Biological Activities in Sapwood and Heartwood Extractives from Paulownia tomentosa

Abstract

Paulownia tomentosa is a representative deciduous tree in South Korea. After 10 years of growth, its wood can be used to make various products through chemical modifications, such as impregnation with a polymer, substitution with chemicals, and physical compression. However, research on the biological resistance of the sapwood and heartwood parts of P. tomentosa xylem is lacking. To ensure the complete utilisation of Paulownia wood, this study aimed to collect baseline data on the necessity of xylem extraction before chemical modification to enhance wood porosity. First, we assessed the decay and termite resistance of sapwood and heartwood blocks. Furthermore, we evaluated the anti-microbial effect of sapwood and heartwood extracts after solvent fractionation. Quantitative and qualitative analyses of the active substances of the fractions with anti-microbial activity were also conducted. The hexane and chloroform solvent fractions of sapwood and heartwood extracts showed fungal resistance against Trametes versicolor and Fomiptosis palustis. Paulownin and sesamin were the main compounds showing anti-microbial activity, and their content in the extracts varied, depending on the wood part. These results provide valuable data for advancing research on porous wood materials and the utilisation of xylem-derived active compounds from Paulownia wood.

1. Introduction

Wood, as a bio-based polymer, is of major importance due to its carbon storage capacity. While wood has the advantage of being an eco-friendly renewable resource, its greater susceptibility to deterioration and lower dimensional stability, compared with materials such as concrete and plastic, are a major disadvantages [1]. Under high humidity conditions in outdoor environments, wood may undergo decay, i.e., decomposition by bio-organisms. Additionally, in dry environments, wood is prone to damage caused by insects, such as termites [2]. Wood comprises cellulose, hemicellulose, and lignin, which constitute the cell walls and serve as major nutrient sources for bio-organisms [3]. White-rot fungi, which are wood-rotting fungi, can decompose all types of polymers found in the cell walls, while brown-rot fungi primarily utilise polysaccharides as their main nutrient source [4,5]. To enhance the durability of wood and prevent damage from bio-organisms, studies have explored various methods, including surface treatment, preservative injection, substitution of functional groups, and impregnation with polymers [6,7,8].

Recently, the balsa tree (Ochroma pyramidale), known for its low specific gravity, has found widespread application in the production of novel functional materials through modification processes [9,10]. Similar to the balsa tree, the empress tree (Paulownia tomentosa) also exhibits a low specific gravity and holds promise for various applications. Consequently, several researchers are exploring alternative tree species [11,12,13]. P. tomentosa is a fast-growing tree species known for its notably low dry specific gravity (~0.25) [14,15]. Mainly distributed in Asia, it has long been a representative species in South Korea, used in the manufacture of furniture and musical instruments [16].

In addition, P. tomentosa contains lignans, including paulownin, sesamin, and β-sitosterol, and is known to be resistant to insects and xylem-rotting fungi [17,18,19]. However, research on the biological resistance of the sapwood and heartwood parts of P. tomentosa xylem is lacking.

Therefore, the present study first differentiated P. tomentosa into sapwood and heartwood. Subsequently, a decay resistance test was conducted, considering the presence or absence of each extracted component to evaluate biotic resistance. Furthermore, data on the extractable components of P. tomentosa regarding microbial inhibition and active compounds were collected. To establish basic data, the active compounds were qualitatively and quantitatively analysed and radical scavenging activities were evaluated to establish the efficient utilisation of P. tomentosa wood.

2. Materials and Methods

2.1. Materials

P. tomentosa wood (approximately 10 years old) was obtained from the Research Forest of Kangwon National University, located in Hongcheon-gun, Gangwon-do, Republic of Korea. To differentiate between the sapwood and heartwood in the xylem, a 3 cm thick section cut from the surface, excluding the bark, was used as sapwood, and a section with a 5 cm radius on the interior, excluding the pith, was used as heartwood.

2.2. Decay Resistance Test

2.2.1. Sample Preparation

Overall, 32 P. tomentosa specimens were prepared (16 sapwood and 16 heartwood), with each cut to a size of 25 × 25 × 9 mm. The specimen for extraction was used in the test 18 h after the 6 h extraction in 60% ethanol, followed by 48 h of drying at 25 ± 2 °C and 24 h of oven-drying at 60 ± 2 °C. The samples containing extractives were considered controls, while those without extractives were considered extractive-free.

