# Development and Evaluation of Topical Formulation Containing _Agrimonia pilosa_ Extract
## 4 Discussion  
It is important to manage and maintain the active ingredients in formulations containing natural products. Moreover, the physicochemical properties during the initial drug development should be maintained, and microbiological contamination should be avoided. However, the evaluation of many natural products is limited to the extent of the appearance and mechanical characteristics. The reason is that it is generally difficult to quantify the component content, and a separate pretreatment technology must be developed. And there are numerous components that cannot be analyzed at once. Therefore, it is reasonable to determine and manage the representative ingredients that can be analyzed. Methods for analyzing these components should be established and continuously evaluated, and various techniques for analyzing ingredients have been studied and developed [35]. An analysis method was developed for one A/OG component and validation was conducted previously [31]. In this study, L/OG was quantified using the same analysis method, and validation was also successfully conducted. This is the first time that L/OG in the formulation has been quantified and validated. In addition, it was expected that the peak of ellagic acid content could be analyzed and quantified, but it was difficult to quantify due to the distribution of each solvent during the pretreatment process. Therefore, this component was excluded as an indicator component. A method of simultaneously analyzing and quantifying low-concentration components was established using a quick and simple pretreatment process. AE has numerous other components, but the amount in this formulation was too low for a UV detector, making it difficult to quantify or not distributed during the pretreatment process. Therefore, A/OG and L/OG were selected as indicators that represent the ingredients in the formulation and managed. Later, various formulations (cream, lotion, and ointment) were prepared to determine the most efficient way to use AE. Topical formulations were selected because they can avoid the first-pass effect in the body [36]. In addition, excipients such as liquid paraffin, propylene glycol, stearyl alcohol, and span 60 were used, which are widely used in topical formulations. The formulations were designed to be manufactured at an economical \begin{table}
\begin{tabular}{c c c c c c c c} \hline  & & **Criteria** & **0 Months** & **3 Months** & **6 Months** & **9 Months** & **12 Months** \\ \hline \multirow{4}{*}{Microbial stability} & Total aerobic bacterial & \multirow{2}{*}{\(<\)10\({}^{5}\) CFU/g} & \multirow{2}{*}{\(<\)1 CFU/g} & \multirow{2}{*}{-} & \multirow{2}{*}{\(<\)1 CFU/g} & \multirow{2}{*}{-} & \multirow{2}{*}{\(<\)1 CFU/g} \\  & & count & & & & & \\ \cline{1-1} \cline{2-7}  & Total yeast count & & & & & & \\ \cline{1-1} \cline{2-7}  & Pathogens & Negative & Not detected & - & & & \\ \hline \end{tabular}
\end{table} 
Table 4: _Cont._  
Figure 5: (**a**) Content of apigenin-7-O-glucuronide (A/OG) in 0.1% AE cream. (**b**) Content of luteolin-7-O-glucuronide (L/OG) in 0.1% AE cream. The grey line indicates the content from the long-term stability test, and the black line indicates the result of the accelerated test. cost by simplifying the manufacturing method. Before manufacturing the formulation, the compatibility study between the excipient and the AE was briefly conducted using HPLC-DAD for two weeks. Since most of the excipients form an oil phase, they were hardly mixed with AE, and that there was no significant change in the peak pattern during the test period. Later cream, lotion, and ointment were classified according to differences in moisture and the composition of ingredients. If the active components are vulnerable to moisture, they can be expected to stabilize in ointments, which have little moisture. However, the composition of the ointment selected in this experiment did not sufficiently dissolve the 0.1% concentration of AE.