Isolation of nematicidal constituents from essential oil of Kaempferia galanga L rhizome and their activity against Heterodera avenae Wollenweber

Purpose: To explore the nematicidal activities of the essential oil of Kaempferia galanga rhizomes and its isolated constituents against Heterodera avenae Methods: Essential oil of K. galanga rhizomes was obtained by hydrodistillation and characterized by gas chromatography/mass spectrometric (GC/MS) analysis using HP-5MS column. Evaluation of nematicidal toxicity was performed against juveniles (J2) of H. avenae. The bioactive constituent compounds were isolated and identified from the oil based on bioactivity-directed fractionation. Results: Forty-one components were identified and the main components of the essential oil of K. galanga are as follows: ethyl-ρ-methoxy cinnamate (34.79 %), ethyl cinnamate (20.72%), 1,8-cineole (8.96 %), trans-cinnamaldehyde (7.03%) and borneol (5.64 %). The essential oil exhibited nematicidal activity against the cereal cyst nematode with an LC50 value of 91.78 μg/mL. Ethyl cinnamate, ethyl ρmethoxy cinnamate and trans-cinnamaldehyde (median lethal concentration LC50 = 100.60 μg/ml, 83.04 μg/mL and 94.75 μg/mL, respectively) exhibited stronger nematicidal toxicity than borneol (LC50 = 734.89 μg/mL) and 1,8-cineole (LC50 = 921.21 μg/mL) against the cereal cyst nematode. Conclusion: The results indicate that the essential oil of K. galanga and its isolated constituents have a potential for development into natural nematicides for the control of cereal cyst nematodes.


INTRODUCTION
During the current author's mass screening program for new agrochemicals from Chinese medicinal herbs, essential oil of Kaempferia galanga L. (Family: Zingiberaceae) rhizomes was found to possess strong nematicidal activity against cereal cyst nematodes (Heterodera avenae Wollenweber).Cereal cyst nematode is one of the most economically damaging endoparasite pests of wheat and causes numerous annual yield losses worldwide.
In China, the area known to be infested by the cereal cyst nematodes with potentially damaging population densities represents about 22 % of the total wheat production area.It is estimated that the overall yield suppression in the infested areas is about 10 % [1].Control of plant nematodes has been provided principally by soil applications of conventional contact nematicides (carbamates and organophosphates) or fumigants [2].Increasing public concern for the environmental effects of pesticides, groundwater contamination, human health effects and undesirable effects on non-target organisms [3,4] becomes more critical on continued applications of conventional nematicides.
Essential oils and their constituents may provide an alternative to currently used nematicides/ fumigants to control plant nematodes because they are often of low mammalian toxicity, readily biodegradable and possess low danger to the environment if used in small amounts [3,4].Many plant constituents and metabolites including essential oils have been investigated for activity against plant-parasitic nematodes [5][6][7][8][9][10][11].
Sand ginger (K.galangal) is found primarily in open areas in Southern China, Indonesia, Cambodia, Malaysia and India, but is also widely cultivated throughout Southeast Asia [12].In China, sand ginger is well known for its popular use in the food spice and medicinal industry, traditionally treating symptoms ranging from hypertension, pectoral and abdominal pains, headache, toothache, rheumatism, dyspepsia, coughs and inflammatory tumour.It also has a long history of fragrance use to help restlessness, stress, anxiety and depression [13].This herb has broad-spectrum biological activities including larvicidal [14], amebicidal [15], nematicidal [16][17][18], antimicrobial [19] and repellent effects [20].However, to the best of our knowledge, there have been no reports to date which describe nematicidal activity of the essential oil of K. galanga rhizomes against the cereal cyst nematodes.Thus, the objective of this study was to investigate the chemical constituents and nematicidal activity of the essential oil against the cereal cyst nematodes and to isolate any active constituent compounds from the oil.

EXPERIMENTAL Plant material and essential oil
The dried rhizomes of K. galanga (5 kg, harvested from Guangxi Zhuang Autonomous Region) were obtained from Anguo Chinese Medicinal Herbs Market (Anguo 071200, Hebei Province, China).The plant was identified, by Dr QR Liu (College of Life Sciences, Beijing Normal University, Beijing 100875, China).The voucher specimen (No. 20150901) deposited at Institute of Genetics and Physiology, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang, China.The sample was ground to powder using a grinding mill (Retsch Muhle, Haan, Germany) and was subjected to hydrodistillation using a modified Clevenger-type apparatus for 6 h and extracted with n-hexane.Anhydrous sodium sulphate was employed to eliminate water.The n-hexane was taken away by distillation under reduced pressure in a rotary evaporator at 35 °C and the concentrated essential oil was saved in airtight containers in a refrigerator at 4 °C for subsequent experiments.

NMR analysis
Structures of the pure compounds was determined based on nuclear magnetic resonance. 1H nuclear magnetic resonance (NMR) spectra were recorded on Bruker ACF300 [300MHz ( 1 H)] and AMX500 [500MHz ( 1 H)] instruments using deuterochloroform (CDCl 3 ) as the solvent with tetramethylsilane (TMS) as the internal standard.

