Allelochemical, Eudesmane-Type Sesquiterpenoids from Inula falconeri

We have identified through bioassay guided isolation an allelochemical, eudesmane-type sesquiterpeniod, 3β-caffeoxyl-β1,8α-dihydroxyeudesm-4(15)-ene (1), from an endemic plant species growing in the Himalayas. In our search for the bioactive subfraction, the hexane one was highly significant, showing 100% inhibition of lettuce seed growth at 100 ppm while other subfractions (chloroform, ethyl acetate, butanol and water) exhibited inhibitory to stimulatory allelopathic effects. The bioactive hexane subfraction was subjected to chromatographic techniques, using lettuce seeds (Lactuca sativa) as indicator species to reveal the bioactive allelopathic fraction. This resulted in the isolation of compound 1, whose structure was elucidated through NMR techniques. The compound presented 92.34% inhibitory effect on the growth of lettuce at 500 ppm. Further field level experiments may help develop an environmentally friendly herbicide from this lead.


Introduction
Allelochemicals are secondary metabolites released by plant species in their environment. They play a pivotal role in agricultural and functional ecology [1,2]. Identification of such chemical constituents has created a thrust to address several problems confronted in plant physiology, pathology, environment and crop production. Pursuing and developing natural products as environmentally friendly herbicides can eliminate damages to human health as well as to ecosystem. Efforts have been made to elaborate the allelopathic effects of various plant species. Genus Inula is famous for its diverse biological activities i.e., anticancer, antibacterial, hepaprotective, cytotoxic, and antiinflammatory, however limited information is available on their role in allelopathy [3]. This is the first report to elucidate an allelopathic interaction of Inula falconeri from the genus. I. falconeri was reported as a potential allelopathic plant species among other four plants while investigating the role of method and concentration in allelopathy [2]. I. falconeri is endemic to the Himalayas and distributed in the northern areas of Pakistan and western Tibet [4]. It grows at 2,400 m altitude, however, it has also been observed at 3,000 m. At lower altitudes, it is found mainly near agricultural fields and household gardens. Due to its colorful flowers, it has been frequently used for decoration purposes. Local people revealed that I. falconeri was abundant at lower altitudes but due to over exploitation the plant population has been reduced considerably over the years as the plant was eradicated as an unwanted species near or in agricultural fields [5]. The aim of this study was to explore and identify the allelopathic potential of this plant species by isolating and characterizing potential allelochemicals through a bioassay guided approach.

Results and Discussion
The structure of compound 1 (Figure 1) was established conclusively by UV, IR, MS and extensive 1 H-and 13 C-NMR spectra analysis and comparison with a published report -identifying it as a known eudesmane-type sesquiterpene (3β-caffeoxyl-1β,8α-dihydroxyeudesm-4(15)-ene) [9]. Pursuing the active allelopathic subfraction after solvent-solvent partition, the hexane subfraction was seen to inhibit the growth of root and hypocotyls of lettuce seedlings. At lower concentrations, it presented higher specific activity i.e., inhibiting 50% growth at 3 ppm and at maximum concentration (300 ppm), no seeds germinated [5]. Chloroform subfraction revealed a dose dependent effect on lettuce seed's growth; however, the effect was insignificant compared to that of hexane subfraction.
Other subfractions (ethyl acetate, butanol and water) exhibited no EC 50 activity and their allelopathic effects ranged from insignificantly inhibitory to stimulatory ( Figure 2) [5]. After column chromatography of bioactive hexane fraction under different solvent regimes, seven subfractions were assayed for their allelopathic behavior. Most subfractions exhibited moderate to high inhibition at maximum concentration (500 ppm) but at lower concentration (30 ppm) none of the subfractions had EC 50 value except 5% MeOH-CHCl 3 . It inhibited lettuce seed germinations at 300 ppm and shown EC 50 activity at 30 ppm ( Figure 3).  The bioactive 5% MeOH-CHCl 3 was further fractionated. The resultant seven subfractions were bio-assayed for their EC 50 activity. Nearly every subfraction presented moderate suppression; however, 5%, 10%, and 20% MeOH-CHCl 3 subfractions had 100% inhibitory effect toward germination of lettuce seeds on all concentrations ( Figure 4).
Subfractions Inhibitory effect (% 30 ppm 100 ppm 300 ppm 500 ppm Due to similar allelopathic effect, 5%, 10%, and 20% MeOH-CHCl 3 fractions were combined and subjected to reverse-phase chromatography, which afforded six new subfractions. Among these, 50% MeOH-CHCl 3 exhibited a significant inhibitory effect. All concentrations of this bioactive subfraction were more than 70% inhibitory towards growth of lettuce seeds ( Figure 5).  Four compounds purified through RP-HPLC were assayed for their EC 50 ( Figure 6). Compound 1 exhibited significant inhibitory effect (92.34%) towards the growth of lettuce seeds at 500 ppm while EC 50 activity was shown at 30 ppm concentration (Figure 7). Due to paucity of material the other three compounds could not be identified. Figure 6. Effect on the growth of lettuce seeds of four purified compounds from 50% methanol: chloroform subfraction using RP-HPLC. Earlier I. falconeri was screened among four plant species for allelopathic potential of leaf leachates [2]. The hexane subfraction of I. falconeri was revealed as the most potent, not only exhibiting allelopathic effects but also displaying a significant zone of inhibition against Alternaria alternata and Rhizoctonia solani and an insignificant effect against Fusarium oxysperum [5]. Previous reports on I. falconeri established the presence of phenolic acids (caffeic acid and chlorogenic acid) and flavonoids (rutin, quercetin and kaempferol) [4], which have already been proved to display allelopathic effects [6][7][8].
Besides broader biological effects of various classes of sesquiterpenoids, these have also been reported to carry allelopathic potential [10,14].
Compound 1 has already been reported to have antibacterial properties against human pathogens [9]. Although eudesmane-type sesquiterpenes have been reported for their cytotoxicity, enzyme inhibitory and antimicrobial effects [9][10][11] however, it is now also reported as a potent allelochemical.

