Ballodiolic Acid A and B: Two New ROS, (•OH), (ONOO−) Scavenging and Potent Antimicrobial Constituents Isolated from Ballota pseudodictamnus (L.) Benth.

Bioassays guided phytochemical investigations on the ethyl acetate-soluble fraction of the root material of Ballota pseudodictamnus (L.) Benth. led to the isolation of two new compounds, ballodiolic acid A (1) and ballodiolic acid B (2), along with three known compounds ballodiolic acid (3), ballotenic acid (4), and β-amyrin (5), which were also isolated for the first time from this species by using multiple chromatographic techniques. The structures of the compounds (1–5) were determined by modern spectroscopic analysis including 1D and 2D NMR techniques and chemical studies. In three separate experiments, the isolated compounds (1–5) demonstrated potent antioxidant scavenging activity, with IC50 values ranging from 07.22–34.10 μM in the hydroxyl radical (•OH) inhibitory activity test, 58.10–148.55 μM in the total ROS (reactive oxygen species) inhibitory activity test, and 6.23–69.01 μM in the peroxynitrite (ONOO−) scavenging activity test. With IC50 values of (07.22 ± 0.03, 58.10 ± 0.07, 6.23 ± 0.04 μM) for •OH, total ROS, and scavenge ONOO−, respectively, ballodiolic acid B (2) showed the highest scavenging ability. Antibacterial and antifungal behaviors were also exposed to the pure compounds 1–5. In contrast to compounds 4 and 5, compounds 1–3 were active against all bacterial strains studied, with a good zone of inhibition proving these as a potent antibacterial agent. Similarly, compared to compounds 3–5, compounds 1 and 2 with a 47 percent and 45 percent respective inhibition zone were found to be more active against tested fungal strains.


Introduction
A variety of foods have been found to contain antioxidants that scavenge active oxygen species (free radicals) and are generally referred to as scavengers [1]. In order to protect plants that are exposed to sunlight and live under severe oxygen stress, many oxidants are phytochemicals and play an important role. Antioxidants also play an important role in human health, because the biologic protective system cannot work under extreme oxygen stress. According to recent studies, activated oxygen is thought to be

General Experimental Procedures
To assess the UV spectrum of compounds, (Shimadzu UV-240, Hitachi U-3200, Kyoto, Japan) UV-240 spectrophotometers were used. On IRA-I and JASCO-320-A spectrophotometers, respectively, IR spectra have been reported. On Bruker AM-400, AM-500, and AM-600 spectrometers (Thermo Fisher Scientific, Waltham, MA, USA) with 400, 500, and 600 aspect data systems, the 1 H-NMR and 13 C-NMR spectra were observed at 400, 500, and 600 MHz z (instrument used Bruker Biospin, Karlsruhe, Germany). As an internal guide, TMS (tetramethylsilane) was used. The JMS-HX-110 data system spectrometer was used to measure the EI-MS spectra of compounds. The Jasco-DIP-360 digital polarimeter (JASCO, Tokyo, Japan) was used to determine the optical rotation of the compounds. TLC was performed with G-25-UV254 pre-coated silica gel plates, and detection was carried out at 254 nm and 10% H 2 SO 4 by ceric sulphate. For column chromatography and flash chromatography, respectively, silica gel (E. Merck,70-230 mesh) and silica gel (E. Merck, 230-400 mesh) were used.

Plant Material
Ballota pseudodictamnus (L.) Benth. plant sample from Latamber, Karak District, Khyber Pakhtunkhwa (Pakistan) was collected in adequate quantity and was described by Dr. Nisar Ahmad, Assistant Professor, Department of Botany, Kohat University of Science and Technology, Kohat, Pakistan.

Extraction and Isolation of compounds
At room temperature, the shade-dried and grinded root material (04 Kg) of Ballota pseudodictamnus (L.) Benth. was exhaustively extracted with methanol. The extract was dried and measured in the (55 g). The crude extract was suspended in water and then was successively partitioned between n-hexane (11 g), chloroform (7 g), ethyl acetate (8 g), n-butanol (12 g), and aqueous extracts (16 g). Ethyl acetate-soluble fraction of the title plant was found to be the most promising for the said activities and is therefore selected for further phytochemical investigation. The fraction of ethyl acetate was subjected to silica gel chromatography using n-hexane with an ethyl acetate gradient of up to 100 percent. Four F1, F2, F3, and F4 sub-fractions were obtained from the compilation process. Furthermore, sub-fraction F1 was subjected to column chromatography and eluted with EtOAc: hexane (1:9) for purification of compound 5 [21]. Similarly, sub-fraction F2 was further referred to column chromatography by eluting the fraction with n-hexane-ethyl acetate (6:4). From this fraction, on successive chromatography with n-hexane-ethyl acetate (2:8 and 1:9) elution, two pure compounds 2 [22] and 3 [22] were extracted, respectively. The sub-fraction F3 was further subjected to column chromatography by carry-out elution with n-hexane-ethyl acetate (5:5). Two pure compounds 1 and 2 were supplied by the fraction. On TLC plates, the purity of the compounds was confirmed.

