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Bioactive Compounds in Tombili Seeds and Tubile Roots as the Alternative for Synthetic Pesticide to Protect Wheats
from Insects and Pests
Weny Musa*, Jusna Ahmad, Chairunisah. J. Lamangantjo
Department of Chemistry, Faculty of Mathematics and Science, State University Gorontalo, 96114, Kotamadya Gorontalo, Indonesia
Abstract: This study was intended to isolate and characterize the pesticide property of secondary metabolite compounds in tombili (Caesalpinia bonduc (L.) Roxb) seed and tubile (Derris elliptica (Roxb) Benth) root. The pure compound isolated from 1.5 kgs tombili seeds and 500 grams tubile roots were macerated using methanol to get methanol extracts of 85.79 and 30 grams, respectively. Phytochemical testing showed positive results for flavonoid, terpenoid, alkaloids, tannin and saponin test. An insecticide bioactivity testing for both plants showed that the most effective result was achieved for ethyl acetate fraction in concentration of 0.05%. IR spectrophotometer for tombili seeds showed functional groups O-H, aliphatic stretching C-H, stretching C=O, aromatic stretching C=C, bending C-H, and stretching C-OH that marked the presence of terpenoid compounds. This result was supported by a UV-Vis spectrophotometer data with 236.00 nm wavelength absorption, possibly correlated with the transition of electrons that not bound to the anti-bonding orbital (n→π*) caused by the presence of the chromophore group C=O. Purity test for tubile roots only resulted in a single spot, therefore the phytochemical test was conducted and showed positive result for flavonoid. This result was supported by two bands resulted from UV-Vis spectrophotometer data with wavelength 280 nm and 235.50 nm for tape-1 and tape-2, respectively. The IR spectrophotometer also showed the functional groups O-H, stretching C-H, stretching C=O, aromatic stretching C=C, bending O-H, bending C-H, and alcoholic stretching C-O, indicating that the isolate was a flavonoid compound.
Keywords: Isolation, characterization, secondary-metabolites pesticide, Caesalpinia bonduc, Derris elliptica.
Introduction
Indonesia has numerous species of flora that can be used to produce a wide range of beneficial compounds.
This biodiversity offers various chemical resources to support human needs like pharmacological substances, insecticides and cosmetics1.
The bioactivity of plants exists in their secondary metabolites, and the secret inside these metabolites always rise interesting challenges for the scientists to isolate, characterize and synthesize them 2. Studies to characterize beneficial properties on these metabolites are highly recommended in order to convert this magnitude of biodiversity into the continuous innovation profitable for human living.
International Journal of ChemTech Research
CODEN (USA): IJCRGG ISSN: 0974-4290 Vol.9, No.04 pp 604-615, 2016
Weny Musa et al/International Journal of ChemTech Research, 2016,9(4),pp 604-615. 605 In Indonesia, particularly in Gorontalo, there are many plants known to exhibit certain advantageous bioactivities. Two of them are tombili seeds (Caesalpinia bonduc (L.) Roxb) and tubile roots (Derris elliptica (Roxb) Benth). Unfortunately, the chemical properties of these plants are barely documented despite the popularity of its traditional usage. Mixed with another plants, tombili seeds and tubile roots are popularly used by farmers as botanical pesticide to protect wheat from insects and pests.
Tombili or Caesalpinia bonduc (L.) Roxb is a Caesalpiniaceae family, can be found worldwide, especially in the tropical area of India, Sri Lanka, Andaman and Nicobar. Indian people also used it to cure some diseases3. Yadav demonstrated that C. bonduc contains cassanediterpenes, a terpenoid compound, using NMR spectroscopy along with IR and UV-Vis spectroscopy4. Kinoshita also found similar compound, cassanefuranoditerpen, using similar method5. Mobasher et.al., also reported another terpenoid, 6β,7β- dibenzodyloxyvouacapen-5α-ol6.
Tubile or tuba roots can be found almost everywhere in Nusantara. Widely used to kill fish (as poison), this plants is also effective as biopesticide at farms7. Gorontalo farmers use this plant to fight insects and pests8.
