Exploration of Preservatives that Inhibit Wood Feeding by Inhibiting Termite Intestinal Enzyme Activity

All termites used in the experiment are Reticulitermes speratus kyushuensis collected in nearby stumps during the investigation of bio-damage related to wooden cultural properties targeting the old house of Kim Hwanki (National Folklore Cultural Heritage No. 251) in Shinan-gun, Jeollanam-do.

The extraction method using solubility was selected as the enzyme extraction method. After selecting worker termites among the termites, ergates that weigh 6.11 g were frozen at -40°C and crushed, 0.1 M sodium acetate buffer (pH 7) was added to the termites for 5 ml per 1 g of weight, and the enzyme was mixed at room temperature for 24 hours, and then filtered with filter paper and used as termite intestinal enzyme.

To measure the protein content of the extracted termite intestinal enzyme, a quantitative method was selected using the Pierce™ BCA protein assay kit (Thermo, USA), and bovine serum albumin (BSA) was selected as a protein for comparative quantification. After reacting the termite intestinal enzyme and the comparative protein BSA with the reaction reagent for 30 minutes at 37 °C according to the quantitative method, absorbance (562 nm) was measured by Microplate readers (TECAN, CHE). The protein of the termite intestinal enzyme was quantified using the comparative protein BSA content as a standard curve.

To determine the activity of termite intestinal enzymes, cellulose and xylan which is hemicellulose were selected as substrates. To measure the content of reducing sugars to be degraded, a quantitative analysis of reducing sugar by DNS (3,5-dinitrosalicylic acid) method of Miller (1959) was conducted. Miller's DNS reducing sugar experiment is a method of quantifying sugar by measuring the absorbance of 3-amino-5-nitrosalicylic acid produced by reacting reducing sugar to DNS (Miller, 1959). DNS reagents were prepared and used by adding DNS 10.6 g and NaOH 19.8 g to 1,416 ml of distilled water, then adding rochelle salts 306 g and phenol 7.6 g and Na+ metabisulfite 8.3 g was added and stirring it for 24 hours.

As a test method for the termite intestinal enzyme activity experiment, add 0.1 M sodium acetate buffer 400 μl to substrate 500 μl such as cellulose and xylan of 1% and preheats at 50°C with water bath for 1 minute. 100 μl of the enzyme was added to the preheated substrate and buffer, and the enzyme and the substrate were reacted at 50 °C for 30 minutes, and then 3 ml of the prepared DNS reagent was added, followed by heating in a hot water bath at 100 °C for 5 minutes. After cooling for about 10 minutes, absorbance was measured at 550 nm.

Two types of cellulose and xylan were used. Regarding Cellulose, Sodium carboxymethyl cellulose (CMC) purchased from Sigma Aldrich with a molecular weight of 250,000 or less and nano-cellulose from hardwood bleached (NC) supported by Changgang Institute of Paper Science & Technology were used. Regarding Xylan, Xylan from oat spelts (Xylan-O) and xylan from beech wood (Xylan-B) purchased from sigma aldrich with a molecular weight of less than 35,000 were selected and experimented.

To check the activity of termite intestinal enzymes against the substrate, the amount of the substrate was increased while checking the content of reducing sugars that are decomposed by the activity of the enzyme, and then the termite intestinal enzyme (T1) was compared with the conventional xylanase (X1) and the activity performance against the substrate was analyzed. The xylanase used as a control was purchased from Sigma Aldrich, and all reagents used in all experiments used a grade of 1 or higher.

An experiment was conducted to select medicinal plant extracts with xylanase-inhibiting performance using extracted termite intestinal enzymes. Medicinal plant extracts were prepared by extracting 628 medicinal plants native to Korea and abroad with methanol. For the termite intestinal enzyme inhibition experiment, a DNS reducing sugar experiment was used as reference. The purpose of this study was to search for medicinal plant extracts that inhibit the enzyme's substrate decomposition activity by adding the medicinal plant extracts during the course of the reaction between the enzyme and the substrate. Through DNS experiments, 10 μl of medicinal plant extract was added to 500 μl of 1% xylan-B (0.1 M sodium acetate buffer pH 7), which is a substrate and 390 μl of 0.1 M sodium acetate buffer, and then preheated at 50 °C for 1 minute in a water bath. Then, after adding 100 μl of the termite intestinal enzyme at a concentration of 300 μg/ml, reacted in a water bath at 50 °C for 30 minutes, added 3 ml of DNS reagent, heated at 100 °C for 5 minutes, then cooled at room temperature for 10 minutes, then absorbance was measured at a wavelength of 550 nm. The control group was measured the amount of decomposed reducing sugar using methanol, an extraction solvent of medicinal plant extract, and the medicinal plant extract, which showed a lower amount than it, was selected as having inhibitory activity.

