Anticancer and antibacterial flavonoids from the callus of Ampelopsis grossedentata; a new weapon to mitigate the proliferation of cancer cells and bacteria

A new flavonoid angelioue (1) together with five known compounds cuminatanol (2), myricetin (3), epigallocatechin (4), taxifolin (5) and dihydromyricetin (6) was isolated from the callus extract of Ampelopsis grossedentata (Hand.-Mazz.) W. T. Wang and the structures were elucidated based on their detailed spectroscopic data. Among the compounds, the new compound angelioue (1) displayed significant antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) with the MIC value of 6.68 μg mL−1 and MBC value of 53.42 μg mL−1; in contrast the other compounds showed moderate to no antibacterial activity. In addition, known dihydromyricetin (6) exhibited potent cytotoxic activities against mouse breast cancer cells (4T1), human lung adenocarcinoma (A549) and human non-small cell lung cancer (NCI-H1975) tumor cell lines with GI50 values of 17.47, 18.91 and 20.50 μM mL−1, respectively. The compounds 1–5 exhibited low micro-molar inhibitory activities. Moreover, the structure–activity relationships of the most active compounds for antibacterial and cytotoxic activities are discussed. The present findings clearly suggest that the A. grossedentata callus is a good source of bioactive compounds.


Introduction
Globally, serious infections by bacteria and the development of resistance due to genetic modication against currently available antibiotics kill around 700 000 patients each year. This is one of the biggest healthcare problems in the community and hospital settings. 1,2 In addition to this, cancer still poses a serious threat to mankind and causes a huge economic burden, although numerous efforts have been made for its prevention and control. In the past year, according to the World Health Organization, 7.6 million deaths were recorded due to cancer worldwide. Globally, the deaths from cancer are projected to continue to increase to over 13.1 million in 2030. 3,4 In order to overcome this serious global threat, there is an urgent need to nd new drugs to effectively treat cancer and drug-resistant bacteria.
Natural compounds produced by plants are a major resource of antibacterial and anticancer drugs. In particular, avonoids constitute one of the most precious and structurally diverse groups of natural compounds which have been shown to possess potent antibacterial activity against drug-resistant bacteria and anticancer activity with minimal toxicity against normal cells. Flavonoids are capable of exerting of antibacterial activities via various mechanisms such as nucleic acid synthesis inhibition, alteration in cytoplasmic membrane function, energy metabolism inhibition, reduction in cell attachment and biolm formation, inhibition of the porin on the cell membrane, changing of the membrane permeability and attenuation of the pathogenicity. 5,6 It also scavenge cancer cell by various molecular mechanisms viz., modulating ROSscavenging enzyme activities, participating in arresting the cell cycle, inducing apoptosis, autophagy and suppressing cancer cell proliferation and invasiveness. 7,8 Biologically derived compounds are proved to be safe to patients and these compounds will not cause any serious damage to normal cells. Moreover, treatment with these bio-compounds against bacterial pathogens and cancer will be cheap and safe. [9][10][11][12] Medicinal plants the principal crasman of novel candidates are well known to prevent and treat human ailments; conventional treatments have been known since ancient times. 13 In addition, plants provide basic raw materials for various medicinal, pharmaceutical, agricultural, cosmetics and food industries. 14 Ampelopsis grossedentata is an important edible medicinal plant commonly called as vine tea belonging to the plant family Vitaceae and is mainly distributed in southern China. 15 Scientically, high levels of avonoids especially dihydromyricetin, myricetin and myricitrin were present in vine tea which were reported to possess antioxidant, 16 antibacterial, 17 antitumor, 18 anti-inammatory, 19 neuroprotective, 20 antihypertensive and antidiabetic activities. 21 To the best of our knowledge the capacity of in vitro shoot culture of A. grossedentata in the production of secondary metabolites has not been studied until now. However, the callus culture could be a realistic source of bioactive secondary metabolites production with no time and space limitation and the technique has been widely used as an alternative for the production of plant secondary metabolites. 22 Therefore, the current research was designed to establish the in vitro callus cultures of this species as an alternative bioactivity-based reproducible approach for extraction and isolation of key bioactive compounds. Further, we assessed the cytotoxicity and antibacterial potential of the callus extract on A549, 4T1 and NCI-H1975 and against a panel of Gram positive bacteria and Gram negative bacterial pathogens, respectively. Moreover, the active ingredients that play a crucial role in the biological activity of the extract were isolated and characterized using detailed spectroscopic analysis.

