Synthesis of biosafe isosorbide dicaprylate ester plasticizer by lipase in a solvent-free system and its sub-chronic toxicity in mice

Abstract

In this study, biosafe isosorbide dicaprylate ester based plasticizer was prepared using renewable feedstock with lipase in a solvent-free system. Different kinds of water removal methods and some important factors including molar ratio of the substrates, reaction temperature and catalyst loading were investigated. Bubbling dried air was determined to be the most effective water removal method. The activation energy (E) of hydrolysis and esterification to form di-substituted isosorbide were examined to determine the limiting step in the synthesis of isosorbide dicaprylate ester. The E_hydrolysis (25.51 kJ mol⁻¹) was found to be higher than E_synthesis (35.65 kJ mol⁻¹), demonstrating that the formation of diester from monoester is the critical step in the process. The lipase can be recycled up to 16 times while 80% diester was maintained. Additionally, the properties of poly(vinyl chloride) PVC blends plasticized with isosorbide ester as secondary plasticizer was studied. The results indicated that the thermal stability of plasticized PVC blends was improved, and the tensile strength was increased. Furthermore, the sub-chronic toxicity study in mice showed that the isosorbide ester was safe, indicating its great potential in industrial applications as a plasticizer.

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I. Introduction

Over the past decade, products which are environmentally and biologically friendly have attracted increased attention.^1–3 Sources of raw materials and production processes are important to the overall quality of such products. Biomass stocks are abundant carbon-neutral renewable resources which can be generated directly and rapidly from solar energy. Specifically, the use of biologically derived raw materials and bio-catalysts are among the most promising approaches for developing bio-friendly products.^4,5 The comprehensive utilization of bio-based materials and the development of bio-refinery processes could minimize waste and protect the environment while simultaneously increasing economic benefit.^6,7

Poly(vinyl chloride) (PVC) is the second largest manufactures resin in the world, and its products are widely used in packaging, toys, flooring, cards and bags. Plasticizer plays an important role in improving the flexibility and process ability of PVC. Phthalates is the main plasticizer additives of PVC, which has been used around the world for many years because of its excellent plasticizing effect, but phthalates may migrate from PVC products to environment when the materials contact with biological fluids, which will take potential risks to human health when it is used in food packing, automotive products, insect repellents, blood storage bags and medical devices.^8,9 Thus the bio-based plasticizer became imperative.

The development of bio-based, especially vegetable and cereal-based, chemicals has the potential to reduce petroleum consumption in the chemical industry and also to open new high-value-added markets.¹⁰ Isosorbide (1,4:3,6-dianhydro-D-glucitol) is an important bio-feedstock as it is chiral, as well as non-toxic.¹¹ The non-toxic character of isosorbide lends it to applications in packaging, cosmetics and medicine.¹² Despite its favorable properties, isosorbide has been utilized in relatively few applications. Currently, its largest commercial use is in the production of isosorbide nitrate,¹³ which is used to treat angina. As it is biodegradable and thermally stable, isosorbide has also been applied as a polyester backbone used in the synthesis of thermosets.¹⁴ Known syntheses of isosorbide-derived products are often dependent on activated di-acid derivatives or Lewis acid catalysts coupled with the removal of low mass condensation products.¹⁵ These processes produce large quantities of waste water and the harmful catalysts and by-products are difficult to remove from the desired products. Thus, environment-friendly bio-refinery processes are the most promising production processes to access to isosorbide-derived chemicals.

Lipase catalyzed esterification, hydrolysis, alcoholysis and acidolysis reactions are among the most promising and environmentally friendly alternatives to traditional chemical methods.¹⁶ Compared to chemical catalysts, lipases react under milder conditions, have broader substrate specificity, reduce undesirable by-products and require lower energy input.^17,18 Candida antarctica lipase B (CALB) in particular has been found to be a versatile biocatalyst in the food, paper, pharmaceutical and cosmetic industries,¹⁹ and CALB-catalyzed esterification has recently grown in popularity.^20–22 The resultant bio-based esters are eco-friendly, since the compounds are generated from renewable resources, the synthesis produces minimal waste, and they are biodegradable.

