Kinetics and thermodynamics study on the extraction process of 10-DAB Ⅲ and Taxol from the branches and leaves of <i>Taxus Yunnanensis</i>

BAI Huilian; ZHU Jingbo

doi:10.11833/j.issn.2095-0756.20250193

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BAI Huilian, ZHU Jingbo. Kinetics and thermodynamics study on the extraction process of 10-DAB Ⅲ and Taxol from the branches and leaves of Taxus Yunnanensis[J]. Journal of Zhejiang A&F University, 2025, 42(X): 1−11 doi: 10.11833/j.issn.2095-0756.20250193

Citation:

BAI Huilian, ZHU Jingbo. Kinetics and thermodynamics study on the extraction process of 10-DAB Ⅲ and Taxol from the branches and leaves of Taxus Yunnanensis[J]. Journal of Zhejiang A&F University, 2025, 42(X): 1−11 doi: 10.11833/j.issn.2095-0756.20250193

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Kinetics and thermodynamics study on the extraction process of 10-DAB Ⅲ and Taxol from the branches and leaves of Taxus Yunnanensis

DOI: 10.11833/j.issn.2095-0756.20250193

BAI Huilian,
ZHU Jingbo^,

School of Food Science, Dalian Polytechnic University, Dalian 116023, Liaoning, China

Received Date: 2025-03-12
Accepted Date: 2025-10-20
Rev Recd Date: 2025-10-15

Abstract

Objective The objective of this study is to investigate the kinetics, thermodynamics, and mass transfer mechanism of extracting 10 deacetylbaccatin Ⅲ (10-DAB Ⅲ) and paclitaxel (Taxol) from the branches and leaves of Taxus yunnanensis by mechanical stirring assistance and water bath heating. Method The first-order and second-order extraction kinetics models were fitted, and the empirical diffusion model was used to estimate temperature-dependent variation laws of the diffusion coefficient (D_e), mass transfer coefficient (K_T) and Biot number (B_iot) of 10-DAB Ⅲ and Taxol in T. yunnanensis, so as to explain the mass transfer behavior during the extraction process. Result (1) The activation energy for the activation of 10-DAB Ⅲ and Taxol by mechanical stirring-assisted extraction was 40.745 and 18.762 kJ·mol⁻¹ respectively, higher than those obtained by water bath extraction (36.428 and 16.668 kJ·mol⁻¹ respectively). The extraction rates of both mechanical stirring-assisted and water bath heating extraction methods increased with the rise of extraction temperature. Among the 2 extraction methods, 10-DAB Ⅲ and Taxol had the highest extraction rates at an extraction temperature of 328 K. The mechanical stirring-assisted extraction rates of the 2 components were 84.40% and 93.88%, respectively, while the water bath heating extraction rates were 71.62% and 93.35% respectively. (2) The results of thermodynamic study on the extraction process indicated that the enthalpy change ($ \Delta H $) and entropy change ($ \Delta S $) values for the extraction of 10-DAB Ⅲ and Taxol by mechanical stirring-assisted extraction and water bath heating were both positive, while the Gibbs free energy ($ \Delta G $) was negative, indicating that the extraction process was endothermic, turbulent, and spontaneous. (3) The results of estimating the diffusion coefficient (D_e), mass transfer coefficient (K_T), and B_iot with the temperature variation using the empirical diffusion model showed that D_e and K_T of 10-DAB Ⅲ and Taxol extracted by mechanical stirring assistance and water bath heating increased with temperature rise, while Biot decreased with temperature rise and was far greater than 100, indicating that the extraction process of 10-DAB Ⅲ and Taxol belonged to internal diffusion. Conclusion The second-order extraction kinetics model better aligns with method of extracting 10-DAB Ⅲ and Taxol from T. yunnanensis by mechanical stirring assistance, while the first-order model is suitable for the slow extraction of 10-DAB Ⅲ and Taxol by water bath. Both the logarithm (lnk) of the solute extraction rate (k) and the logarithm (lnK_e) of the equilibrium rate constant (K_e) exhibit a linear relationship with the reciprocal of the extraction temperature. The extraction process of 10-DAB Ⅲ and Taxol from T. yunnanensis belongs to internal diffusion. [Ch, 6 fig. 3 tab. 32 ref.]
- 10-deacetylbaccatin Ⅲ,
- Taxol,
- dynamics,
- thermodynamics,
- Taxus Yunnanensis