Results and discussion
Preliminary antibacterial activity and cytotoxicity The antibacterial susceptibility of the callus extract (EtOH-AG) was determined and the results with zones of inhibition are tabulated (Table 2). Fascinatingly, the extract showed varying degrees of antibacterial activity against the tested bacterial pathogens. Particularly, the extract was found to be effective against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis with highest zones of inhibition ranging from 10.88 AE 0.07-10.35 AE 0.07 mm. Further, EtOH-AG exhibited activities depending on bacterial strains with the MIC and MBC values in the ranges of 24.15-48.30 mg mL À1 and 96.60 mg mL À1 , respectively. A previous report by Vignesh et al. (2022) 23 stated that the callus extract of Saraca asoca showed remarkable antibacterial activity against Enterococcus faecium and Salmonella typhi with zones of inhibition of 17 and 14 mm, respectively. In addition, the extracts also exhibited maximum inhibitory effects at 75 mg mL À1 against the tested Gram positive and Gram negative bacterial pathogens. On the other hand, EtOH-AG exhibited substantial cytotoxicity against NCI-H1975, 4T1 and A549 cell lines with the GI 50 values of 63.89, 69.10 and 74.00 mg mL À1 as shown in Fig. 1 Compound 2 was obtained as a white powder and identied as acuminatanol (Fig. 2b2). 25 Compound 3 was isolated as a yellow amorphous powder and identied as myricetin ( Fig. 2b3). 26 Compound 4 was obtained as a white amorphous powder and identied as epigallocatechin (Fig. 2b4). 27 Compound 5 was obtained as a green amorphous powder and identied as taxifolin (Fig. 2b5). 28 Compound 6 was obtained as a white amorphous powder and identied as dihydromyricetin (Fig. 2b6). 29 The NMR, HRESIMS data and HPLC chromatograms of compounds 1-6 are given as supplementary les (Tables S1 and S2, Fig. S10-S35 †).

Cytotoxicity and structure activity relationship of avonoids
Flavonoids are specialized plant secondary metabolites which are ubiquitous throughout the plant kingdom. They belong to a chemically heterogeneous group of small molecules found in sufficient amounts in fresh fruits, green leafy vegetables, cocoa, tea, seed, tubers and peel with diverse biological activities. As part of our continuous search for potent anticancer compounds, the isolated avonoids were evaluated for their cytotoxic efficiency against the aforementioned cell lines. All the tested compounds showed strong to weak anticancer activity in a concentration-dependent manner with lower GI 50 values ranging from 6.93 to 98.36 mM mL À1 in comparison with standard drug doxorubicin against human lung adenocarcinoma A549 cells (A549), mouse breast cancer cells (4T1) and human non-small cell lung cancer (NCI-H1975). These data are consistent with preliminary cytotoxic potential of the extract; such a trend reported previously by Wang et al. (2016) 30 in which the total avonoid content of Cotinus coggygria Scop., signicantly increased the tumor cell killing efficacy in a concentration-dependent manner against human glioblastoma cell lines compared with the control. Interestingly, among the compounds screened, dihydromyricetin (6) Fig. 3a-f). On the other hand angelioue (1), acuminatanol (2), myricetin (3), epigallocatechin (4) and taxifolin (5) exhibited moderate to weak inhibitory activities with low micro-molar concentration. The cytotoxicity was further validated by comparing with the reported results of Liu et al. (2014) in which dihydromyricetin (6) showed no cytotoxic effect against the normal human hepatic cell (HL7702 and L-02). 9 Similarly another study conducted by Han et al. (2021) reported that dihydromyricetin showed no activity against MRC-5 fetal lung broblast and 267B1 prostate epithelial cells. 10 In addition, Zhou et al. (2014) has reported that dihydromyricetin showed no cytotoxic effect on normal human breast epithelial cells (MCF-10A). 11 Correlating the earlier reports obtained for the normal cells which showed that the dihydromyricetin was found to be selective towards cancer cells. Therefore, this study also conrmed their selective anticancer activity. 9-11 Based on the current interest in avonoids, the cytotoxic potential of the most active compound was further validated by structure-activity relationships (SAR) as predictors of cytotoxicity with most well-known mechanism of action involved in the anticancer activity of avonoids. In this line, the SAR of the dihydromyricetin (6) can be explained as follows. It has been proposed that the carbonyl group (C]O) located at C4 position in the C ring showed a trend towards higher cytotoxicity of avonoids at low micromolar concentration than other compounds without a carbonyl group in C4. The importance of the C4 carbonyl group for cytotoxicity is further underlined by the fact that compound 4 (epigallocatechin), the only compound in our study without a carbonyl group in C4 position, exhibited weak cytotoxicity among all tested compounds. These data are in conjunction with the previous report of  32 in which the presence of oxo group at C4 position of the C ring and a low electron density of 5 and 7 oxygen atoms in the hydroxyl group of the A ring were important in the suppression of cyclooxygenase-2 (COX-2) transcriptional activity that played an important role in carcinogenesis. 30 In addition by correlating the previous report of Jacob et al. reported that the ortho-hydroxylated avonoid (quercetin) was about three times more cytotoxic than the meta-hydroxylated avonoid (morin) signifying that the hydroxylation pattern of the B ring was involved in cytotoxic activity of orthohydroxylated compound (dihydromyricetin). 31 In addition, this fact was also further conrmed by another report by Lopez-Lazaro, (2002) 34 in which the number of hydroxyl groups on the B-ring was related to a molecular conformation that inuenced the interactions between enzymes such as protein kinase C and tyrosine kinase with avone thereby increasing the transcriptional activity of COX-2. Therefore, our results clearly suggest that an oxo group at 4 position of the C ring, the number of OH groups located on the B ring and the low electron density of the oxygen at 5 and 7 position of the A ring are important features of dihydromyricetin structure that afford increased cytotoxicity activity associated with cell shrinkage, cytoplasmic condensation and distortion which prevent tumor cell's metabolism and nutrient uptake leading to amplication of autophagy.
Antibacterial activity and structure activity relationship of avonoids   (1) can be inferred from the results of signicant anti-MRSA activity with some regular patterns of SAR, which are: the presence of adjacent hydroxyl group at C-3 0 , C-4 0 and C-5 0 in the B ring greatly reduced the growth of bacteria. The antibacterial activity of the avonoids has been proposed to be linked to their chemical structure especially the position and of number hydroxyl groups. [35][36][37] Additionally, the presence of two hydroxyl substituents on C-7 and C-3 of A and C rings is presumed to possess good antibacterial activity. 38 The previous research by Wu et al. (2013) 39 reported that hydroxyl groups are an important aspect of antibacterial activity related to DNA gyrase inhibition of avonoids.

