Int. J. Mol. Sci. 2014, 15, 16302-16319; doi:10.3390/ijms150916302 OPEN ACCESS
International Journal of
Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article
Extraction, Preliminary Characterization and Evaluation of in Vitro Antitumor and Antioxidant Activities of Polysaccharides from Mentha piperita Xin Liu 1,†, Zhen-Liang Sun 2,†, Ai-Rong Jia 1, Ya-Ping Shi 1, Rui-Hong Li 3,* and Pei-Ming Yang 2,* 1
2
3
†
Biology Institute of Shandong Academy of Sciences/Key Laboratory for Applied Microbiology of Shandong Province, Jinan 250014, China; E-Mails: [email protected] (X.L.); [email protected] (A.-R.J.); [email protected] (Y.-P.S.) State Key Laboratory of New Drug & Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China; E-Mail: [email protected] Fengxian Hospital Affiliated to Southern Medical University, 6600 NanFeng Road, Shanghai 201499, China These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: [email protected] (R.-H.L.); [email protected] (P.-M.Y.); Tel./Fax: +86-21-5742-3899 (R.-H. L.); +86-21-6247-9808 (P.-M.Y.). Received: 26 May 2014; in revised form: 25 August 2014 / Accepted: 3 September 2014 / Published: 15 September 2014
Abstract: This study describes the extraction, preliminary characterization and evaluation of the in vitro antitumor and antioxidant activities of polysaccharides extracted from Mentha piperita (MPP). The optimal parameters for the extraction of MPP were obtained by Box-Behnken experimental design and response surface methodology (RSM) at the ratio of water to raw material of 20, extraction time of 1.5 h and extraction temperature at 80 °C. Chemical composition analysis showed that MPP was mainly composed of glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the molecular weight of its two major fractions were estimated to be about 2.843 and 1.139 kDa, respectively. In vitro bioactivity experiments showed that MPP not only inhibited the growth of A549 cells but possessed potent inhibitory action against DNA topoisomerase I (topo I), and an appreciative antioxidant action as well. These results indicate that MPP may be useful for developing safe natural health products.
Int. J. Mol. Sci. 2014, 15 Keywords: Mentha antitumor; antioxidant
16303 piperita;
response
surface
methodology;
polysaccharide;
1. Introduction Cancer is a leading cause of death worldwide and a diverse group of diseases characterized by the uncontrolled proliferation of anaplastic cells, leading to invasion of surrounding tissues and metastasis to other organs [1]. It is one of the most lethal diseases severely threatening to human life. Chemotherapy is one of the most frequently used therapeutic modalities for the treatment of cancer, but the therapeutic outcome is usually unsatisfactory due to serious and intolerable adverse side effects. Therefore, it is very important to develop novel antitumor substances with little toxicity to the host. In recent decades, numerous polysaccharides isolated from natural materials have been proven to have few adverse effects and possess a wide range of biological functions such as antitumor and antioxidant properties [2–5]. Natural polysaccharides may prove to be important potential pharmaceuticals for the prevention and treatment of cancer in the future. Mentha piperita L., belonging to the family Lamiaceae, is a perennial herb native to Europe, sparingly naturalized in the northern USA and Canada, and cultivated in many parts of the world. It used to be known as a hybrid of M. spicata L. (spearmint) and M. aquatic (water mint) [6]. It is best known for its fragrance and flavor compositions. In Eastern and Western traditional medicines, peppermint and its oil have been used in antispasmodics, aromatics, antiseptics or even medications for the treatment of colds, cramps, indigestions, nausea, sore throat, toothache or even cancer [7]. Modern pharmacology research has demonstrated that M. piperita possesses antioxidant, antitumor, antiallergenic, antiviral and antibacterial activities [8–12]. So far, research on M. piperita has focused on peppermint oil and small molecules, and there is no published information about the biological and physiochemical properties of the polysaccharides extracted from M. piperita. Polysaccharides are polymeric carbohydrate structures, formed of repeating units joined together by glycosidic bonds. Aqueous extraction is the most common method of extraction of plant polysaccharides, and the extraction process is usually optimized by mathematics. Response surface methodology (RSM) is an empirical statistical technique for multiple regression analysis by using quantitative data obtained from properly designed experiments to solve multivariate equations simultaneously. This methodology consists of a group of mathematical and statistical procedures that can be used to evaluate the functional form of a process, involving one or more dependent variables influenced by various factors or independent variables. This methodology has been widely used to optimize the polysaccharides extraction process [13–16]. Knowing that RSM can simplify the complexity of experimental trials needed to evaluate multiple variables and their interactions, the objective of this study was to optimize the extraction process of polysaccharides from M. piperita using RSM, and to investigate their antitumor and antioxidant activities in vitro.
