Purification of Tea Saponins and Evaluation of its Effect on Alcohol Dehydrogenase Activity

Tea saponins, extracted from a Camellia oleifera cake, were found to have a potent effect on de-alcoholic activity. To obtain highly pure tea saponins, which can better maintain the activity of alcohol dehydrogenase (ADH), this paper presents an extraction method for tea saponins using deionized water as the extraction agent and a two-stage precipitation method, including ethanol precipitation and CaO precipitation. The optimum conditions for ethanol precipitation were 95% alcohol, a duration of 1.5h and a solid/liquid ratio of 1:4; while the optimum conditions for CaO precipitation were a duration of 2h and an NH4HCO3/CaO ratio of 2:1. Under the optimum conditions, the content of saponins was 87.58%. The results showed that the greater the amount of tea saponins and the higher its purity, the more significant its activating effect on ADH. When the purity of tea saponins was above 75%, it activated ADH. It indicated that the de-alcoholic mechanism of tea saponins is associated with the activity of ADH. Furthermore, the study characterized the structure of tea saponins by UV absorption and Fourier Transform Infrared (FTIR) spectrometry and LC-MS.

Camellia oleifera is known as a high-grade oil plant that is widely grown in China. After oil-pressing, much seed cake is left behind, containing approximately 10~18% saponins [1]. Saponins are a group of compounds which have a number of pharmacological activities including anti-inflammatory, antioxidant, anticancer, insecticidal, anthelmintic and antimicrobial [2]. Saponins from Polygonum hydropiper L. show high antiangiogenic, anti-tumor, brine shrimp, and fibroblast NIH/3T3 cell line cytotoxicity [3, 4]. Saponins were observed to be the most effective showing 93.3 % tumor inhibition at 1000 μg/ml with IC50 values of 18.1 μg/ml by potato tumor assay. Similarly, saponins also excelled in anti-angiogenic evaluation, exhibiting 78.9 % (IC50 = 64.9 μg/ml) at 1000 μg/ml respectively. These studies show that crude saponins exhibited notable anti-tumor and anti-angiogenic activities. Tea saponins also have surface activities, such as emulsification, dispersion and foaming [5]. Thus, it is necessary to establish a high purity extraction and purification method for tea saponins.

There are many studies on the extraction and purification methods of tea saponins, including n-butanol extraction, recrystallization, and the use of macroporous resin [6]. However, these methods have deficiencies, such as the colour of tea saponins obtained by n-butanol extraction being too deep and the extraction resulting in more impurities. Precipitation methods have the advantages of a simple operation and are suitable for industrial production. They are widely used in the extraction and purification of polysaccharides and proteins [7]. Additionally, different types of tea saponins have shown different activities [8, 9]. Acetone precipitation and two-stage purification were compared. According to this comparison, water extraction was used as the first step. Then, a two-stage purification of tea saponins was devised and optimised. The two-stage purification includes ethanol precipitation and CaO precipitation [10]. The purpose of ethanol precipitation is to achieve an initial purification and separate some tea polysaccharides, and the purpose of the second step is to complex CaO with tea saponins and then release tea saponins using NH4HCO3. The activity of tea saponins purified in this way and its effect on ADH activity have not been reported. The tea saponins obtained by this purification method have an effect on ADH activity.

Chinese wine culture is a traditional culture; however excessive alcohol intake can lead to alcohol poisoning, causing symptoms such as nausea, vomiting and alcoholic liver disease [11]. Hence it is essential to study the de-alcoholic activity of tea saponins and find an effective way to maintain its activity. The saponins fraction from the seeds of the tea plant was found to show a potent protective effect against gastric mucosal lesions induced by ethanol in rats [12, 13]. However, there are no reports about the de-alcoholic mechanism of tea saponins. In vivo ethanol is first oxidized to acetaldehyde, and 80% of the ethanol is converted into acetaldehyde by ADH in the process of hepatic 

metabolism. Referring to the method used by Valle & Hoch in the in vitro experiments, this paper compared acetone precipitation [6] and the two-stage purification to obtain tea saponins and the effect of tea saponins on ADH activity was investigated, showing that tea saponins obtained by the two-stage purification method can dispel the effects of alcohol to some extent, providing guidance for the further study of the de-alcoholic activity mechanism of tea saponins and product development. Hereby, it is hypothesized that tea saponins reduce the absorption metabolism of alcohol by influencing ADH activity. According to some reports, tea saponin carbohydrates could inhibit the activity of ADH, and different purities of tea saponins have different effects on ADH. An additional purpose of this study was to identify the de-alcoholic mechanism of tea saponins and develop a new de-alcoholic product based on tea saponins.

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