As presented in
The importance of the C4 carbonyl group for anti-MRSA is further emphasized by the fact that avones are less active than corresponding avanone angelioue > dihydromyricetin > epigallocatechin > myricetin. This result may indicate that the saturation of the C2]C3 double bond increases the antibacterial activity. 38 On the other hand, the presence of 5-gallate in the A ring offered a stronger antibacterial activity. 40 Incidentally, the puried compounds exhibited higher cytotoxicity and antibacterial activities than crude extract; this may be due to the fact that purication process would have freed the active compounds which mask the biological activity of its chemical constituents on certain therapeutic targets. In this regard, Azizah et al. (2020) as reported that the mass spectrometry-based molecular networking is a powerful dereplication strategy that identies known and unknown compounds in crude extract but also suggests the presence of related analogues for further drug discovery paradigms. 41

Experimental
General experimental procedures UV-Vis Spectroscopy was taken on Shimadzu UV 2550 spectrophotometer in analytical grade methanol. The optical rotation of pure compound was measured using digital polarimeter MCP 100 in CH 3 OH. FTIR and HR-ESI-MS spectra were recorded on a Nicolet iS20 FTIR Spectrometer and maXis II Q-TOF mass instrument (Bruker, Germany), respectively. NMR spectra were obtained on a Bruker NMR 600 spectrometer operated at 600 MHz ( 1 H) and 150 MHz ( 13 C) with TMS as an internal standard (Bruker, Germany). Thin-Layer Chromatography (TLC) was performed on 0.2 mm thick silica gel plates (HS-GF254). Analysis and preparation of compounds were performed using an Agilent 11260 Innity II Prime with an ODS column (YMC, 250 Â 10 mm, 5 mm). The organic solvents used for extraction and purication were procured from Beijing chemical factory (Beijing, China).

