Moldavica acid A, a new salicylic acid derivative from Dracocephalum moldavica

Abstract In this paper, we present the discovery of a novel salicylic acid derivative, moldavica acid A (1), and a new natural dibenzo[b,f]oxepin, moldavica acid B (2), together with four known phenylpropionic acids (3-6) and protocatechuic acid (7) that were isolated from Dracocephalum moldavica L. Their structures were elucidated by comprehensive spectroscopic methods, including infrared and nuclear magnetic resonance. Compound 1 is the first example of salicylic acid linking a carboxylated α-pyrone via an ethyl bridge. Beyond expanding the knowledge of the chemical diversity of D. moldavica, both compounds 1 and 2 were shown to upregulate the expression of Kruppel-like factor 2, which could serve as a prospective therapeutic target for the treatment of atherosclerosis. Graphical Abstract


Introduction
Dracocephalum moldavica (D. moldavica) L. (Labiatae), called "Xiang Qing Lan" in Chinese, is an annual plant, mainly distributed in the northwest of China. The dried aerial parts of the plant have long been used as traditional medicine for the treatment of cardiovascular and cerebrovascular diseases in Xinjiang Uygur Autonomous Region, China [1]. More recently, scientific studies have identified a number of chemical compounds found within D. moldavica that can explain some of its medicinal qualities and potential applications. Phytochemical investigation of D. moldavica led to some flavonoids [2,3], phenylpropanoids [3][4][5], and terpenoids [6][7][8]; compounds that display anti-oxidative [2], anti-inflammatory [8], antiapoptotic [4], and anti-complementary activities [6,7]. In particular, tilianin, a flavonoid glycoside in D. moldavica, is considered as a potential natural lead compound for the treatment of cardiovascular disorders [9]. Other studies have focused on the bioactivity of total flavonoids contained within D. moldavica and have demonstrated their anti-atherosclerotic [10], anti-myocardial ischemic [11], anti-cerebral ischemic [12], as well as potential therapeutic effects on chronic mountain sickness [13]. In addition to these intriguing properties, the ethyl acetate (EtOAc) fraction of D. moldavica ethanol (EtOH) extract displays antimicrobial activity against MRSA bacteria [14], antioxidant activity, and cardioprotective effect [15].
One potential therapeutic application of compounds from D. moldavica is the treatment of atherosclerosis (AS), a common pathophysiological basis of many ischemic cardiovascular diseases. Its occurrence and development are closely related to inflammation and lipid metabolism disorders, and as such, an effective treatment for AS could have significant and far-reaching benefits for patients. The Krueppel-like factor 2 (KLF2), belonging to a subclass of the zinc finger family of transcription factors, exhibits several important functions, including the protection of endothelial cells, prevention of thrombosis, and anti-inflammation [16]. Thus, it has been proposed that the upregulation of the KLF2 expression could serve as a prospective therapeutic target for the treatment of AS [17].
In this study, as part of a program to discover the bioactive components from traditional Chinese medicines, we investigated the EtOH extract of the aerial part of D. moldavica. Seven phenolic acids were isolated in this work, including a new salicylic acid derivative, moldavica acid A (1), a new natural dibenzo[b,f]oxepin, moldavica acid B (2), together with four known phenylpropionic acids (3)(4)(5)(6), and protocatechuic acid (7) (Figure 1). Compounds 1 and 2 could significantly upregulate the expression of KLF2.

Results and discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula was determined to be C 15   (1H, d), 6.71 (H, d), 7.18 (H, dd), two isolated aromatic protons at d H 7.06 (1H, s) and 6.30 (1H, s), and two linked methylenes at d H 2.89 (2H, brt) and d H 2.73 (2H, brt). In addition, the 13 C NMR and DEPT spectra indicated the presence of eight quaternary (three carbonyl, three olefinic, and two oxygenated olefinic) carbons ( Table 1).
In the preliminary bioactivity test, both 1 and 2 significantly upregulated the expression of KLF-2 at 5.1 and 10.6 folds, with EC 50 values at 59.5 and 23.0 lM, respectively.
In conclusion, a new salicylic acid derivative, moldavica acid A (1), and a new natural dibenzo[b,f]oxepin, moldavica acid B (2), together with four known phenylpropionic acids (3)(4)(5)(6) and protocatechuic acid (7), were isolated from the EtOH extract of D. moldavica. Moldavica acid A is the first example of a salicylic acid linking a carboxylated a-pyrone via an ethyl bridge. Furthermore, both moldavica acids A and B were shown to enhance the upregulation of the KLF-2 expression. This discovery expands the chemical diversity of known D. moldavica compounds and also provides an anti-AS target for synthetic chemistry.

Plant material
The dried aerial part of Dracoceph moldavica L. was collected in September, 2020, from Changji, Xinjiang Uygur Autonomous Region, China. Plant identity was verified by Jiang He, associate researcher of Xinjiang Key Laboratory of Uighur Medicine. A voucher specimen (No. XQL-2020) was deposited at Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Extraction and isolation
The dried aerial part of D. moldavica (40 kg) was extracted with 40% ethanol (120 L, 3 Â 1 h) using a heated refluxed method. The extract was then concentrated, and the EtOH removed completely to a 20 L suspension. The suspension was dissolved in H 2 O (40 L) and subjected to macroporous adsorption resin chromatography using the gradient elution with H 2 O, 30% EtOH, 50% EtOH, and 95% EtOH to give four fractions. After removing EtOH under reduced pressure, the 50% fraction was extracted three times with EtOAc to give an organic phase, which was concentrated under vacuum to yield a residue (41.1 g).
The EtOAc extract was separated using silica gel column chromatography and eluted with a stepwise gradient elution of CH 2 Cl 2 -MeOH (from 100:0 to 0:100, v/v),

Kruppel-like factor 2 (KLF2) luciferase assay
Briefly, COS-7 cells were cultured in DMEM with 10% Fetal Bovine Serum (FBS). Then, the cells were transfected with KLF2 plasmid in 96-well plates (5 Â 10 5 cells/ml, 150 ml/well) for 6 h. The test compounds were diluted to different concentrations at 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.078 lM in 200 ll/well with DMEM containing 10% FBS, and incubated for 24 h [17]. The activity of KLF2 luciferase reporter gene was detected using EnVision Multifunctional Assay System. The luciferase activity was calculated as the ratio of compound to blank (DMSO) firefly luciferase value. The EC 50 value was generated by GraphPad Prism 8 software.

Disclosure statement
No potential conflict of interest was reported by the author(s).