This study was aimed to isolate and characterize the secondary metabolites in tombili seeds and tubile roots, and to test their effectiveness against pests in farm.
Methods
Tools and materials. Plant materials used are tombili seeds and tubile roots. Chemicals used include:
methanol, n-hexane, ethyl acetate, MTC, Mayer reagent, Wagner reagent, Hager reagent, distilled water, concentrated HCl, Mg powder, NaOH, concentrated H2SO4, chloroform, acetone, silica gel and GF254 TLC plate. Tools used are: blender, analytical balance, UV lamp, microwave, tools to visualize H2SO4 spots, filter papers, glass set, rotary vacuum evaporator, thin-layer chromatography and column chromatography equipments, cutter, test tubes, and test tube rack.
Extraction. Fresh samples of tombili seeds and tubile roots were dried by aeration in the open air, direct sunlight avoided. Dry samples were macerated using methanol for 3 × 24 hours. The solvent was replaced every 24 hours until the filtrate is colorless. The filtrate then evaporated, resulting in a thick methanol extract. This condensed extract was mixed with distilled water with ratio of methanol in water was 1: 2, then partitioned with n-hexane and ethyl acetate, successively, to obtain each partition of both fractions. Each partition was evaporated at 30 – 40 °C to obtain the extract from n-hexane, ethyl acetate and water.
Flavonoid test. About 0.1 gram of condensed extract was dissolved in 10 ml of methanol and distributed into four test tubes. The first tube was used as the control tube, and solution in three other tubes were mixed with NaOH, concentrated H2SO4, and Mg powder plus concentrated HCl, respectively. This test yield positive result (and flavonoid compound detected) if there were a marked differences between the color of solution in the three last tubes compared with the color of solution in the first tube9.
Alkaloid test. About 0.1 gram of condensed extract was dissolved with 10 ml of ammoniac chloroform and distributed into two test tubes. The first tube was tested with Hager reagent, while the second was mixed with 0.5 ml of H2SO4 2 N. The acid layer was separated then distributed into three test tubes. Each tube was tested with Mayer, Dragendorff, and Wagner reagent, respectively. The test yield positive result (or the solution is an alkaloid solution) if sedimentation was detected.
Steroid,terpenoid, and saponintest. About 0.1 gram of condensed extract dissolved with 10 ml of diethyl ether. The dissolved part was dripped on a drop plate, with additional 2 drops of anhydride acetic acid and 1 drop of H2SO4. The remaining undissolved part was mixed with a small volume of hot distilled water, moved to a test tube, mixed with hot distilled water again, and then shaken vigorously for 15 minutes. The filtrate under the foam was taken and placed on a vaporizer cup, mixed with HCl and evaporated until it dried as a crust. A drop of diethyl ether was dripped into the crust, plus 2 drops of anhydride acetic acid and 1 drop of strong H2SO4. The presence of steroid substance in the crust would change its color into bluish green; any terpenoid would change its color into brownish red, while saponin would produce foams above the crust.
Weny Musa et al/International Journal of ChemTech Research, 2016,9(4),pp 604-615. 606 Tanin test. A condensed extract as much as 0.1 gram was dissolved with alcohol in a test tube. FeCl3 1% then was dropped into the tube. This test yield positive result,in the presence of tanin, if the color changed into dark blue or dark green.
Insecticide bioactivity test. Eight groups of wheat plants chosen randomly were treated with different dose of tombili and tubile extracts to observe their survival against insects and/or pests. The type of treatments and dosage are listed in Table 1. This treatment and observation was done periodically during the whole season.
Table 1. Type of extract and dosage used in insecticide bioactivity test.
Group Type of extract Concentration(%) Tombili
1 Methanol 0.25
2 n-Hexane 0.05
3 Ethyl acetate 0.05
4 H2O 0.25
Tubile
5 Methanol 0.25
6 n- Hexane 0.05
7 Ethyl acetate 0.05
8 H2O 0.25
Purification and Separation
The purification and separation procedures were done using thin-layer and columnar chromatography.
TLC was first used to choose the best extract to be used for separation. With a mix of n-hexane and ethyl acetate in ratio 8:2 as the mobile phase, and the silica gel as the static phase, all fractions were tested for their purity and the best would underwent the separation process. Subsequent purity tests then were conducted by means of TLC.
