Pesticidal activity

In our experiment, at 24 h duration of exposure, chloroform soluble fraction of the plant was observed for significant pesticidal activity against T. castaneum (Table 1). With the increment of doses the mortality record was up-regulated i.e. highest mortality record (96.60%) was observed for dose 1.77 mg/cm² and lowest mortality record (20.00%) was observed for the dose 0.22 mg/cm² (Table 1, 2, Figure 1). LD₅₀ of chloroform fraction for 24 h duration of exposure was 0.40 mg/cm² (Table 2). When duration of exposure was increased (48 h), mortality record was little increased and LD₅₀ little reduced 0.37 mg/cm² (Table 2). No pesticidal activity was found for petroleum ether, ethyl acetate and methanol soluble fractions of the plant. The compound 3,4-dihydroxybenzoic acid that was isolated from ethyl acetate fraction (also detected in chloroform fraction) did not showed pesticidal activity against T. castaneum.

Dose (mg/cm2)	#	Mortality record for applied pests
		Record after 24 hours	Average ± SD record after 24 hours	Record after 48 hours	Average ± SD record after 48 hours
1.77	10	10	9.66 ± 0.57	10	9.66 ± 0.57
1.77	10	10		10
1.77	10	9		9
0.88	10	10	9.00 ± 1.00	10	9.00 ± 1.00
0.88	10	9		9
0.88	10	8		8
0.44	10	5	5.33 ± 0.57	6	5.66 ± 0.57
0.44	10	6		6
0.44	10	5		5
0.22	10	2	2.00 ± 1.00	2	2.33 ± 0.57
0.22	10	1		2
0.22	10	3		3
Control	10	0	0.00	0	0.00

# = Number of pests applied per petridish.

Recording time	Dose (mg/cm2)	% of Average mortality	% Corrected mortality	Regression equation	LD50(mg/cm2)	95% Confidence limits
						Lower	Upper
Record after 24 hours	1.77	96.66	97	Esimate 1	0.40	7.99	6.41
	0.88	90.00	90	Y = 1.72 + 3.10X
	0.44	53.33	53	Esimate 2
	0.22	20.00	20	Y = 1.61 + 3.19X
Record after 48 hours	1.77	96.66	97	Esimate 1	0.37	7.33	15.65
	0.88	90.00	90	Y = 1.93 + 2.97X
	0.44	56.66	57	Esimate 2
	0.22	23.33	23	Y = 1.88 + 3.02X

Pest repellency activity

In pest repellency study, chloroform fraction showed good repellency property at all tested doses (0.94 to 0.23 mg/cm²) (Table 3, Figure 2). Observations of first hours not significantly differ from the observations of subsequent hours (second, third, fourth and fifth hours). Like pesticidal activity, pest repellency activity also increased with doses. Petroleum ether, ethyl acetate and methanol soluble fractions as well as 3,4-dihydroxybenzoic acid were also subjected to pest repellency test, but no activity was found.

Dose (mg/cm2)	#	Repellency record
		Hourly observation					Average of hourly observation (Nc)					Percent repulsion (PR) PR = (Nc - 5) х 20%
		1 h	2 h	3 h	4 h	5 h	1 h	2 h	3 h	4 h	5 h	1 h	2 h	3 h	4 h	5 h
0.94	10	9	10	10	10	10	9.6	9.6	9.6	10.0	10.0	93.2%	93.2%	93.2%	100%	100%
0.94	10	10	10	10	10	10
0.94	10	10	9	9	10	10
0.47	10	9	10	10	10	10	9.0	9.3	9.3	9.6	9.6	80.0%	86.6%	86.6%	93.2%	93.2%
0.47	10	10	8	8	9	10
0.47	10	8	10	10	10	9
0.23	10	6	8	8	8	8	8.0	8.6	8.3	9.3	9.3	60.0%	73.2%	66.6%	86.6%	86.6%
0.23	10	10	9	9	10	10
0.23	10	8	9	8	10	10

# = Number of pests applied.

Plant materials

The fresh rhizomes of D. quercifolia J. Smith was collected in the month of October from mango trees of Khamar Bari, Lakshmidurpara, Lakshmipur, Bangladesh (year). The plant was taxonomically identified by Professor A. T. M. Naderuzzaman, Department of Botany, University of Rajshahi, Rajshahi, Bangladesh and its voucher specimen (No. 1939) had been deposited.

The rhizomes were first washed with water to remove adhering dirt, cut into small pieces, sun dried for three days and finally dried at 45°C for 36 h in an electrical oven [18]. After complete drying, the entire portions were pulverized into a coarse powder with the help of a grinding machine (FFc-15, China) and were stored in an air tight container for further use [19].

Extraction of plant materials

The powder materials (600 g) were extracted with ethanol (3 L) in a Soxhlet apparatus (Quickfit, England). The extraction was continued for 72 h at 65°C. The extract was filtered through filter paper. The filtrate was concentrated under reduced pressure at 50°C in a rotary vacuum evaporator to afford a blackish green mass (52.4 g). The blackish green mass was further extracted with petroleum ether, chloroform, ethyl acetate and methanol, and dried under reduce pressure to afford petroleum ether (7.5 g), chloroform (7.8 g), ethyl acetate (5.5 g) and methanol (8.4 g) fractions, respectively [20].