2.2.2. Fungal Culture

The microorganisms used in the decay resistance test included brown-rot fungi Fomitopsis palustris (FOM) and white-rot fungi Trametes versicolor (TRA) obtained from the National Institute of Forest Science. After 5 days of culturing in a Petri dish, the outermost part of the mycelia was collected and placed in culture medium containing 25 g glucose, 5 g peptone, 10 g malt extract, 3 g phosphoric acid, 3 g potassium hydroxide, and 2 g magnesium sulphate (Daejung, Seoul, Republic of Korea) for 1-week culture in an incubator at 26 °C.

2.2.3. Decay Resistance Test

To conduct the decay resistance test on P. tomentosa wood, the Korean Industrial Standard KS F 2213 [20] was used with some modifications to the decay periods. Briefly, 250 g of silica sand was placed in a culture bottle, and 80 mL culture solution (25 g glucose, 5 g peptone, 10 g malt extract, 3 g phosphoric acid, 3 g potassium hydroxide, and 2 g magnesium sulphate dissolved in distilled water to 1000 mL) was added. Subsequently, the bottle was autoclaved at 121 °C for 30 min. After 1 week, 3 mL microbial culture solution was added to sand and cultured in an incubator. After 2 weeks, the controls and extractive-free P. tomentosa specimens, which were sterilised at 121 °C for 30 min, were added to the culture medium to start the decay experiment. The decay resistance test lasted 10 weeks at 26 °C, and, at the end of the experiment, the following calculation was conducted:

Weight loss rate after decay (%) = (W₁ − W₂)/W₁ × 100

(1)

where W₁ is the weight of the oven-dried sample before rotting and W₂ is the weight of the sample after decay.

2.2.4. Scanning Electronic Microscopy

To examine the cell walls of the control and extractive-free P. tomentosa specimens, the specimens were cut to 5 mm × 5 mm × 5 mm and coated with platinum. The cross-, radial, and tangential sections were comparatively analysed using scanning electron microscopy (SEM) (SUPRA55VP; Carl Zeiss AG, Oberkochen, Germany).

2.3. Termite Resistance Test

The specimens for the termite resistance test were prepared in the same manner as those for the decay resistance test. The termite breeding container was an acryl cylinder with 8 cm width, 6 cm length, and a bottom closed using ~5 mm thick dental gypsum. Ten to fifteen cylinders were placed in advance in a box with a lid and a ~2 cm thick wet pad. The wood specimens were vertically placed on the top of the gypsum plaster in the container, and, after supplying 200 worker and 20 soldier termites (Reticulitermes speratus kyushuensis), the resistance test was performed for 21 days. After 21 days of termite breeding, the specimens were dried at 60 ± 2 °C to a constant weight, and the weight was measured to a precision of 0.01 g. The number of dead termites was counted every 2 days of breeding, and, at the end of the experiment, the rate of weight loss of the treated specimens and five untreated specimens was estimated as follows:

Weight loss rate (%) = (W₁ − W₂)/W₁ × 100

(2)

where W₁ is the sample weight before the termite resistance test (g), and W₂ is the sample weight after the test (g).

$$\mathrm{A}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{m}\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e} (\%)=\frac{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{e}\mathrm{a}\mathrm{d} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s}}{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l} \mathrm{n}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{i}\mathrm{n}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{s}}\times 100$$

(3)

2.4. Anti-Fungal and Anti-Oxidant Activities

2.4.1. Preparation of Crude Extract and Solvent Fractionation

To obtain the crude extract, the sapwood and heartwood parts of P. tomentosa were ground using 0.425 mm-sized mesh to prepare the wood samples. Extraction was conducted using a solution of ethanol:distilled water (6:4, v/v) with 48 h of immersion at 25 ± 2 °C. The resulting extract was filtered, and the solvent was completely vaporised in a vacuum evaporator (Eyela, Tokyo, Japan), after which the extract was freeze-dried. Each freeze-dried extract of sapwood and heartwood was dissolved in distilled water for fractionation in hexane solvent, and the undissolved distilled water layer in hexane was used for the fractionation in chloroform in ethyl acetate. Each fraction was concentrated in a vacuum evaporator and freeze-dried to prepare the fraction powders.

