Pimarane Diterpenoids from Aerial Parts of Lycopus lucidus and Their Antimicrobial Activity

The ethyl acetate fraction obtained from aerial parts of L. lucidus was subjected for isolation of new bioactive compounds, which enabled isolation of five new pimarane-type diterpenoids, namely, 3β, 8β, 12β, 18-tetrahydroxy pimar-15-ene (10), 7α, 8β, 12β, 18-tetrahydroxy pimar-15-ene (11), 3β, 8β, 11β, 12α, 18-pentahydroxy pimar-15-ene (12), 12β acetoxy, 8β, 3β, 18-trihydroxy pimar-15-ene (13), and 3β acetoxy, 8β, 12β, 18-trihydroxy pimar-15-ene (14), along with nine known compounds. The structures were elucidated by spectroscopic analysis and comparison with literature data. The isolated new pimarane diterpenoids were examined for antimicrobial activity against Gram-negative and Gram-positive bacteria strains. Among them, the compound 3β, 8β, 12β, 18-tetrahydroxy pimar-15-ene (10) was most effective, exhibiting minimum inhibitory concentration (MIC) values of 15.62 µg/mL against Staphylococcus epidermidis, 31.25 µg/mL against Staphylococcus aureus, 62.5 µg/mL against Pseudomonas aeruginosa, and 125 µg/mL against Escherichia coli.


Introduction
e genus Lycopus of family Lamiaceae (Labiatae) contains around 16 species with wide distribution in Europe, Asia, and North America [1]. In Asia, Lycopus lucidus is widely distributed species in Korea, China, Japan, Russia, and Taiwan. It is most abundant in Korea. L. lucidus is a flowering perennial glabrous herb occurring in aquatic environment and grows up to 0.6-1.2 m height at an altitude of 320 m to 2100 m [2][3][4]. L. lucidus is one of the popular edible plants with its long history as a folk remedy in traditional medicinal system of China, Japan, and Korea [5].
is plant has been used as both traditional and official formulations, as they are potent source of bioactive tannins, coumarins, flavonoids, terpenoids, and essential oils. Major bioactive compounds that have been isolated from this plant are flavonoid and its esters, rosmarinic acid derivatives, phenylpropanoids, steroids, pentacyclic triterpenes, essential oils, oligosaccharides, polysaccharides, and diterpenoid glycosides [3,[6][7][8].
e leaf and stem (aerial part) of L. lucidus have been extensively used for the treatment of inflammation, cardiovascular problem, insomnia, menstrual problems, and thyroid problem, as a sedative, wound healing, pain reliving agents, herbal tea, and useful tonic [6,8,9]. e root of L. lucidus is known as small ginseng in China and widely used as dietary supplement [10]. Many biological activities such as inhibition of superoxide radical [4], nitric oxide scavenging effect [6], inhibition of hypercholesterolemia and atherosclerosis [11], acaricidal activities [10], and hyaluronidase inhibition [5] have been explored from this plant.
In the current scenario, bacterial infectious diseases are a serious worldwide public health problem due to an increase in their resistance towards antibiotics, which have ultimately given on to the birth of multiresistant bacterial strains. Increased rates of mortality and morbidity are due to the lack of long-term effective drugs and the unaffordable cost of new generation antibiotics. e problem of microbial resistance is growing and the prospect of the use of antimicrobial drugs is uncertain.
is disastrous situation has compelled us to explore more successful antimicrobial agents using plant resources so that they will serve as an active therapeutic ingredient, as well as leading molecule for the synthesis of optimized new drugs. e plant species have always been serving as a major source of novel and potent antimicrobial constituents, as they possess the capability to synthesize secondary metabolites to combat diverse pathogenic microorganisms available in the environment [12]. In this context, our study is mainly focused on isolation and structure elucidation of possible new compounds from the aerial parts of L. lucidus followed by screening their antibacterial properties against pathogenic Gram-positive and Gram-negative bacterial strains.

