Journal of Applied Botany and Food Quality 86, 33 - 36 (2013), DOI:10.5073/JABFQ.2013.086.005 Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi, India Plant growth inhibitory terpenes from Eupatorium adenophorum leaves A. Kundu*, S. Saha, V. Ahluwalia, S. Walia (Received October 15, 2012) * Corresponding author Summary Eupatorium adenophorum is commonly known as Crofton weed, growing widely throughout the northern hilly terrains. Bioactive potential of E. adenophorum leaves was investigated through phytochemical study and in vitro plant growth inhibitory. Essential oil was hydrodistilled from leaves and analyzed by GCMS. Leaf material was extracted using cold extraction process followed by evaporation of solvent under reduced pressure. Five cadinene sesquiterpenes and one sterol were isolated from hexane and EtOAc concentrates and their structures were established spectroscopically. Plant growth inhibitory activity of extractives and isolated terpenes were studied against different seeds of weed and crops. Essential oil was moderate seedling growth inhibitor. Among various extracts, EtOAc extract was found to be most inhibitory to Phalaris minor seeds (EC50 117 μg mL-1). Among the sesquiterpenes, 5,6- dihydroxycadinan-3-ene-2,7-dione was found to be most active and inhibited both shoot and root growth of Phalaris minor seed (EC50 97 μg mL-1) and Polygonum plebejum (EC50 117 μg mL-1). Introduction Eupatorium adenophorum Spreng (syn. Ageratina adenophora King and Robinson) is fast growing perennial herbaceous plant, belonging to the family Compositae (Duan, 2003). The plant flourishes abundantly forested and cultivated lands, and is widely distributed from tropical to temperate region such as America, Europe, Australia, South Africa, India, Thailand and China (AULD, 1966). The plant is introduced in India in 19th century since then widely proliferating in the hilly terrain of northern, north eastern region and other lower hilly regions of southern India (BORTHAKUR, 1977). Due to significant essential oil content in its aerial parts (WEYERSTAHL et al., 1998; PALA-PAUL et al., 2002), it is considered as a valuable raw material for perfumery industry (SHARMA et al., 1998; ADHIKARI and KRAUS, 1994). The plant is reported to contain an array of bioactive constituents like monoterpenes, sesquiterpenes, flavonoids, phenyl propanoids and their derivatives (PROKSCH et al., 1983). Sesquiterpenoids are the major constituent of leaves and flowers (DING et al., 1999; ZINGHUI and JINGKAI, 1999). Cadinene sesquiterpenes were also identified in leaves (LAN et al., 2006). The plant is also well known for its insect repellent activities (SOOD et al., 2000; LI et al., 2001; MEHTA et al., 2002; WANG, 2002; RAJMOHAN and RAMASWAMY, 2007). Potential molluscicidal activity of aqueous extract of the plant was reported against Oncomelania hupensis (ZOU et al., 2009). Besides, growth inhibitory activity of E. adenophorum litter was also studied on survival and growth of Lantana camara (KAUL and BANSAL, 2002). However, except some sporadic attempts on plant growth inhibitory activity (ZHENG and FENG, 2005), detailed growth inhibition assay of cadinenes from E. adenophorum has not been studied. As far as our literature survey could ascertain, there is no report on antifungal activity of the cadinene derivatives isolated from leaves of E. adenophorum. The present study was therefore aimed to isolate and evaluate major bioactive constituents of E. adenophorum leaves for plant growth inhibitory activity against seeds of three weeds and two crops. Materials and methods Reagents and instruments All solvents and reagents were of the highest purity needed for each application. Solvents and chemicals were purchased from Sigma® (USA) and Merck® India Ltd. and used without further purification. Materials used for column chromatography was silica gel (100- 200 mesh; Merk Specialities Private Ltd. Mumbai, India). HSGF254 silica gel TLC plates (Merck Specialities Private Ltd. Mumbai, India) were used for analytical TLC and spots were detected under UV light or by heating after spraying with 98% H2SO4. Preparative TLC (0.4-0.5 mm) was performed on glass plates precoated with silica gel GF254. UV and IR spectra were recorded respectively with a Jasco V-650 spectrophotometer and an FTS-40 infrared spectro- meter with KBr pellets. NMR spectra were recorded on a Brucker 400 AC (400 and 75.5 MHz) NMR