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沈阳化工大学材料科学与工程学院沈阳 110142

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紫杉醇(Taxol)由美国化学家WANI等^[1]于1971年在太平洋红豆杉Taxus brevifolia中发现。美国食品药品管理局(FDA)分别于1992、1994年批准了天然紫杉醇和基于10-去乙酰基巴卡亭Ⅲ(10-DAB Ⅲ)的半合成紫杉醇用于临床^[2−3]。1998、2010年基于10-DAB Ⅲ半合成的多烯紫衫醇、卡巴他赛等抗癌药物用于临床^[4]。这些药物的抗癌机制是选择性破坏微管、抑制有丝分裂等使癌细胞死亡，已被用于乳腺癌、卵巢癌、宫颈癌和前列腺癌等癌症的治疗^[5−8]。红豆杉植物枝叶和树皮是天然Taxol和10-DAB Ⅲ的主要来源，但红豆杉植物中Taxol质量分数及提取率均极低。太平洋红豆杉树皮中Taxol质量分数为0.010 0%~0.080 0%，平均分离得率仅为0.014%~0.017%^[9]。Taxol在云南红豆杉T. yunnanensis和东北红豆杉T. cuspidata树皮中质量分数为0.005 0%~0.030 0%，枝叶中质量分数0.001 3%~0.013 7%^[10]。随着Taxol及其他半合成紫杉烷类药物如多烯紫衫醇、卡巴他赛等的广泛应用，仅从红豆杉植物中提取的Taxol已经不能满足日益增长的医药市场需求，而合成其他紫杉烷类药物需要大量的10-DAB Ⅲ。虽然已经对从生物催化合成和从红豆杉植物的内生菌中发现紫杉烷类化合物及从内生菌中的分离10-DAB有所研究，但离产业化需要仍有距离^[11−12]。以Taxol质量分数为红豆杉中3~10倍的10-DAB Ⅲ 作为原料进行半合成是目前生产Taxol及其他紫杉烷类药物的主要途径。1988年DENIS等^[13]报道了有效获取天然紫杉醇的方法。半合成、全合成方法由OJIMA等^[14]和HOLTON等^[15]在1992年和1994年进行报道。从红豆杉植物中提取10-DAB Ⅲ和Taxol对后续采用10-DAB Ⅲ半合成Taxol及其他紫杉烷类药物具有重要意义。

植物活性物质提取是指应用溶剂浸提法将植物中的可溶性组分提取出来的过程，常采用静态和搅拌提取2种方式。以往的植物成分提取多集中于从工艺角度进行比较研究，确定最佳工艺，很少涉及从传质及动力学角度进行深入探讨。粒径较小时搅拌提取优于微波辅助提取，具有操作简便省时、高效、节能的特点^[16]。机械搅拌辅助提取因其方便快速、提取率高等优点被广泛应用在天然产物的提取。机械搅拌辅助提取可以结合罐组式逆流提取，具有溶媒和能源用量少，药材受热均匀、受热时间短，有效成分转移率高，可不间断连续生产，可更好地保留热敏性有效成分等优点^[17−18]。对于这种基于搅拌辅助提取连续工业化的提取设计，传质规律研究更具有现实意义。

随着提取工艺的不断优化，红豆杉植物中10-DAB Ⅲ和Taxol的提取过程动力学研究对紫杉烷类化合物工业化生产具有重要意义，目前尚未见关于云南红豆杉10-DAB Ⅲ和Taxol提取动力学相关报道。本研究通过分析机械搅拌辅助和水浴加热提取紫杉烷类化合物过程中的动力学、菲克第二扩散定律和热力学，估算提取过程中的活化能、热力学参数、扩散系数、传质系数和毕奥数，并使用标准差和相关系数表示动力学模型与实验动力学数据的拟合状况，进一步阐述植物成分提取的传质机制，为相关植物功能成分的提取研究和工业化生产提供理论基础。