Int. J. Mol. Sci. 2014, 15
16304
2. Results and Discussion 2.1. Statistical Analysis and Model Fitting On the basis of preliminary experiments (data not shown), the three parameters: ratio of water to raw material (A), extraction time (B) and extraction temperature (C) were selected as the variables to optimize the extraction conditions of polysaccharide from M. Piperita (MPP). A 17-run Box-Behnken design (BBD) was applied statistically to optimize Y (MPP yield) under different experimental combinations and is presented in Table 1. There was a considerable variation of Y depending on the different extraction conditions. MPP yields ranged from 5.140% to 8.452%. Table 1. Box-Behnken Design (BBD) experimental design with the independent variables. Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
A 15 25 15 25 15 25 15 25 20 20 20 20 20 20 20 20 20
B (h) 1.0 1.0 2.0 2.0 1.5 1.5 1.5 1.5 1.0 2.0 1.0 2.0 1.5 1.5 1.5 1.5 1.5
C (°C) 80 80 80 80 70 70 90 90 70 70 90 90 80 80 80 80 80
Y MPP Yield (%) 5.140 7.376 5.769 6.588 5.592 7.162 5.838 7.463 7.486 7.058 7.152 7.324 7.989 8.452 8.328 8.021 8.371
Using multiple regression analysis on the experimental data, the response variable and the test variables were related by the following second-order polynomial equation: Y = 82.32 + 7.81A − 0.52B + 0.60C − 3.54AB + 0.14AC + 1.50BC − 13.78A2 − 6.36B2 − 3.41C2
(1)
The coefficient of determination (R2 = 0.9862), the adjusted coefficient of determination (R2Adj = 0.9684) and the coefficient of variation (C.V. = 2.55%) are shown in Table 2, where the values indicate that the accuracy and the general availability of the polynomial model are adequate, and the R2Pred of 0.9345 is in reasonable agreement with the R2Adj. The “Adequate Precision” of 22.21 indicated that this model could be used to navigate the design space. The significance of each coefficient was checked using the F-test and the p-value (Table 2). The Model F-value of 55.48 implied that the model was significant, and there was only a 0.01% chance that a “Model F-value” that was this large occurred because of noise. The “Lack of Fit F-value” of 0.38 implied that the Lack of Fit was not significant relative to the pure error, and there was
Int. J. Mol. Sci. 2014, 15
16305
a 22.27% chance that a “Lack of Fit F-value” of such a size could have occurred because of noise. As shown in Table 2, the linear coefficients (A), a quadratic term coefficient (A2, B2, C2) and cross product coefficients (AB) were significant, with very small p-values (p < 0.05). The other term coefficients were not significant (p > 0.05). Therefore, A, A2, B2, C2 and AB were important factors in the extraction process of the polysaccharides. Table 2. Analysis of variance (ANOVA) of the experimental results of the BBD. Source Model A B C AB AC BC A2 B2 C2 Residual Lack of Fit Pure Error Cor Total Standard Deviation 0.18
Sum of Squares 16.51 4.88 0.022 0.029 0.50 7.563 × 10−4 0.090 7.99 1.71 0.49 0.23 0.051 0.18 16.74
Degree of Freedom 9 1 1 1 1 1 1 1 1 1 7 3 4 16
Mean Square 1.83 4.88 0.022 0.029 0.50 7.563 × 10−4 0.09 7.99 1.71 0.49 0.033 0.017 0.045 -
F-Value 55.48 147.70 0.65 0.87 15.18 0.023 2.72 241.71 51.58 14.80 0.38 -
Mean
C.V.%
Press
R2
R2Adj
R2Pred
7.12
2.55
1.10
0.9862
0.9684
0.9345
p-Value
International Journal of
Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article
Extraction, Preliminary Characterization and Evaluation of in Vitro Antitumor and Antioxidant Activities of Polysaccharides from Mentha piperita Xin Liu 1,†, Zhen-Liang Sun 2,†, Ai-Rong Jia 1, Ya-Ping Shi 1, Rui-Hong Li 3,* and Pei-Ming Yang 2,* 1
2
3
†
Biology Institute of Shandong Academy of Sciences/Key Laboratory for Applied Microbiology of Shandong Province, Jinan 250014, China; E-Mails: [email protected] (X.L.); [email protected] (A.-R.J.); [email protected] (Y.-P.S.) State Key Laboratory of New Drug & Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China; E-Mail: [email protected] Fengxian Hospital Affiliated to Southern Medical University, 6600 NanFeng Road, Shanghai 201499, China These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: [email protected] (R.-H.L.); [email protected] (P.-M.Y.); Tel./Fax: +86-21-5742-3899 (R.-H. L.); +86-21-6247-9808 (P.-M.Y.). Received: 26 May 2014; in revised form: 25 August 2014 / Accepted: 3 September 2014 / Published: 15 September 2014
