Catechol-O-methyltransferase Inhibitors from Calendula officinalis Leaf

Calendula officinalis is commonly known as marigold and its flowers are used in herbal medicines, cosmetics, perfumes, dyes, pharmaceutical preparations, and food products. However, the utility of its leaves has not been studied in depth. The purpose of the present study was to identify the major compounds in C. officinalis leaves and to determine the inhibitory properties of the isolated compounds toward human catechol-O-methyltransferase (COMT), a key neurotransmitter involved in Parkinson’s disease and depression. We isolated and identified ten compounds, including two phenylpropanoids and seven flavonoids, from C. officinalis leaf extracts, of which four flavonoids were identified from C. officinalis leaves for the first time. Eight compounds exhibited COMT inhibitory activities with IC50 values of less than 100 μM. Our results indicate that compounds in C. officinalis leaves are potentially effective for preventing Parkinson’s disease and depression. Thus, C. officinalis leaves may hold promise as dietary supplements.


Introduction
Calendula officinalis is a medicinal plant belonging to the Asteraceae family and is distributed mainly over large areas of the Mediterranean [1,2]. The petals of C. officinalis exhibit a broad range of biological activities, including antioxidant [3], antitumor [4,5], antibacterial [4], and anti-inflammatory activities [5,6]. Oleanane-type triterpene glycosides [5,7], triterpene alcohols [8,9], flavonoid glycosides [5], and carotenoids [10] have been found in the petals of C. officinalis and contribute to various biological activities. The petals of C. officinalis have been used in Europe since the 13th century for treating wounds, and a variety of cosmetics have been developed from the plant [11,12].
Although C. officinalis petals are used medicinally, the other parts of the plant are presently not utilized. Several recent studies have drawn attention to the effective use of waste-or by-products arising from productization processes [13,14]. Effective utilization of the currently unused parts of C. officinalis requires information on their chemical composition and biological activities. Although the leaves of C. officinalis are used as a traditional treatment for varicose veins in India [1], other biological uses have not been investigated. We therefore investigated the usability of C. officinalis leaves by isolating the major components and determining their structures by spectroscopic analysis. In addition, the catechol-O-methyltransferase (COMT) inhibitory activities of both the leaves of C. officinalis and the isolated compounds were evaluated. COMT is a target enzyme of Parkinson's disease and depression [15,16]. Current anti-parkinsonian drugs exhibit severe toxicity, whereas natural sources could provide many potentially safe COMT inhibitors [17]. The leaves of C. officinalis and the isolated compounds exhibited remarkable COMT inhibitory activities, indicating their utility as bioactive ingredients toward Parkinson's disease.

Total Polyphenol Content and COMT Inhibitory Activity of C. officinalis Leaves
First, we compared the total polyphenol content of each part of C. officinalis (petal, leaf, and stem) using the Folin-Ciocalteu colorimetric method. As shown in Figure 1, the leaves showed the highest content. Next, the COMT inhibitory activities of various parts of C. officinalis were evaluated ( Figure 2). Consistent with the total polyphenol content, the ethanol extracts of leaves exhibited the highest activity and were three times that of the petals, suggesting that Calendula leaves can be used as a source of natural compounds effective toward Parkinson's disease. [17]. The leaves of C. officinalis and the isolated compounds exhibited remarkable COM inhibitory activities, indicating their utility as bioactive ingredients toward Parkinson disease.

Total Polyphenol Content and COMT Inhibitory Activity of C. officinalis Leaves
First, we compared the total polyphenol content of each part of C. officinalis (peta leaf, and stem) using the Folin-Ciocalteu colorimetric method. As shown in Figure 1, th leaves showed the highest content. Next, the COMT inhibitory activities of various par of C. officinalis were evaluated ( Figure 2). Consistent with the total polyphenol conten the ethanol extracts of leaves exhibited the highest activity and were three times that the petals, suggesting that Calendula leaves can be used as a source of natural compound effective toward Parkinson's disease.

Component Analysis of the Leaves of C. officinalis
The major components of Calendula leaves were determined by isolating ten know compounds using various chromatography techniques (Figures 3 and 4).  [17]. The leaves of C. officinalis and the isolated compounds exhibited remarkable COMT inhibitory activities, indicating their utility as bioactive ingredients toward Parkinson's disease.

Total Polyphenol Content and COMT Inhibitory Activity of C. officinalis Leaves
First, we compared the total polyphenol content of each part of C. officinalis (petal, leaf, and stem) using the Folin-Ciocalteu colorimetric method. As shown in Figure 1, the leaves showed the highest content. Next, the COMT inhibitory activities of various parts of C. officinalis were evaluated ( Figure 2). Consistent with the total polyphenol content, the ethanol extracts of leaves exhibited the highest activity and were three times that of the petals, suggesting that Calendula leaves can be used as a source of natural compounds effective toward Parkinson's disease.

Component Analysis of the Leaves of C. officinalis
The major components of Calendula leaves were determined by isolating ten known compounds using various chromatography techniques (Figures 3 and 4). The compounds were identified by NMR and MS to be [20], chlorogenic acid (8) [21], 3,4-dicaffeoylquinic acid (9) [22], and syringic acid (10) [23]. NMR and MS spectra of 1-10 were indicated in Figures S1-S46. Compounds 1-3 and 8-10 were previously isolated from C. officinalis petals [24] but the present study is the first to the finding of 4-7 from Calendula.
Compounds 1-3 and 8-10 were previously isolated from C. officinalis petals [24] but the present study is the first to the finding of 4-7 from Calendula.
Tolcapone, entacapone, and opicapone are COMT inhibitors used to treat Parkinson's disease [17]. However, tolcapone exhibits severe hepatotoxicity [28], and the toxicity of opicapone has not been evaluated in detail [29]. Therefore, COMT inhibitors with low toxicity and good safety profiles are required. Natural sources of pharmaceuticals could offer many potentially safe COMT inhibitors because of their low toxicity, and therefore Calendula leaves might be a source of safe and effective ingredients for the prevention and treatment of Parkinson's disease.

Biological Material
C. officinalis leaves were collected in Hokkaido, Japan, in October 2019 and July 2020. The voucher numbers of each sample are 201910 and 202007, respectively.

Extraction and Isolation of Compounds in Calendula Leaves
The structures of 1-10 were determined based on 1D and 2D NMR, HRMS, and comparisons with data from previous studies.
Author Contributions: W.K. and R.M. performed the structural analysis of the isolated compounds and COMT inhibitory assay; M.F. and Y.S. collected the C. officinalis samples; the manuscript was prepared by R.M; and S.K. supervised all the experiments and the manuscript preparation. W.K. and R.M. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author.