Few papers have referred to the isosorbide diester as plasticizer and the enzymatic esterification of isosorbide with aliphatic acids: D. Mukesh et al. reported lipozyme IM-20-catalyzed esterification of isosorbide oleate in the presence of silica gel to remove water.²³ C. Cecutti et al. reported the reaction of isosorbide with fatty acyl chlorides to yield the diester without catalysts in pyridine and DMF.²⁴ More recently, El Boulifi et al. reported enzymatic production of the monoester from isosorbide and ricinoleic acid.²⁵ In these studies, the restricted water adsorbing capacity of silica gel and the solvent (pyridine and DMF) limits the scalability of this method to industrial applications, and the fatty acyl chloride reagent is not a desired substrate for the synthesis of esters because the operation process is not safe.

To overcome the limitations introduced by the use of solvents and silica gel and to better understand the critical factor for synthesizing isosorbide diester, we present an eco-friendly synthesis of isosorbide diester. Isosorbide and caprylic acid were esterified under enzymatic solvent-free conditions using Novo 435 as the catalyst. The experiment route is illustrated in Scheme 1. During the esterification, several factors were found to have significant influence over the reaction content. These factors are the method for removing water, the stoichiometry of caprylic acid and isosorbide, the reaction temperature, and the enzyme loading. The activation energy for the hydrolysis and esterification of isosorbide dicaprylate ester was determined to explain the limiting factor in achieving high ester content and therefore to optimize the reaction while reducing costs. The recyclability and operational stability of Novo 435 under the solvent-free conditions were also investigated. The properties of poly(vinyl chloride) PVC blends plasticized with isosorbide ester as secondary plasticizer were studied. Finally, the sub-chronic toxicity of isosorbide ester was tested in mice.

Scheme 1 The experiment route way.

II. Materials and methods

(A) Materials and equipment

The catalyst Novozym 435 was purchased from Novo Nordisk A/S (Denmark). Isosorbide (purity: 98%) was supplied by Sinopharm Chemical Reagent Co., Ltd. Caprylic acid (99%) was from Beijing Chemical Factory, Beijing, China. All the reagents and solvents used in the experiments were of analytical grade. The esterification and hydrolysis reactions were carried out in parallel. Polyvinyl chloride (PVC) was obtained by Shandong jiqing Chemical Co., Ltd. Kunming mice were purchased from Experimental Animal Center of Chinese Drug and Biological Products.

(B) Enzymatic synthesis of isosorbide diester

Experiments were carried out in a 100 mL two-neck flask. The suitable esterification reactions of isosorbide and caprylic acid with Novo 435 lipase were examined under different water removal conditions (the reaction exposed to air, the reaction under vacuum, and the reaction bubbled with dried air), caprylic acid to isosorbide molar ratios in the solvent-free system (2, 2.5, 3, and 4), reaction temperatures (50, 60, 70, and 78 °C), and lipase loadings (3.5, 5, 6.5, and 8% (w/w)). For hydrolysis experiments, the esterification products were reacted with water under the optimized esterification conditions (29.3 g diester, 3 g water, 1.97 g Novozym 435, 300 rpm for 105 min at 60 °C). When determining the activation energy of the esterification and hydrolysis reactions, the reactions were conducted continuously in the reactor for 60 min. The reaction condition of the esterification was as follows: caprylic acid to isosorbide molar ratio 1 : 2.5, 8% (w/w) lipase loading amount, 300 rpm with bubbling dried air at 40, 50 or 60 °C. And the reaction condition of the hydrolysis reactions was as follows: diester to water molar ratio 1 : 2.5, 8% (w/w) lipase loading amount, 300 rpm with bubbling dried air at 30, 43 or 50 °C. The water activity and the temperature of the air at the inlet and outlet were measured in the gaseous phase by HygroClip 2 sensors (Rotronic, Switzerland) and monitored on a computer. The monoester and diester contents were analyzed by gas chromatography (GC).

(C) GC analysis

Aliquots were taken from the reactions at scheduled times and immediately heated to ensure enzyme deactivation. The levels of fatty acid, mono-, and di-substituted esters in the reaction mixture were quantified using a Trace 1300 gas chromatograph (Thermo scientific, USA) equipped with a DB-1ht capillary column (30 m × 0.25 mm × 0.1 μm; J&W Scientific, USA) and a flame ionizing detector (FID). The column temperature was held at 120 °C and then ramped to 180 °C at 30 °C min⁻¹ and to 320 °C at 45 °C min⁻¹ where it was held for 2 min. The temperatures of the injector and detector were both set at 320 °C. The retention time of caprylic acid, isosorbide, and monoester and diester was 1.387 min, 1.533 min, 3.560 min and 5.308 min, respectively. The molar fractions of fatty acid, isosorbide, monoester and diester were calculated.