The separation was run using columnar chromatography. First, a cotton filter was used to suspend the silica from dropping down with the solvent. The preparation of column was done with dry method using n- hexane as solvent and hot silica gel previously prepared with microwave for about 20 minutes. The best extract previously selected from TLC mixed with silica gel and then dropped into the column in open state. The gravitation forced the formation of certain number of fractions to be analyzed individually using TLC with a mixed of n-hexane, MTC and acetone as eluent in ratio 6:2:2. Nodes were grouped and symbolized as fractions.
The purity of fractions then was analyzed by examining node patterns and calculating their Rf values.
Result and Discussion
Phytochemical tests for tombili seeds.
Phytochemical test was a qualitative chemical test for a plant to determine the presence of certain secondary metabolite inside its extract. This study involved some routine phytochemical tests such as flavonoid test, alkaloid test, steroid, terpenoid, and saponin test, and also a tanin test.
This study demonstrated that all extracts (in methanol, ethyl acetate, n-hexane and distilled water) contain flavonoid substance marked with certain color changes when mixed with flavonoid reagents.
The alkaloid test for methanol, ethyl acetate and distilled water extract yield positive results, marked with a sedimentation formed when the extracts were mixed with alkaloid reagents, while such sedimentation was not seen in n-hexane fraction.
Steroid and terpenoid tests for tombili seeds done using Lieberman-Bouchard reagent resulted in brownish red coloration indicating the presence of terpenoid and the absence of steroid substance. The saponin test yielded negative results for n-hexane and ethyl acetate fractions, but positive for methanol and distilled
Weny Musa et al/International Journal of ChemTech Research, 2016,9(4),pp 604-615. 607 water extracts, marked with foam formation caused by the glycoside ability to form foam in water . The addition of hot distilled water into saponin test was ended in hydrolysis reaction.
The tanin test yielded positive results for all extracts, marked with dark blue or dark green coloration because of the polifenolic property in tanin (Harborne, ; in Darmawijaya and Yudha)10.
Phytochemical tests for tubile roots.
Phytochemical test for tubile root extract demonstrated that all fractions (methanol, n-hexane, ethyl acetate and distilled water) contain flavonoid substance.
Insecticide bioactivity test.
As seen in Table 1, the observation for insecticide bioactivity was conducted by dividing a wheat farm into eight areas or groups, while each group received different extract fraction and dosage of tombili seeds or tubile roots.There are two type of concentration used: 0.05 and 0.25%. Both were created by dissolving 0.5 and 2.5 grams (respectively) of extract in 1,000 ml distilled water.
By a serial observation, this study demonstrated that the most effective treatment was achieved using the ethyl acetate fraction at concentration 0.05%. This was proven by the total volume of rice produced from that area, 24 sacks, compared with only 10 sacks produced from the control area. Other 7 areas only produced 18 – 20 sacks of rice, but still superior compared with the production from control area.
Separation and purification of tombili seeds.
TLC was used to choose which of four extracts to be used for separation with columnar chromatography. This selection demonstrated that the methanol extract showed the best node pattern and separation, therefore it would be used for the separation phase.
About 10.6 grams of methanol extract mixed with silica gel and poured into the column in open state.
Elution with ethyl acetate forced the separation of extract from the eluent and formed a gradient of concentration from 100% ethyl acetate to 100% methanol extract. This then was analyzed with TLC technique again using eluent of n-hexane : MTC : acetone in order to separate the nodes. Similar nodes grouped as fractions and this process produced 14 fractions with 58 node groups(Figure 1).The most crystallized fraction suitable for next purification was A1.
The A1 fraction then was separated again with columnar chromatography using the same eluent and produced 62 node groups. Tested with TLC, this resulted in five fractions (A11through A15) as shown in Figure 2. The fraction A12 was found to be the best subject for the next purification because it was the most crystallized fraction and has a single node pattern.
A1A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14
Figure 1. The result of columnar chromatography and TLC using eluent n-hexane : MTC : acetone (6:2:2).
