Extraction of High-Purity Native State Gutta-Percha from Enzymatic Hydrolyzed Eucommia ulmoides Pericarps by Ultrasound Treatment and Surfactant Aqueous Phase Dispersion

Abstract

Herein, a method of ultrasound treatment combined with surfactant aqueous phase dispersion was proposed for the extraction of high-purity gutta-percha in its native state from enzymatic hydrolyzed Eucommia ulmoides pericarps. Firstly, the plant tissues wrapped around gutta-percha were destructed through enzymatic hydrolysis, then the plant tissues debris still attached to gutta-percha were further stripped off by ultrasound. Finally, under the “amphiphilic” action of sodium dodecyl sulfate (SDS), the entangled gutta-percha was untwined, allowing the residual plant tissue debris to be released and precipitated, thus high purity gutta-percha was obtained. The process parameters were optimized through single factor and response surface experiments. The optimal parameters for ultrasonic treatment were displayed as follows: frequency of 40 kHz, power of 320 W, time of 7.3 h, temperature of 50 °C, and material-to-liquid ratio of 1:70 g/mL, and for the aqueous phase dispersion of surfactant were SDS concentration 1.7%, temperature 80 °C, stirring speed 1200 rpm, solid-to-liquid ratio 1:60 g/mL, and time 60 h. Under optimal conditions, the purity of gutta-percha reached 95.4 ± 0.31% and its weight average molecular weight (Mw) was 20.85 × 10⁴. Moreover, the obtained gutta-percha maintained its native filamentous form. The obtained products were characterized by IR, NMR, XRD, TGA, DSC, and tensile experiments, which showed that the obtained product was gutta-percha and maintained the natural α- and β- crystal structure. The proposed method overcomes the disadvantages of the traditional organic solvent method, which has great environmental pollution and destroys the gutta-percha structure. This is also the first reported method to obtain high purity gutta-percha while maintaining its native state.

1. Introduction

Eucommia ulmoides Oliv (E. ulmoides) is a peculiar, rare tree species in China. Its leaves, bark, and samara all contain gutta-percha [1], commonly known as balata rubber (Figure 1). Gutta-percha is a white filamentous high-molecular polymer with the structural formula of trans-1,4-polyisoprene, which is the isomer of natural hevea rubber [2]. Therefore, gutta-percha is the best substitute and supplement for hevea rubber with considerable development potential [3,4].

Besides, gutta-percha can be applied in many fields such as the rubber industry, chemical industry [5], medical [6], and sports [7] due to its rubber-plastic dual characteristics [8,9], good anti-acid and anti-alkaline properties, tear resistance and shape memory function [10]. Meanwhile, gutta-percha is also an ideal ergonomic material, which can be used to prepare artificial blood vessels and skin, etc. [6,11]. However, the research and application of gutta-percha mostly rest in the laboratory stage at present, without large-scale research or industrialized application. The key reason lies in the lack of a high-quality and high-purity gutta-percha extraction technology.

So far, the reported gutta-percha extraction methods include mechanical crushing, organic solvent extraction, steam explosion, microbial fermentation, and enzymatic hydrolysis method [12,13,14]. Among them, the enzymatic hydrolysis method was first proposed by our laboratory [15], which utilizes the biocatalytic action of appropriate enzymes to degrade the plant tissues wrapped around gutta-percha, thus achieving the separation of gutta-percha from the plant tissues. Compared with other methods, enzymatic hydrolysis uses enzymes to specifically degrade plant tissues under mild conditions, so that the natural structure of gutta-percha and the physiological activity of the medicinal components in Eucommia are maintained at the same time, thus allowing for the simultaneous extraction of gutta-percha and natural medicinal components, and enabling the production costs of gutta-percha to be reduced. However, due to the complexity of plant tissue components, the plant tissues cannot be completely removed so far just by the enzymatic hydrolysis alone. Gutta-percha products after enzymatic hydrolysis still contain many plant tissue debris. Hence, it is necessary to combine other technologies to continuously remove the remaining plant tissue debris.

In this study, the ultrasonic treatment combined with surfactant aqueous phase dispersion was proposed for the first time to further purify gutta-percha from enzymatic hydrolyzed E. ulmoides pericarps. The adhesive force between the gutta-percha and the residual plant tissues was weakened by ultrasound. Next, the intertwined gutta-percha was untwined under the “amphiphilic” action of surfactants. Subsequently, the relieved plant tissue debris sank to the bottom, while gutta-percha floated to the water surface, thus obtaining gutta-percha with high purity (>95%) in a pollution-free way. The extraction scheme is shown in Scheme 1.