2.4.2. Thin-Layer Chromatography

To examine the distribution of compounds in the solvent fractions from the crude extract, thin-layer chromatography (TLC) was performed using a silica TLC plate (Merck, Darmstadt, Germany) as the stationary phase. A solution of chloroform:methanol (3:1, v/v) was prepared as the developing solvent, and, after development, the spots in each ultraviolet (UV) area (254 and 365 nm) on the TLC plate were identified.

2.4.3. Anti-Fungal Activity

To evaluate the anti-microbial effects of the crude extract and solvent fractions obtained from the sapwood and heartwood of P. tomentosa wood, wood decay fungi (TRA and FOM) previously used in the decay resistance test were employed. Additionally, mould—Penicillium glabrum, Trichoderma harzianum, and Aspergillus niger—were included in the study.

For the anti-fungal activities against wood decay fungi, the paper disc diffusion method of Lim et al. [21,22] was used, with some modifications. A paper disc (Ø 8 mm; Advantec MFS, Inc., Dublin, CA, USA) was placed on potato dextrose agar (Daejung, Republic of Korea), and the crude extract and solvent fractions at varying concentrations (100,000, 50,000, and 10,000 ppm) were applied to the paper disc for absorption. After 7 days of culturing the wood-rotting fungi, a cork borer (Ø 8 mm) prepared to a size similar to the paper disc size was placed on the disc. As a control, dimethyl sulfoxide was added to the paper disc. After 4 days of culturing in an incubator, mycelial growth was examined.

To determine anti-fungal activity against mould, agar well diffusion was used. Mould samples (P. glabrum, T. harzianum, and A. niger) were spread on the potato dextrose agar, and a well was formed using a cork borer (Ø 8 mm), into which the crude extract and solvent fractions at varying concentrations (100,000, 50,000, and 10,000 ppm) were added. After 4 days of culturing, growth inhibition rings were examined.

2.4.4. Anti-Oxidant Activity

• 2,2-Diphenyl-1-picrylhydrazyl(DPPH) radical-scavenging activity

To test DPPH radical scavenging activity, a 0.2-mM DDPH reagent was prepared using 0.04 g DDPH in 500 mL MeOH. The crude extract and the hexane, chloroform, ethyl acetate, and water fractions were prepared to concentrations of 31.25, 62.5, 125, 250, 500, and 1000 ppm. Thereafter, 20 μL of each sample and 180 μL of the reagent were added to a 96-well plate, and, after 30 min of reaction in the dark, UV (517 nm) measurements were taken.

2.

2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity

To test ABTS radical scavenging activity, a solution of 7.0 mM ABTS and 2.45 mM potassium persulfate was prepared in a 1:1 ratio and left to react in the dark for 12–16 h. The crude extract and the hexane, chloroform, ethyl acetate, and water fractions were prepared to the same concentrations as those for DPPH radical scavenging activity. Further, 20 μL of each sample and 180 μL of the reagent were added to a 96-well plate, and, after 15 min of reaction in the dark, UV measurements were performed.

2.4.5. Quantitative and Qualitative Analyses

Gas chromatography/mass spectroscopy (GC/MS) was performed using a GC 7890/5975C System (Agilent Technologies, Santa Clara, CA, USA) to analyse the compounds in the crude extract of the sapwood and heartwood of P. tomentosa. The column used was a DB-5 ms column (Agilent Technologies), and the temperatures at the ion source and transfer line were maintained at 200 and 275 °C, respectively. The column oven temperature was maintained at 200 °C for 2 min and was later increased at a rate of 10 °C/min to 300 °C, which was maintained for 10 min. The carrier gas was He, which was supplied at a flow rate of 35 cm/s. The GC injection rate and split ratio were 1 μL and 10:1, respectively.