(D) Preparation of blends and measurements

PVC blends were melted at 165 °C for 5 min at 50 rpm using a Poly Lab Torque rheometer (Hakke Instrument Crop., Germany). PVC resin was pre-treated by drying at 60 °C for 2 h to eliminate possible absorbed water on the surface of particle. Formulations used for preparing plasticized PVC blends were 70 g PVC and 30 g isosorbide diester. Dumbbell-shaped samples of blends were molded on a MiniJetII Micro-injection molding machine (Hakke Instrument Crop. Germany) according to GB/T 17037.1-1997 (China). Mounding conditions were set at 165 °C for 5 min at 550 bar.

TGA was carried out in a TG209F1 TGA thermal analysis instruments (Netzsch Instrument Crop., Germany) in N₂ atmosphere (50 mL min⁻¹) at a heating rate of 10 °C min⁻¹. The samples were put into platinum pans and scanned from ambient temperature to 600 °C.

Tensile modulus, tensile strength, and elongation at break were determined according to GB/T 1040.1-2006 (China) under ambient conditions, using E43.104 Universal Testing Machine (MTS Instrument Crop., China). The reported values were the average of at least three samples. Hardness was determined using a HT-6510D Shore durometer (Shanghai Jielun, China) according to GB/T 3398.2-2008 (China).

(E) Sub-chronic toxicity study

The mice (both male and female) were weighed upon arrival, and then randomly distributed into four groups. The diester was administered to the mice once a day. The mice in control group only received vehicle (0.5% CMC-Na in distilled water), whereas other groups were treated daily with diester at 5, 10 or 20 μL per g mice per day for 28 days, respectively (Table 1). After 28 days, the blood routine, prothrombin time (PT), activated partial clotting enzyme live time (APTT), thrombin time (TT), fibrinogen (FIB) and blood biochemical ALT and BUN were determined. Mice liver and male mice testis were observed by optical microscope. All the experiments were performed in compliance with the relevant laws and institutional guidelines, the institutional committee has approved the experiments, and the informed consent was obtained for experimentation with human subjects.

Table 1 The project of the isosorbide diester toxicity in mice

	High dose (20 μg per g mice per day)	Middle dose (10 μg per g mice per day)	Low dose (5 μg per g mice per day)
Control (0.5% CMC-Na in distilled water)	3 female mices	3 female mices	3 female mices
	3 male mices	3 male mices	3 male mices
Sample	3 female mices	3 female mices	3 female mices
	3 male mices	3 male mices	3 male mices

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III. Results and discussions

(A) Effect of the water removal methods

In organic systems, the hydration of lipase is necessary for catalytic function. However, water is a by-product of esterification reactions and the reaction rate and ester content will decrease if water is accumulated in the reaction medium. It is thus necessary to remove water from the esterification to obtain high reaction rates and ester content. As shown in Fig. 1A, different water removal methods were used, and they increased the efficiency of diester synthesis as compared to the reaction without water removal. Specifically, the diester content (64%) is higher when the reactor is bubbled with air than when it is exposed to air (23%) and held under vacuum (21%) after 24 h, and it is much higher than without removal of water (6%). Fig. 1B showed the time-course profiles of the water activity in different reaction systems. It is obvious that the water activity was increased in the first 30 minutes with or without dried air bubbled into the system. However, as the reaction time increasing, the water activity continued to increase to the equilibrium (42%) about 2 h without dried air bubbling into the system, and it decreased to 12% and kept stable when the dried air was bubbled into the system. It proved that the dried air could remove the produced water efficiently, and regulate the esterification in solvent-free system,²⁶ therefore, the bubbled reactor is the optimal one. After 24 h, the reaction reaches equilibrium so a reaction time of 24 h was used to optimize reaction conditions and enzyme loading.

Fig. 1 (A) Effects of the water removal methods on diester content. (B) The water content of the reaction exposed to air and the reaction bubbled with dried air. Reaction conditions: 9.855 g isosorbide 24.3 g caprylic acid, 1.97 g Novozym 435, at 300 rpm for 50 h at 50 °C.