2. Materials and Methods

2.1. Materials

E. ulmoides pericarps obtained by mechanical de-seeding of Eucommia samara was provided by Zhejiang Xuyuan Eucommia Biotechnology Co. Ltd. (Jiaxing, China). Cellulase (ZH-1) was purchased from Daheng Biotechnology Company (Zhangjiagang, China). The special enzymes for paper pulping, MTPE-90, and OCE-90, were purchased from Sukohan Biological Engineering Co. Ltd. (Weifang, China). 4.0 g/L complex enzyme solution was obtained by adding appropriate amounts of ZH-1, OCE-90, and MTPE-90 (mass ratio 1:1:1) to 1000 mL of citric acid-sodium citrate buffer at pH 6.0, stirred at 50 °C for 30 min and then filtered. Polyethylene glycol octylphenyl ether (S1), polyoxyethylene lauryl ether (S2), polyethylene glycol PEG-400 (S3), dioctyl sodium sulfosuccinate (S4), alkylphenol polyoxyethylene ether (S5), fatty alcohol polyoxyethylene ether (S6), sodium dodecyl sulfate (S7), hexadecyltrimethylammonium bromide (S8), octadearyl dimethyl ammonium chloride (S9) were purchased from De-Zhong Chemical Technology Co. Ltd. (Foshan, China).

2.2. Enzymatic Pretreatment of Raw Materials

The E. ulmoides pericarps obtained by mechanical de-seeding of Eucommia samara were used as raw material, which was ultrasonicated for 8 h in water and then dried at 60 °C under vacuum. After that, 10 g of the above treated E. ulmoides pericarps were accurately weighed and put into a triangular flask, 300 mL of 4.0 mg/mL complex enzyme solution was added and the enzymatic hydrolysis was carried out at 50 °C for 4 h. The enzymatic solution was filtered. The solids were collected and washed repeatedly with water and dried under a vacuum at 60 °C to obtain the enzymatic hydrolyzed E. ulmoides pericarps.

2.3. Ultrasonic Treatment of Enzymatic Hydrolyzed E. ulmoides Pericarps

5.0 g of enzymatically hydrolyzed E. ulmoides pericarps were sonicated in water for a certain time, the solids were collected and dried under vacuum at 60 °C to obtain crude gutta-percha. Five experimental factors (material-to-liquid ratio, sonication frequency, sonication power, sonication temperature, and sonication time) were optimized by single-factor experiments. Based on the results of a single-factor test, the factors were further optimized by the Box-Behnken Design (BBD) with gutta-percha purify as the index, and the levels of experimental factors are shown in

Table 1. Codes and levels of independent variables for response surface analysis during ultrasonication.

	Factors
Level	X1	X2	X3	X4	X5
	Frequency	Power	Time	Temperature	Material-to-Liquid Ratio
	kHz	W	h	°C	g/mL
−1	33	280	4	40	1:60
0	40	320	6	50	1:70
1	59	360	8	60	1:80

.

2.4. Purification of Crude Gutta-Percha

10.0 g of crude gum was weighed accurately, stirred magnetically at a certain temperature for a certain time in an aqueous solution containing surfactant, then the solid was collected, washed several times with double distilled water to remove residual surfactant and dried under vacuum at 60 °C to obtain gutta-percha.

Six experimental factors (surfactant type, surfactant concentration, water bath temperature, water bath time, stirring rate, and material-to-liquid ratio) affecting the purification of gutta-percha were investigated. According to the single factor test results, the surfactant was fixed as S7, and BBD was used to optimize the experimental factors with gutta-percha purity as the index, the factors and levels are shown in

Table 2. Codes and levels of independent variables during surfactant aqueous phase dispersion.

	Factors
Level	X′1	X′2	X′3	X′4	X′5
	Concentration	Temperature	Time	Stirring Speed	Material-to-Liquid Ratio
	%	°C	h	rpm	g/mL
−1	1	80	40	800	1:40
0	2	85	50	1200	1:50
1	3	90	60	1600	1:60

.

