Enzymatic preparation and anti cervical cancer activity of Chamagu isothiocyanate

In recent years, the incidence rate of cancer is increasing rapidly in China, and the prevention and treatment of cancer is urgent. Research shows that increasing the intake of cruciferous vegetables can effectively reduce the incidence rate of cancer. Brassicaceae vegetables contain abundant glucosinolates, also known as glucosinolates (GLs). When plants are mechanically damaged, GLs can be hydrolyzed by their own myrosinase to produce isothiocyanates. Isothiocyanates are a class of compounds containing conjugated double bonds, with a molecular formula of R-N=C=S (R represents different types of alkyl groups). In recent years, isothiocyanates have become a research hotspot due to their outstanding biological activity. Research has shown that isothiocyanates have various biological activities such as anti-tumor, antibacterial, free radical scavenging, and pathogen resistance. It has been developed and used in many fields such as healthcare, food, and agriculture.

Brassica rapa L., scientific name “turnip”, is a biennial herbaceous plant belonging to the Brassicaceae family. It is mainly distributed in the southwest of Tianshan Mountains and northwest of Tarim in Xinjiang, and is a local specialty vegetable commonly consumed by people of all ethnic groups in Xinjiang. Research has found that Chamagu roots contain abundant GLs. Zhuang et al. used ultra-high liquid chromatography technology to analyze the types and contents of GLs in different developmental stages and growth parts (roots, stems, leaves) of Xinjiang turnips during their nutritional growth period. They found that there were certain differences in the types and mass fractions of glucosinolates during different developmental stages of nutritional growth. The differences in the types and contents of GLs in plants will lead to variations in the types and contents of isothiocyanates, resulting in different biological activities. Sun has reported that Qiamagu seeds contain four types of isothiocyanates: phenethyl isothiocyanate, tert butyl isothiocyanate, isopropyl isothiocyanate, and allyl isothiocyanate.

At present, the preparation of isothiocyanates mainly involves chemical synthesis and enzymatic hydrolysis. Among them, the chemical synthesis method has complicated steps, many intermediate products, is not environmentally friendly, and has a low synthesis rate; Enzymatic hydrolysis utilizes the glucosinolate myrosinase system in cruciferous plants to extract isothiocyanates, which has the advantages of simple operation and relatively high yield. Enzymatic hydrolysis is divided into endogenous myrosinase degradation method and exogenous myrosinase degradation method. Using endogenous enzymatic hydrolysis to prepare isothiocyanates, although the process is simple, the endogenous myrosinase in plants cannot be fully released, resulting in long enzymatic hydrolysis time and low efficiency. Ding compared the amount of isothiocyanates generated by GLs degradation by endogenous and exogenous enzymes in rapeseed and found that the amount of isothiocyanates generated by exogenous myrosinase was 7.6 times that of endogenous myrosinase. GC-MS identification and analysis showed that exogenous myrosinase had a variety of degradation products, relatively high content, and fewer impurities.

This study used Chamagu root as the raw material and used exogenous myrosinase degradation method to prepare Chamagu isothiocyanate (BRICe). On the basis of single factor screening, enzyme hydrolysis time, temperature, pH, and ascorbic acid concentration were identified as significant influencing factors. Four factor three-level orthogonal experiment was used to optimize the extraction process. Finally, its chemical composition was analyzed by gas chromatography-mass spectrometry (GC-MS), and the anti-cancer effect of isothiocyanate was studied.

Cancer is one of the malignant diseases that threaten human life safety. Multiple epidemiological studies have found that consuming more cruciferous vegetables can effectively reduce the risk of cancer. Isothiocyanate is one of the products of self enzymatic hydrolysis in cruciferous vegetables. Research has shown that isothiocyanates can activate phase II enzymes in the human body, allowing the products of phase I reactions to bind with glucuronic acid and glutathione, thereby promoting the rapid elimination of carcinogens from the body. Therefore, isothiocyanates can be used as a cancer inhibitor in medicine. Qiamagu is a medicinal and edible plant in Xinjiang, containing various active ingredients such as glucosinolates, flavonoids, isothiocyanates, and polysaccharides. However, the development and utilization of Qiamagu isothiocyanates are currently insufficient. Enzymatic hydrolysis is currently an environmentally friendly method for extracting isothiocyanates. By optimizing enzymatic hydrolysis conditions, not only can the production of other secondary metabolites be reduced, but the production rate of the target product can also be accelerated, thereby increasing yield. Jang prepared isothiocyanates using endogenous myrosinase in the Brassicaceae family. Although the endogenous enzyme method has a simple process, the myrosinase in plants cannot be fully released, resulting in long enzymatic hydrolysis time and low efficiency. Compared to endogenous enzymes, using exogenous enzymes to prepare isothiocyanates is easier to control the enzymatic hydrolysis conditions of exogenous enzymes, which is beneficial for directing changes in the types of enzymatic hydrolysis products and increasing the amount of products. Therefore, optimizing the external enzymatic hydrolysis conditions is the key to preparing isothiocyanates.