Determination of Minimum Inhibitory Concentration (MIC).
e twofold serial broth microdilution technique was adopted to calculate the MIC values of new isopimarane diterpenoids, against four different test organisms. A total of 10 vials were labeled and sterilized, then 960 µL of sterilized Mueller-Hinton Broth (MHB) was transferred into each vial. For the sample solution preparation, 25000 µg/mL of stock solution was prepared in DMSO, subjected to serial dilution, using a 1 : 1 mixture of DMSO and water to prepare sample solutions of 10 different concentrations (25000 µg/mL-48.8298125 mg/mL). After that, 40 µL of sample solution was transferred into a corresponding vial containing 960 µL of MHB, so that the final concentration of sample ranged from 1000 µg/mL to 1.95 µg/mL. Bacteria with an inoculum of about 1 × 10 5 CFU/mL were loaded into each vial. For the preparation of microorganism inocula, broth culture was incubated for 12 h, and turbidity of the suspension was adjusted to the turbidity of 0.5 McFarland standards. One inoculated vial was used as a negative control, to ensure suitability broth for growth of microorganism growth. Also, 4% DMSO was tested as a blank control. Streptomycin sulfate and Vancomycin were considered as a positive control for Gram-negative and Grampositive microorganisms, respectively. After the incubation of the sample containing broth media for 24 h at 37 0 C, the MIC value was determined. e MIC value was considered as the minimum concentration of compound that prevented the microorganism growth. e bacterial cell viability was determined by using 3(4,5 dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide (MTT) by incubating at 37°C for further 2 h and visual inspection of formazan formation [13].

Identification and Structure Elucidation of Noble
Compounds.

Antimicrobial Activity of Isolated Pimarane Diterpenoids.
e isolated new pimarane diterpenoids were examined for their antimicrobial potency in response to Grampositive bacteria S. epidermidis (ATCC 12228) and S. aureus (ATCC 9144) and Gram-negative bacteria P. aeruginosa (ATCC 27853) and E. coli (ATCC 14948), using a twofold serial broth dilution technique and MIC was evaluated. As shown in Table 3, the most significant compound was 10, which manifested MIC values of 15.62 and 31.25 µg/mL against S. epidermidis and S. aureus, respectively. Also, this compound was effective against P. aeruginosa and E. coli with MICs of 62.5 µg/mL and 125 µg/mL, respectively. It is to be noted that compounds 13, 14, and 15 were ineffective against both Gram-negative strains at examined concentrations.
In the search for natural products as effective antimicrobial agents, numerous investigations have proved the promising bactericidal effect revealed by diterpenoids. However, very limited researches have been reported for the antibacterial potency of pimarane diterpenoids [25]. It has been reported that the presence of a decalin ring system in pimarane diterpenoids fascinates its penetration into the lipophilic cell membrane of bacteria to induce bacterial lysis. Furthermore, an appropriately positioned hydrophilic functional group (hydrogen bond donor group; HBD) is capable of interacting with phosphorylated groups of the bacterial cell membrane [26]. In this study, MIC values of two isomers, i.e., compounds 10 and 11, were slightly different. e structural difference between these compounds is only the position of one hydroxyl group (3 rd position OH group in compound 10 is shifted to 7 th position in compound 11). Although the exact structureactivity relationship is not known, this may signify that a change in position of the same functional group may also alter the antibacterial effect. On the other hand, the antibacterial activity of compounds 13 and 14 was reported to be reduced, in which -OH group was replaced by an electron-withdrawing acetyl group. In a previous study, 3and 19-hydroxyl groups of isopimarane compounds were acetylated to investigate the role of substituents on antimicrobial activity. e results indicated that if acetylation occurs in the 3-hydroxyl position or both 19-and 3-hydroxyl groups, the antibacterial activity gets reduced significantly [27]. Hence, our study also revealed a similar result. Furthermore, this study suggested that a slight variation in the position and/or nature of the oxygenated moiety can result in a substantial change in antibacterial activity. Similar results were also found in previous studies, regarding the biological activities of other pimarane diterpenoids [25,28,29].

Conclusion
Five new pimarane diterpenoids along with nine known compounds (polyphenol and flavonoids) were isolated from the ethyl acetate fraction of L. lucidus and their chemical structures were elucidated completely through instrumental data including 2D-NMR.
Among them, compound 6 (rosmarinic acid) was isolated in the largest amount. Compound 2 (protocatechualdehyde) and compound 4 (methyl 3, 4, dihydroxy benzoate) were isolated for the first time from this plant. Besides, antibacterial activity screening for newly isolated compounds showed that pimarane diterpenoids are more sensitive towards Gram-positive bacteria. Among the isolated new diterpenoids, compounds 10 and 11 were found to be most effective with 15.62 µg/mL and 31.25 µg/mL MICs values, respectively, against S. epidermidis. In addition, this screening showed that small changes in position and/or nature of the oxygenated functional group in diterpenoids can result in a significant variation in antibacterial activity.

Data Availability
All the data used to support the result of this research are available from Jitendra Pandey.

Conflicts of Interest
All the authors in this article have on conflicts of interest.

Authors' Contributions
Amrit Poudel and Hyeong Kyu Lee conceived the experiment. Jitendra Pandey designed and performed the experiment. Jitendra Pandey and Bang Yeon Hwang analyzed the Evidence-Based Complementary and Alternative Medicine data. Jitendra Pandey and Amrit Poudel wrote the manuscript. Jitendra Pandey revised the manuscript.