2.5. Determination of Gutta-Percha Purity

M₀ g of gutta-percha was weighed accurately and placed in a triangular flask, then 200 mL of mixed xylene containing 0.25% of 2-thiolylbenzothiazole was added. The gutta-percha was fully dissolved by heating to 125~130 °C and then the hot solution was filtered by a clean and dry stainless steel mesh sieve (45 μm pore size) with known mass. The residual impurities in the triangular flask were also washed into the mesh sieve with hot solvent. Subsequently, the sieve containing impurities was washed twice with petroleum ether and dried to a constant weight. The purity of gutta-percha can be calculated according to the following formula.

$$Y=\left(1-\frac{M_2-M_1}{M_0}\right)\times 100\%$$

(1)

where, M₀ (g) is the weight of the gutta-percha, M₁ (g) is the weight of the mesh sieve and M₂ (g) is the total weight of the mesh sieve and impurities.

2.6. Identification and Characterization of Gutta-Percha

Fourier transform infrared (FT-IR) spectra were collected on an IRAffinity-1S FT-IR spectrometer (Shimadzu, Kyoto, Japan). Gutta-percha was dissolved with petroleum ether and then evenly coated on KBr tablets. The ¹H-NMR spectra were collected using AVANCE III 500 MHz nuclear magnetic resonance spectrometer (Bruker, Fällanden, Switzerland) with CDCl₃ as the solvent and TMS as the internal standard (δ_H = 0). X-ray Diffraction Analysis was performed with Empyrean X-ray Diffractometer (PANalytical B. V., Almelo, The Netherlands). The 2θ angle range was 5°~35°. Gel permeation chromatography (GPC) analysis was carried out on a Waters GPC system including a Plgel 5 um MIXED-C column (Agilent Technologies Co., Ltd., Beijing, China). CHCl₃ was used as the mobile phase at a flow rate of 1 mL/min. STA 449 F5 Thermogravimetric analyzer (NETZSCH, Selb, Germany) was used for Thermo gravimetric (TGA) analysis in the range of 28~600 °C. Tensile tests were performed using CMT5304 (Sansi, Shenzhen, China) at room temperature. DSC analysis was performed with a TA Q2000 DSC instrument (TA, New Castle, DE, USA), and the scanning temperature was −75 °C~125 °C.

2.7. Data Processing and Analysis

Excel 2019 was used to process the data and Origin 8.5 (Northampton, MA, USA) was used for graphical plotting. Box-Behnken response surface experimental design, ANOVA, and significant difference analysis were performed by Design-Expert 8.0.6 (Minneapolis, MN, USA). FT-IR was analyzed by OMNIC software 8.2 (Madison, WI, USA) and ¹H-NMR spectra was analyzed by MestReNova software 12.0.1 (Santiago de Compostela, Spain).

3. Results

3.1. Enzymatic Hydrolysis of E. ulmoides Pericarps

Gutta-percha grows in E. ulmoides and is wrapped by complex plant tissues (the main components are cellulose, pectin, and lignin). Gutta-percha can be released to some extent by destroying the plant tissues outside gutta-percha through the degradation of specific enzymes. Figure 2 compares the E. ulmoides pericarps before and after enzymatic hydrolysis. It can be observed that after enzymatic hydrolysis, the plant tissues wrapped at the outer layer of gutta-percha were pertinently degraded to a great extent, so gutta-percha was exposed. In addition, it can be seen from Figure 2c that gutta-percha still kept its original filamentous state after enzymatic pretreatment. However, a lot of plant residues were still wrapped in the enzymatically hydrolyzed E. ulmoides pericarps. Hence, the plant residues were required to be further removed.

3.2. Ultrasonic Stripping of Plant Tissue Debris

The adhesive force between gutta-percha and the plant debris can be weakened by the cavitation effect of ultrasound, thus enabling the debris to be stripped from the gutta-percha, the corresponding mechanism of which was discussed in the Supplementary Materials (Scheme S1).