(B) Effect of reaction conditions on diester content

(1) Effect of substrates molar ratio. The effect of changing the ratio of caprylic acid to isosorbide over the range of 2 : 1 to 4 : 1 on diester content was investigated. Fig. 2A shows that the highest content of diester was observed using a molar ratio (caprylic acid to isosorbide) of 2 : 1. When the molar ratio was higher than 2 : 1, with the increase of the molar ratio, the formation of diester decreased, possibly due to the enzyme inhibition by excess fatty acid. The diester content was 74%, 64%, 56%, and 48% when the molar ratio was 1 : 2, 1 : 2.5, 1 : 3, and 1 : 4. The amount of isosorbide used was held constant in these reactions, while the caprylic acid amount varied with variations in substrate molar ratio. Therefore, with a fixed molar ratio, the relatively higher isosorbide concentration led to a greater conversion of isosorbide to ester. The optimal molar ratio was determined to be 2 : 1 for the formation of isosorbide diester.

Fig. 2 Effect of reaction conditions on diester content. (A) Effect of substrates molar ratio on diester content. Reaction conditions: 9.855 g isosorbide, 19.44 g, 24.3 g, 29.16 g, 38.88 g caprylic acid, 1.97 g Novozym 435, at 300 rpm for 24 h at 50 °C. (B) Effect of esterification temperature on diester content. Reaction conditions: 9.855 g isosorbide, 19.44 g caprylic acid, 1.97 g Novozym 435, at 300 rpm for 24 h under 50, 60, or 70 °C. (C) Effect of enzyme loading on diester content. Reaction conditions: 9.855 g isosorbide, 19.44 g caprylic acid, at 300 rpm for 24 h at 60 °C with 3.5%, 5%, 6.5% and 8% (w/w) Novozym 435. (D) The content of isosorbide, caprylic acid, monoester and diester in the esterification process. Reaction conditions: 9.855 g isosorbide, 19.44 g caprylic acid, at 300 rpm for 24 h at 60 °C, with 1.97 g Novozym 435.

(2) Effect of temperature. In enzyme catalyzed reactions, temperature plays an important role. Reaction temperature affects the solubility of the substrates and products, mass transfer, reaction rates, reaction equilibria, as well as the stability of the enzyme. As shown in Fig. 2B, increasing the reaction temperature above 50 °C, resulted in a corresponding increase in the rate of esterification and in product content with the maximum content observed at 60 °C (80%). Increasing the temperature beyond 60 °C resulted in a little decrease in product content, and when the reaction temperature reached 70 °C, the diester content had a little decrease (1.7%). Therefore the optimal reaction temperature was 60 °C.²⁷

(3) Effect of enzyme loading. Increased catalyst loading also had a positive influence on ester content over the experimental range, where the enzyme loading was increased from 3.5% to 8% (w/w). The effect of increasing lipase loading is illustrated in Fig. 2C. The content of diester and the reaction rate were maximized when the lipase concentration (8%) was the highest. Under these conditions, the reaction reached equilibrium quickly, with a diester content of 80%. This illustrates that high catalyst loadings are necessary for the synthesis of a dipolar ester with hydrophilic and hydrophobic compounds, as has been reported previously.²⁸ As shown in Fig. 2D, the bell-type curve of monoester means that the diester is generated stepwise. Before 1.25 h, due to the high isosorbide concentration, the system contains a large amount of monoester (28%). However, only a small amount of isosorbide caprylate diester formed in this time (9%). As the concentration of monoester increases, the production rate of diester also increases and ultimately reaches an 81% content.

(C) Determination of activation energy

The activation energy for the hydrolysis and the esterification of di-substituted isosorbide esters by Novozym 435 were studied under optimized reaction conditions over a temperature range of 40–60 °C using the Arrhenius equation as given in eqn (1), which represents the temperature dependence of reaction rate:where k is the reaction rate constant, A₀ is a pre-exponential factor, E is the activation energy (kJ mol⁻¹), R is the gas constant (8.314 J mol⁻¹ K⁻¹), and T is the temperature (K). The activation energy for the hydrolysis of the diester was calculated using reaction temperatures of 30, 43 and 50 °C and 29.3 g diester, 3 g water and 1.97 g Novozym 435 monitored from 0 to 60 min. The activation energy for the synthesis of diester was calculated using reaction temperatures of 40, 50 and 60 °C and 19.44 g caprylic acid, 9.85 g isosorbide and 1.97 g Novozym 435 monitored from 0 to 120 min. In each case, the diester content (%) was calculated by GC.