For GC analysis, to construct the calibration curves for the main compounds, the reference compounds sesamin and paulownin (Sigma-Aldrich, St. Louis, MO, USA) were prepared to varying concentrations (12.5–200 μg/mL). Crude extracts of the sapwood and heartwood of P. tomentosa were dissolved in methanol, and the hexane and chloroform fractions were dissolved in chloroform to a concentration of 125 μg/mL and then analysed by GC. The GC equipment employed a flame ionisation detector and a gas chromatograph (Agilent 6890; Agilent Technologies) equipped with an autosampler; the GC column used an HP-5 fused silica capillary (30 m 0.25 mm 0.25 μm; Agilent Technologies). The temperature was maintained at 200 °C for 2 min and then raised at a rate of 10 °C per min to 300 °C and maintained for 10 min. Nitrogen gas was used as the carrier gas at a flow rate of 25 cm/s. The GC injection volume was 1 μL, and the split ratio was 10:1.

2.5. Statistical Analysis

Significant differences in the control/extracted specimens between sapwood and heartwood were statistically examined using ANOVA followed by Duncan’s multiple range test. The statistical analyses were performed using SPSS software (SPSS ver. 29; SPSS Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Decay Resistance Test

3.1.1. Weight Loss

Visual examination revealed that the extracted specimens were more susceptible to decay (Figure 1). The rate of weight loss was higher in the extracted sapwood and heartwood specimens than in the control (

Table 1. Mass loss rate (%) after decay resistance test from P. tomentosa.

	Fomitopsis palustris	Trametes versicolor
Control—heartwood	28.77 ± 3.25	12.37 ± 3.64 *
Control—sapwood	16.63 ± 1.54 *	31.75 ± 6.26
Extracted—heartwood	44.93 ± 16.66	31.52 ± 2.65 *
Extracted—sapwood	53.54 ± 4.53 *	35.51 ± 10.58

* indicates statistical significance at p < 0.05.

). The weight loss rate against TRA was 31.8 and 12.4% for the sapwood and heartwood control specimens, respectively. According to the decay grades in

Table 2. KS F 2213 grades for the decay resistance test.

Average Weight Loss (%)	Class of Resistance
0–10	Highly resistant
11–24	Resistant
25–44	Moderately resistant
>45	Slightly resistant or nonresistant

, the sapwood control was moderately resistant to decay, while the heartwood control was resistant to decay. The weight loss rate against FOM was 16.6 and 28.8% for the sapwood and heartwood control specimens, respectively; according to Table 1, the sapwood and heartwood controls were resistant and moderately resistant, respectively.

The high weight loss rates of the extracted P. tomentosa specimens due to FOM and TRA are consistent with the findings of Kirker, who conducted experiments without distinguishing between sapwood and heartwood [23]. This suggests that the P. tomentosa wood extract contained a compound that exhibited decay resistance.

In the case of FOM, the sapwood control specimen showed a lower weight loss rate than that of the heartwood control specimen. Extractable components of wood are generally found in the heartwood, while sapwood is more prone to wood decay. However, the results of this study indicated a higher level of decay in the heartwood.

3.1.2. Cell Wall Changes

SEM images of the specimens at the end of the decay resistance test are shown in Figure 2, Figure 3 and Figure 4. The cell walls in the cross-, radial, and tangential sections of the decayed specimens varied significantly from those of the control specimens before decay. Mycelia were detected in the cross sections of the decayed specimens, confirming traces of decay on the uneven cell wall surface.

The three specimen sections decayed by FOM showed a higher level of damage on the cell walls due to the removal of extracts. In the radial section, in particular, large pores were observed on the walls of the extractive-removed specimens (Figure 3e,f). Further, the cell walls in the heartwood were thinner than those in the sapwood and deteriorated more.

TRA, which decomposes sugars and lignins, caused bore holes in the radial sections of decayed heartwood specimens, which are characteristic of decayed woods (Figure 3g,h) [24]. The cross-section of the extracted sapwood specimens also showed extreme damage on the cell walls (Figure 2j).

3.2. Termite Resistance Test

3.2.1. Weight Loss

A termite resistance test was performed on the extracted and non-extracted P. tomentosa sapwood and heartwood specimens, and the corresponding results are shown in Figure 5 and

Table 3. Mass loss rate and mortality (%) of P. tomentosa after the termite resistance test.

	Weight Loss Rate	Mortality
Control—heartwood	12.6 ± 2.27 *	16.3 ± 6.17 *
Control—sapwood	9.38 ± 0.80 *	24 ± 1.77 *
Extracted—heartwood	45.95 ± 4.17 *	2.10 ± 3.13 *
Extracted—sapwood	31.00 ± 1.27 *	6.20 ± 2.91 *

* indicates statistical significance at p < 0.05.