General
The 1 H and 13 C NMR, COSY, HMQC and HMBC spectra were recorded in CD 3 OD using TMS as internal standard on a Bruker spectrometer operating 500/125 MHz. The chemical shift values are reported in ppm (δ ) units and coupling constant (J) in Hz. EI, CI MS were recorded using JMS-HX-110 with data acquisition system and on JMS-DA 500 mass spectrometer. HPLC (TOSOH, Tokyo, Japan; UV PD 8020) was performed equipped with a reversed-phase column (Inertsil1 ODS-3, 10 × 250 mm, GL Sciences Inc., Tokyo). Methanol and milli-Q water were used as elution solvents in HPLC with a flow rate of 2.8 mL/min.

Extraction and Isolation
Dried plant material of I. falconeri (1.20 kg) was soaked in pure MeOH (2.5 L) and extracted for 30 days at 5 ºC in dark. MeOH extract was evaporated to dryness in vacuo. It was subjected to solventsolvent partition into hexane, chloroform, ethyl acetate, butanol and water subfractions. All subfractions were screened for their allelopathic effect. The hexane subfraction revealed higher EC 50 activity. This subfraction (8 g) was extracted five times with hexane to get polar compounds. It was later subjected to column chromatography using silica gel-60 (Wako Pure Japan; 19 mm i.d. 300 mm) in successive elution with hexane-CHCl 3 (5%, 10%, 20% and 50%) and CHCl 3 -MeOH (5%, 10% and 20%) (300 mL each).

Allelopathic Effects
The allelopathic potential of each fraction isolated from chromatographic column was assayed [12,13]. Lettuce seeds (Lectuca sativa, Takii Seed Co. Ltd., Japan) were used as indicator species to know the effective concentration of extract/compound that induces 50% inhibition of tested organism (EC 50 ) when exposed to various concentrations of the fractions [12]. The inhibitory effects of fractions were studied by the specific activity method. Five concentrations viz. 3, 10, 30, 100 and 300 ppm of hexane, chloroform, ethyl acetate, butanol and water subfractions were used for EC 50 bioassay. Four concentrations i.e., 30, 100, 300 and 500 ppm of every successive subfraction of the bioactive hexane subfraction were then used for the EC 50 bioassay. The concentrations were dissolved in 2% DMSO before application to the lettuce seeds. Initial concentration was 1,000 ppm. A filter paper (27 mm, Type Roshi Kaisha, Tokyo) was placed in a glass Petri dish. The dilutions were applied on filter paper and thus allowed to spread over it. Seven lettuce seeds were placed on it and dishes were sealed and packed for incubation for 72 hours at room temperature. For each fraction, mean, SD variance [13] and standard error were calculated to determine inhibition pattern at various concentration levels. While using EC 50 , specific activity of each extract was determined. Specific activity of subfractions was measured using specific activity = 1/EC 50 × Concentration [12].