Plant material and callus induction
Ampelopsis grossedentata explants were obtained from Central China Medicinal Botanical Garden (Enshi, China, E109 45 0 24 00 ; N30 10 0 51 00 ) (Fig. S37a †). The identity of this plant material was veried by Dr Meijun He, Hubei Academy of Agricultural Sciences. The collected young shoot tips were washed in running tap water followed by rinsing with sterile water and soaked in 75% ethanol for 45 s with intermittent soaking in 0.1% HgCl 2 for 20 min. The treated explants were washed with sterile water and then were cut into small pieces under aseptic condition. About 5-10 mm long shoot discs of in vitro explants were inoculated on 40 mL of sterile Murashige & Skoog medium (MS medium) with 3% sucrose, 1-naphthaleneacetic acid (NAA) (0.25 mg L À1 ), 6-benzylaminopurine (6-BA) (1.5 mg L À1 ) and 0.8% agar. The medium was adjusted to pH-5.8 before sterilization (121 C, 15 psi, for 15 min). The cultures were kept at 25 AE 2 C under photoperiod of 16 hours with white uorescent light (50 mmol m À2 s À1 ) for 14 days.

Extraction and isolation
The healthy callus tissues were collected at the maximum biomass production phase (four weeks of culture, Fig. S37b †) and used for extraction of secondary metabolites. Briey, the harvested callus was dried in a hot air oven at 45 C. The dried biomass of A. grossedentata callus weighing 324.25 grams was extracted thrice with ethanol (5L). The solvent was evaporated under reduced pressure to obtain yellowish green gum (6.48 g). The extract was fractionated over silica gel chromatographic column and eluted with solvents of increasing polarity of 3 mg) and compound 6 (7.5 mg). The purity of the isolated compounds was tested by HPLC, and the purity of which exceeded 98% was further identied (Fig. S35 †). USA) and allowed to dry for 5 minutes. Aer drying, the precoated paper discs containing extracts (10 mL per disc) and compounds (5 mL per disc) were placed on the surface of the test plates and kept at 37 C overnight. Kanamycin (50 mg per disc) DMSO (5 mL per disc) was used as positive and solvent controls, respectively. The antibacterial activity was perceived as a zone of clearing in the turbid medium around the disk containing inhibitory activity. The clear zones were measured using a zone scale from Hi-media and expressed in millimeters.

Determination of minimum inhibitory concentration (MIC) and minimum bacteriostatic concentration (MBC)
The MIC and MBC values of extract and compounds were determined by using broth microdilution assay in sterile 96-well microtiter plate according to Li et al. (2022) and Rozman et al. (2017). 24,43 About 25 mL of extract and puried compounds were serially diluted in 75 mL of MHB to obtain 0-200 mg mL À1 and 0-100 mM mL À1 , respectively and 5 mL of freshly grown bacterial cultures were added to each well. DMSO and kanamycin were added as solvent and positive controls. The treated plates were kept at 37 C for 24 hours followed by addition of 40 mL of iodonitrotetrazolium chloride (200 mg mL À1 ) into each well and re-incubated at 3 C for 30 min. The colour changed from yellow to purple indicating the microbial growth in the well. To check the viability of the tested bacterial pathogens, the mixture in each well was streaked on MHA and incubated overnight at 37 C.

Statistical analysis
All statistical analyses were performed using the SPSS soware 24.0 (Systat Soware, Systat, Evanston, IL, USA). Data were presented as mean AE SEM. The signicance among all groups was evaluated with one-way ANOVA. P < 0.05 was considered statistically signicant. The Anova analyses were carried out using Graphpad version 8.4.3 for Windows version 7.

Conclusions
In summary, the study of Ampelopsis grossedentata callus extract led to the isolation and characterization of one novel compound along with ve known compounds with narrow spectrum antibacterial and cytotoxic activities. It is noteworthy that the new compound angelioue (1) showed signicant anti-methicillinresistant Staphylococcus aureus activity and the known compound dihydromyricetin (6) exerted signicant cytotoxicity against mouse breast cancer cells (4T1), Human lung adenocarcinoma A549 cells (A549) and human non-small cell lung cancer (NCI-H1975) tumor cell lines with very low micro-molar concentrations. In addition, the structure-activity relationship (SAR) provided vital information regarding the types of moieties that were involved in the suppression of bacteria and cancer cells. Therefore, the results of this study clearly demonstrate that the observed antibacterial and cytotoxic activities of these compounds may justify the use of the extracts of this plant in folk medicine for the treatment of bacterial infections and cancer. This warrants further attention in the future for the drug development.

Author contributions
Conceptualization

Conflicts of interest
There are no conicts to declare.