spectrometer with TMS as an internal standard. ESI-MS/MS were measured on a Thermo LC/ MSD Trap XCT mass spectrometer (Finnigan MAT Incos 50). Plant material Leaves of E. adenophorum were collected during 2009 from forest area of Kangra, Himachal Pradesh, India. After identification of specimen, a voucher sample (EA-EAA-05-09) was deposited in the herbarium of Department of Botany, Himachal Pradesh Agricultural University, Himachal Pradesh, India. Essential oil extraction and GC-MS analysis Shade dried leaf powder (5 kg) was subjected to hydrodistillation for 4 hours using a Clevenger apparatus to obtain essential oil. Essential oil was collected and dried over anhydrous sodium sulfate and preserved in a sealed vial at 4 °C until analysis. GC-MS analysis was carried out on a Thermo Fischer capillary gas chromatograph equipped with flame ionization detector (FID) for FI-detection, which is further directly coupled to the mass spectrometer system and HP-5MS capillary column (30m × 0.25 mm i.d. × 0.25 µm film thickness). Temperature programming was done 60-125 °C at 2 °C/min hold time 2 min., 125-160 °C at 0.5 °C /min hold time 5 min. and 160-240 °C at 5 °C/min. Injector and detector temperatures were maintained at 220 °C and 290 °C, respectively. Helium was used as carrier gas with a flow rate of 1 mL/min. Ion source temperature was 270 °C and mass transfer line 250 °C with split ratio 1:20. Identification of the constituents of essential oil was performed by comparison of their retention times, retention indices and comparison of mass spectral fragmentation pattern. Cold extraction of leaves Shade dried leaves (5 kg) of E. adenophorum was extracted se- parately with MeOH (10 L). The solvent was filtered and evaporated 34 A. Kundu, S. Saha, V. Ahluwalia, S. Walia in vacuuo at 42 °C to yield methanolic concentrates. Chlorophyll was removed from methanolic concentrate (920 g) by precipitation with lead acetate (5 %). Methanolic concentrate (10 g) was dissolved in aqueous ethanol (70 %), lead acetate solution (5 %) was added and kept undisturbed for 10 minutes. Chlorophyll lead acetate complex precipitated was filtered to remove undesired precipitates and the filtrate was partitioned sequentially with hexane, EtOAc and n- BuOH to obtain respective concentrates. Isolation of terpenes EtOAc concentrate (70 g) was subjected to silica gel column chromatography (100-200 mess particle size, pre-activated at 110 °C) using eluent mixtures of EtOAc in hexane (20-100 %, v/v) to obtain 50 fractions. Fractions 15-23 showing four major spots on TLC plates were combined and re-chromatographed using hexane- EtOAc (10 %) followed by preparative-TLC to give EA-1 (75 mg) and EA-2 (55 mg). Fractions 37-46 showing similar spots on TLC were combined and re-chromatographed using a gradient of hexane- EtOAc (50-80 %) as eluent to provide 16 fractions (Fr. I-Fr. XVI). Fraction IX-XII were combined and subjected to preparative TLC to obtain comparatively less polar EA-3 (45 mg) and more polar EA-4 (55 mg). On the other hand, hexane concentrate (50 g) was silica gel column chromatographed with the gradient of increasing hexane in EtOAc (0-50 %). Total number of 32 fractions was collected and finally 5 sub-fractions obtained on combining the eluates based on their similar behavior on TLC. Sub-fraction 1 was subjected to preparative-TLC with hexane-EtOAc (80 %) to obtain EA-5 (60 mg) and EA-6 (32 mg). Plant growth inhibition assay Seeds of three weeds Phalaris minor, Polygonum plebejum, Chenopodium album and two field crops wheat (Triticum aestivum L.) cv. HD 2329 and chickpea (Cicer arietinum L.) cv. BGD72 were collected from Seed Technology Centre, Division of Seed Science and Technology, IARI, New Delhi. Test solutions of essential oil, extracts and terpenes were prepared using DMSO (0.1 %, v/v) as the initial solvent carrier followed by diluting with distilled water to a final concentration of 25 μg mL-1. Other test solutions (i.e., 50, 100, 250, 500 μg mL-1) were prepared by dilution with an aqueous solution of DMSO (5 %). Seeds were washed with ethanol (70 % v/v) for 2 min and surface sterilized using sodium hypochlorite (0.5 % v/v) for 2 min, followed by three washes with sterile distilled water. After sterilization seeds were stored in a refrigerator at 4 °C for 3 days before use. Three layers of filter papers were put in 6 cm diameter glass Petri plates, and the filter papers were