3.2.1. Single-Factor Optimization of Ultrasonic Experimental Parameters

The ultrasonic experimental parameters were optimized using gutta-percha purity as the index, and the results are shown in Figure 3. The influence of ultrasonic frequency was shown in Figure 3a. The results showed that the purification effect was the best at 40 kHz, and then weakened at 59 kHz. A possible reason for this is that too high frequencies lead to the attenuation of ultrasonic vibrations, which weakens the cavitation intensity. The influence of ultrasonic power was shown in Figure 3b. As the power was elevated, the ultrasound energy increased, which was good for stripping off plant debris. The purity of gutta-percha was the highest at 320 W. However, the purity was reduced if the power was continuously increased, indicating that the separation of gutta-percha was not further promoted by continuously increasing the ultrasonic power, and excessive ultrasonic power would also increase the energy consumption. As shown in Figure 3c, the purity increased with increasing sonication time, but after 6 h, the purity changed very little. Considering the time cost, the sonication time of 6 h was chosen. The influence of ultrasonic temperature on the purity is exhibited in Figure 3d. The purity was the highest (60.5%) at 50 °C. As the temperature was further elevated, the purity declined, since excessive temperature resulted in the decline of impact force of cavitation microbubbles. The material-to-liquid ratio was also optimized (Figure 3e). In the range of 1:50 g/mL to 1:70 g/mL, the purity increased with the increase of material-to-liquid ratio, but further decreased, which was possibly due to the thermal effect, mechanical effect, and cavitation effect of ultrasonic waves on unit volume of extraction solution being weakened when the material-to-liquid ratio was too large [16,17]. According to the results in Figure 3, the optimization central points for BBD of frequency, power, time, temperature, and material-to-liquid ratio were 40 kHz, 320 W, 6 h, 50 °C, and 1:70 g/mL, respectively.

3.2.2. BBD Optimization and Verification of Ultrasonic Parameters

Forty-six groups’ tests of 5-factor at 3-level were designed based on the principle of BBD, and the results are listed in Table S1. The multiple regression analysis was performed to obtain the following regression equation:

$$\begin{array}{c}Y=66.12+0.73X_1+1.65X_2+3.37X_3+1.70X_4+0.78X_5+0.59X_1{X}_2+0.34X_1{X}_3-\\ 1.45X_1{X}_4-0.20X_1{X}_5+1.05X_2{X}_3+1.71X_2{X}_4-0.54X_2{X}_5-0.39X_3{X}_4-0.85X_3{X}_5-\\ 0.91X_4{X}_5-6.94X_1{}^2-5.77X_2{}^2-4.51X_3{}^2-3.84X_4{}^2-3.52X_5{}^2\end{array}$$

(2)

ANOVA results were listed in Table S2. The p-value (<0.0001) suggests that the model is significant. The coefficient of determination (R²) was 0.9655, indicating that this model could explain 96.55% of the variation in response values. The p-value of lack-of-fit was not significant (0.1499 > 0.05), indicating that the regression model adequately reflected the actual situation. It can also be seen from Table S2 that the one-order term (X₂, X₃, X₄), the quadratic term (X₁², X₂², X₃², X₄², X₅²), and the interaction term (X₁X₄, X₂X₄) exerted extremely significant influences on gutta-percha purity (p < 0.01). the one-order term (X₁ and X₅) had a significant effect on gutta-percha purity (p < 0.05). According to the F value, the influences of different factors on gutta-percha purity were sorted as: Time > Temperature > Power > Frequency > Material-to-liquid ratio.

To visualize the effect of the interaction between the ultrasonic factors on Y, the 3D surface charts were drawn (Figure 4) [18]. The steeper surface and the higher slope indicate a stronger influence of the interaction between two factors on gutta-percha purity [19]. It could be observed that the interaction between frequency (X₁) and temperature (X₄) and that between power (X₂) and temperature (X₄) had greater impacts on the removal of plant tissue fragments.

The optimal ultrasonic experimental parameters predicted through BBD were frequency 44.83 kHz, power 325.33 W, time 7.31 h, temperature 49.28 °C, and material-to-liquid ratio 1: 66.73 g/mL. Under such conditions, the predicted purity was 65.9%. Three parallel validation tests were carried out at 40 kHz, 320 W, 7.3 h, 50 °C, and 1:70 g/mL. The purity of the obtained gutta-percha was 64.8 ± 0.55%, which only deviated slightly from the predicted value, thus indicating the reasonability and reliability of the constructed model.

The morphology of the crude gutta-percha obtained after ultrasonic treatment is presented in Figure 5a. It can be observed that the adhesion of the plant tissue debris to the gutta-percha became looser after sonication, and the amount of debris wrapped in the crude gutta-percha was obviously reduced. Figure 5b shows that the gutta-percha still remained in the filamentous state after sonication.