. Kirker found no weight loss in P. tomentosa wood, while Kim reported a weight loss rate of 5.8% [23,25]. The weight loss rates estimated in the current study demonstrated that the sapwood control (~9.38%) was less prone to damage than the heartwood control (~12.6%). According to the JIS K 1571 (2004) [26], a weight loss rate of ≥3% caused by termites indicates ‘Not resistant’ and a rate of <3% indicates ‘Resistant’; accordingly, the P. tomentosa control specimens in the present study could be classified as ‘Not resistant’.

Additionally, compared with the control specimens, the extracted specimens showed a higher weight loss rate similar to that in the decay resistance test, thus highlighting the importance of identifying the extractable components. Similar to the decay resistance test, heartwood exhibited a higher weight loss rate despite the presence of extractable components in the termite test.

3.2.2. Mortality

In the present study, despite comparatively low mortality, termite-related mortality was higher in the control specimens than that in the extracted specimens of both P. tomentosa sapwood and heartwood. This indicated an insecticidal effect of a compound in P. tomentosa wood. Notably, the mortality was higher in the sapwood control specimen (~24%) than that in the heartwood control specimen (~16.3%). Heartwood is generally known to contain higher contents of extractable components compared with sapwood; moreover, heartwood has higher contents of phenolic compounds that protect against microorganisms [27,28]. Kim [25] reported a mortality rate of 96% for control wood specimens, while Lee [29] reported a considerably high mortality of P. coreana wood (86%). Kim reported that termites mostly preferred sapwood [5]. However, contrasting results were observed in the present study; thus, the extracts acquired from sapwood and heartwood were further analysed.

3.3. Evaluation of Biological Activities

3.3.1. Crude Extract and Solvent Fraction Yield

Table 4. Yield of solvent fractions from Paulownia tomentosa (%).

	Sapwood	Heartwood
Crude (Total extractives)	8.43 ± 0.05	7.59 ± 0.63
Hexane	4.03 ± 0.02	2.80 ± 0.36
Chloroform	15.57 ± 1.70	12.92 ± 3.51
Ethyl acetate	11.61 ± 0.92	9.74 ± 1.56
Water	50.68 ± 2.09	58.70 ± 6.68
Residue *	18.12	15.85

* The yields (%) of residues were calculated as: 100 − (Hexane + Chloroform + Ethyl acetate + Water).

presents crude extract and solvent fraction yields from P. tomentosa sapwood and heartwood specimens. P. tomentosa crude extract yields in the sapwood and heartwood were approximately 8.43 and 7.59%, respectively. Esteves et al. reported a total content yield of 7.36% of extractable components in P. tomentosa, which was similar to the yield obtained in this study [30].

The yield of the P. tomentosa solvent fractions was in the order of water > chloroform > ethyl acetate > hexane, at 50.68% ± 2.09%, 15.57% ± 1.70%, 11.61% ± 0.92%, and 4.03% ± 0.02%, respectively, in sapwood, and 58.70% ± 6.68%, 12.92% ± 3.51%, 9.74% ± 1.56%, and 2.80% ± 0.36%, respectively, in heartwood.

3.3.2. Extract Distribution Using TLC

Figure 6 presents the TLC results for the crude extract and solvent fractions from P. tomentosa sapwood and heartwood specimens. In the presence of the chloroform:methanol developing solvent, the compounds that dissolved in hexane and chloroform migrated to the top of the TLC profile, indicating the distribution of non-polar compounds with the highest density. Various compounds were distributed in the same developing solvent in a belt-like form for the ethyl acetate and water fractions. Notably, high amounts of compounds were found at the bottom of the TLC profile without development. No significant variation between sapwood and heartwood was observed.

3.3.3. Anti-Fungal Activity

Table 5. Diameter (mm) of the mycelium in anti-fungal test results for the crude extracts and solvent fractions obtained from the sapwood and heartwood of Paulownia tomentosa against Trametes versicolor and Fomitopsis palustris (values ≤ 30 are displayed in bold).