1. 材料与方法

1.1. 仪器与材料

仪器包括AC Chrom S6000型高效液相色谱分析仪(北京华谱新创科技有限公司)、600Y型多功能粉碎机(广州旭朗机械设备有限公司)、W2S型升降恒温水浴锅(巩义市英裕高科仪器厂)、JJ-1型精密增力电动机械搅拌器(常州国华电器有限公司)、BS223S型电子天平(北京赛多利斯仪器系统有限公司)、KQ-500B型超声波清洗器(巩义市英裕高科仪器厂)。

材料分别为10-DAB Ⅲ和Taxol标准品(纯度≥99%)购于西安天丰生物科技有限公司；乙腈(色谱纯)、甲醇(纯度≥98%)购于天津康科德科技有限公司；乙腈(分析纯)、甲醇(分析纯)购于天津市科密欧化学试剂有限公司；云南红豆杉枝叶采自云南保山市。

1.2. 样品制备

将云南红豆杉枝叶在粉碎机中粉碎和筛分，原料粒度为150~250 μm。过筛后的原料在50 ℃烘箱中干燥至恒量并保存于玻璃干燥器中。

1.3. 色谱条件

采用C18色谱柱(4.6 mm×250 mm，5 μm)进行分析，流动相A为乙腈，流动相B为超纯水。梯度洗脱程序如下：0~20 min：15%~50%A；20~50 min：50%~100%A；50~65 min：100%A；65~67 min：100%~15%A；67~80 min：15%A。流动相流速为0.8 mL·min⁻¹，检测波长为227 nm，柱温为30 ℃，进样量为10 μL。

1.4. 10-DAB Ⅲ和Taxol分析的标准品溶液制备

称取适量10-DAB Ⅲ和Taxol标准品溶于甲醇，配制不同质量浓度梯度的标准溶液，用0.22 μm微孔滤膜过滤，采用高效液相色谱-二极管阵列检测器(HPLC-DAD)测定并得到10-DAB Ⅲ和Taxol标准曲线。10-DAB Ⅲ标准曲线的回归方程为：y=1.798×10⁷x+2.353×10⁵，R²=0.998 9；Taxol标准曲线的回归方程为：y=2.707×10⁷x+1.649×10³，R²=0.999 8。其中：x为标准品质量浓度(mg·mL⁻¹)；y为采用HPLC-DAD测得的峰面积(mV·s)，分别用于后续液体提取物中10-DAB Ⅲ和Taxol质量浓度的测定。

云南红豆杉枝叶中10-DAB Ⅲ和Taxol的质量浓度经液相分析和标准曲线方程计算。公式如下：

式(1)~(2)中：C₁为样品质量浓度(mg·mL⁻¹)；C₂为标准品质量浓度(mg·mL⁻¹)；A₁为样品峰面积(mV·s)；A₂为标准品峰面积(mV·s)；M为样品总量(mg)；V为样品体积(mL)。

1.5. 提取方法

1.5.1. 机械搅拌辅助提取

称取50 g云南红豆杉原料加入250 mL甲醇，设定电动搅拌器转速为136 r·min⁻¹，以1∶5料液比进行机械搅拌辅助提取，在搅拌提取时间为2~120 min，提取溶剂甲醇沸点为64.7 ℃，提取温度分别为15、35、55 ℃。提取液中10-DAB Ⅲ和Taxol质量浓度通过标准曲线计算得到。

1.5.2. 水浴提取

称取5 g云南红豆杉于250 mL锥形烧瓶中，以云南红豆杉滞液量1.7 mL·g⁻¹甲醇浸润，以1∶10料液比进行水浴提取，并控制提取时间为5~240 min，提取溶剂甲醇沸点为64.7 ℃，提取温度分别为35、45、55 ℃。提取液中10-DAB Ⅲ和Taxol质量浓度通过标准曲线计算得到。