In order to measure the contact insecticidal concentration of the medicinal plant extract against termites, it was investigated whether the selected medicinal plant extract showed activity against termites. 30 termites were used, and medicinal plant extracts were dispensed into filter paper at concentrations of 50, 10, 5, 1, and 0.5 mg/ml, dried to remove the solvent sufficiently and supplied moisture. Moisture was supplied in a spray form using distilled water. The death of termites was observed and counted for 96 hours, and the experiment was conducted without supplementing dead termites.

In order to confirm the feeding inhibition rate of the selected medicinal plant extract on the termites, the medicinal plant extract at a concentration of 0.5 mg/ml was dispensed on filter paper and dried to remove the solvent. While moisture was supplemented with a spray form, the feeding amount of termites was set to methanol as the control group. The termite count was selected as 30 termites, and the termite count was checked every 24 hours to maintain a constant termite count. After the final 120 hours, the weight of the filter paper was measured to compare the feeding amount of termites.

Borneolum Syntheticum, Ephedra sinica extracts that inhibit the activity of termite intestinal enzymes were analyzed by GC-MS (GC : 7890A/MSD : 5975C inert, agilent technologies). As a pre-treatment of GC-MS analysis, the extract was filtered using a syringe filter (25 mm MFS, JA), and the injection amount per one time was determined to be 1 μl for Borneolum Syntheticum extract and 0.3 μl for Ephedra sinica extract and injected using the liquid type direct injection method. DB-1HT (30 m × 250 mm × 0.1 mm) was used as the column, and the conditions of the oven were the same as those in Table 1.

As a result of measuring the enzyme protein content of termites extracted by the extraction method using solubility with the BCA protein diagnostic kit, the concentration of total protein content was 2.33 mg/ml. It was confirmed that the amount of 381.87 μg/g could be extracted when termites were frozen and extracted with a buffer (Table 2).

In order to confirm the activity of the extracted termite intestinal enzyme, its properties were identified through various substrates. Cellulose and xylan were used as substrates, and two types (4 types in total) were selected for the measurement of DNS activity using the activity of the extracted enzyme and in the result. It was confirmed that the activity increased in xylan (Fig. 1). It was confirmed that the amount of reducing sugar produced increased as the amount of substrate addition increased, but in the case of cellulose, the amount of reducing sugar produced was changed little even if the amount of substrate increased. It seems that the enzyme activity is low since cellulose, a substrate, is composed of polysaccharides with a large molecular weight, which makes it difficult to decompose into monosaccharides, reducing sugars.

Fig. 1. Termite intestinal enzyme activity on substrate type (cellulose, xylan) and content (200~900 μl). Confirm the activity of the enzyme by using the separated enzyme and increasing the activity according to the substrate and the amount of the substrate. Values are expressed as mean±standard deviation.

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In the previous experiment, as cellulose was found to have a low decomposition rate with the termite enzyme, xylan, which is hemicellulose, was selected as an additional substrate and compared with xylanase that is sold on the market. When the protein concentration of the enzyme was set to 300 μg/ml and the activity against cellulose and xylan was compared, the activity of the termite intestinal enzyme was less active against xylan than xylanase, but it showed similar results, i.e., it has decomposition activity against xylan. (Fig. 2). In particular, it was confirmed that the activity was higher in xylan (Xylan-B) isolated from chestnut than xylan (Xylan-O) isolated from oatmeal. Based on the above, with experiment conditions for identifying medicinal plant extracts that inhibit the activity of termites intestinal enzymes, xylan-B, separated from chestnut trees, was selected as the substrate and 300 μg/ml was determined as the concentration of termites intestinal enzymes.

Fig. 2. Comparison of activity on substrates (cellulose, xylan) between xylanase (X1) and termite intestinal enzyme (T1) after protein quantification. Comparative analysis of termite intestinal enzyme and xylanase activity on substrate by measuring the same amount of protein content (300 μg/ml). Values are expressed as mean±standard deviation.

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In order to select medicinal plant extracts that inhibit termite enzyme activity, we explored 628 medicinal plants native in Korea, China, and Japan to find the material that the xylan-B and enzyme protein 300 μg/ml reaction result produced less reducing sugar than the control group, that is, the material that the inhibitory activity of the extract is high. As a result, Borneolum Syntheticum, Ephedra sinica, Menthol were evaluated to be high in inhibitory activity. As a result of treating the three screened extracts with concentrations (0.05, 0.1, 0.5 mg/ml), the activity inhibition rate of termite enzyme increased as the concentration increased. At 0.5 mg/ml concentration, the activity of termite intestinal enzymes was inhibited 16.06% with Borneolum Syntheticum extract (BSM), 17.73% with Ephedra sinica extract (ESM), and 25.66% with Menthol (MTM). (Fig. 3).