Gas chromatography-mass spectrometry (GC-MS)
Analyses of volatile constituents were determined using an Agilent 5973 GC-MS system operating in the EI mode at 70 eV [equipped with a 30 m HP-5MS column (0.25 mm × 30 m × 0.25 μm) and coated with 5 % phenylmethylpolysiloxane using a HP-5MS (df = 0.25 μm) (Agilent J&W Scientific, USA)].The temperature program employed for the analysis was as follows: first temperature at 60 °C, maintained for 1 min, increased at 4 °C /min to 290 °C and remained there for 15 min.Helium was the carrier gas at 1.0 mL/min; the sample (1 μL 1/100, v/v, in acetone) was introduced in the split mode (1:10).The injector and detector temperatures were operated at 230 and 300 °C, respectively.Most constituents were recognized by gas chromatography by comparison of their retention indices with those of the literature or with those of authentic compounds available in our laboratories.The retention indices were estimated corresponding to a homologous series of n-alkanes (C 8 -C 24 ) under the same operating conditions.Further identification was taken by comparison of their mass spectra with those saved in NIST 05 and Wiley 275 libraries or with mass spectra from literature [21].

Nematodes
Cysts of H. avenae were taken out from rhizosphere soil of wheat roots collected in Zhengzhou city (34.44 N and 112.56 E), Henan Province, China.Cyst masses were stocked at -4 C for a month firstly and were maintained in Petri dishes at 15 C during 3-7 days for the juvenile eclosion.Only freshly hatched second stage juveniles (J 2 ) were applyed in the experiments [22].

Nematicidal toxicity bioassay
Range-finding studies were carried on to find out the appropriate testing concentrations.A serial dilution of the essential oil and its five constituents (six concentrations, ranging from 50-1,600 µg/mL) was prepared in H 2 O solution with 2 % DMSO.Aliquots of H 2 O (20 µL) containing ca. 100 juveniles (J2) were moved to vials to which 980 µL of the solution containing the essential oil was put.The vials were kept in a hood at 25 C.The nematodes were counted every 24 for 48 h.The nematodes were regarded as dead if the nematodes kept not moving and stiff after put in 1 -2 drops of 1 % NaOH solution.Six repetitions for each treatment were carried out using H 2 O and a 2 % DMSO in H 2 O solution as a control.The experiments were replicated three times.Fosthiazate was purchased from National Center of Pesticide Standards (8 Shenliao West Road, Tiexi District, Shenyang 110021, China) and employed as a positive control.

Data analysis
The results from all replicates were subjected to probit analysis using PriProbit Program V1.6.3 to estimate LC 50 values and their 95 % confidence intervals [23].Samples for which the 95 % fiducial limits did not overlap were considered to be significantly different.
Sand ginger (K.galangal) is well known for its popular use in the food spice and medicinal industry [13].It is suggested that this Chinese medicinal herb (spice) is safe for human consumption because it has been used as a spice and medicinal herb for hundreds of years.Moreover, an acute toxicity study of crude extracts of K. galanga and ethyl p-methoxy cinnamate validated that K. galanga extracts were safe at a dose level of 5,000 mg/kg, and LD 50 value was estimated to be higher than 5,000 mg/kg and ethyl p-methoxy cinnamate had a LD 50 value of higher than 2,000 mg/kg [30].The toxicity of crude rhizome extract of K. galanga using the Hippocratic screening test and acute and subacute toxicities in rats were also measured [31].The results showed that extract of K. galanga rhizomes demonstrates less toxicity.However, no information on toxicity of the essential oil and the other constituents to human were available.Thus, to establish a practical application for the essential oil and the isolated constituents as novel nematicides, further research into the safety of the essential oil/compounds in humans is required.Additional studies on the development of formulations are also necessary to enhance the efficacy and stability and to cut down cost.

CONCLUSION
The findings of the present work suggest that the essential oil of K. galanga rhizomes and its isolated constituents, especially the three cinnamate derivatives, demonstrate strong nematicidal activity against the cereal cyst nematode, H. avenae.Thus, the essential oil and the five isolated ingredients, may find application in pest control, but further studies to determine safety in humans and to enhance its activity is required.

Table 1 :
Composition of the essential oil of Kaempferia galanga rhizome * Identification by co-injection of authentic compounds

Table 2 :
Nematicidal activity of the essential oil derived from Kaempferia galanga rhizomes and its isolated constituents against Heterodera avenae * * * * * * Significant at p < 0.05 level Figure 1: Constituents isolated from the essential oil of Kaempferia galangaDISCUSSIONEthyl-ρ-methoxy cinnamate, ethyl cinnamate, 1,8-cineole, trans-cinnamaldehyde and borneol were the major compounds in the essential oil of K. galanga rhizomes.Chemical composition of the essential oil of K. galanga in the present study was the same as that reported in previous studies[20,25,26]which ethyl ρ-methoxy cinnamate and ethyl cinnamate were always two main constituent compounds in the essential oil.