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A Paulownia coreana Box for Storage of Annals of Joseon Dynasty: Its Efficacy and Functionality Evaluations of Temperature and Relative Humidity Control, and Microbe and Insect Repellent Activity

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT Paulownia coreana has various advantages as a convenient workability, aesthetic outlook, beautiful patterns, low weight and high strength, and its permeability. P. coreana has been widely used for storage but there are no empirical researches proving its functionality in a field of conservation science until now. In this study, the seasonal and daily temperature and relative humidity control, and microbe and insect repellent activity were evaluated under the controlled and uncontrolled circumstances from 2015 to 2016. The results showed to be mainly excellent in relative humidity control and the buffering effect was good to adjust the average daily relative humidity range from the outside. With respect to the antimicrobial properties of P. coreana, we observed that its water-soluble extract produced visible zones of inhibition against five bacteria. However, it was difficult to predict the antimicrobial and/or insecticidal properties.


INTRODUCTION
The storage boxes made with wood materials have been traditionally used to preserve the paper and textile objects for a long-term storage in not only Korea but also near countries as China and Japan. The wood has been used as a variety of household goods, furnitures, and architecture materials since ancient period due to its various advantages as convenient workability, aesthetic outlook and outstanding patterns, low weight and high strength, and its permeability. Following the analysis results of eighty-two wooden species from the furnitures dated from late Joseon Dynasty and possessed by the Seoul Museum of History, 7% of wooden species was identified as Paulownia spp. It is mainly used for inner drawers' material because of its lightweight, low-specific gravity and low strain rate (Song et al., 2010). Among them, P. coreana belongs to the family of Scrophulariaceae with a very clear ring and the colour is either light purple or pale rose pink with unclear distinction between the sapwood and heartwood. P. coreana is widely used in various appliances and furnitures because of its fast growth and low absorbency.
Following the published researches, it is known well that the air-dried gravity of P. coreana is low to 0.24 and also easy to control humidity (Lee et al., 2004). So it has been applied Research Article 256 | Journal of Conservation Science Vol.36, No.4, 2020 for making the boxes and furniture in storage and/or display spaces to preserve the small-sized artifacts such as costumes, papers, archives and paintings (Park, 2005;Chung et al., 2008;Cho, 2011).
Even though it is known well that it has peculiar characteristics such as low weight, little cracking and/or splitting, relative humidity control and insect repellent, an empirical researches has not been reported to probe its functionality of the material itself. In this study, we tried to verify empirically its functionality by evaluating the temperature and relative humidity control, and microbe and insect repellent activity of P. coreana box.

Temperature and Relative Humidity Control
Two comparative spaces were set up to identify the temperature and relative humidity control of P. and inner sealed P. coreana furniture as a close space ( Figure 1). Also, 5 locations were chosen to identify its self-regulating control under uncontrolled circumstances without any thermohygrostat equipment at the Anguk-dong detached palace: inner newly-fabricated P. coreana and P. menziesii wooden box located in room, inner room, inner newly-fabricated P.
coreana box located outdoor, and the outside (Figure 2).

Active ingredient extraction from wood materials
Prior to evaluating the microbe and insect repellent activity of P. coreana, active ingredient was extracted from wood materials and P. menziesii was used as a control group.  Before extraction process, the wood materials (sap wood) were sufficiently dried, cut into 1 to 3 cm, and then powdered up into 700 meshes with a universal cutting mill (PULVERISETTE 19,FRITSCH,DEU) for an experiment.
The extracting process was divided into water-soluble extraction and volatile extraction to extract the active ingredient (Jeong et al., 2018). The water-soluble extraction was made by mixing 100 g of dried wood powder and 1000 mL of distilled water at 30℃ for 72 hours. Then, the mixture of dried powers and extract was separated from the filter and volatile condensation was conducted with evaporator under the 50 to 70℃. For volatile extraction, 100 g of wood powder was distilled into 125 mL of dichloromethane (CH 2 Cl 2 ) that had been re-extruded from the SDE device. And then sodium anhydrous sulfate (Na 2 SO 4 ) was added into the extract to remove the moisture and then condensed with N 2 gas. coreana furniture as a close area. The collected microbe was cultured in a 28℃ incubator for 3 to 5 days; and after extracted DNA was analyzed with PCR and Automatic DNA sequencer, BlastN search was conducted at the genebank (NCBI, National Center for Biotechnology Information).

