Five previously undescribed compounds from Ajuga lupulina Maxim. and their in vitro activities

Abstract A new biflavone (philonotisflavone-3′′′-methyl ether), three diterpenes (lupulin G, lupulin H, lupulin I), a new ecdysteroid (ajugasterone E), and four known compounds were isolated from the whole plant of Ajuga lupulina Maxim. Their structures were determined by spectroscopic analysis, including MS, NMR and ECD spectral data. Compounds 1-3 has DPPH radical scavenging ability, and compound 1 has stronger activity than vitamin C. Compounds 2, 3, 7 and 8 have potential cytotoxic activity against Hela, with IC50 values less than 20.0 μM. Abietane diterpenes 2, 3, 7 and 8 are also found to have NO inhibitory effects with IC50 values less than 40.0 μM. Graphical Abstract


Introduction
Ajuga Linn., annual or biennial to perennial herbs belonging to the Labiatae family, is distributed in Asia, North African and South European countries (Qing et al. 2017).There are more than 300 species of Ajuga plants, such as Ajuga forrestii Diels, Ajuga nipponensis Makino, Ajuga bracteosa Wall ex.Benth, Ajuga pantantha Hand.-Mazz,Ajuga ovalifolia var.calantha, Ajuga decumbens Thunb.and Ajuga iva (L.) Schreber.The genus Ajuga has been used to treat a variety of inflammatory diseases (Wang et al. 2012;Ma et al. 2019;Dong et al. 2020).The researches about chemical composition and activity of the genus Ajuga began in the 1970s (Kubo et al. 1976).The chemical constituents and bioactivities of Ajuga genus are varied, and diterpenes are one of the main bioactive ingredients (Dong et al. 2020;Wang et al. 2021aWang et al. , 2021b)).
Ajuga lupulina Maxim., mostly grows in Sichaun, Qinghai and Tibet Province.The whole plant has been used to treat anthrax, epilepsy, inflammations and so forth.Compounds isolated from A. lupulina include diterpenoids (Chen et al. 1996(Chen et al. , 1997(Chen et al. , 1999)), flavonoids (Wang et al. 1991), steroids (Wang et al. 1991), iridoids (Li et al. 2000), and the results of the biological activity studies showed that diterpenoids had antibacterial activity.Besides, the ethanol extract of A. lupulina showed antioxidant activity (Lin et al. 2006).
During our present exploration from the EtOH extract of A. lupulina, five previously undescribed compounds were isolated and identified for the first time.The electronic circular dichroism (ECD) method was used in structural research to define their absolute configurations.In vitro biological activities of the compounds were examined.

Structural elucidation
Compound 1 was obtained as a brownish red solid with a molecular formula of C 31 H 20 O 12 based on the positive ion at m/z 585.1020 [M þ H] þ (calcd.for 585.1028) in its HRESIMS data.The 1 H NMR spectrum has given a methoxy group d H 3.75, two alkene protons (d H 6.05, 6.65), together with eight protons (d H 7.34, 7.27, 7.11, 7.04, 6.85, 6.30, 6.10, 5.84) ), and d C 149.9(C-4 0 ); another one was also confirmed based on HMBC correlations (Figure S5).The complete data of 1 H NMR and 13 C NMR spectra (the chemical shifts are given in Tables S1 and S2) showed a similarity to the data of philonotisflavone-4 0 ''-methyl ether (Brinkmeier et al, 1999).The same mass spectrometry data was also available for both compounds.The HMBC correlation of d H 3.75 (-OMe) with d C 149.3(C-3 0 '') determines the substitution position of the methoxy group.The 2 D spectrums showed the location of the methoxyl group position at C-3 0 '' rather than C-4 0 '' of the known compound.It was finally determined that compound 1 is philonotisflavone-3 0 ''-methyl ether.

Results of in vitro activities
Reference to reports (Huang et al. 2011;Ma et al. 2019), DPPH radical scavenging assay is commonly used to evaluate the antioxidant activity of compounds.The result showed that the IC 50 value of the positive control vitamin C was 44.34 lM.Compounds 1-3 showed antioxidant activity with IC 50 values 36.01 lM, 47.65 lM and 48.41 lM.Compared with the positive control vitamin C, compound 1 showed stronger antioxidant activity.Compounds 1-9 were tested for cytotoxic activity against human cancer cells lines Hela (cervical) using MTT cell viability assay (Shima et al. 2021).Rearranged abietane diterpenes 2, 3, 7 and 8 showed cytotoxic activity for Hela with IC 50 values 4.01, 6.77, 12.17, 16.33 lM.According to reports, Nitric oxide (NO) was regarded as an indicator of inflammatory response (An et al. 2019;Dong et al. 2020;Liu et al. 2020;Wang et al. 2021b).Rearranged abietane diterpenes 2, 3, 7 and 8 also showed potential effects against NO production in LPS-induced RAW 264.7 cells, with IC 50 values 22.37, 23.96, 16.73, 14.54 lM.The effects of the substituent position were not obvious in C-3, C-4, C-15, C-16.The structure of compound 6 was similar to 2, 3, 7 and 8, and no activity of 6 was shown in results of in vitro activities.The results indicated that the carbonyl substituent in C-2 largely affected the in vitro activities.

