Sugar Containing Compounds and Biological Activities of Lagochilus setulosus

Phytochemical investigation of the methanolic extract obtained from the aerial parts of Lagochilus setulosus (Lamiaceae) afforded the new compound 1-methoxy-3-O-β-glucopyranosyl-α-l-oliose (1) together with five known glycosides, namely sitosterol-3-O-β-glucoside (2), stigmasterol-3-O-β-glucoside (3), pinitol (4), 6β-hydroxyl-7-epi-loganin (5), and chlorotuberoside (6). The structures of these compounds were elucidated by extensive spectroscopic analyses, especially HR-MS, 1D and 2D NMR spectroscopy. The in vitro cytotoxic activity of the methanolic extract and the isolated compounds was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and crystal violet (CV) staining assays. In addition, the antifungal activities of the components were evaluated against Botrytis cinerea, Septoria tritici, and Phytophthora infestans. The anthelmintic potential was determined against Caenorhabditis elegans nematodes. Neither the extract nor the isolated compounds showed promising activity in all the bioassays.


Introduction
The genus Lagochilus (Lamiaceae) is native to central, south-central, and eastern Asia. It consists of 46 species with 18 of them growing in Uzbekistan. Taxa of the genus Lagochilus basically occur throughout the territory of Uzbekistan, starting from the deserts to Tian-Shan and Pamir-Alay mountain systems. The majority of species can be found in the Pamir-Alai Mountain, southwest of Tian-Shan and Turanian lowland. Species from the genus Lagochilus belong to the most vulnerable plant species from the Lamiaceae family [1], with a decoction of herb and roots of Lagochilus species used in folk medicine mainly as styptics, hemostatics, and tranquilizers, and also for skin conditions, stomach pain, hemorrhages, and inflammation. Previous phytochemical investigations on Lagochilus species revealed the presence of essential oils, flavonoids, polysaccharides, sterols, iridoids, terpenoids, and more frequently diterpenes, which are considered as chemical markers for this genus. About 150 metabolites have been reported from all previous classes in the genus [2][3][4].
The species of Lagochilus setulosus Vved. is endemic of the southwestern area of Tian-Shan, and it can be found distributed in low mountains between the border of the regions Chimkent (Kazakhstan) and Tashkent (Uzbekistan) [1]. A literature survey reveals that there are no reports on traditional use, biological activity, or chemical investigations of L. setulosus, except our previous report on the volatile components of the aerial parts and their antioxidant and enzyme inhibitory activities [5]. In continuation of our phytochemical studies on L. setulosus, herein, we report the isolation and structure identification of a new disaccharide 1-methoxy-3-O-β-glucopyranosyl-α-L-oliose (1) (Figure 1) together with two known sterol glucosides: sitosterol-3-O-β-glucoside (daucosterol) (2) and stigmasterol-3-O-β-glucoside (3), as well as cyclitol-pinitol [3-(O-methyl-D-chiro-inositol)] (4), and the two iridoid glycosides 6β-hydroxyl-7-epi-loganin (5) and chlorotuberoside (6) ( Figure 1). In addition, the anthelmintic, antifungal, and cytotoxic effects of the isolated compounds 1, 4, a mixture of 5 and 6, and the methanolic extract of L. setulosus have been evaluated.

Chemical Composition of L. setulosus
Since no publications were found regarding the chemical composition of the aerial parts of L. setulosus, this is the first report on the occurrence of sugar-containing compounds 1-6. The chromatographic fractionation of the methanolic extract of this species afforded sitosterol-3-O-β-D-glucoside (2) and stigmasterol-3-O-β-D-glucoside (3) as an inseparable mixture (with a ratio of 1:1), and the new compound 1. The chemical structure of compounds 2 and 3 was determined based on spectral analysis and comparisons with relevant literature [6]. These compounds were also found in other Lagochilus species viz. L. inebrians and L. gypsaceus [7]. Further investigations of the methanolic extract of L. setulosus afforded pinitol (4) and an inseparable mixture (with a ratio of 1.2:1) of 6β-hydroxyl-7-epi-loganin (5) and chlorotuberoside (6). Most recently, the iridoid glycoside 5 has been reported from several species of Eremostachys [8,9] and Picconia excelsa [10]. Chlorotuberoside (6) was previously isolated from different genera of Lamiaceae, such as Phlomis [11-

Biological Activities
The cytotoxicity of the crude methanolic extract of L. setulosus, compounds 1, 4, and the mixture of 5 and 6 was evaluated in MTT and CV assays against the human cancer cell lines PC-3 (prostate cancer) and HT-29 (colon adenocarcinoma). Results of the tests showed weak effects. In addition, the methanolic extract and isolated compounds were tested against the phytopathogenic ascomycetes Botrytis cinerea Pers. and Septoria tritici Desm. and the oomycete Phytophthora infestans (Mont.) de Bary., but the samples did not show any promising activity up to the highest tested concentration of 125 ppm. The results of anthelmintic assays showed that the samples did not significantly kill Caenorhabditis elegans under the testing concentration of 500 μg/mL.