impregnated with test solutions. To avoid toxic effect of organic solvent, filter paper treated with DMSO solution was placed in a fume hood for 1 h to allow complete solvent evaporation. Fifty seeds of each test crop were evenly placed on the moist filter paper in each Petri plates. Two controls (filter paper treated with 3 mL of DMSO and filter paper without any treatment) were set. Each treatment had three duplicates. Seeds were allowed to germinate under 12 hrs light at 25 °C. The root and shoot length was measured in both treated (T) and control (C) plates (after 7 days) until all the seeds in the control Petri plates were fully germinated. Percentage inhibition of growth (I %) and ED50 (μg mL-1) were determined. Statistical analysis Experimental data were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) to determine significant differences among treatment means. Dif- ferences were considered significant at p levels of 0.05 and 0.01. Results and Discussion Chemical constituents Light yellowish essential oil (0.90 % v/w, dry weight basis) with strong aromatic odour was hydrodistilled from leaves. GC-MS spectrum indicated total twenty six volatile compounds, representing 83.5 % of total composition. About 71.9 % sesquiterpenes and 11.3 % monoterpenic constituents were identified in essential oil (Tab. 1). γ-Cadinene (13.56 %) and germacrene-D (10.03 %) were the major constituents. Two cadinene sesquiterpenes were isolated and purified from hexane concentrate. Similarly, EtOAc fraction was further investigated for its phytochemical compositions. Four cadinene derivatives were isolated and their structures were de- termined by NMR and mass fragmentation pattern. Further iden- tification of these cadinene derivatives were also done by com- parison with the literature data. Cadinene derivatives were iden-tified as 7-hydroxycadinan-3-ene-2-one (EA-1) (BOHLMANN and GUPTA, 1981), 5,6-dihydroxycadinan-3-ene-2,7-dione (EA-2) (ZHAO et al., 2009), 2-acetyl-cadinan-3,6-diene-7-one (EA-3) (BARUAH et al., 1994), stigmasterol (EA-4), cadinan-3-ene-2,7-dione (EA-5) (LAN et al., 2008) and cadinan-3,6-diene-2,7-dione (EA-6) (LAN et al., 2008) (Fig. 1). Copies of the original spectra are obtainable from author. Plant growth inhibition activity A number of studies have been reported in literature which showed that plant allelochemicals interfere with the growth and establish- Tab. 1: Essential oil constituents of E. adenophorum leaves. Compounds RT RI Content (%) Cymene 9.10 1026 0.3±1.2 Camphor 14.95 1100 1.4±1.1 Borneol 16.20 1155 1.8±0.3 Camphene 17.66 1162 2.2±0.6 Verbenol 18.19 1171 0.3±0.9 Bornyl acetate 23.36 1268 1.7±1.3 Geranial 27.17 1294 2.2±2.6 Limonene 31.35 1352 1.5±2.5 Aromadendrene 32.53 1408 3.1±0.7 β-Cedrene 32.94 1418 1.3±1.4 α-Bergamotene 33.32 1433 0.7±2.6 β-Farnesene 34.20 1445 6.7±1.0 Elemene 35.56 1449 9.8±3.6 Caryophyllene 35.87 1457 4.6±1.2 α-Humulene 36.52 1462 2.6±1.8 Germacrene-D 38.03 1464 10.0±1.1 α-Curcumene 38.27 1479 0.7±0.6 Bicyclogermacrene 39.66 1489 3.0±3.7 Cadina-1,4-diene 40.72 1496 2.3±4.1 β-Bisabolene 40.98 1503 1.5±0.4 Sesquiphellandrene 42.19 1508 0.7±1.5 Spathulenol 47.44 1519 1.6±1.0 γ-Cadinene 50.60 1529 13.6±1.2 Cadinol 53.29 1653 0.7±1.8 Farnesol 58.17 1656 5.4±1.5 Nerolidol 59.40 1677 3.8±0.7 RT=Retention time (min) RI = Relative index to n-alkanes (C9-C21) on HP-5 MS column Terpenes of Eupatorium adenophorum 35 ment of other crops or weeds (WHITTAKER, 1971; ENS et al., 2009). Several allelopathic compounds such as catechin, juglone, apigenin, gallic acid derivatives have been discovered and investigated against various weed/crop species. Our study indicated that both hexane and EtOAc concentrate was effective on P. minor and P. plebejum. Essential oil was moderately active on shoot and root growth inhibition of weed seeds. However, seedling growth of crop seeds was not hampered. Growth inhibition percentage was dose dependant and detailed lethal median concentration was depicted in Tab. 2. Shoot length of T. aestivum seed was also found to be inhibited by the extracts. MeOH concentrate was active against P. minor and C. album. There was no growth inhibition effect observed with n-BuOH extract. Root and shoot growth inhibition of C. arietinum was significantly less. Since, solvent concentrates were found to be active; it was investigated in details to isolate bioactive principle. Among the cadinene constituents, 5,6-dihydroxycadinan-3-ene-2,7- dione exhibited highest shoot and root growth inhibition against P. minor (Tab. 2). Similar trend was observed in growth inhibition of P. plebejum. However, C. album and C. arietinum growth was not much hampered at the same concentration. As evident from the data, growth inhibition of C. arietinum was below 20 %. 