The diester content was plotted with respect to time to estimate the rate constant (k) at different temperatures for hydrolysis and for the synthesis of diester, respectively. The linear Arrhenius plot was used to calculate the activation energy (E) for both the hydrolysis and synthesis of isosorbide diester with lipase according to eqn (1) using the values given in Table 2. Activation energy (E) of 25.51 kJ mol⁻¹ and 35.65 kJ mol⁻¹ were calculated for the hydrolysis and synthesis of di-substituted isosorbide, respectively. The E for hydrolysis of the diester was higher than that for synthesizing diester, and there was no hydrolysis reaction without enzyme. This indicates that the formation of diester requires more energy than the diester hydrolysis reaction, and that the hydrolysis of diester is much easier than its synthesis.²⁹

Table 2 Rate constant (k) for hydrolysis and synthesis of diester by Novozym 435 at different temperatures

	T (K)	k (mmol h^−1)	ln k	Ea (kJ mol^−1)
Hydrolysis reaction	303	10.91	2.39	25.51
	316	17.29	2.85
	323	20.19	3.01
Esterification	313	1.01	0.0075	35.65
	323	1.74	0.55
	333	2.29	0.83

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As shown in Fig. 3, in the process of isosorbide diester hydrolyzation, the content of diester decreased quickly with reaction time, the content of caprylic acid and monoester also increased quickly. However, the reaction reached equilibrium 50 min later when the content of the caprylic acid, monoester, and diester became about 30%, 51%, and 18%. Therefore the monoester is stable and does not undergo hydrolysis to caprylic and isosorbide easily. The contents in the hydrolysis system and the activation energy of the hydrolysis reaction demonstrate that the diester can hydrolyze to the monoester and caprylic acid easily, whereas the synthesis of diester is more difficult. Together, these results demonstrate that synthesizing isosorbide dicaprylate ester from the corresponding monoester is the critical step in the synthesis.

Fig. 3 Effect of hydrolysis reaction on caprylic acid, monoester, isosorbide, and diester content. Reaction conditions: 29.3 g diester, 3 g water, 1.97 g Novozym 435, at 300 rpm for 105 min at 60 °C.

(D) Reusability of enzyme

The operational stability of the lipase is very important for the practicability of the synthesis of isosorbide diester in industrial applications. The reusability of the lipase used in this reaction was investigated. Multiple reactions were conducted at 50 °C for 24 h in a solvent-free system. After the completion of each run, the catalyst was filtered and washed three times with ethyl acetate to remove the residual substrates and the reaction was repeated using fresh substrates. As shown in Fig. 4, the contents of isosorbide and caprylic acid were approximately zero and below 5% respectively after reused 16 batches. This demonstrates that the conversions in these reactions were higher than 95%. The contents of monoester and diester in these cycles were balanced at about 10% and 80%. It is demonstrated that the product water can be removed efficiently, and bubbling dried air to the system is an effective method for esterification.³⁰

Fig. 4 Repetitive synthesis of isosorbide dicaprylate ester by Novezym 435. Reaction conditions: 9.855 g isosorbide, 19.44 g caprylic acid, 1.97 g Novozym 435, at 300 rpm for 24 h at 60 °C.

(E) Properties of plasticizer on PVC

TGA was used to study the thermal degradation of PVC plasticized with isosorbide diester. The parameters and curves of TGA are presented in Fig. 5. As shown in Fig. 5, all of the PVC blends were thermally stable in N₂ atmosphere below 90 °C and were divided into a three-stage thermal degradation process above the temperature. The first stage degradation at around 90–260 °C is corresponding to evaporation and decomposition of water, HCl and small molecules. The second stage at around 260–452 °C is the fastest and could be attributed to the formation and stoichiometric elimination of HCl. The last stage at above 452 °C is attributed to cross linking containing C=C bonds. Thermal degradation of polyenes involves cyclization and splitting of chains.

Fig. 5 TGA curves of PVC blends.

Mechanical properties of PVC blends were characterized by using E43.104 Universal Testing Machine (MTS Instrument Crop., China) according to GB/T 1040.1-2006 (China) under ambient conditions. Table 3 summarizes the values of the mechanical properties of PVC blends. The addition of isosorbide diester in the PVC blends caused increment in tensile strength and reduction in elongation at break. It is accordance with the common rule that the plasticizer can increase the elongation at break. The reason may be that the compatibility between plasticizer molecular and PVC molecular is well, so isosorbide diester could be used as an effective plasticizer for PVC.