The formation of enzymatic hydrolysis products is not only related to the type of glucosinolate, but also includes hydrolysis time, hydrolysis temperature, pH, pressure, and the presence of Fe2+. Latt é found that when the pH of the solution is neutral, it is favorable for glucosinolates to form isothiocyanates. However, when the pH is acidic/alkaline, or when cyclic sulfur characteristic proteins or Fe2+are present, the enzymatic hydrolysis products will form a large amount of nitriles. Yang used response surface methodology to analyze the factors of pH, EDTA, and ascorbic acid, and found that these three factors have a significant impact on the extraction amount of isothiocyanate. Burmeister found that ascorbic acid replaced the catalytic base function in the active site of myrosinase, exhibiting catalytic properties of myrosinase. Adding an appropriate amount of ascorbic acid to fresh broccoli can significantly enhance the activity of myrosinase, thereby increasing the production of isothiocyanates. This study used exogenous myrosinase (derived from white mustard seeds) degradation method to prepare BRIe. The enzymatic hydrolysis conditions were optimized through single factor and orthogonal experiments, and four factors that had a significant impact on the extraction of BRIe were screened: enzymatic hydrolysis time, enzymatic hydrolysis temperature, ascorbic acid concentration, and pH. The optimal enzymatic hydrolysis conditions for BRIe were determined as follows: myrosinase addition of 20 μ L, reaction temperature of 70 ℃, reaction time of 3h, pH of 6.5, ascorbic acid concentration of 0.1mg/g, and GLs freeze-dried powder of 2g. It is interesting that the optimal enzymatic hydrolysis temperature for this study is 70 ℃. There are research reports that the temperature range for enzymatic hydrolysis of plant isothiocyanates from broccoli is 20-60 ℃, with the optimal enzymatic hydrolysis temperature being 40 ℃; The temperature range for extracting isothiocyanates from wasabi roots is 30-90 ℃, and the optimal enzymatic hydrolysis temperature is 70 ℃. Therefore, the optimal temperature for myrosinase varies depending on the species of origin.

Usually, the types of isothiocyanates formed vary depending on the conditions of enzymatic hydrolysis. Chen et al. used endogenous enzymatic hydrolysis to identify the enzymatic hydrolysis products in broccoli as allyl isothiocyanate, isobutyl isothiocyanate, 1-butenyl isothiocyanate, 4-methylthionitrile, 5-methylthiopentonitrile, 3- (methylthio) propyl isothiocyanate, butyl isothiocyanate, radish thiocyanate, and radish sulforaphane using GC-MS, with radish sulforaphane having the highest content. Yang used exogenous enzymatic hydrolysis to identify the enzymatic hydrolysis products in broccoli as allyl isothiocyanate, phenethyl isothiocyanate, cyclopentyl isothiocyanate, phenylpropanonitrile, 2,1-phenyloxazolidinone, and thyroid stimulating hormone. This study analyzed the composition of the optimal enzymatic hydrolysis conditions for BRIe using GC-MS, and identified seven enzymatic hydrolysis related products, namely 3-amino-2-oxazolidinone, 2-oxazolidinethione, 3-buten-1-ylisothiocyanate (BITC), phenyl isothiocyanate (PEITC), 1-isothiocyanate propyl ester, isothiocyanate isopropyl ester, and 2-phenylisothiocyanate (2-PITC), of which 4 were isothiocyanate types.

Many studies have shown that isothiocyanates can prevent the occurrence of various cancers. Research has found that PEITC dose dependently inhibits the proliferation of mouse osteosarcoma cell line K7M2 and can cause G2/M cell cycle arrest and apoptosis; BITC can significantly reduce the cell viability of gastric adenocarcinoma cell AGS and induce apoptosis and autophagy. However, there are currently few reports on the effects and mechanisms of isothiocyanates on cervical cancer. This study found through an in vitro cell model that BRIe significantly inhibited the proliferation of cervical cancer cells Hela, Siha, and U14 in a time – and dose-dependent manner. Comparing the 24-hour IC50 values of these three types of cells, it is concluded that Hela cells are the most sensitive to BRIe. Uncontrolled cell proliferation is the fundamental cause of cancer, which is due to the disruption of the cell cycle. Flow cytometry analysis revealed that BRIe can arrest the Hela cell cycle in the S and G2/M phases. The metastasis of cancer cells has always been a research hotspot and clinical treatment challenge. Through scratch experiments, it was found that BRIe can effectively inhibit the migration of Hela cells. Hochest 33258 staining revealed that BRIe can induce DNA condensation in Hela cell nuclei, and visible granular fluorescence can be observed in the nuclei, suggesting that it is an apoptotic body. Further analysis using Annexin V-FITC double staining revealed that the apoptosis rate of Hela cells treated with BRIe increased with increasing concentration after 24 hours, indicating that BRIe induced apoptosis in Hela cells. In summary, BRIe exhibits significant anti cervical cancer activity. Based on GC-MS data, it is speculated that BITC, PEITC, isopropyl isothiocyanate, and 2-PITC may be its main active ingredients, which need to be further validated through in vitro and in vivo experiments.

In summary, this study has for the first time determined the optimal preparation process for exogenous enzymatic hydrolysis of isothiocyanates in Chamagu. Seven enzymatic hydrolysis related products were identified through GC-MS analysis, and the enzymatic hydrolysis product BRIe obtained has the ability to inhibit the proliferation of cervical cancer cells, suppress cell migration, induce cell cycle arrest and apoptosis. The research provides a reference basis for further development and utilization of Chamagu plant resources. The research results were published in the 6th issue of Natural Product Research and Development in 2024