Abstract: This study describes the extraction, preliminary characterization and evaluation of the in vitro antitumor and antioxidant activities of polysaccharides extracted from Mentha piperita (MPP). The optimal parameters for the extraction of MPP were obtained by Box-Behnken experimental design and response surface methodology (RSM) at the ratio of water to raw material of 20, extraction time of 1.5 h and extraction temperature at 80 °C. Chemical composition analysis showed that MPP was mainly composed of glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the molecular weight of its two major fractions were estimated to be about 2.843 and 1.139 kDa, respectively. In vitro bioactivity experiments showed that MPP not only inhibited the growth of A549 cells but possessed potent inhibitory action against DNA topoisomerase I (topo I), and an appreciative antioxidant action as well. These results indicate that MPP may be useful for developing safe natural health products.
Int. J. Mol. Sci. 2014, 15 Keywords: Mentha antitumor; antioxidant
16303 piperita;
response
surface
methodology;
polysaccharide;
1. Introduction Cancer is a leading cause of death worldwide and a diverse group of diseases characterized by the uncontrolled proliferation of anaplastic cells, leading to invasion of surrounding tissues and metastasis to other organs [1]. It is one of the most lethal diseases severely threatening to human life. Chemotherapy is one of the most frequently used therapeutic modalities for the treatment of cancer, but the therapeutic outcome is usually unsatisfactory due to serious and intolerable adverse side effects. Therefore, it is very important to develop novel antitumor substances with little toxicity to the host. In recent decades, numerous polysaccharides isolated from natural materials have been proven to have few adverse effects and possess a wide range of biological functions such as antitumor and antioxidant properties [2–5]. Natural polysaccharides may prove to be important potential pharmaceuticals for the prevention and treatment of cancer in the future. Mentha piperita L., belonging to the family Lamiaceae, is a perennial herb native to Europe, sparingly naturalized in the northern USA and Canada, and cultivated in many parts of the world. It used to be known as a hybrid of M. spicata L. (spearmint) and M. aquatic (water mint) [6]. It is best known for its fragrance and flavor compositions. In Eastern and Western traditional medicines, peppermint and its oil have been used in antispasmodics, aromatics, antiseptics or even medications for the treatment of colds, cramps, indigestions, nausea, sore throat, toothache or even cancer [7]. Modern pharmacology research has demonstrated that M. piperita possesses antioxidant, antitumor, antiallergenic, antiviral and antibacterial activities [8–12]. So far, research on M. piperita has focused on peppermint oil and small molecules, and there is no published information about the biological and physiochemical properties of the polysaccharides extracted from M. piperita. Polysaccharides are polymeric carbohydrate structures, formed of repeating units joined together by glycosidic bonds. Aqueous extraction is the most common method of extraction of plant polysaccharides, and the extraction process is usually optimized by mathematics. Response surface methodology (RSM) is an empirical statistical technique for multiple regression analysis by using quantitative data obtained from properly designed experiments to solve multivariate equations simultaneously. This methodology consists of a group of mathematical and statistical procedures that can be used to evaluate the functional form of a process, involving one or more dependent variables influenced by various factors or independent variables. This methodology has been widely used to optimize the polysaccharides extraction process [13–16]. Knowing that RSM can simplify the complexity of experimental trials needed to evaluate multiple variables and their interactions, the objective of this study was to optimize the extraction process of polysaccharides from M. piperita using RSM, and to investigate their antitumor and antioxidant activities in vitro.