1.6. 传质研究

1.6.1. 动力学研究

参照文献[19−20]，基于一阶动力学模型和二阶动力学模型分别对2种云南红豆杉枝叶中紫杉烷类物质的提取动力学进行研究。一阶提取模型公式(3)可写成公式(4)；二阶提取模型公式(5)可以写成公式(6)，也可以写成如公式(7)所示线性形式。具体公式如下：

式(3)~(7)中：C_t为时间t (s)云南红豆杉提取物溶液中紫杉烷类物质的质量浓度(mg·mL⁻¹)；$ {C}_{0} $为云南红豆杉提取物溶液中紫杉烷类物质的初始质量浓度(mg·mL⁻¹)； C_∞、$ {C}_{\mathrm{e}} $为一阶提取模型和二阶提取模型紫杉烷类物质的平衡质量浓度(mg·mL⁻¹)；t为时间(s)；k₁为一级速率常数(s⁻¹)；k₂为二级速率常数(mL·mg⁻¹·s⁻¹)，提取速率常数受温度的影响。

根据Arrhenius方程可以推导出热量与提取速率常数之间的关系，利用公式(8)计算活化能^[21]。

式(8)中：k为提取速率常数(mL·mg⁻¹·min⁻¹)；E_a为活化能(kJ·mol⁻¹)；T为温度(K)；R为通用气体常数(8.314 J·mol⁻¹·K⁻¹)；$ {k}_{0} $为指前因子，也称为频率因子。

1.6.2. 热力学研究

热力学参数对于确定反应发生的能量变化至关重要。应用Van’t Hoff方程计算化合物的标准焓和标准熵，确定化学提取过程，在恒温恒压的热力学系统中计算基本组分所得吉布斯自由能。焓(ΔH，kJ·mol⁻¹)和熵(ΔS，kJ·mol⁻¹·K⁻¹)的由公式(9)通过绘制平衡常数速率(lnK_e)与温度(1/$ T $)之间的曲线计算得到。吉布斯自由能(ΔG)由公式(10)计算^[21]：

式(9)~(10)中：K_e是平衡常数速率，即已提取的紫杉烷类物质质量浓度与未提取的残留紫杉烷类物质质量浓度的关系^[21]：

式(11)中：Y_s为饱和10-DAB Ⅲ或Taxol产量(mg·g⁻¹)；Y_max为最大10-DAB Ⅲ或Taxol产量(mg·g⁻¹)。

1.6.3. 传质研究

基于以下假设^[22]和Fick第二定律扩散模型研究10-DAB Ⅲ和Taxol的有效扩散系数。①10-DAB Ⅲ和Taxol的提取被认为是由细胞壁中10-DAB Ⅲ、Taxol的快速溶解和溶质从固体颗粒缓慢扩散到提取液中的2步过程。②10-DAB Ⅲ和Taxol最初均匀地包含在对称的球体固体中，粒子平均直径为200 μm。③10-DAB Ⅲ和Taxol分子的转运是一种扩散现象。它由恒定的且与时间无关的扩散系数(D)描述。10-DAB Ⅲ、Taxol和其他化合物的扩散是同时发生且没有相互作用的。④在机械搅拌器的帮助下溶液是完全紊流和混合的。忽略液相中传质阻力，10-DAB Ⅲ和Taxol的质量浓度仅取决于时间。⑤在界面处，假设孔隙中液体与外部溶液的溶质平衡质量浓度相等。⑥水浴提取实验的平衡质量浓度是用时间为240 min来计算的。机械搅拌提取的10-DAB Ⅲ和Taxol化合物无降解现象。

固体和液体提取过程中的毕奥数(B_iot)是确定和验证传质内外部阻力相对量的主要方法^[23]。球形颗粒提取化合物根据Fick第二定律。

式(12)中：M为云南红豆杉颗粒中紫杉烷类化合物的质量浓度(kg·m⁻³)；r为云南红豆杉颗粒与球形红豆杉颗粒中心的距离(m)；D_e为紫杉烷类化合物的有效扩散系数；t为时间(s)。