	Concentration (ppm)	T. versicolor		F. palustris
		Sapwood	Heartwood	Sapwood	Heartwood
Crude	0	40	40	45	35
	10,000	40	40	40	35
	50,000	35	40	40	35
	100,000	25	40	35	35
Hexane	0	40	45	40	35
	10,000	40	38	35	30
	50,000	30	30	25	30
	100,000	25	20	25	18
Chloroform	0	40	40	45	40
	10,000	35	38	35	38
	50,000	30	30	30	30
	100,000	20	26	26	25
Ethyl acetate	0	40	45	45	40
	10,000	40	45	45	40
	50,000	35	40	45	40
	100,000	35	30	40	35
Water	0	35	40	35	40
	10,000	35	40	35	40
	50,000	35	40	35	40
	100,000	35	40	35	40

presents the results of the decay resistance test using the test strains FOM and TRA for evaluating the anti-microbial effects in varying concentrations of the P. tomentosa crude extract and solvent fractions. Although the observed growth inhibition effect was not absolute, both FOM and TRA showed inhibited growth in response to the crude extract, as well as the hexane and chloroform fractions from sapwood and heartwood. Li et al. [21] reported a substantial anti-fungal effect of the chloroform extract of Cinnamomum camphora on Coriolus versicolor. Thus, it is surmised that an active compound in the extract inhibited the growth of FOM and TRA using a non-polar solvent.

Table 6. Measurement of inhibition zone diameter (mm) from the anti-fungal test results of mould for crude extract and solvent fractions from the sapwood and heartwood of P. tomentosa (values ≥ 20 are displayed in bold).

	Concentration (ppm)	T. harzianum		A. niger		P. glabrum
		Sapwood	Heartwood	Sapwood	Heartwood	Sapwood	Heartwood
Crude	0	0	0	0	0	0	0
	10,000	0	0	0	0	0	13
	50,000	0	15	0	0	0	15
	100,000	0	20	0	0	0	15
Hexane	0	0	0	0	0	0	0
	10,000	30	20	11	0	15	13
	50,000	30	30	11	0	20	15
	100,000	30	30	11	0	20	20
Chloroform	0	0	0	0	0	0	0
	10,000	25	25	0	0	15	14
	50,000	30	30	0	0	15	15
	100,000	30	30	0	0	20	20
Ethyl acetate	0	0	0	0	0	0	0
	10,000	0	0	11	0	0	0
	50,000	0	0	11	0	0	0
	100,000	0	0	11	0	0	0
Water	0	0	0	0	0	0	0
	10,000	0	0	0	0	0	0
	50,000	0	0	0	0	0	0
	100,000	0	0	0	0	0	0

presents the results using the three surface contamination species. For P. glabrum, growth-inhibition rings were observed with the crude extract and the hexane and chloroform fractions. For T. harzianum, growth-inhibition rings were observed with the hexane and chloroform fractions. In contrast, for A. niger, no distinct growth-inhibition rings were detected with the crude extract or the solvent fractions. Tabassam et al. [31] reported high anti-fungal activity in the hexane extract of Picrorhiza kurroa root against Aspergillus flavus and Fusarium oxyporum. Jameel et al. [32] also showed that the hexane extract of Capparis decidua had the largest anti-fungal effect. Based on these findings, the hexane and chloroform fractions were predicted to contain compounds that exhibited anti-microbial effects, and the potential active compounds from the TLC results were qualitatively and quantitatively analysed.

3.3.4. Anti-Oxidant Activities

Table 7. Anti-oxidant activity of Paulownia tomentosa crude extract and its fractions.

Sample		IC50 * (µg/mL)
		DPPH Radical-Scavenging Activity	ABTS Radical-Scavenging Activity
P. tomentosa Heartwood	Crude	37.84	18.00
	Hexane	130.95	507.43
	Chloroform	95.63	30.42
	Ethyl acetate	25.39	8.61
	Water	27.83	17.24
P. tomentosa Sapwood	Crude	55.78	15.36
	Hexane	90.42	418.43
	Chloroform	86.19	17.63
	Ethyl acetate	24.93	8.11
	Water	42.09	15.69
Ascorbic acid (Standard compounds)		6.01	5.78

* IC₅₀: a measure of the potency of a substance in inhibiting a specific biological or biochemical function.

presents the evaluation results for DPPH and ABTS radical-scavenging activities for the investigation of the anti-oxidant activity of P. tomentosa wood extracts.

