Profiling Rosmarinic Acid and Sinensetin Content of Orthosiphon aristatus. from Three Different Locations with Variety Ethanol Concentration

: Orthosiphon aristatus is a well-known medicinal plant acknowledged for its therapeutic effect in treating urinary tract diseases, hypertension, diabetes mellitus, and arthritis. It is widely used as an ingredient in herbal medicine and distributed throughout the world, including China, Europe, and Indonesia. Sinensetin is normally used as a chemical marker to evaluate and control the quality of O. aristatus . However, in 2021, the European Medicines Agency changed the marker to rosmarinic acid. To determine the levels of rosmarinic acid and sinensetin in O. aristatus as well as the correlation between the two compounds, this study used high-performance liquid chromatography (HPLC) with a UV detector to analyze O. aristatus extract from three distinct locations with four different ethanol concentrations (p.a, 75%, 50%, and 25%). The results showed that the combination of solvent concentration and growing location had a significant effect on the levels of rosmarinic acid and sinensetin, with a p-value < 0.05. The Spearman test obtained a correlation coefficient (r) of - 0.423. It can be concluded that there is no correlation between the content of rosmarinic acid compounds in O. aristatus leaves and the content of sinensetin compounds


INTRODUCTION
Orthosiphon aristatus (Blume) Miq., locally known "cat's whiskers" or as "kumis kucing", is a medicinal plant from the Lamiaceae family distributed widely throughout China, Europe and Southeast Asian countries, including Indonesia.The leaves of its plant have been used empirically to treat kidney stones and urinary tract diseases [1].Furthermore, pharmacological studies have verified that this plant is therapeutically effective in treating diabetes mellitus [2], decreasing blood pressure [3], [4], [5], treating urinary tract diseases [6], and managing arthritis due to its anti-inflammatory properties [7].Due to its potency, O. aristatus is widely used as an ingredient in herbal medicine product.According to the Indonesia Food and Drug Administration (BPOM), more than three hundreds herbal medicine products have been registered for containing O. aristatus [8].In addition to being an economic opportunity, the growing interest in herbal medicine is also a challenge in the field of quality assurance.Quality assurance is essential to ensure the safety and quality of the products.
The quality of herbal medicine can be assessed by a number of methods, such as fingerprinting and marker compound analysis.Although fingerprint-based systems have gained more attention, the conventional technique of using chemical makers is commonly chosen by herbal pharmacopeia in many countries [9].Phytochemical studies discovered that the ethanol extract of O. aristatus leaves contained 34 compounds, and 14 of them were successfully absorbed and retained in mice's blood plasma.Among the fourteen phytochemical constituents, tanshinone IIA, salvianolic acid B, salvigenin, sinensetin, and rosmarinic acid are the compounds that are responsible for the pharmacological activity and are suggested to be the marker compounds of O. aristatus [10].
Sinensetin has been used as chemical marker of Orthosiphon aristatus by Indonesia's Herbal Pharmacopoeia (2017) and European Medicines Agency (2010) [11], [12].However, in 2021, the European Medicines Agency updated the O. aristatus marker into rosmarinic acid [13].This change is undoubtedly a challenge in the analysis field.Therefore, it is necessary to know the level of rosmarinic acid and sinensetin in O. aristatus along with the correlation between the two.
The content of sinensetin and rosmarinic acid in Orthosiphon aristatus extract is variable and can be influenced by some factors such as the origin and extraction methods.Phytochemical study reported that the different location of O. aristatus effect of the level of sinensetin [14].On the other hand, the choice of solvents is important due to the targeted compound to be extracted from the plant.
The most common solvents used for O. aristatus extraction are methanol, ethanol, and distilled water [11], [15], [16].Although methanol gives the high yield of rosmarinic acid and sinensetin than ethanol, but the use of methanol is frequently questioned because of its toxic to humans.Thus, the industry finds ethanol more appealing for the extraction because less toxic and shares similar chemical properties with methanol [17].According to a literature search, no one has reported the effect of the combination of ethanol concentration and growing location on the content of rosmarinic acid and sinensetin in cat whisker extract or the relationship between the two compounds.Therefore, this study aims to determine the levels and correlation of rosmarinic acid and sinensetin in O. aristatus from various locations and ethanol concentrations.

Materials 2.1.1 Plant materials
The Ortoshipon aristatus leaves were collected from 3 different locations (Sleman, Bantul, Klaten) on March 2024.The third to eights leaves from the shoots were picked by hand.The leaves are green, with a lanceolate leaf blade and a serrate margin.The venation is reticulate-pinnate, the petiole is reddish purple in colour, and the leaves range in size from 7 to 11 cm (Figure 1).The leaves were dried in an oven at 45 O C for 24 hours.Afterward, the dried leaves were ground and sifted with a 40-mesh sieve to obtain brownish green powder of O. aristatus.

Sample extraction
The preparation of extract was modified according to previous report [14].Approximately 500 mg of O. aristatus leaves powder was added with 5 mL of ethanol (p.a., 75%, 50% and 25%), then extracted using ultrasound-assisted extraction method for 15 minutes at room temperature and filtered through a 0.45 PVDF syringe filter.After that, each O. aristatus extract solution was dissolved in ethanol to obtain a concentration 10 mg/ml.The sample solution was filtered through a 0.45 PTVE syringe filter, and 10 µL was injected into HPLC.

