Studies on chemical constituents of Picrasma javanica, Globba sherwoodiana and Kaempferia candida collected in Myanmar and their anti-Vpr activities

概要

1. Introduction
Human immunodeficiency virus (HIV) is a virus that causes Acquired Immunodeficiency Syndrome (AIDS). This is a disease of the cell-mediated immune system or T-lymphocytes of the human body. The main treatment for HIV is antiretroviral therapy (ART), a combination of daily medications that reduces viral reproduction. Despite the success of this therapy, ART-resistant viruses continue to emerge to threatening loss of effectiveness of the treatment. Thus, researches are shifting their focus to alternative target for novel drugs. Viral protein R (Vpr) is a small basic protein (14 kDa) of 96 amino acids, found in HIV-1, HIV-2, and simian immunodeficiency virus (SIV), and plays an important role in the virus life cycle. Vpr seems to be essential for viral replication in non-dividing cells such as macrophages. Based on the functions, the Vpr inhibitor is one of the possible target molecules for anti-HIV drugs. In our previous studies, we reported the chemical constituents such as isopimarane diterpenoids, quassinoids, terpenoids, iridoids, and bis-iridoid glycosides with potent anti-Vpr activities. Based on aforementioned results, including those obtained from Myanmar medicinal plants, I was motivated to continue the search for new Vpr inhibitors as a part of the anti-HIV drug discovery from Myanmar natural resources. Therefore, in this study, in order to search for new Vpr inhibitor compounds, eleven medicinal plants were collected from Myanmar. The methanol and chloroform extracts of all samples were tested separately for their anti-Vpr activities on the TREx-HeLa-Vpr cells. The screening results revealed that the chloroform extracts of Picrasma javanica wood, Globba sherwoodiana rhizomes, and Kaempferia candida roots and rhizomes showed the most potent anti-Vpr activities without any cytotoxicity on the Vpr-induced cells with the cell proliferation percentages of 178% ± 0.48, 160% ± 2.26, and 160% ± 1.06 at 10 μg/mL, respectively.

2. Constituents of Picrasma javanica collected in Myanmar and their anti-Vpr activities1,2)
P. javanica Blume is a medium-sized tree of the Simaroubaceae family and is found spanning in Southeast Asia, Papua New Guinea, India, Indonesia, the Solomon Islands, and Myanmar. Its bark and leaves are widely used for the treatment of malaria in traditional medicine in Myanmar, Indonesia, Thailand, and northern India. The bark and leaves contain quassinoids, indole alkaloids, coumarins, sesquiterpenes, and triterpenes. Previous our studies revealed that some quassinoids isolated from the P. javanica bark possess anti-Vpr activities.
The investigation of the Vpr inhibitors from the active chloroform extract of the P. javanica wood led to isolation of five new quassinoids, (16R)-methoxyjavanicin B (1), (16S)-methoxyjavanicin B (2), javanicinol A (3), javanicinol B (4), and 4-keto-(16S)-methoxyjavanicin B (5), together with seven known quassinoids (6−12), one alkaloid (13), one triterpenoid (14), and one coumarin (15) (Figure 1). The structures of all isolated compounds were determined by analyses of their spectroscopic data (1D and 2D NMR, MS, CD) and by comparing these data with those reported in the literature.
All isolated compounds showed the potent anti-Vpr activities without showing any cytotoxicity at the concentration of 5 μM, except for 5, which had insufficient yield (Figure 2). The anti-Vpr activities of 1 (158%) and 2 (159%) were found to be similar to that of positive control damnacanthal (Dam) (157%) at the 2.5 μM concentration and were higher than those of 3 (148%) and 4 (147%) at the same concentration, respectively. The anti-Vpr activities of isolated compounds 1−4 and 13−15 was investigated for the first time in this work. Moreover, the structure-activity relationship (SAR) study of C-20 type quassinoids, neoquassin and quassin revealed that the presence of the methoxy group at C-16 and the absence of the β-methoxy group and/or the hydroxy group at C-2 were essential for Vpr inhibitory activities. Our finding suggested that the quassinoids from P. javanica could be candidates for anti-Vpr active drugs.