Biological Activities
The cytotoxicity of the crude methanolic extract of L. setulosus, compounds 1, 4, and the mixture of 5 and 6 was evaluated in MTT and CV assays against the human cancer cell lines PC-3 (prostate cancer) and HT-29 (colon adenocarcinoma). Results of the tests showed weak effects. In addition, the methanolic extract and isolated compounds were tested against the phytopathogenic ascomycetes Botrytis cinerea Pers. and Septoria tritici Desm. and the oomycete Phytophthora infestans (Mont.) de Bary., but the samples did not show any promising activity up to the highest tested concentration of 125 ppm. The results of anthelmintic assays showed that the samples did not significantly kill Caenorhabditis elegans under the testing concentration of 500 µg/mL.

Plant Material
The aerial parts (flowers, leaves, and stems) of L. setulosus were collected in Oqtepa Yunusabad, Tashkent region (Uzbekistan), in May 2019. A voucher specimen (N273) is deposited at the Herbarium the Botany Institute of Uzbekistan and verified by Dr. Akbar Akhmedov. The aerial parts were air-dried in shadow and powdered in a mortar before use (moisture content was 11%, w/w).

Biological Assays
The anthelmintic, antifungal, and cytotoxic effects of all isolated compounds were determined by the procedures described below.

Cytotoxic Activity
The cytotoxicity of the methanolic extract and isolated compounds was evaluated against the human tumor PC-3 (prostate cancer) and HT-29 (colon adenocarcinoma) cell lines. The extract and isolated compounds were tested at the concentrations of 0.05 and 50 µg/mL, and 0.01 and 10 µM, respectively. The cell maintenance and assay procedure were performed as described by Dos Santos et al. [20]. The viability of the cells was determined by MTT and CV assays after 72 h incubation time. The absorbance was measured with an automated microplate reader at 540 nm with a reference wavelength of 670 nm [21]. The results are presented as a percentage of control values obtained from untreated cultures.

Antifungal Activity
The methanol extract and isolated compounds were tested in 96-well microtiter plate assays against the phytopathogenic ascomycetes Botrytis cinerea Pers. and Septoria tritici Desm. and the oomycete Phytophthora infestans (Mont.) de Bary according to the monitoring methods approved by the fungicide resistance action committee (FRAC) with minor modifications as described before [22]. Crude extracts and fractions were examined at a final concentration of 125 µg/mL, while pure compounds were tested in a serial dilution, ranging from 100 to 0.1 µM. The solvent DMSO was used as negative control (max. concentration 2.5%), and the commercially used fungicides epoxiconazole and terbinafine (Sigma-Aldrich, Darmstadt, Germany) served as positive controls. Five to seven days after inoculation, pathogen growth was evaluated by measurement of the optical density (OD) at λ 405 nm with a TecanGENios Pro microplate reader (5 measurements per well using multiple reads in a 3 × 3 square). Each experiment was carried out in triplicates.

Anthelmintic Activity
The Bristol N2 wild type strain of Caenorhabditis elegans was used in the anthelmintic assay. The nematodes were cultured on NGM (Nematode Growth Media) petri plates using the uracil auxotroph E. coli strain OP50 as a food source according to the methods described before [20]. The anthelmintic assay was carried out following the method developed by Thomsen et al. [23]. Briefly, after 4 days of cultivation, the nematodes were transferred from the Petri plate to a 15 mL falcon tube by rinsing each plate twice with 2 mL M9 buffer. The worm suspension was centrifuged for 1 min at 800 G. After removal of the supernatant, the nematodes were washed again with 2 mL M9 buffer under the same conditions and, depending on the number of individuals, re-suspended in 2 to 8 mL M9 buffer. To this suspension, 10 µL penicillin-streptomycin-solution (10 mg/mL) was added. After triply counting the nematodes in 10 µL solution droplets under a stereo microscope (Olympus SZX12), the worm number was adjusted to 20-30 animals per 20 µL. The assay was performed in 384 well plates. The outer wells were filled with water to minimize evaporation. To the test wells, 20 µL worm suspension was added and the number of living and dead animals in each well were counted using the cell culture microscope Olympus CKX41. The number of living nematodes was set at 100%. At staggered intervals, 20 µL test solution (test compound in 4% DMSO in M9 buffer) was added followed by a microscopic enumeration of living and dead test organism after 30 min of incubation. For all test plates, the solvent DMSO (2%) and the standard anthelmintic drug ivermectin (10 µg/mL) were used as negative and positive controls, respectively. All the assays were done in triplicate.

Conclusions
From the aerial parts of L. setulosus, a new disaccharide, 1-methoxy-3-O-β-glucopyranosylα-L-oliose, and five known glycosides were isolated and identified. All compounds were isolated for the first time from this species. The results of anthelmintic, antifungal, and cytotoxic assays demonstrated that the methanolic extract and isolated compounds of L. setulosus were not toxic in general in in vitro assays.