Isolation of 3, 4-dihydroxy benzoic acid

The ethyl acetate soluble fraction was subjected to column chromatography using chloroform and methanol of increasing polarity. Column chromatography yielded 32 fractions. The fractions eluting with 10-25% methanol in chloroform were subjected to preparative TLC (mobile phase 15% methanol in chloroform) to give compound 1 (89 mg). In solubility test, compound 1 was sparingly soluble in water and freely soluble in ethyl acetate, methanol and acetone. The liquid chromatography/electrospray-mass spectroscopy (LC/ES-MS) in the positive ion mode of compound 1 showed molecular [M + H]⁺ peak at m/z 154.8 corresponding to a molecular formula of C₇H₆O₄. The IR spectrum exhibited bands at 1240, 1375, 1739, 2877, 2908 and 2985 cm^-1. The ¹H-NMR, ¹³C-NMR, HSQC and HMBC spectral data of compound 1 was in good agreement with spectral data of 3,4-dihydroxybenzoic acid (Figure 3) published in literature [21].

Collection and maintenance of pest

The Tribolium species, T. castaneum (Herbst) used in the present experiment was originally received from the Crop Protection Department of the University of Newcastle, U.K. and were reared in the Crop Protection and Toxicology Laboratory, Department of Zoology, University of Rajshahi, Bangladesh.

T. castaneum were maintained in 1 L glass jar containing food medium. A filter paper was placed inside each jar for easy movement of the pest. The jar was covered with a filter paper at the top and kept in an incubator at 30 ± 0.5°C.

A standard mixture of wheat flour and powdered brewers yeast in the ratio of 19: 1 was used as food medium to culture the pest. Both flour and yeast were previously passed through a 250 micrometer aperture sieve and mixed thoroughly using an electric blender. The food was sterilized in an oven at 120°C for 6 h. Food was not used until at least 15 d after sterilization to allow its moisture content to equilibrate with that of environment.

Screening for pesticidal activity

Screening for pesticidal activity was carried out using surface film method [22–24], is a simple and widely used method. The working solution was prepared by dissolving 100 mg experimental sample in 2 ml mixed solvent (50% chloroform + 50% methanol) in a vial. For each sample similar three vials was prepared.

Thirteen clean and dried petridishes (size of each is 60 mm, area of each is 28.26 cm²) were taken for each sample. Four petridishes were marked by 50, 25, 12.5 and 6.25 mg. One ml working solution (prepared previously) was poured into the 50 mg petridish and agitated clockwise, anticlockwise, left to right and right to left to further confirm the uniform dispersion. One ml solvent (50% chloroform + 50% methanol) was added to that vial from which 1 ml had been used and mixed uniformly. From this vial, 1 ml solution was poured into the 25 mg petridish and agitated similarly for uniform dispersion. Using this serial dilution technique, likewise sample was poured into 12.5 mg and 6.25 mg petridishes and agitated similarly for uniform dispersion. The above processes were continued two times further using two remaining vials of working solution and eight remaining petridishes. Then the layers of dispersed sample into the petridishes were air dried. One ml solvent (50% chloroform + 50% methanol) was poured and dispersed into control petridish and air dried.

The pests were collected by sieving and ten pests were applied on each layer of dispersed sample into the petridish. This process is continued for each petridish. Then the number of pests have died were recorded after passing 24 and 48 h.

Pest Repellency Test

Pest repellency test was conducted using filter paper disc method [22, 24, 25]. The working solution was prepared by dissolving 60 mg experimental sample in 2 ml mixed solvent (50% chloroform + 50% methanol) in a vial. For each sample similar three vials was prepared.

Nine clean and dried petridishes (size of each is 90 mm) and Nine filter papers (size-90 mm) were taken for each sample. Three petridishes were marked by 30, 15 and 7.5 mg. Three filter papers were taken for these three petridishes and each filter paper was cut (by scissors) into equal two parts through centre where one part can be used as control part and other part can be used as treated part. For 30 mg petridish with its filter paper, treated part of filter paper was taken at outer background of the petridish and one ml working solution (prepared previously) was dispersed uniformly thorough out this part of filter paper and air dried. Then this part of filter paper was joined with its control part using transparent adhesive tape and placed into the 30 mg petridish using forceps. For 15 mg petridish with its filter paper, treated part of filter paper was taken at outer background of 15 mg petridish. One ml solvent (50% chloroform + 50% methanol) was added to that vial from which 1 ml had been used and mixed uniformly. From this vial, 1 ml solution was dispersed uniformly throughout the treated part of filter paper and air dried. Then this part of filter paper was joined with its control part using transparent adhesive tape and placed into the 15 mg petridish using forcep. Similar works was done for 7.5 mg petridish with its filter paper. The above processes were continued two times further using two remaining vials of working solution and six remaining petridishes and filter papers.

The pests were collected by sieving and ten pests were applied on the filter paper at the center of the petridish. This process was continued for each petridish. Then the number of pests have repelled were counted per hour interval up to 5 h. The percentages of repellency were determined and results were provided through ANOVA after transforming them into arcsin percentage value.

Statistical analysis

The percent mortality was subjected to statistical probit analysis [26] and the dose-mortality relationship was expressed as a median lethal dose (LD₅₀). The repellency values in the recorded data were calculated for percent repellency, which was again transformed by arcsine transformation for the calculation of analysis of variances (ANOVA). Means values were compared using ANOVA (two factors without replication) (Additional File 1: Supplementary Table 1).