Preparation of The Standard Solutions
A standard solution was prepared for rosmarinic acid and sinensetin.Each compound was made into a stock solution with concentration of 500 µg/ml in methanol.The combined working solution was obtained by mixing the stock solution of rosmarinic acid and sinensetin for 120 µL and 20 µL, respectively.The combined working solution was further diluted to five series concentration 3-60 µg/ml (rosmarinic acid) and 0.5-10 µg/ml (sinensetin).

HPLC Conditions
The analysis conditions for the quantification of rosmarinic acid and sinensetin compound in O. aristatus leave were conducted according to Ramadhani et al [18].A Shimadzu prominence-high performance liquid chromatography system (Shimadzu Corporation, Kyoto, Japan) equipped with ultraviolet (SPD-20A/20AV) detector was used for the analysis.Separation was archived on cosmosil C18-MS-II column (4.6 i.d x 250 mm, 5.0 µm) using a 0.1% TFA in water (A) and acetonitrile (B) as a mobile phase

Data analysis
A nested analysis of variance (nested-ANOVA) was performed to determine the significant effect of the ethanol concentration on sinensetin and rosmarinic acid from three different locations of O. aristatus.Then, a spearman correlation analysis using Minitab 21.4.1 software was performed to determine the association between rosmarinic acid and sinensetin levels in O. aristatus at different ethanol concentrations and locations.

RESULTS AND DISCUSSION
In this study, extraction was performed using four different ethanol concentrations (p.a, 75%, 50%, and 25%) and three growing locations (Sleman, Bantul, and Klaten).The fact that some peaks are absent in the beginning retention times and present in the end retention time indicates that the use of ethanol p.a tends to extract non-polar compounds rather than polar compounds.Meanwhile, the use of binary solvents (ethanol and water) significantly extracted polar compounds, as evidenced by the appearance of many peaks at the initial retention time.This may indicate that a binary solvent is suitable for optimizing the extraction of polar compounds.Sinensetin is a flavone group compound with five methoxy groups bound to the benzene ring (Figure 2).The methoxy groups cause sinensetin to be relatively less polar than rosmarinic acid, which has four hydroxyl groups.The use of a C18 reversed phase column and acetonitrile as a solvent causes less polar compounds bond to the stationary phase more strongly and move more slowly through the column, so the peak could be seen at the end of the retention time.The chromatogram showed that the peak for sinensetin was visible at a retention time of 21.827, whereas the peak for rosmarinic acid was apparent at 10.995.

Figure 2. Structure of Rosmarinic Acid (A) and Sinensetin (B)
The comparation of rosmarinic acid and sinensetin content in O. aristatus from three different location with varying ethanol concentration are presented in Figure 3 (A) and (B).A nested ANOVA revealed that the solvent-nested growth location variable had a p-value less than 0.05.These results indicate that the ethanol concentration and growing location had a significant effect on the rosmarinic acid and sinensetin content.
Among all location, hydro-ethanol achieved a significantly higher rosmarinic acid content than ethanol p.a.The results of this study are similar to those of Suhaimi [19], who found that variation in the concentration of ethanol had a significant effect on the levels of rosmarinic acid in the O. aristatus extract.The content of rosmarinic acid increases as the ratio of water to ethanol increase.
However, when the ethanol percentage was less than 70%, rosmarinic acid levels decreased.The presence of water in the extraction acts as a swelling agent, which can increase extraction efficiency by increasing the contact surface area of the plant material and solvent [20].The high presence of water in the solvent reduces the rosmarinic acid content because rosmarinic acid is a relatively polar compound but insoluble in water [21].The Hildebrand solubility value (δ) of 21.18 tends to make rosmarinic acid more soluble in ethanol (δ = 26.6)than in water (δ = 47.8)[22].Taken together, in this study, we conclude that binary ethanol (50-75%) was the most efficient solvent to extract rosmarinic acid.
Among all locations, O. aristatus from Klaten had a higher sinensetin content compared to others.A nested-ANOVA revealed that the growing locations variable had the most significant effect on sinensetin content.Therefore, it can be concluded that the growing location influences the level of sinensetin in O. aristatus.These results are similar to the previous study, which showed that the level of sinensetin in O. aristatus varies depending on the growing location [14], [16], [23].The amounts of sinensetin found in this study are similar to those found by Kartini et al. (2022), who analysed O.
aristatus samples from 14 different areas in Indonesia and found levels ranging from 0.0238 to 0.1533 mg/g.

CONCLUSION
Variations in ethanol content and growing location significantly influenced the levels of rosmarinic acid and sinensetin in cat's whisker extract.The concentration of ethanol is the primary factor that has a significant impact on rosmarinic acid extraction.Binary ethanol (50-75%) was the most efficient solvent to extract rosmarinic acid.Meanwhile, the growing location variable had the most significant effect on sinensetin content.In addition, this study discovered that sinensetin levels had no correlation with rosmarinic acid levels (r = -0,423).To verify current results, studies for additional places should be carried out in future research.
Funding: This research was funded by Lembaga Pengelola Dana Pendidikan (LPDP, Indonesia Endowment Fund for Education)

Figure 3 .Figure 4 .
Figure 3.Effect of ethanol concentrations and locations on rosmarinic acid content (A) and sinensetin content (B)

Table 1 .
A details of the O. aristatus locations