Abstract

Background: Prunella vulgaris (PV) is a low-growing perennial herb, which can be found in different parts of the world as Asia, Europe and North America. It is traditionally used for medicinal treatment in various cultures in India, China, Japan, Korea, Russia, and Eastern Europe for treating different ailments, such as fever, and healing wounds. In our previous article, we showed the anti-tumorous effect of the volatile organic compounds (VOCs) of PV and characterized the steam distillation process in the extraction of VOCs from PV. This has never been done before as we are aware of. To use the VOCs as drugs, there is a question of how much of the VOCs are lost before the prepared drugs reach the patients. Thus, the first aim of the present article is to try to explore the time depletion effect on the VOCs in the PV extracts. Then, the second aim is to extend the work in the previous paper and further understand the dynamics of the distillation process of PV by changing the steam flow rate in the extraction process.

Methods: To achieve the first aim to explore the aging effect of how much VOCs are depleted after they are extracted, the VOCs were first extracted by the same method as before, i.e., using steam distillation. Then, tubes of the aqueous solution containing the VOCs were then stored in a 5°C refrigerator. They were then taken out for GC-MS analysis according to a preplanned schedule up to 8 weeks after the VOCs were extracted. The chemical composition of the distillate could then be evaluated. This revealed the changes in the abundance of VOCs with aging. At the same time, the cell viability of SCC154 oral squamous cells treated by these herbal solutions, which were at different aging stages, was evaluated using a tetrazolium-based colorimetric reagent, Cell Counting Kit-8. To achieve the second aim of exploring the dynamics of the steam distillation process, the steam flow rate was adjusted by changing the temperature setting of the hot plate. GC-MS was again used to quantify the chemical constituents of the distillates.

Results: By using GC-MS to measure the abundance of volatile compounds at different time points after the distillation process, it was found that the volatile compounds persist for a very long time, or over 8 weeks, which was the longest period of our experiment. The aging of the distillates also did not depreciate much the cell cytotoxicity of the PV distillate on the cancer cells. With respect to the dynamics of the steam distillation process, it was found that, at a low steam flow rate, volatile compounds of lower molecular weight are more efficient to be extracted, while at a high steam flow rate, volatile compounds of higher molecular weight are more efficiently extracted.

Conclusion: Our findings demonstrate that the VOC compounds extracted and present in aqueous form do not deplete much for at least 2 months after the extraction process, neither they exhibit cell cytotoxicity. The experiments on the dynamics of the steam distillation process demonstrate that the mass of herb present in the flow path of the steam has significant effects on the relative amounts of VOCs extracted.

Keywords: Prunella vulgaris, Xia Ku Cao, volatile organic compounds, steam distillation, GC-MS, time depletion effect, cell cytotoxicity, extraction dynamics, Chinese herbal medicine, Cell Counting Kit-8.

Graphical Abstract