Measurement of Total ROS Generation Inhibition
Rat kidney homogenates prepared from the kidneys of newly killed male Wistar rats, 130-180 g in weight, were mixed with or without the suspension of extracts or compounds, which were dissolved in 10% EtOH (final concentration: 0.4%). The mixtures were then incubated with 12.5 mM 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA, Molecular Probes Inc., Eugene, Oregon), which was dissolved in 100% EtOH (final concentration: 0.2%), at 37 • C for 30 min. A 50 mM phosphate buffer (Wako Pure Chemical Industries, Osaka, Japan) solution at pH 7.4 was also used. DCFH-DA is a stable compound that is hydrolyzed by intracellular esterase to create a reduced, nonfluorescent compound of 2 ,7 -dichlorodihydrofluorescein (DCFH). The ROS produced by the homogenates oxidizes the DCFH to a highly fluorescent 2 ,7 -dichlorofluorescein (DCF). A microplate fluorescence spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT) with excitation and emission wavelengths of 460 and 530 nm, respectively, was used to track the fluorescence intensity of the oxidized DCF [23]. As a positive regulation, Trolox has been used as an important standard oxidant.

Measurement of Hydroxyl Radical Generation inhibition
First, 1 mM H 2 O 2 and 0.2 mM FeSO 4 (Fisher Science, Fair Lawn, NJ, USA) were applied to extracts or compounds dissolved in 10 percent EtOH (final concentration: 0.4 percent) and incubated at 37 • C for 5 min. Then, 2 M DCFH-DA (Molecular Probes Inc., Eugene, Oregon) treated with esterase in 100 percent EtOH was introduced, and fluorescence changes were tracked on a microplate fluorescence spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA) with 460 and 530 nm excitation and emission wavelengths, respectively, for 30 min [24]. As a positive regulation, Trolox, an efficient standard oxidant, was used.

Measurement of ONOO − Scavenging Activity
The ONOO − scavenging activity was calculated by monitoring the oxidation of dihydrorhodamine 123 (DHR 123, Molecular Probes Inc., Eugene, OR, USA) using a slight modification of the reported method [25]. At 80 • C, DHR 123 (5 mM) in DMF, purged with N 2 , was stored as a stock solution. Prior to the analysis, this solution was then put on ice and held in the dark. The buffer consisted of 90 mM NaCl, 50 mM Na 3 PO 4 , 5 mM KCl at pH 7.4, and 100 M diethylenetriaminepentaacetic acid (DTPA), each of which was prepared and purged with N 2 with high quality deionized H2O.The final DHR 123 concentration amounted to 5 M. The context and final fluorescent intensities were assessed with and without the authentic ONOO − 5 min after treatment. The authentic ONOO − oxidized DHR 123 was rapidly oxidized, and the final fluorescent strength of the oxidized DHR 123 was determined using the FL 500 microplate fluorescence reader (BioTek Instruments Inc., Winooski, VT, USA) at 480 and 530 nm excitation and emission wavelengths, respectively. For the final fluorescence intensity minus background fluorescence, the results are expressed as the mean ± standard error (n = 3). The results are expressed as DHR 123 oxidation percent inhibition, and DLpenicillamine, the normal oxidant, has been used as a positive regulation. In all three scavenging experiments, the IC 50 was classified as sample concentrations showing 50 percent scavenging activity and was calculated from a triplicate experiment.

Anti-Bacterial Assay
To test the antibacterial activities of isolated compounds, the process of Agar diffusion was used. In the antibacterial bioassay, five bacterial strains were used, i.e., Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilis, and Staphylococcus aureus. The various bacterial strains were sub-cultivated for a second time to obtain new and fresh culture of each strain in order to test the antibacterial activities of acquired compounds. The nutrient broth was inoculated with a single colony of each strain, then it was incubated for 24 h at 37 • C. Nineteen grams of nutrient agar was dissolved in 1L of distilled water, and the resulting solution was autoclaved for 30 min at 121 • C. Then, 75 mL of solid media was prepared after cooling by pouring the media into Petri dishes (14 cm). Four holes per plate were made with sterile cork borer in these media (8 mm). For inoculation of all bacterial strains, a single Petri dish was used. DMSO (dimethyl sulfoxide) was prepared in the stock solution of compounds 1-3. Two solutions of DMSO (negative control) and that of levofloxacin (positive control) were used in these specific holes for 100 µL of each solution. The activity of each hole zone was calculated in mm when the generic drug inhibition zone (Levofloxacin) was compared using the protocol [26].