For DPPH free-radical scavenging, the highest activity was found in the ethyl acetate fractions of P. tomentosa sapwood and heartwood (25.39 and 24.93 µg/mL, respectively). The activity was in the following order: water > crude > chloroform > hexane. Si et al. [33] reported that the IC₅₀ of the ethyl acetate fractions of P. coreana was ~16 µg/mL, while Meng et al. [34] reported an IC₅₀ of 71.35 µg/mL for the 60% ethanol fractions of P. tomentosa flowers.

Similarly, for ABTS free-radical scavenging, the highest activity was found in the ethyl acetate fractions of P. tomentosa sapwood and heartwood (8.61 and 8.11 µg/mL, respectively). The water fraction and crude extract showed a similar level of activity, while the hexane fraction displayed a markedly low level.

These findings suggest the potential utility of the ethyl acetate and water fractions and crude extract. However, the IC₅₀ was rather low compared with that for the ascorbic acid used as the control.

3.3.5. Quantitative and Qualitative Analyses

The crude extract and the hexane and chloroform fractions that demonstrated a growth inhibition effect on the test strains in the evaluation of anti-microbial effects were analysed through GC/MS. As shown in Figure 7, the analysis results of the crude extract from sapwood and heartwood dissolved in the methanol solvent revealed two distinct peaks. Based on the MS and reference compound analyses, the compounds detected at 17.5 and 19.4 min were identified as sesamin and paulownin, respectively (Figure 8).

When the quantified values of the crude extract and the hexane and chloroform fractions were applied to the calibration curves constructed in advance for two reference compounds, the sesamin content, as the representative lignan in P. tomentosa, was the highest in the hexane fraction for both sapwood and heartwood, followed by the chloroform fraction. The paulownin content, contrastingly, was similar in the hexane and chloroform fractions for both sapwood and heartwood and was approximately two-fold higher than the content in the crude extract.

Table 8. Qualitative analysis of the crude extract and the hexane and chloroform fractions of P. tomentosa using gas chromatography.

Part	Sample	Sesamin	Paulownin
		Content (µg/mL)	Content (µg/mL)
Sapwood	Crude	13.94	25.71
	Hexane	37.60	63.69
	Chloroform	17.96	68.23
Heartwood	Crude	13.00	25.68
	Hexane	44.92	66.71
	Chloroform	19.20	62.17

presents the quantitative analysis results of the two active compounds in the crude extract and the hexane and chloroform fractions that demonstrated an anti-microbial effect.

Sesamin can reduce the survival of the caterpillar Anticarsia gemmatalis, while paulownin exhibits an anti-fungal effect against T. versicolor [35]. These findings suggest that the reduced microbial growth induced by the hexane and chloroform fractions in the previous anti-microbial test could be attributed to the paulownin and sesamin contents. Therefore, it is necessary to evaluate the growth-inhibitory ability of each of paulownin and sesamin. Additionally, as growth was inhibited in a concentration-dependent manner, further studies should identify an adequate dose for use as an anti-microbial agent.

4. Conclusions

In this study, the biological durability of wood was evaluated and the utility of P. tomentosa extracts was verified. The crude extract and the hexane and chloroform fractions of P. tomentosa sapwood and heartwood exhibited a growth inhibition effect on the mycelia of the test fungal strains, namely, wood decay fungi F. palustris and T. versicolor. The hexane and chloroform fractions also led to the growth inhibition of P. glabrum and T. harzianum.

The search for active compounds exhibiting inhibitory effects on microbial growth in the crude extract and the hexane and chloroform fractions revealed sesamin and paulownin as the active compounds, with the highest contents observed in the hexane fraction for both sapwood and heartwood. The contents did not vary significantly between the sapwood and heartwood of P. tomentosa.

In summary, in the utilisation of P. tomentosa xylem, a potential approach to the process was introduced, prompted by the findings that certain extracted compounds demonstrated efficacy in biological resistance. This involved incorporating a pre-extraction step to utilise active components before wood modification. Therefore, our next step is to apply pre-extracted P. tomentosa wood to modification research.