Studies on the active ingredients, mode of insecticidal action
and their structure-activity relationships of the yellow azalea,
Rhododendron molle G. Don
Zhong Guohua
(Laboratory of Insect Toxicology, South China Agricultural University,
Guangzhou 510642 P. R. China)
Abstract
The yellow azalea, Rhododendron molle G. Don (Ericaceae), is a Chinese
plant known for its medical and insecticidal uses for long time. This paper
reported the results on the isolation and identification of the active ingredients,
insecticidal activities and mode of action against Spodoptera litura, Pieris rapae,
Ostrina farnacalis, Plutella xylostella, Phyllotreta strilata, Lipalus erysimL and
their strucuture-activity relationships of these compounds from R.molle.
The highest activity partition of methanolic extract was obtained by ethyl
acetate (EtOAc). Rhodojaponin-Ⅲ(Abbr.R-Ⅲ) was isolated from EtOAc extract
through silica gel column for several times and its structure was elucidated on
the basis of spectroscope data interpretation. In addition, compounds 1~13 were
isolated from other fractions from EtOAc extracts.
The bioassay showed that R-III, compound 7, 8 and 10 possessed more
oviposition deterrent properties than other tested compounds. After 72h treated
with 50mg/L R-III, compound 7,8 and 10 in choice tests, the oviposition
deterrent rates were 65.22%, 60.64%, 65.28% and 53.28% against P. rapae, and
were 77.48%, 67.98%, 71.64% and 69.61% against P. xylostella, respectively.
They also could change the ratio between the number of eggs on leaves’ surface
and back, increase the mortality of the 1st instar larvae and strengthen the
repellence to the larvae of P. rapae and 3rd instar larvae of P. xylostella when
treated with 50 mg/L R-III, compound 7,8 and 10.
There were significantly differences in antifeeding property for all tested
compounds: R-III> compound 8,10>compound5,6,7,9,11,12,13>compound 1, 2,
3, 4. The values of AFC50 (antifeeding median concentration) against P. rapae
5th instar larvae were 18.69, 44.80 and 57.21 mg/L for R-III, compound 8, 10,
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and were 187.88, 117.52, 132.10, 129.11, 171.05, 148.56 and 121.68 mg/L for
compound 5,6,7,9,11,12,13 in no-choice test. Results of laboratory also showed
that R-III, compound 8, 10 possessed more antifeeding property than other tested
compounds.
The insect growth regulating properties included inhibiting larvae’s weight
increase and growth, reducing the generation potency and affecting growth rate,
etc. The values of LC50 inhibiting larval weight increase were 9.91, 15.59, 16.86
and 15.64 mg/L for R-III, Compound 8,10 and 12 against P. rapae 3rd instar
larvae, and the EC50 for inhibiting growth were 2.32, 9.40, 11.15 and 8.44 mg/L,
respectively. The pupating rate and emergence rate and the number of laid eggs
from 2nd instar larvae of P. xylostella treated with 20mg/L R-III, compound 5
and 10 decreased greatly.
R-III and compound 4,8,10 possessed strongest contact toxicity and the
values of LD50 were 2.18, 13.91, 9.06, 10.92 g/g against P. rapae after dropping
application 72h. As to stomach poison, the LD50 for R-III and compound 5, 6, 7,
8, 9,10 were 6.31,45.36, 21.59, 26.22, 15.41, 23.87 and 12.09 g/g against 5th
instar larvae of P. rapae. By diet of treated leaves with tested compounds for 96h,
the values of R-III, compound 8 and 10 were 7.14, 11.00, 18.68 mg/L against 3rd
instar larvae of P. rapae and were 9.76, 29.32, 31.84 mg/L against 4th instar
larvae, respectively.
What’s more, the egg hatch rate reduced 43.51%, 39.85%, 46.39%,57.69%
and 45.82% when eggs of P. rapae were treated with R-III, compound 7, 8, 10,
11. The results were similar to those of treated eggs of P. xylostella.
On the research of biochemical mechanism, the mode of action of active
ingredients was invested by feeding larvae of P. rapae, S. liture, O. farnacalis,.
The preliminary studies showed that the content of protein in integument of
4th instar larvae of S. liture when treated with R-III, compounds 5,7,8,10
decreased significantly.
The inhibition rates of the activity of -carboxylesterase in midgut were
much more than that in haemolymph in vitro. The values of Km and Vmax were
significantly different from those of CK. The activities of alkaline phosphatase
and acid phosphatase could be inhibited by R-III and other tested compounds as
well.
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ATPase was one of the important target enzymes to most of active
compounds. Most of tested compounds could significantly inhibit the activity of
Na-K-ATPase and Ca-Mg-ATPase. The inhibition median concentration (I50) of
them to Na-K-ATPase was coherent to the LD50 by dropping application.
The optimum reaction condition of Na-K-ATPase, the protein
concentration(6g/mL), pH(7.0), temperature(35C), reaction time(10 min) and
the concentration of substrate(0.5mmol/L ATP) were obtained by uniform
experimental design. A screening model for natural products pesticides was
suggested.
Three new compounds, rhodomolin A (5), rhodomolin B（7）,rhodomolin C
（8）and six known compounds, such as rhodojaponin-III (R-III), rhodomollein-I
(6), rhodomollein XVIII (10), were Iisolated from the tested flowers of R.
molle. By means of spectroscopic analysis such as UV, IR, MS, 1DNMR, 2D
NMR (COSY, NOESY), their structures were identified.
The quantitative measure methods of R-III in reference were corrected and
a new method with UV thin scan method was set up.
The structure-activity relationship, both qualitative and quantitative analysis,
was invested by qualitative compare the groups and activity, Free-Wilson
model compare molecular field analysis (CoMFA). The 2D, 3D-QSAR models
for the contact, antifeeding toxicity, growth inhibiton and poison action were set
up, respectively.
Based on the experimental results, mode of action of R-Ⅲ, ATPase as target
enzyme for screening model of natural products pesticide, application prospect
of R. molle and R-III, and quantitative structure-activity relationship (QSAR) in
pesticides designs were discussed in this thesis.
Key words
Rhododendron molle G.Don, active ingredient, mode of action
Structure-activity relationship
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