3.3. Purification of Crude Gutta-Percha

Under the “amphiphilic” action of surfactant, the entangled gutta-percha unentangled and released the plant tissue residues wrapped in gutta-percha. The related mechanism was discussed in the supplementary material (Figure S1).

3.3.1. Single-Factor Optimization of Surfactant Dispersion Conditions

The effect of the aqueous phase dispersion conditions on gutta-percha purity is shown in Figure 6. First, the surfactant species were chosen (Figure 6a). It was found that SDS (S-7) had the best effect with a purity of 84.4%. SDS is an anionic surfactant with superior wettability and permeability and is inexpensive. Therefore, SDS was chosen in the subsequent experiments.

Next, the influence of SDS concentration on purity was explored (Figure 6b). The results showed that the purity tended to increase in the concentration range of 0.5 to 2.0%, and reached 85.0% at 2.0%. As the concentration was further increased, the purity decreased, probably due to the formation of too dense micelles, which were detrimental to the dispersion of gutta-percha.

The effect of temperature on the purification of gutta-percha is exhibited in Figure 6c. The purity of gutta-percha reached a maximum (86.4%) at 85 °C.

The influence of extraction time is displayed in Figure 6d. As the time was lengthened, SDS exerted a more sufficient effect, and the purity showed a rising trend. The purity increased to 87.8% at 50 h, but it was almost unchanged after then. Considering the time cost, the extraction time was chosen as 50 h for the subsequent experiment.

The influence of agitating rate is as shown in Figure 6e. As the agitating rate increased, the purity was enhanced, reaching 93.6% at 1200 rpm. When the stirring speed was further accelerated, the purity declined, which might be related to the fact that the too fast agitation was not good for the stability of micelles. Besides, it could be found from Figure 6f that the purity was the highest (94.7%) at the material-to-liquid ratio of 1:50 g/mL.

3.3.2. BBD-Based Optimization of Surfactant Dispersion Conditions and Verification

Based on the preliminary experimental results, 46 groups’ tests of five independent variables at three levels were designed. The experimental results are displayed in Table S3. The experimental data were fitted by multi-regression analysis to obtain the following second-order polynomial equation:

$$\begin{array}{c}Y=94.71+1.47{X^{\prime }}_1+0.88{X^{\prime }}_2+3.58{X^{\prime }}_3+1.90{X^{\prime }}_4-0.56{X^{\prime }}_5+1.16{X^{\prime }}_1{X^{\prime }}_2+\\ 0.21{X^{\prime }}_1{X^{\prime }}_3+0.77{X^{\prime }}_1{X^{\prime }}_4-0.44{X^{\prime }}_1{X^{\prime }}_5-4.49{X^{\prime }}_2{X^{\prime }}_3+0.18{X^{\prime }}_2{X^{\prime }}_4-0.06{X^{\prime }}_2{X^{\prime }}_5-\\ 3.04{X^{\prime }}_3{X^{\prime }}_4-1.96{X^{\prime }}_3{X^{\prime }}_5-0.42{X^{\prime }}_4{X^{\prime }}_5-4.13{{X^{\prime }}_1}^2-3.21{{X^{\prime }}_2}^2-4.65{{X^{\prime }}_3}^2-3.53{{X^{\prime }}_4}^2-\\ 0.94{{X^{\prime }}_5}^2\end{array}$$

(3)

The ANOVA of the experimental results were presented in Table S4. The p-value of the model is <0.0001, indicating that the regression model is highly significant. the coefficient of determination (R²) was 0.9529, indicating that this model could explain 95% of the variation in response values. The p-value of lack-of-fit was insignificant (0.0567 > 0.05), manifesting that the actual situation could be reflected by the regression model.

The one-order term (X′₁, X′₃, X′₄), quadratic term (X′₁², X′₂², X′₃², X′₄²), and interaction term (X′₂X′₃, X′₃X′₄, X′₃X′₅) in the regression model all generated extremely significant (p < 0.01) influences on the purity of gutta-percha, the one-order term (X′₂) and quadratic term (X′₅²) had a significant effect on gutta-percha purity (p < 0.05). Meanwhile, it could be known from the F value that the influences of different factors on the purity of gutta-percha were sorted as Time > Stirring speed > Concentration > Temperature > Material-to-liquid ratio. The response surface plots were presented in Figure 7 [18]. It can be seen that the interaction between temperature (X′₂) and time (X′₃) and between time (X′₃) and stirring speed (X′₄) has greater effect on the purity of gutta-percha.

