant activity (Wijeratne et al., 1996(Wijeratne et al., , 2001Puvenendran et al., 2008Puvenendran et al., , 2010.

Journal of the National Science Foundation of Sri Lanka 41(4)
December 2013 In conclusion, this study reveals the richness of bioactivity of the endemic Annonaceae of Sri Lanka, and  its very promising potential as a source of plant medicines. However, it also highlights the grim conservation status of the endemic Annonaceae. If urgent remedial action is not undertaken towards their conservation, these plants will be well on their way towards extinction. This work also highlights the importance of screening the endemic flora of the South Asian region for potential drugs before they are threatened by deforestration and other anthropogenic factors.