Microbe repellent activity and antimicrobial activity test
To evaluate the microbe repellent activity test, 150 µl of a microbial suspension was inoculated on PDA and NA medium of which P. coreana samples (20 × 20 × 15 mm) were placed in middle and cultured to confirm the inhibition zone of the microbe formed around it. To assess the antimicrobial activity test with extract, 150 µl of a microbial suspension was applied soaking on a paper disc and cultured in 28℃ to confirm the inhibition growth formed around the paper disc.

Insect repellent activity test
Two typical species of insects were provided and used at the Institute of Preventive Conservation for Cultural Property, Korea National University of Cultural Heritage for insect repellent activity test: Reticulitermes speratus kyushuensis and Sitophilus oryzae. R. speratus kyushuensis was known as one of the main pests that have damaged the wooden cultural heritage and S. oryzae was often used for judging the fumigation effect in a field of conservation science. R. speratus kyushuensis was collected from Hapjeong-ri, Buyeo on April of 2016 and only repletes were used after breeding at room temperature and RH 90±5%. S. oryzae was selected without distinction of their gender at 25±3℃ and RH 75±5% at a laboratory.
In this study, the insect repellent activity tests were divided into contact toxicity, fumigation toxicity and insect repellent tests. 0.5 µl of extract was directly contacted to the thorax of 40 insects to identify whether they are confronted by death for the contact toxicity test. A close container method was applied for fumigation toxicity test; a filter paper soaking 100 µl of extract was put in the bottom of lower plastic container (65 × 65 × 100 mm) and then 40 insects and their small amount of their food (R. speratus kyushuensis: 2 × 2 cm filter paper, S. oryzae: grains of rice) were put together to the upper container; test was conducted at RH 90±5% for 72 hours with 24 hours intervals with 3 times repeats. In a T-shaped container, both treated area with water-soluble and volatile extracts and untreated area with distilled water and dichloromethane were placed on both sides. And then 40 insects were placed in the middled of container for the repellent tests. The absorption process with extracts upon the treated and untreated area was carried out in same way with a fumigation toxicity test. The repellent test was conducted on water-soluble and volatile extract of P. coreana in the dark room for 24 hours with 3 repeats.

Temperature and relative humidity control
A national storage room of the Kyujanggak Institute for Korean Studies showed little monthly average temperature difference because the temperature and relative humidity have 258 | Journal of Conservation Science Vol.36, No.4, 2020 kept constant with thermo-hygrostat equipment from October of 2015 to September of 2016. The monthly average temperature ranged minimum 19.0℃ to maximum 21.3℃; and monthly average relative humidity was in range of 52.6% to 60.7% inside the controlled storage room. There was little temperature difference depending on its locations from uncontrolled traditional space without any thermo-hygrostat systems at Anguk-dong detached palace and it indicates the outside circumstances influenced the monthly average temperature of the indoor and outdoor spaces (Figure 3).
Measuring the monthly average relative humidity for 4 seasons under uncontrolled spaces, inner P. coreana boxes exposed to the outside followed the relative humidity of the outside. They are measured higher in fall season (77.7%) than hot and humid summer season (72.7%) and the lowest was in spring season (61.0%). And under the controlled circumstances, there was no significant difference between the old P. coreana box and the newly fabricated P. coreana and P. menziesii box (Figure 4).
The monthly average daily temperature range of the outside was the highest in spring (March) and lowest in fall season (September). On spring of 2016, the average daily temperature range was 13.5℃ in outside and 11.4℃ in the inner P. coreana boxes exposed to the outside; that of inner P. coreana boxes installed in the uncontrolled room was 9.8℃; and those of inner P. coreana furniture and boxes installed in a controlled storage room ranged from 0.5℃ to 0.9℃. It indicates that this was based on the outside weather (13.5℃)   (3.7% of average daily temperature range) was confirmed to 0.5% lower than that of the P. menziesii storage box (3.2%).
In March, the humidity control capacity was increased under the controlled storage in the order of the newly fabricated P. coreana box (1.0%), new P. menziesii box (0.9%) and old P. coreana box (0.7%). In the spring season when the daily relative humidity range was the highest, buffering capacity for a daily humidity range showed 41.0% of the inner newly-fabricated P. coreana box installed in the outside as an uncontrolled space while daily relative humidity variability of the outdoor was 48.6%; and those of inner newlyfabricated P. coreana and P. menziesii box in an uncontrolled room were 43.6% and 43.9%. And the humidity control capacity of the P. coreana corridor where the indoor air moved inside the controlled storage was 0.6% lower than inner P. coreana furniture or box in a controlled storage room during the summer season of 2016 ( Figure 6).