Table 3 Mechanical properties of PVC blends

Yield stress	Elongation	Rupture stress	Elongation at break
16.525 MPa	102.128%	14.66 MPa	236.704%

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Maximum load	Tensile strength	Largest strain elongation	Given elongation stress
106.39 N	24.06 MPa	227.08%	16.36 Mpa

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(F) Sub-chronic toxicity

The safety of isosorbide ester was evaluated by testing the sub-chronic toxicity in mice with a 28 day treatment with different doses of isosorbide diester.

(1) Body and behavior observation. As shown in Table 4, the appearance, activities, and psychosis of all the mice in both isosorbide diester treatment and control groups were all normal, and there were no abnormal discharge and death. There were no significant differences in mice body weight between control and treatment groups.

Table 4 The body and behavior observation of mice

	Mice		Appearance	Activities	Psychosis	Abnormal discharge	Death	Weight (g)
	Female	Male						Start	End
Control (H)	3		Normal	Normal	Normal	None	None	19.0	30.9
Control (H)		3	Normal	Normal	Normal	None	None	18.3	40.8
Control (M)	3		Normal	Normal	Normal	None	None	19.1	32.2
Control (M)		3	Normal	Normal	Normal	None	None	19.7	40.5
Control (L)	3		Normal	Normal	Normal	None	None	18.8	32.4
Control (L)		3	Normal	Normal	Normal	None	None	19.2	40.7
Sample (H)	3		Normal	Normal	Normal	None	None	19.4	31.9
Sample (H)		3	Normal	Normal	Normal	None	None	19.6	39.6
Sample (M)	3		Normal	Normal	Normal	None	None	18.6	30.3
Sample (M)		3	Normal	Normal	Normal	None	None	18.6	40.3
Sample (L)	3		Normal	Normal	Normal	None	None	19.0	32.9
Sample (L)		3	Normal	Normal	Normal	None	None	19.3	40.0

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(2) Hematological, liver and kidney analysis. The analysis of blood parameters is relevant to risk evaluation because the changes in the hematological system have a higher predictive value for human toxicity when the data are translated from animal studies.³¹

As shown in Table 5, at the end of the experiment (after 28 days), the WBC, RBC, HGB and PLT showed no significant differences between control and isosorbide diester treatment groups. It is indicated that the isosorbide diester did not affect the hematological system of mice.

Table 5 Hematological analysis

	Mice		WBC (×10^9 L^−1)	RBC (×10^12 L^−1)	HGB (×g L^−1)	PLT (×10^9 L^−1)
	Female	Male
Control (H)	3		5.4 ± 0.7	5.5 ± 0.5	142.0 ± 14.7	182.0 ± 36.7
Control (H)		3	5.9 ± 0.8	5.8 ± 0.3	134.0 ± 13.9	175.6 ± 29.9
Control (M)	3		5.7 ± 1.5	4.7 ± 0.5	134.7 ± 13.6	151.7 ± 26.7
Control (M)		3	5.9 ± 0.4	6.2 ± 0.7	133.3 ± 10.3	174.7 ± 15.6
Control (L)	3		5.7 ± 0.6	4.6 ± 0.6	142.3 ± 6.7	178.3 ± 12.0
Control (L)		3	5.9 ± 0.5	5.9 ± 0.2	130.0 ± 2.0	173.6 ± 8.6
Sample (H)	3		5.1 ± 0.7	5.8 ± 0.4	135.6 ± 14.4	167.0 ± 16.6
Sample (H)		3	6.0 ± 1.7	5.1 ± 0.8	134.0 ± 11.5	172.6 ± 16.6
Sample (M)	3		4.8 ± 0.3	5.5 ± 0.5	135.3 ± 12.7	161.0 ± 14.2
Sample (M)		3	4.5 ± 0.9	6.7 ± 1.2	133.0 ± 15.7	157.0 ± 2.6
Sample (L)	3		5.3 ± 0.6	6.5 ± 0.5	129.7 ± 2.5	155.3 ± 17.6
Sample (L)		3	5.1 ± 0.6	5.8 ± 0.3	131.7 ± 6.1	153.7 ± 16.9

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The analysis of liver and kidney are shown in Table 6. There were no significant differences in liver and kidney after treatment with isosorbide diester. After 28 days treatment with isosorbide diester, the liver and kidney of the treatment groups were almost the same with those of the control groups, illustrating that the isosorbide diester was not harmful to mice liver and kidney.

