Chemical Constituents from Ethanoic Extracts of the Aerial Parts of Leea aequata L., a Traditional Folk Medicine of Myanmar

Abstract We aimed at reporting the chemical constituents and antimicrobial activities of Leea aequata L., a traditional folk medicine used in Myanmar for the treatment of wounds and skin diseases. A new neolignan, (7S,8R)-9′-O-acetylcedrusin (1), a new lactam, (3S,4S)-4-chloro-3-hydroxypiperidin-2-one (2), along with 21 known compounds, including five lignans (3–7), four flavonoid glycosides (8–11), and others (12–23), were isolated from the ethanoic extract of the aerial parts of L. aequata. The structures of the new compounds were determined by NMR, MS, and ECD spectra. For all the antimicrobial tests of the 23 compounds, only 3,4,5-trihydroxybenzoic acid ethyl ester (17) showed weak inhibitory activities against Escherichia coli and Salmonella enterica subsp. enterica. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-019-0209-y) contains supplementary material, which is available to authorized users.


Introduction
Medicinal plants and their traditional knowledge are important source for modern drug development. In Myanmar, majority of the populations had been relying on traditional herbal remedy for treatment of various diseases for generations. However, in the past several decades, Myanmar is behind the development of science and technology due to political unease and backward of social economic development, which kept the valuable knowledge and resources on medicinal plants un-known to the world; and thus rarely applied at international scale. Recently, Defilipps and Krupnick summarized the medicinal plants used in Myanmar, showing a total number of 472 plant species from 123 families used as herbal medicine [1]. Medicinal Plant List of Myanmar, a book published by FAME Company in Myanmar, recorded more than 1500 medicinal plant species [2]. Few ethnobotanical studies documented various list of medicinal plants used locally such as in southern Shan State [3], at Natma Taung National Park [4], and in southern Chin State [5]. These publications provide pilot investigations and fundamental information to understand the value of these wealthy biodiversity and culture for medicinal plants in Myanmar. Apart from these, applying modern technology, such as phytochemistry and pharmacology for Myanmar medicinal plant research were also surging. For example, Nwet Nwet Win published a series of papers on natural compounds isolated from Kaempferia pulchra [6], Premna integrifolia [7], and Kayea assamica [8,9]. Other examples include extracts with anti-influenza virus property from Jatropha multifida [10], along with evaluation of antioxidant and antimicrobial activities of indigenous medicinal plants [11,12]. However, the discovery of the traditional knowledge, biological constituents, and pharmacological properties of the vast pool of Myanmar medicinal plants just commences, and intensive field explorations and scientific validations are much needed.
The genus Leea belongs to the family of Vitaceae. Species of Leea are distributed from Africa to Asia, northeastern Australia, New Guinea and islands of the Pacific (Fiji, Solomon Islands, Caroline Islands) [13]. Some species are used as traditional folk medicines. For example, the roots of L. asiatica (L.) Ridsdale are used to treat icteric hepatitis in China [14], the roots of L. macrophylla Roxb. ex Hornem are used in medication for guineaworm in Myanmar [1], the leaves of L. guineense G. Don are used against cancers in Guinea [15], and the roots of L. thorelii Gagnep. are used as a tonic in Thailand [16]. Flavanoids and flavanoid glycosides are found to be the major constituents of the genus [16,17].
Leea aequata is usually a shrub, less often a small tree, distributed in Bangladesh, Bhutan, Cambodia, China, India, Malaysia, Myanmar, Nepal, Philippines, Thailand, and Vietnam [18]. A previous research showed that the seeds, stems, and roots of L. aequata have antibacterial activity [19]. However, no knowledge is available on the chemical constituents of this species. In Mandalay, Myanmar, it is locally known as Kya-petthein (naga-mauk). The fresh leaves of the plant are crushed and externally used for treating wounds and skin diseases by Bamar people. During our field visit for inventory of medicinal plants in central Myanmar in Dec 2015, we collected the specimen of L. aequata and documented the traditional uses by local people around Myingyan, Kyaukpadaung, Po-pa Mountain in Mandalay. In Feb 2018, we continued a further investigation of ethnobotanical knowledge and collected the aerial parts of the species from the same site for phytochemical analysis. We aimed at isolating and understanding the chemical constituents and at testing the antimicrobial activities of this species.

Structure Elucidation
Two undescribed compounds (1 and 2, Fig. 1) and 21 known compounds  were isolated from the ethanoic extracts of L. aequata by silica gel and Sephadex LH-20 column chromatography and semipreparative HPLC.   3 methylenes, and two sp 3 methines, which implied that compound 1 might be an acetylated neolignan. By comparing its NMR data with those of cedrusin [20], 1 was deduced to be an acetylated derivative of cedrusin, which was confirmed by COSY and HMBC correlations (Fig. 2). The acetoxyl group was located at C-9′ based on the HMBC correlations from H 2 -9′ to C-1″. The trans relationship between H-7 and H-8 was elucidated by the ROESY correlations of H-7/ H 2 -9 ( Fig. 2), as well as the chemical shift of C-8 (δ C 55.7) [21]. In this type of neolignans, the chemical shift of C-8 is approximately 54 ppm in the trans isomers and is approximately 49 ppm in the cis analogues [21]. The absolute configurations of dihydrobenzofuran neolignans are usually determined by the signs of the band 1 L b (270-300 nm) or 1 L a (220-240 nm) in their ECD spectra. The positive signs of the 1 L b band in the ECD spectra predict the absolute configuration of 7,8-trans-3-methoxydihydrobenzofuran neolignans to be 7S,8R [21][22][23]. The ECD spectrum of 1 showed a positive Cotton effect at 294 nm (Δε + 0.52). Therefore, the structure of 1 was determined to be (7S,8R)-9′-O-acetylcedrusin.

In Vitro Antimicrobial Assays
The bacterial strains, E. coli ATCC25922, S. aureus subsp. aureus ATCC29213, S. enterica subsp. enterica ATCC14028, and P. aeruginosa ATCC27853, and the fungal strain, C. albicans ATCC10231, were purchased from China General Microbiological Culture Collection Center. The antimicrobial assays were performed according to modified versions of the CLSI (formerly NCCLS) methods as described previously [43,44]. Ceftazidime and benzylpenicillin sodium were used as the positive control drugs in the antibacterial assay and amphotericin B was used as the positive control in the antifungal assay.

ECD Calculations
Computational methods are presented in Supplementary Material.

Conclusion
Twenty-three compounds including one new lignan, one new lactam, five known lignans, four flavonoid glycosides, and other compounds were isolated from the ethanol extracts of the aerial parts of L. aequata collected from Myanmar. 3,4,5-Trihydroxybenzoic acid ethyl ester (17) showed the weak inhibitory activities against E. coli and S. enterica subsp. enterica.