Microbe repellent activity and antimicrobial activity tests
Fungi was inoculated in a PDA medium and bacteria was conducted in a NA (Nutrient Agar) for 3 to 5 days in 28℃ incubator until the microbial growth was visible to the naked eye. Through morphological separation, airborne microorganisms were isolated into 4 fungi and 5 bacteria, and the genetic analysis showed that the analyzed species were Phaeosphaeriaceae sp., Staphylococcus aureus and so on ( Table 1).
The microbe repellent activity test was conducted to evaluate that the extracted materials can control the growth of microorganisms that target the surface wood, leading to contamination and/or discoloration. The result of the microbe repellent activity confirms that P. coreana and P. menziesii    Antimicrobial activity tests were performed to confirm whether the wood extract inhibited the growth of microorganisms. In case of the fungi tested, the water-soluble extracts of P. coreana and P. menziesii did not produce clear zones of growth inhibition, indicating that these extracts do not have any antifungal properties. Meanwhile, the antibacterial activity tests revealed growth inhibition of five bacteria by the water-soluble extract of P. coreana, with clear zones ranging from 0.05 to 0.5 cm around the paper disc containing the extract (Figure 8). However, no antibacterial activity was observed for the water-soluble extract of P. menziesii. Notably, the volatile extracts from P. coreana and P. menziesii, including dichloromethane, did not produce growth inhibition zones in the antimicrobial activity tests. Thus, based on the antifungal and antibacterial activity tests, it was concluded that the volatile extracts from both P. coreana and P.
menziesii do not affect the growth of fungi and bacteria ( Figure 9, Table 2). Moreover, we determined that antifungal substances were not present in these extracts.

Insect repellent activity
In the contact toxicity test, it was clear that R. speratus kyushuensis and S. oryzae were not identified toward the water-soluble extract whereas a large number of insects were identified with volatile extracts. This seems to be influenced by the solvent rather than the extract components since there is no significant difference from a control group, dichloromethane itself. The contact toxicity with both R. speratus kyushuensis and S. oryzae was equally ineffective in watersoluble extracts, and the effect from the volatile extracts was confirmed to be due to extracting solvent ( Figure 10).
Monitoring for 3 days with fumigation toxicity tests of water-soluble extracts, R. speratus kyushuensis was active after 3 days and S. oryzae was reduced to average 3.2 insects after 3 days. This was confirmed that the numbers of both Dermacoccus sp.
---+ + ----   R. speratus kyushuensis and S. oryzae were reduced by natural death, not by extracts; volatile extract is also considered to have no fumigation toxicity effect due to the small numbers of dead insects ( Figure 11). Also, both R. speratus kyushuensis and S. oryzae freely moved toward both areas where the water-soluble and volatile extracts were absorbed and control areas with distilled water or dichloromethane in the repellent test. Therefore, these extracts from P. coreana and P. menziesii do not have any insect repellent so R.
speratus kyushuensis and S. oryzae do not avoid the extracts.

CONCLUSION
The temperature and relative humidity control of P. However, growth inhibition sized to 0.05 to 0.5 cm was observed around the paper disc applying with the watersoluble extract of P. coreana toward 5 bacteria; in particular, JB3 and JB5 showed growth inhibition zones so that this indicates water-soluble extract of P. coreana had a weak antibacterial activity.
The water-soluble extract of P. coreana wasn't effective to 2 types of insects as R. speratus kyushuensis and S. oryzae in the contact toxicity test; and water-soluble and volatile extracts also could not affect the fumigation effect to insects in the fumigation toxicity test. And insects can approach freely toward the material itself extracted from P. coreana without any repellent in a repellent test. Following the published research, it coincides the conventional heritage storage box material, P. coreana, was not good enough for antimicrobial property (Chung et al., 2008). Additionally, despite its customary use, it reported there are many disadvantages on electromagnetic wave blocking, gas absorption, alkali resistance, low strength for storing heavy materials, weather resistance and gas adsorption (National Research Institute of Cultural Heritage, 2009).
Summarizing these results, it can be judged that P.
coreana does not prevent the biological deterioration when the pest comes into contact and/or attempted to attack it.
Based on the integrated results, we cannot expect P. coreana boxes, which are widely used for the preservation of heritage materials, to have insecticidal or antibacterial properties. So we are planning to study further on the correlation between humidity and microbial growth in the future.