General experimental procedures
Optical rotations were measured with a MCP200 automatic polarimeter (Anton paar, Germany).High-performance liquid chromatography (HPLC) data were recorded on an Agilent 1260 instrument equipped with a photo-diode array (PDA) (Agilent Technologies, Ltd., USA) and a YMC C18 column (250 Â 4.6 mm, 5 lm).HRESIMS data were obtained with an Agilent 1290 Infinity liquid chromatography system and an Agilent 6540 UHD Accurate-Mass Q-TOF mass spectrometer (Agilent Technologies, Ltd., USA). 1 D and 2 D NMR spectra were recorded on a Bruker Avance 600 spectrometer with solvent peaks as references(Bruker, Switzerland).Preparative HPLC was a performed on Sanotac instrument China with a UV detector and a YMC C18 column (250 Â 20 nm, 5 lm).TLC was conducted with glass precoated with silica gel GF254 (Yantai Chemical Industrial Institute, Yantai, China).Column chromatographic separations were carried out with ODS (50 lm, YMC, Kyoto, Japan), Middle Chromatogram Isolated gel(MCI) (70-150 lm, SEP, Beijing, China) and silica gel Qingdao Marine Chemical Group Corporation,Qingdao,China).UV spectra were recorded on a U-T6A UV spectrophotometer.Chromatographic grade methanol and acetonitrile were purchased from Fisher.All other solvents were of chemical grade (Da Mao Chemical Co. Ltd., Tianjin, China).DPPH (Sigma Aldrich, USA); cancer cell lines (Shanghai Institutes for Biological Sciences, China); RAW264.7 cells (National Collection of Authenticated Cell Cultures, China).

ECD calculations
The ECD spectra were calculated according to the reported methods (Zhu, 2015;Luo et al, 2016).The computational ECD data were fitted in the SpecDis software package.The computational data were fitted in the Origin 2021.

Antioxidant activity assays
The DPPH stock solution (1 mmol/L) was diluted with ethanol to a concentration of 200 lmol/L.Sample stock solutions (20 mmol/L) were diluted to final concentrations of 200, 100, 50, 25, 12.5, 6.25 and 3.125 lmol/L in ethanol.100 lL of DPPH ethanol solution was added to 100 lL of sample solutions of different concentrations, and allowed to react at room temperature.After 30 min the absorbance values were measured at 517 nm.Ethanol (200 lL) was used as a blank.Ethanol (100 lL) plus sample solution (100 lL) was used as a sample blank.DPPH solution (100 lL) plus ethanol (100 lL) was used as a negative control.The positive controls were those using the standard solutions.
olefinic carbons d C 173.1 and d C 114.5, seven oxygen-containing substituent carbons d C 71.5, d C 71.5, 71.0, 65.5, 64.2, 62.2, 49.3 and 15 aliphatic carbons.Two acetyl oxygen groups (d C 170.4, 169.9, 21.2, 21.1) were found based on the data from HSQC and HMBC.The carbon signals at d C 173.7, 114.5, 173.1, and 71.0, together with HSQC and HMBC data, suggested an a,b-unsaturated lactone moiety consisting of C-13 À C-16.The structure was determined by 1 H-1 H COSY and HMBC correlations (Figure Compound 5 was obtained as white needles.Its molecular formula of C 31 H 20 O 12 was determined from the positive ion at m/z 557.2726 [M þ Na] þ (calcd.for 557.2721) in its HRESIMS data.According to the 1 D NMR and HSQC data, twenty-nine carbons were identified, including three carbonyl groups d C 215.7, 206.3 and d C 180.7, two olefinic carbons d C 167.5 and 122.2, six oxygen-containing substituent carbons d C 85.1, 82.2, 79.6, 76.2, 68.7, 68.5, three methine carbons at d C 51.8, 51.8, 49.5, five methyl carbons d C 25.5, 24.4,20.1, 19.5, 17.9, and other ten aliphatic carbons.The structure of compound 5 was established from 1 H-1 H COSY and HMBC correlations (Figure S5).The carbon signals at d C 49.5, 76.2, 180.7, 19.5, 79.6 and 20.1, together with HMBC data, from H-24 to C-25 and C-28, from Me-29 to C-28, from H-27 to C-25 and C-26, suggested lactone moiety consisting of C-24 À C-29.The complete data of 1 H NMR and 13 C NMR data were given (Tables