08(완료)149~152KJAE 12-02(박일권).pdf216.0KB

• Materials and Methods
• Sample preparation
• Chemicals and insects
• Bioassay
• Statistical analysis
• Results and Discussion

 Piper nigrum, known as black pepper, has been widely used as spices. Its taste is strongly pungent, and the odor is aromatic. Black pepper is native to India, and harvested in East and West Indies, Brazil, United States, Taiwan, Indonesia, Malay peninsula, and China. The fruit of P. nigrum has been used for prescribing for cholera, dyspepsia, flatulence, diarrhoea, various gastric ailments, and paralytic and arthritic disorders (Siddiquiet al., 1997). As well as used as medicine, the Piperaceae (Pepper family) has long been used in traditional agriculture as an insect-control agent (Schultes and Raffauf, 1990). This plant species has been known to be toxic to several insect species (Freeborn and Wymore, 1929; Lathrop and Keirstead, 1946; Synerholmet al., 1945; Su, 1977; Scott and Mckibben, 1978; Miyakado and Yoshioka 1979; Miyakado et al., 1979, 1989; Park et. al., 2002). The insecticidal principles of black pepper such as pipercide, dihydropipercide, guineensine, pellitorine or retrofractamide A were identified in several studies (Miyakado and Yoshioka, 1979; Miyakado et al. 1979; Park et al., 2002). This laboratory study examined the methanol extract of the fruit from P. nigrum for insecticidal constituents against the diamondback moth, Plutella xylostella larvae.

Materials and Methods

Sample preparation

 Sample preparation. The fruits (3.6 kg) of Piper nigrum purchased as a commercially available product were powdered with a blender, extracted twice with methanol (20 L) at room temperature for 2 d and filtered (Toyo filter paper No. 2, Toyo Roshi, Japan). The combined filtrate was concentrated in vacuo at 35℃ to give a yield of 11% (based on the dry weight of the fruit). The extract (396 g) was sequentially partitioned into hexane (63.3 g), chloroform (249.5 g), ethyl acetate (19.8 g), butanol (13.8 g) and watersoluble (43.56 g) portions for subsequent bioassay with the insects used. The organic solvent portions were also concentrated to dryness by rotary evaporator at 35℃.

Chemicals and insects

 Chemicals and insects. In a primary screening, hexane and chloroform solvent portions showed potent insecticidal activity against diamondback moth larvae, Plutella xylostella (Table 1). The chloroform fraction was selected as target fraction because of its relatively higher yield than the hexane fraction. Isolation of pellitorine, guineensine, pipercide, and retrofractamide A was well described in our previous study (Park et al., 2002). Piperine was purchased from Sigma (St Louis, MO, USA). P.xylostellalarvae (3rd instar) were used in this experiment. The larvae were reared on Brassi cacampestris subsp. napus var. nippooleifera (MAKINO) seedlings. They have been maintained in the laboratory without exposure to any insecticide at 24±2℃, 50-60 relative humidity (RH), and a photoregime of 16 :8 h (L : D).

Bioassay

 Bioassay. Leaf dipping method was used to investigate the insecticidal activity. Chinese cabbage leaves were punctured with a cork borer (Ø3 cm), and test materials suspended in distilled water with Triton X-100 (Coseal Co., Seoul) added at the rate of 1 mL/Lwere used. Two concentrations, 5.0 and 2.5mg/mL, for methanol extract were prepared, while the other organic fractions were tested at 2.5 mg/mL.Leaf discs were dipped in test solution for 30 sec. Controls received distilled water with Triton X-100 added at the rate of 1 mL/L. After evaporation in a draft for 2 h, twenty insects were separately placed onto the treated and the control leaf discs. Total replications were four times. Mortalities were determined 2 d after treatment. Seven to five doses (from 0.5 mg/mL to 0.007 mg/mL) of each compound were used to obtain LC50 values.

Statistical analysis

 Statistical analysis. The insecticidal activity was determined and transformed to arcsine values for analysis of variance (ANOVA). Treatment means were compared and separated by Scheffe's test. Means ( S.E.) of untransformed data are reported. LC50 values were calculated by probit analysis (SAS, 2004).

Results and Discussion

 Table 1 showed the insecticidal activity of methanol extract and solvent fractions of P. nigrum against P. xylostella at 5.0 and 2.5 mg/mL concentration. Methanol extract of P. nigrum showed 100 and 96% insecticidal activityat 5.0 and 2.5 mg/ml, respectively. Hexane and chloroform fractions showed 100% mortality at 2.5 mg/mL. However, ethyl acetate, butanol and water-soluble fractions exhibited 13, 7 and 3% mortality, respectively. The larvicidal activities of compounds purified from the Piper fruit against 3rd instar larvae of P. xylostella were examined by leaf dipping method (Table 2). Guineensine(LC50, 0.013 mg/mL) was much more toxic than retrofractamide A, pipercide, and pellitorine. On the basis of LC50 values, guineensine was about 3.5 times stronger in insecticidal activity than pellitorine. Piperine showed very low larvicidal activity against 3rd instar larvae of the diamondback moth, whereas isobutyl-containing amides pellitorine, guineensine, pipercide, and retrofractamide A produced potent larvicidal activity. These results indicated that methylenedioxyphenyl group was not essential for the toxicity against the diamondback moth larvae but isobutyl amine might play a great role in larvicidal activity. Miyakadoet al. (1989) and Park et al. (2002) already reported that the isobutylamine moiety appeared to be essential for insecticidal activity againstadzuki bean weevil (Callosobruchuschinensis) and three mosquito species such as Culex pipiens pallens, Aedes aegypti and Aedes togoi. Additionally, there was a difference in insecticidal activity between the isobutylamides with and without a methylenedioxyphenyl moiety. Pellitorine without methylenedioxyphenyl moiety was the least toxic followed by pipercide, retrofractamide A and guineensine with methylenedioxyphenyl moiety. Park et al. (2002) already reported that difference in insecticidal activity between pellitorine and other isobutylamides such as pipercide, guineensine and retrofractamide A was attributed to chemical stability. Compounds with a conjugated dienamide chromophore (-C=C-C=C-CONH-), such as pellitorine, are quite unstable owing to their unsaturation (Miyakadoet al., 1989). Our results and early study indicate that isobutylamine moiety appeared to be essential for insecticidal activity, and the methylenedioxyphenyl moiety improved insecticidal activity of isobutylamides by stabilizing the chemical structure. In conclusion, the Piper fruit-derived materials might be useful for managing P. xylostella larvae. Further studies on the exact mode of Piper derived compounds, their effect on other insect pests of the active compounds from the Piper fruit, formulation improving the insecticidal potency, and the safety and stability of a selected formulation for commercialization might be conducted for its practical use as a naturally occurring insectcontrol agent.

Table 1. Insecticidal activity of a methanol extract and solvent fractions of Piper nigrum fruit against Plutella xylostella larvae using the leaf dipping method, at 48 h

Table 2. LC50 values of compounds from Piper nigrum fruit against Plutella xylostella larvae using the leaf dipping method, at 48 h.