Anti-Fungal Assay
For the determination of antifungal activities of isolated compounds, the Disk diffusion method was used. In an antifungal bioassay of isolated compounds, five fungal strains were used: Aspergillus flavus, Fusarium solani, Aspergillus fumigatus, Aspergillus niger, and Canadida glabrata. The old fungal strains were subcultivated using nutrient broth medium in order to evaluate the antifungal activities of the obtained compounds and to obtain the fresh culture of each fungal strain. For seven days at 28 • C, the new cultures were incubated. Every fungal strain was inculcated through point inoculation on a separate potato dextrose agar plate (PDA). Then, 100 µL of stock solution of compounds 1-3, pure DMSO (negative control) and Miconazole (positive control) were used for experiments. After incubation for seven days, the activity of each compound was measured at a temperature of 28 • C [27].
Alkaline hydrolysis of 1 and 2 provided p-hydroxy cinnamic acid and 3,4-dihydroxy cinnamic acid, respectively (characterized through m.m.p., Co-TLC, superimposable IR), providing conclusive evidence for the presence of these moieties in the respective compounds. The important HMBC correlations were in complete agreement to the assigned structures of compounds 1 and 2.
For the isolated compounds 1-5 and different solvent soluble extracts, we investigated the general antioxidant effects of the compounds and extracts to inhibit OH and absolute ROS and to scavenge genuine ONOO − . The different solvent soluble fractions of root, stem, and leave parts of Ballota pseudodictamnus exhibited remarkable anti-oxidant activities ( Table 2). The ethyl acetate soluble fraction of root part of the plant showed stronger anti-oxidative activity than that of the other fractions in all the three scavenging tests with the IC 50 values of 19.10 ± 0.05, 65.13 ± 0.05, and 20.18 ± 0.05 for • OH, ROS, and ONOO − , respectively. The chloroform soluble fraction of root part of the plant observed the • OH, ROS, and ONOO − scavenging activities with IC 50 values of 47.28 ± 0.07, 80.19 ± 0.03, and 41.23 ± 0.07 µg/mL, respectively (Table 2). In those three studies, Compounds 2, with one aliphatic hydroxyl, one carboxylic acid, and 3,4-dihydroxy cinnamoyl moieties and 1, with 3-hydroxy cinnamoyl, one aliphatic hydroxyl, one carboxylic acid moieties, showed greater potential to scavenge • OH, total ROS, and ONOO − with the IC 50 values (07.22 ± 0.03, 58.10 ± 0.07, and 6.23 ± 0.04 µM and 09.15 ± 0.07, 64.09 ± 0.02, and 8.16 ± 0.01 µM), respectively. Compound 3 with two free hydroxyl groups and carboxylic acid moiety in its structure showed good antioxidant activity ( Table 2). Lactone and carboxylic acid moiety compounds 4 displayed moderate activity with IC 50 values (19.08 ± 0.05, 69.15 ± 0.08, and 21.13 ± 0.09 µM) for • OH, total ROS, and ONOO − , respectively, for scavenge. β-amyrin (5) with one hydroxyl moiety showed lower activity, but still significant activity in all the scavenging activity tests (Table 3). The antioxidative activity was also examined in terms of the chemical structures including those of functional radical and its orientation. Hydroxyl groups in the 1,4-and 1,3orientation at cinnamoyle are mainly involved in the scavenging potential of compounds 1 and 2. Based on these results, a benzene ring where the hydroxyl radical is in 1,4-orientation allow the oxygen atom to share a positive charge, thereby causing stabilization through delocalization and hence increasing the antioxidant activity as was observed in compound 1. Because of the electron donating effect of the hydroxyl group in 1,3-orientation, it helps to stabilize positive charge, and this is taught to influence the scavenging ability. This was observed in case of compound 2 where further enhanced scavenging activity was observed as this compound bear 3,4-dihydroxy cinnamoyl moieties.
The antibacterial and antifungal activities of compounds (1)(2)(3)(4)(5) against the various bacterial strains Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilis, and Staphylococcus aureus (Table 4) were observed in this study. Table 4, displays the compounds (1-5) from the test samples in terms of diameters of inhibition zones. Against the examined microorganisms, such as Escherichia coli, Salmonella typhi, and Staphylococcus aureus, the compounds were found to be prominently active. In contrast to compound 3-5, compounds 1 and 2 were active against all bacterial strains studied, with an inhibition zone of 12 ± 0.03 and 11 ± 0.01 mm, respectively. In the same concentration range, compound 3 demonstrated good antibacterial activity against the test species. Antifungal activity of these compounds (1)(2)(3)(4)(5) against Aspergillus flavus, Fusarium solani, Aspergillus fumigatus, Aspergillus