The optimal aqueous phase dispersion conditions predicted by BBD were concentration 1.69%, temperature 80.40 °C, time 59.94 h, stirring speed 1120.94 rpm, and material-to-liquid ratio 1:58.96 g/mL, under which the predicted purity of gutta-percha was 95.8%. The verification experiments were performed under practical experimental conditions (concentration1.7%, temperature: 80 °C, time 60 h, stirring speed 1200 rpm, and material-to-liquid ratio 1:60 g/mL), and the purity of gutta-percha was 95.4 ± 0.31%, which was approximate to the predicted value, thus proving the reasonability and reliability of the constructed model. Figure 8 shows the gutta-percha obtained after surfactant treatment. It can be seen that the purity of the gutta-percha was significantly improved, only very few impurities existed in the obtained gutta-percha. The gutta-percha still remained in its native filamentous state. Figure 8e also shows gutta-percha prepared by extraction with petroleum ether. It can be seen that the gutta-percha obtained is in the form of lumps and the natural morphology of gutta-percha has been destroyed.

3.4. Identification and Characterization of the Extracted Gutta-Percha

3.4.1. Identification of Molecular Structure

FT-IR spectroscopy and ¹H-NMR spectrum were used to identify the molecular structure of the obtained gutta-percha (Figure 9). In Figure 9a, the asymmetric stretching vibration peak of -CH₃ was observed at 2965 cm⁻¹, and the peaks at 2912 cm⁻¹ and 2847 cm⁻¹ correspond to the asymmetric stretching vibration and symmetric stretching vibration of -CH₂-, respectively. The symmetric and asymmetric deformation vibration peaks of -CH₃ occurred at 1383 cm⁻¹ and 1445 cm⁻¹, respectively. Peaks at 799 cm⁻¹ and 1664 cm⁻¹ are characteristic absorption peaks of the trans-C(CH₃)=CH- group, corresponding to the deformation vibration of C-H and the stretching vibration of C=C double bond, respectively. From the literature [20], the characteristic absorption peaks at 843, 910, and 890 cm⁻¹ in the IR spectra of polyisoprene are attributed to the 1,4-link, 1,2-link, and 3,4-link, respectively. In Figure 9a, only the absorption peak at 843 cm⁻¹ was found, indicating that the obtained gutta-percha has only 1,4-link. Figure 9a is in general agreement with previous literature [21,22] and shows the IR spectrum of trans-1,4-polyisoprene.

Figure 9b displays the NMR spectrum of the as-prepared gutta-percha. The peak signal is strong at chemical shift 1.60 ppm, which can be attributed to the methyl proton peak in the trans-1,4- structure, while the absence of the methyl proton peak in the cis-1,4- structure at chemical shift 1. 67 ppm indicates the absence of cis-1,4 -polyisoprene [23,24]. The two peak signals at chemical shifts of 2.06 ppm and 1.96 ppm correspond to two methylene proton absorption peaks. In addition, the peak at 5.12 ppm is the characteristic peak of the unsaturated proton on the C=C double bond in the trans-1,4- structure. No signal peaks for protons in the 1,2-structure and 3,4-structure were observed. In combination with the results in Figure 9a,b, the structure of the extracted gutta-percha was confirmed to be trans-1,4 polyisoprene [8].

3.4.2. XRD Analysis

There are two crystalline forms in gutta-percha [25,26], namely α- and β-crystalline forms. As shown in Figure 10a, the peaks at 11.4°, 17.9°, 26.9°, 30.8°, and 33.5° are the characteristic peaks of the α-crystalline form of gutta-percha, while the peaks at 19.1° and 22.8° are the characteristic peaks of the β-crystalline form [27,28], showing that the extracted gutta-percha maintains its natural crystalline structure.

3.4.3. Analysis of Molecular Weight and Molecular Weight Distribution

The molecular weights and molecular weight distributions of the extracted gutta-percha were measured by GPC (Figure 10b). The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of gutta-percha obtained by enzymatic hydrolysis combined with ultrasound treatment and surfactant aqueous phase dispersion were 20.85 × 10⁴ and 5.37 × 10⁴, with a polydispersity index (Mw/Mn, PDI) of 3.88. Also, the Mw and Mn of gutta-percha extracted with petroleum ether were 13.25 × 10⁴ and 5.88 × 10⁴, respectively, with a PDI of 1.91 (

Table 3. Comparison of molecular weights and DPIs of gutta-percha extracted by different methods.