参考文献[24]，公式(12)在初始条件和边界条件下的解分别见公式(13)和(14)：

式(13)~(14)中：Y_s为饱和目标化合物产量(mg·g⁻¹)；Y_t为t时刻的目标化合物产量(mg·g⁻¹)；r_p为颗粒半径(m)；D_e为扩散系数(m²·s⁻¹)。

B_iot根据公式(15)计算。传质系数根据各温度值下ln[C_∞/(C_∞－C_t)]与时间的关系应用公式(16)计算^[25]：

式(15)~(16)中：K_T为传质系数(m·s⁻¹)；A为粒子总表面面积(m²)；V_s为溶液体积(m³)。

1.7. 数据分析

使用WPS 2021处理数据，采用SPSS 22进行单因素分析(one-way ANOVA)和多重比较(邓肯法)，显著性水平为0.05，使用Origin 2017作图。

3. 讨论

3.1. 动力学模型的比较

植物成分提取动力学研究是指导工业生产的理论基础。植物活性成分提取的动力学模型大部分基于一阶动力学模型和二阶动力学模型。它们可简化植物活性成分提取过程中所涉及的生物、化学、物理及其相互影响的复杂过程，模型构式可线性化处理而使得参数求解简单，有利于促使模型应用于包括食品、植物、中药有效成分提取及组合提取工艺，如罐组逆流提取等^[30−32]。本研究表明：一阶动力学模型和二阶动力学模型均可以用来描述植物中机械搅拌辅助提取和水浴提取动力学。针对机械搅拌辅助提取，二阶动力学模型具有更好的拟合度(R²≥0.997)，水浴提取既适用于一阶动力学模型(R²≥0.931)也适用于二阶动力学模型(R²≥0.923)，但较一阶动力学模型拟合度差。一阶动力学模型不能很好地描述成分提取过程^[31]。

3.2. 提取温度对植物成分提取的影响

表1中较高的决定系数(R²≥0.923)显示温度是影响植物中活性成分提取的重要因素，提取速率常数随着温度升高而升高，在温度较高时可以获得较好的提取率。10-DABⅢ和Taxol的机械搅拌辅助提取率分别为84.40%和93.88%；水浴提取的提取率分别为71.62%和93.35%。

本研究结果表明：温度升高可以提高10-DAB Ⅲ和Taxol的提取速率，动力学参数由Arrhenius方程确定，且提取速率与温度关系符合Arrhenius方程。在后续研究中仍需进一步考察搅拌速率与提取速率间的定量关系。动力学模型拟合得到提取动力学常数与温度符合Arrhenius方程，间接表明模型拟合结果是合理的。

本研究结果表明：模型使用广，可为提高云南红豆杉中10-DAB Ⅲ和Taxol的提取和后续罐组逆流联合机械搅拌辅助提取的动力学模型提供理论依据，为工业化生产提供参数依据。

4. 结论

机械搅拌辅助提取符合二阶动力学方程，水浴提取符合一阶动力学方程。动力学分析结果表明：10-DAB Ⅲ和Taxol在机械搅拌辅助提取和水浴提取中的提取速率均随温度升高而增大，且呈线性关系(R²＞0.905 5)。热力学分析结果显示：机械搅拌辅助提取和水浴提取的平衡常数速率 (K_e)随着温度升高而增大呈线性关系(R²＞0.921 6)。通过Van’t Hoff方程得到2种提取方法的焓变值为正值(ΔH＞0)，熵变值为正值(ΔS＞0)，吉布斯自由能为负值(ΔG＜0)，表明提取过程是吸热的、湍流的且2种提取过程是自发进行的。通过Fick第二定律得到2种提取方法的有效扩散系数(D_e)和传质系数(K_T)随温度升高而增大，而B_iot随温度升高呈下降趋势且远大于100，表明2种提取方法均为内扩散。