Int. J. Mol. Sci. 2014, 15
16304
2. Results and Discussion 2.1. Statistical Analysis and Model Fitting On the basis of preliminary experiments (data not shown), the three parameters: ratio of water to raw material (A), extraction time (B) and extraction temperature (C) were selected as the variables to optimize the extraction conditions of polysaccharide from M. Piperita (MPP). A 17-run Box-Behnken design (BBD) was applied statistically to optimize Y (MPP yield) under different experimental combinations and is presented in Table 1. There was a considerable variation of Y depending on the different extraction conditions. MPP yields ranged from 5.140% to 8.452%. Table 1. Box-Behnken Design (BBD) experimental design with the independent variables. Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
A 15 25 15 25 15 25 15 25 20 20 20 20 20 20 20 20 20
B (h) 1.0 1.0 2.0 2.0 1.5 1.5 1.5 1.5 1.0 2.0 1.0 2.0 1.5 1.5 1.5 1.5 1.5
C (°C) 80 80 80 80 70 70 90 90 70 70 90 90 80 80 80 80 80
Y MPP Yield (%) 5.140 7.376 5.769 6.588 5.592 7.162 5.838 7.463 7.486 7.058 7.152 7.324 7.989 8.452 8.328 8.021 8.371
Using multiple regression analysis on the experimental data, the response variable and the test variables were related by the following second-order polynomial equation: Y = 82.32 + 7.81A − 0.52B + 0.60C − 3.54AB + 0.14AC + 1.50BC − 13.78A2 − 6.36B2 − 3.41C2
(1)
The coefficient of determination (R2 = 0.9862), the adjusted coefficient of determination (R2Adj = 0.9684) and the coefficient of variation (C.V. = 2.55%) are shown in Table 2, where the values indicate that the accuracy and the general availability of the polynomial model are adequate, and the R2Pred of 0.9345 is in reasonable agreement with the R2Adj. The “Adequate Precision” of 22.21 indicated that this model could be used to navigate the design space. The significance of each coefficient was checked using the F-test and the p-value (Table 2). The Model F-value of 55.48 implied that the model was significant, and there was only a 0.01% chance that a “Model F-value” that was this large occurred because of noise. The “Lack of Fit F-value” of 0.38 implied that the Lack of Fit was not significant relative to the pure error, and there was
Int. J. Mol. Sci. 2014, 15
16305
a 22.27% chance that a “Lack of Fit F-value” of such a size could have occurred because of noise. As shown in Table 2, the linear coefficients (A), a quadratic term coefficient (A2, B2, C2) and cross product coefficients (AB) were significant, with very small p-values (p < 0.05). The other term coefficients were not significant (p > 0.05). Therefore, A, A2, B2, C2 and AB were important factors in the extraction process of the polysaccharides. Table 2. Analysis of variance (ANOVA) of the experimental results of the BBD. Source Model A B C AB AC BC A2 B2 C2 Residual Lack of Fit Pure Error Cor Total Standard Deviation 0.18
Sum of Squares 16.51 4.88 0.022 0.029 0.50 7.563 × 10−4 0.090 7.99 1.71 0.49 0.23 0.051 0.18 16.74
Degree of Freedom 9 1 1 1 1 1 1 1 1 1 7 3 4 16
Mean Square 1.83 4.88 0.022 0.029 0.50 7.563 × 10−4 0.09 7.99 1.71 0.49 0.033 0.017 0.045 -
F-Value 55.48 147.70 0.65 0.87 15.18 0.023 2.72 241.71 51.58 14.80 0.38 -
Mean
C.V.%
Press
R2
R2Adj
R2Pred
7.12
2.55
1.10
0.9862
0.9684
0.9345
p-Value