Table 6 Liver and kidney analysis

	ALT (U L^−1)	BUN (mmol L^−1)	PT (S)	APTT (S)	TT (S)	FIB (g L^−1)
Control (H)	24.0 ± 7.5	6.6 ± 0.8	10.7 ± 0.6	27.3 ± 2.5	16.1 ± 1.2	2.4 ± 0.5
Control (H)	26.3 ± 4.0	6.4 ± 1.2	11.1 ± 1.2	25.6 ± 3.0	16.9 ± 1.0	2.3 ± 0.3
Control (M)	19.3 ± 3.8	5.9 ± 1.2	10.8 ± 1.0	26.9 ± 3.7	17.4 ± 3.2	2.2 ± 0.2
Control (M)	25.1 ± 6.6	4.9 ± 1.0	11.7 ± 0.4	26.3 ± 2.4	16.2 ± 1.7	2.3 ± 0.3
Control (L)	25.3 ± 8.5	6.1 ± 0.8	11.2 ± 1.0	26.5 ± 2.2	17.2 ± 1.3	2.2 ± 0.3
Control (L)	25.7 ± 6.0	5.9 ± 0.6	11.5 ± 0.5	27.3 ± 1.4	16.2 ± 0.8	2.4 ± 0.4
Sample (H)	26.0 ± 7.8	5.3 ± 1.5	10.9 ± 0.6	27.2 ± 1.7	16.9 ± 1.0	2.1 ± 0.3
Sample (H)	23.3 ± 1.5	6.0 ± 1.0	11.7 ± 0.4	26.8 ± 1.0	16.6 ± 0.6	2.3 ± 0.4
Sample (M)	22.3 ± 2.5	5.4 ± 0.5	11.5 ± 0.5	26.7 ± 1.1	16.6 ± 1.5	2.5 ± 0.2
Sample (M)	25.0 ± 5.0	5.3 ± 1.5	11.0 ± 1.0	26.6 ± 1.3	16.3 ± 2.3	2.4 ± 0.6
Sample (L)	24.3 ± 5.1	5.6 ± 0.5	11.1 ± 1.2	26.8 ± 2.0	17.5 ± 0.5	2.3 ± 0.5
Sample (L)	21.7 ± 3.8	6.3 ± 0.6	11.3 ± 0.8	28.2 ± 1.2	17.0 ± 1.0	2.0 ± 0.1

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(3) Organ histopathology. As shown in Fig. 6, after treatment with isosorbide diester, there were no significant pathological changes in liver. The liver cells in the liver tissue were normal and the liver lobules were tidy and complete. Similarly, isosorbide diester treatment didn't induce significant changes in testis of mice. The chamber of the seminiferous tube arranged regularly, the wall of the basement membrane was integrity, and the lumens were full of sperms. In summary, isosorbide diester treatment did not induce significant histopathological changes in mice.

Fig. 6 Organ histopathology photograph. (A) The liver structure of control mice (H). (B) The liver structure of isosorbide diester treatment mice (H). (C) The kidney structure of control mice (H). (D) The kidney structure of isosorbide diester treatment mice (H).

IV. Conclusions

In this study, isosorbide diester was synthesized with middle chain caprylic acid under solvent-free conditions with low levels of monoester formation. The critical parameters in the process of esterification were discussed and the optimal conditions were determined. Based on activation energy analysis, it was determined that synthesizing diester from the monoester is the limiting step in the synthesis. The discovery of the limiting step is significant for industrial production. The property of isosorbide diester as plasticizer on PVC was determined. Furthermore, the sub-chronic toxicity of isosorbide diester was tested in mice, and the results showed that the diester was biosafe. So isosorbide diester has great potential for industrial application as a plasticizer.

Acknowledgements

This work was supported by the National Basic Research Program of China (973 program) (2013CB733600, 2012CB725200), the National Nature Science Foundation of China (21576019, 21436002), National High-Tech R&D Program of China (863 Program) (2014AA022100), and Ningbo Natural Science Foundation of China (2011A610028).

Notes and references

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