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提取方法	紫杉烷类化合物	T /℃	料液比	一阶动力学模型			二阶动力学模型
提取方法	紫杉烷类化合物	T /℃	料液比	k₁/min	R²	活化能/(kJ·mol⁻¹)	k₂/(×10⁻⁵ L·mg⁻¹·s⁻¹)	R²
机械搅拌辅助提取	10-DAB Ⅲ	15	1∶5	0.022±0.002	0.958	40.745±1.855	1.860±0.046	0.999 8
		35		0.040±0.002	0.967		1.947±0.073	0.999 7
		55		0.178±0.002	0.950		2.641±0.026	0.999 5
	Taxol	15		0.024±0.002	0.982	18.762±1.211	5.676±0.051	0.997 0
		35		0.031±0.001	0.996		6.445±0.233	0.999 0
		55		0.064±0.000	0.982		7.538±0.016	0.998 0

水浴提取	10-DAB Ⅲ	35	1∶10	7.744±0.089	0.979	36.428±0.282	7.273±0.353	0.970 0
		45		13.723±0.028	0.931		8.785±0.008	0.962 0
		55		18.388±0.091	0.988		14.319±0.528	0.986 0
	Taxol	35		7.537±0.012	0.956	16.668±0.256	0.300±0.0001	0.923 0
		45		9.792±0.011	0.950		0.315±0.0001	0.958 0
		55		11.201±0.009	0.996		0.348±0.0004	0.949 0
说明：k₁为一级速率常数；k₂为二级速率常数。

提取方法	紫杉烷类化合物	T /℃	料液比	K_e	ΔH/(kJ·mol⁻¹)	ΔS/(J·mol⁻¹·K⁻¹)	ΔG/(kJ·mol⁻¹)	R²
机械搅拌辅助提取	10-DAB Ⅲ	15	1∶5	1.756±0.001	38.840±0.305	138.240±1.034	−0.976±0.007	0.922
		35	1∶5	3.088±0.005			−3.741±0.013
		55	1∶5	12.977±0.299			−6.506±0.034
	Taxol	15	1∶5	3.256±0.053	30.359±0.186	115.045±0.460	−2.774±0.044	0.997
		35	1∶5	6.926±0.207			−5.075±0.044
		55	1∶5	15.330±0.094			−7.376±0.035

水浴提取	10-DAB Ⅲ	35	1∶10	2.240±0.003	4.981±0.031	22.819±0.087	−1.591±0.006	0.958
		45	1∶10	2.331±0.004			−2.047±0.004
		55	1∶10	2.523±0.002			−2.504±0.002
	Taxol	35	1∶10	2.338±0.002	71.958±0.291	239.765±0.942	−1.889±0.001	0.949
		45	1∶10	4.019±0.005			−4.287±0.009
		55	1∶10	13.068±0.101			−6.685±0.018
说明：K_e为平衡常数速率；ΔH为焓变；ΔS为熵变；ΔG为Gibbs自由能变化。

提取方法	提取物	提取温度/℃	料液比	D_e/(×10⁻¹³ m²·s⁻¹)	B_iot/×10⁸	K_T/(m·s⁻¹)
机械搅拌辅助提取	10-DAB Ⅲ	15	1∶5	3.616±0.495	1.919±0.088	0.692±0.063
		35	1∶5	6.770±0.351	4.361±0.065	2.953±0.197
		55	1∶5	30.169±0.394	3.551±0.328	10.720±1.129
	Taxol	15	1∶5	0.127±0.008	61.933±2.211	0.283±0.021
		35	1∶5	4.833±0.251	2.191±0.163	1.057±0.024
		55	1∶5	11.972±0.011	1.859±0.002	2.225±0.005

提取方法	提取物	提取温度/℃	料液比	D_e/(×10⁻¹³ m²·s⁻¹)	B_iot/×10⁵	K_T/(×10⁻⁴m·s⁻¹)
水浴提取	10-DAB Ⅲ	35	1∶10	1.308±0.014	3.865±0.116	5.057±0.097
		45	1∶10	2.297±0.036	2.650±0.027	6.155±0.062
		55	1∶10	3.109±0.022	2.528±0.018	7.857±0.110
	Taxol	35	1∶10	0.933±0.003	3.539±0.003	3.300±0.007
		45	1∶10	1.655±0.002	2.669±0.006	4.417±0.006
		55	1∶10	1.894±0.002	3.048±0.006	5.771±0.007
说明：D_e. 扩散系数；K_T. 传质系数。