Method	Eucommia Organ	Mw (×104)	Mn (×104)	PDI	Ref.
Enzymatic pretreatment combined with ultrasound and surfactant aqueous phase dispersion	pericarps	20.85	5.37	3.88	This work
Petroleum ether extraction	pericarps	13.25	5.88	1.91	This work
Petroleum ether extraction	pericarps	5.6	5.1	1.1	[29]
Toluene extraction followed by ethanol purification	pericarps	8.4	3.9	2.18	[30]
Turpentine extraction	bark	4.9	1.9	2.54	[31]
Steam explosion followed by petroleum ether extraction	bark	20.46	12.64	2.2	[32]
Alkali treatment combined with enzymatic hydrolysis and petroleum ether extraction	leaves	6.21	1.0	6.2	[33]

). It can be noted that the gutta-percha extracted by the proposed method has higher Mw than the traditional organic solvent method, indicating that the proposed method in this work better preserves the molecular structure of gutta-percha. PDI is an indicator to assess the distribution width of a polymer’s molecular weight. Compared to petroleum ether extraction, the gutta-percha obtained by the proposed method has a wider molecular weight distribution, indicating that gutta-percha with smaller or larger molecular weights was also extracted efficiently. Moreover, the molecular weights and DPIs of gutta-percha in other reported literature are also listed in Table 3. It can be observed that the gutta-percha extracted by the proposed method has a higher molecular weight and broader molecular weight distribution. More importantly, the gutta-percha in this work maintained its natural filamentous form.

3.4.4. Thermo Gravimetric Analysis

The thermal stability properties of the purified gutta-percha were tested. As can be seen in Figure 11, the TG curve shows a thermal weight loss step, and the corresponding DTG curve shows only one distinct peak, indicating that the thermal-oxidative degradation reaction of gutta-percha is a one-step reaction, which is attributed to the degradation of the isoprene linkages [34]. The decomposition of gutta-percha began at around 300 °C, followed by a steep and pronounced weight loss step with increasing temperature, and the TG curve stabilized at around 500 °C, indicating that the decomposition of gutta-percha was complete. The maximum peak value of the DTG curve occurred at around 387.5 °C, indicating a maximum thermal degradation rate at this temperature. The main degradation process of gutta-percha occurred at 330–460 °C, which is consistent with that reported in other literature [9].

3.4.5. DSC Analysis

The crystallization behavior of purified gutta-percha was studied by DSC (Figure 12). It can be seen that there were two endothermic peaks in the process of heating, among which the absorption peak at 43.15 °C is the melting peak of β-crystal, and 58.78 °C is the melting peak of α-crystal [7,34]. During the cooling process of gutta-percha, there was a sharp crystallization peak in the range of 20–25 °C, and the maximum crystallization rate temperature was 23.2 °C. The crystallinity of purified gutta-percha was 48.3%.

3.4.6. Mechanical Tensile Properties Analysis

Figure 13 shows the stress-strain curves of the gutta-percha extracted by the proposed method and by petroleum ether. It can be found that the tensile strength of the gutta-percha extracted by the proposed method was 26.5 Mpa and the elongation at break was 441.1%, while the tensile strength and elongation at break of the gutta-percha extracted by petroleum ether was 17.14 Mpa and 303.1%, indicating that the gutta-percha obtained by the proposed method showed better tensile properties.

4. Conclusions

Gutta-percha in E. ulmoides is in solid form and does not flow automatically from plant tissues like hevea rubber, so it must be extracted by physical, chemical, or biological methods. In this study, specific enzymes were used to hydrolyze and destroy the plant tissues outside gutta-percha, and then ultrasound treatment and surfactant aqueous dispersion was used in turn to further remove the residual plant tissue debris, finally yielding gutta-percha with a purity of 95.4 ± 0.31%, which retained its native filamentous structure and no chemical residues. The proposed method features a simple process and low production cost without needing any expensive instruments. Therefore, the method can be easily applied to the industry for the continuous large-scale production of high-purity, native state gutta-percha.