3. Constituents of Globba sherwoodiana collected in Myanmar and their anti-Vpr activities3)
G. sherwoodiana (Zingiberaceae) is a small perennial herb with rhizomes cultivated in Myanmar, Thailand, and India. To the best of our knowledge, no previous phytochemical and biological studies have been reported from any part of G. sherwoodiana. Investigation of the Vpr inhibitors from the active chloroform extract of the G. sherwoodiana rhizomes led to isolation of two new homodrimane sesquiterpenes, globbatone A (16) and globbatone B (17), and one new 16-norlabdane diterpenoid, globbatone C (18), along with thirteen known sesquiterpenoids (20−21), diterpenoids (19, 22−30), and one isospongian diterpenoid (31) (Figure 3). Among these, 19 and 20 were isolated as new naturally occurring compounds. The anti-Vpr assay showed that 16, 18−27, and 29−31 had moderate anti-Vpr activities, although 31 possessed weak cytotoxic effect at the concentration of 10 μM (Figure 4). In contrast, 17 and 20 inhibited the Vpr-activity, without any cytotoxicity. The SAR study of homodrimane sesquiterpenes suggested that the presence of the hydroxypropionate group at C-12 was important for increasing the activities. In addition, the 8(17)-exomethylene labdane diterpenoids revealed that the presence of the lactone ring and carbonyl group could be the important functionalities to enhance the activity. The occurrence of sesquiterpene and diterpene types of compounds from G. sherwoodiana could be potential chemotaxonomic marker for this species.

4. Constituents of Kaempferia candida collected in Myanmar and their anti-Vpr activities4)
K. candida is a perennial herb with numerous inflorescences that emerges directly from rhizomes and is distributed in Myanmar and Thailand where it grows in the burnt bamboo forest. However, no previous phytochemical and biological studies on K. candida have been reported so far. The chloroform extract of the roots and rhizomes of K. candida showed the potent anti-Vpr activities on the TREx-Hela-Vpr cells. The investigation of the Vpr inhibitors from the active chloroform extract of K. candida led to isolation of three new compounds including one copaene type sesquiterpene, 7-hydroxymustakone (32), one nerolidol type sesquiterpene, 15-hydroxynerolidol (33), and one 15-norlabdane diterpene, kaempcandiol (34), one copaene sesquiterpene (35), three known labdane diterpenoids (36−38), and one phenolic derivative (39) (Figure 5). Compounds 32 and 34−39 possessed potent anti-Vpr activities at the concentration of 5 μM, without showing any cytotoxicity (Figure 6). Compound 33 exhibited moderate anti-Vpr activities at the 5 μM concentration, although this compound possesses weak cytotoxic effect. The SAR study suggested that tricyclic sesquiterpenes with α,β-unsaturated ketone group at C-2 could be potential candidate for anti-Vpr activities and the presence of tertiary carboxylic acid and exomethylene and/or hemiacetal groups at C-8 on labdane diterpenes may play essential roles for their anti-Vpr activities.

Conclusion
In this study, the chemical investigation of the active chloroform extracts of the P. javanica wood, G. sherwoodiana rhizomes, and K. candida roots and rhizomes led to the isolation of 39 compounds including five new quassinoids 1−5, two new homodrimanes 16 and 17, two new norlabdanes 18, 34, one new copaene 32, one new nerolidol 33, and two natural new terpenoids 19 and 20, together with 26 known compounds 6−15, 21−31, and 35−39. The phytochemical and biological studies of the G. sherwoodiana and K. candida species are investigated here for the first time. The SAR suggested that the most of the quassinoids, bicyclic and/or tricyclic sesquiterpenes, and labdane diterpenes showed potent anti-Vpr activities. These results suggested that the three skeletons could be crucial for development of anti-HIV-1 drugs. The findings will contribute to the scientific development of Myanmar traditional medicine, especially in the areas concerned with the disease of AIDS.

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