Time Depletion Effects on the Volatile Compounds from the Distillation Extracts of Prunella vulgaris and the Dynamics of their Extraction
Authors: Mak William Chi Keung 
Published: April 2022

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.