5,6- Dihydroxycadinan-3-ene-2,7-dione was most effective in inhibiting coleoptile growth of P. minor (EC50 97 μg mL-1) and P. plebejum (EC50 117 μg mL-1) than C. album (EC50 516 μg mL-1). It was also highly inhibitory of root growth of P. minor (EC50 190 μg mL-1). 7-Hydroxycadinan-3-ene-2-one was found to be active in reducing the shoot length of P. plebejum (EC50 147 μg mL-1) and P. minor (EC50 169 μg mL-1) than C. album (EC50 601 μg mL-1). Root growth of these weeds was not much affected by 7-hydroxycadinan-3-ene- O O OHOH OH O O O O O HO EA-1 EA-2 EA-3 EA-4 EA-5 EA-6 O O O Fig. 1: Structures of terpenes isolated from E. adenophorum leaves. (EA-1), 7-hydroxycadinan-3-ene-2-one; (EA-2), 5,6-dihydroxycadinan-3-ene-2,7-dione; (EA-3), stigmasterol; (EA-4), 2-acetyl-cadinan-3,6-diene-7- one; (EA-5), cadinan-3-ene-2,7-dione; (EA-6), cadinan-3,6-diene-2,7-dione Tab. 2: Plant growth inhibitory activity of extracts and cadinene sesquiterpenes. Compounds / Extracts EC50 (µg mL-1) please round the values P. minor P. plebejum C. album T. aestivum C. arietinum Root Shoot Root Shoot Root Shoot Root Shoot Root Shoot Essential oil 375 252 468 265 636 412 841 740 755 617 Hexane extract 216 161 427 186 456 517 346 226 678 521 MeOH extract 346 257 527 397 338 277 457 307 735 616 EtOAc extract 270 117 312 190 534 435 345 155 789 630 n-BuOH extract 982 790 858 690 975 757 >1000 992 >1000 876 EA-1 279 169 327 147 639 501 334 198 637 519 EA-2 190 97 200 117 435 516 291 111 601 576 EA-3 287 144 300 162 574 413 421 169 620 525 EA-4 590 437 802 679 945 583 622 480 >1000 947 EA-5 432 279 489 311 767 570 410 301 790 657 EA-6 468 333 429 353 672 520 424 361 724 580 36 A. Kundu, S. Saha, V. Ahluwalia, S. Walia 2-one. On the other hand, stigmasterol showed lowest shoot and root growth inhibition against all test species. 2-Acetyl-cadinan-3,6- diene-7-one was also exhibited shoot length inhibition of P. minor (EC50 144 μg mL-1). In general, shoot growth inhibition was more as compared to root growth inhibition. Cadinan-3-ene-2,7-dione and cadinan-3,6-diene-2,7-dione exhibited moderate growth inhibition of test seeds. Our study indicated that 5,6-dihydroxycadinan-3-ene-2,7-dione was most potential plant growth inhibitor against P. minor seeds. Higher plant growth inhibitory activity of 5,6-dihydroxycadinan-3-ene- 2,7-dione was attributed to the fact that this compound is relatively more polar than the other isolated compounds. In general crude extracts were also active towards the weed seeds. Further, the relative effect on coleoptile growth was greater than that of root growth. These results are in agreement with the previous reports (TRIPATHI et al., 1981). Furthermore, coleoptile and root growth of the test species exhibited different responses towards cadinene derivatives, confirming the contention of WHITTAKER, 1971 that plant growth inhibitory activity depends on the nature of test species and concentration of the compounds. Experimental results suggested that the cadinenes are the major constituents of E. adenophorum leaves. Chemical constituents and possible growth inhibitory activity of leaves of E. adenophorum are most important examined features. In view of botanical origin of active ingredient and huge availability of the plant material makes it potential source of naturally occurring bioactive constituents. Acknowledgements Authors are thankful to Head, Division of Agricultural Chemicals, Indian Agricultural Research Institute, India for providing necessary facilities for carrying out the research work. References ADHIKARI, R., KRAUS, W., 1994: Bioactive constituents of Eupatorium adenophorum Spreng (Banmara). J. Nepal Chem. 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ZOU, F., DUAN, G., XIE, Y., ZHOU, Y., DONG, G., LIN, R., ZHU, X., 2009: Molluscicidal activity of the plant Eupatorium adenophorum against Oncomelania hupensis, the intermediate host snail of Schistosoma japonicum. Ann. Trop. Med. Parasitol. 103, 549-553. Address of the corresponding author: Dr. Aditi Kundu, Scientist, Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi-110012