Fig. 3. Inhibition of activation of termite intestinal enzyme substrates by treatment of BSM ESM and MTM. The inhibition rate of termite intestinal enzyme activity was 0.5 mg/ml to 16.06% (BSM), 17.73% (ESM) and 25.66% (MTM) respectively. Values are expressed as mean±standard deviation. (BSM : Borneolum Syntheticum methanol extract, ESM : Ephedra sinica methanol extract, MTM : Menthol methanol extract).

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In order to measure the contact insecticidal concentration of Borneolum Syntheticum, Ephedra sinica, Menthol extracts, the dead number of insects by concentration was investigated. In the case of Borneolum Syntheticum extract, all termites died after 48 hours at a concentration of 10 mg/ml or more and in the case of Ephedra sinica extract at a concentration of 50 mg/ml. In particular, in the case of Menthol, they all died within 1 hour at a concentration of 1 mg/ml or more and the contact insecticidal effect is high-efficiency on termites even at a small concentration of it (Table 3).

In the feeding amount research conducted with 0.5 mg/ml concentration of extracts to measure the inhibition rate of feeding of the extracts of Borneolum Syntheticum, Ephedra sinica, Menthol, the feeding amount of termites was significantly more reduced in the group treated with the extracts than that in the control group (Fig. 4). In the case of Borneolum Syntheticum extract, it showed a feeding amount of about 73.0% compared to the control group, 72.2% for Ephedra sinica, and 67.4% for Menthol extract. It was inferred that each of the extracts contained components that inhibit the termite feeding activity by reducing the amount of feed by the three extracts. In the course of the termite feeding activity experiment, the termite feeding amount was greatly affected by the condition of the termite and the external environment. Therefore, it is important to maintain the environment so that they can be compared with each other by maintaining a constant temperature and humidity when studying the termite feeding amount (Zukowski AND Su, 2019).

Fig. 4. Termite filter paper intake was measured by treating BSM, ESM and MTM. After incubating the termites for 120 hours, the weight of the filter paper was measured to confirm the intake. As a result of confirming the intake of termites, 72.96% of BSM, 72.18% of ESM and 67.42% of MTM were consumed compared to control. Values are expressed as means ± standard deviation. (BSM : Borneolum Syntheticum methanol extract, ESM : Ephedra sinica methanol extract, MTM : Menthol methanol extract).

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One of the medicinal plant extracts selected in this study, Menthol, has already been reported. Menthol is a crystal obtained by refining and cooling Mentha arvensis. The constituents are reported to be Menthol (Shasany et al., 2000), and the activities of Menthol are reported to have insecticidal effects, antioxidant effects, and antibacterial effects against ticks (Karamaouna et al., 2013; Rozza et al., 2014; Talebi et al., 2014). Therefore, the composition analysis was performed on two substances except Menthol.

As a result of GC-MS analysis, the main components of each Borneolum Syntheticum, Ephedra sinica extracts were clearly identified. As the main components of Borneolum Syntheticum extract, Camphene, Isoborneol, 1-Methyl-4-(1-methylethyl)-1,3-cyclohexadiene, Borneol, (1S)-2,2-Dimethyl-3-methylene–bicyclo[2.2.1]heptane, Anthraquinone-2-carboxylic acid etc. were identified (Table 4).

Camphene is a substance extracted in the form of essence oil, and its antibacterial activity and insecticidal effect against beetles have been reported (Feng et al., 2020; Ibrahim et al., 2020; Sayout et al., 2020) and Isoborneol is known to have activity on a dry eye syndrome among diabetes complications (Ai et al., 2019). In the case of Borneol, it has been reported to have an antioxidant effect and an insecticidal effect on beetles as an aromatic substance present in herbs (Kasrati et al., 2015; Mühlbauer et al., 2003). In the case of 1-Methyl-4-(1-methylethyl)-1,3-cyclohexadiene, the insecticidal effect on the beetle has also been reported, and in the analysis results of Borneolum Syntheticum extract, the substances that reported the insecticidal effect on the beetle accounted for the majority.

As the main components of Ephedra sinica extract, 4-Methyl-1-Indanone, (Z)-9-Octadecenamide, 17-Penta triacontene, Phenmetrazine, 7-Methoxy-2H-1-benzo pyran- 2-one, Benzaldehyde, etc. were identified (Table 5).

Among the substances analyzed in Ephedra sinica, 4-Methyl-1-Indanone is known as a substance found in the essential oil part extracted from avocado seeds (Durak and Aysu, 2015), and in the case of (Z) -9- Octadecenamide, it has been reported to have a protective effect on nerve cells (Gugnani et al., 2018). 17-Pentatriacontene is primarily known as a substance found in plant root extracts and has been reported as an aromatic compound (Sen et al., 2017; Tayade et al., 2013).