niger, and Canadida glabrata, as shown in Table 5, with an inhibition zone of 7% to 47%. Compound 2 showed the highest antifungal activity with a 47% to 25% inhibition zone, while the other two compounds 1 and 3 showed good antifungal activity against the fungal strains listed above. In recent years, while technology and medicine have grown widely, due to a reduction in natural wealth and disadvantages, some countries have found it necessary to use natural products for several different purposes. As in several other nations, plants known to individuals with health benefits are collected and used in Pakistan for the treatment of various diseases. The antimicrobial and antifungal capabilities of compounds (1-5) isolated from Ballota pseudodictamnus have been identified in this study. Owing to the strength of widespread resistance to them, the use of certain antibiotics is no longer recommended. As shown in Table 4, these compounds can also be used instead of antibiotics; they have shown visible antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Bacillus subtilis, and Staphylococcus aureus. In addition, against different strains such as Aspergillus flavus, Fusarium solani, Aspergillus fumigatus, Aspergillus niger, and Canadida glabrata, the antifungal activity of these compounds was prominent (Table 5).  The established antimicrobial mechanisms associated with each group of chemicals to which the isolated compounds belong can explain the antimicrobial potential of Ballota pseudodictamnus solvent soluble fractions and compounds 1-5. Disruption of the membrane has been proposed as one of the possible mechanisms of action [29,30]. The antimicrobial activities of compounds 1-5 may also be clarified by this. The bacterial and fungal organisms differed in their antimicrobial activities. The genetic differences between the microorganisms [29,30] may be the explanation for these variations. This is interesting in view of the possibility of new antimicrobial medicines being produced from natural products.
Based on structure-activity relationships, the hydroxyl substitution pattern on benzene ring is an indicator of antimicrobial activity for compounds, and the additional hydroxyl group at 4 -position significantly increases the activity, while the methylation of these hydroxyl groups reduces the antibacterial potential at different levels. Besides this, the addition of the 3-hydroxyl group seems to enhance the activity [31][32][33]. Here in our reported compounds, 2 and 3 have 3-hydroxyl group and 3,4-dihydroxyl substitution pattern, respectively, and hence the compound 3 was found more active than compound 2, followed by compounds 1, 4, and 5 (Tables 4 and 5).
The in vitro antioxidant function of ethanol extracts extracted from 21 aromatic plants belonging to the family Lamiaceae has been investigated by Couladis et al. Salvia ringens, Stachys spruneri, Phlomis lanata, Salvia pomifera, Origanum dictamnus, Ballota pseudodictamnus, Ballota acetabulosa, Teucrium polium, Calamintha glandulosa, and Micromeria graeca exhibited the same behavior as α-tocopherol among the extracts tested [35]. The authors also studied the chemical composition of the Ballota pseudodictamnus essential oil obtained by GC/MS from the aerial components. Aryophyllene oxide, phytol, and γ-muurolene were the major components of the 52 reported constituents of the crude. In addition, the essential oil for its antimicrobial activity has been investigated [36].
Previously, our research group reported in vitro antimicrobial, antiprotozoal activities and heavy metals toxicity of different parts of Ballota pseudodictamnus (L.) Benth. [20]. The analysis was conducted to verify the function of different sections of Ballota pseudodictamnus (L.) Benth. in antimicrobial and antiprotozoal terms. These behaviors were then compared with the toxicity of heavy metals in various parts, plants accumulating in different amounts in different parts. Ethanolic extract, chloroform, and ethyl acetate fractions in the Ballota pseudodictamnus (L.) Benth. roots showed antileishmanial activity in in-vitro antileishmanial results. The fraction of ethanol, n-butanol, and ethyl acetate in the stem showed inhibition of leishmania amastigote type. Inhibition of the leishmanial parasite was seen in the ethanol extract, chloroform, and n-butanol fraction in the leaves. Results have shown that different parts of the plant have different properties of inhibition [20].
Diterpenes and diterpenoids have antioxidant activity. Carnosol and carnosic acid are inhibitors of lipid peroxidation; they prevent the oxidation of fatty acids, triglycerides, and low-density lipoproteins in human aortic endothelial cells [37,38]. Under oxidative stress, nematodes treated with carnosol had a 21% increase in lifespan compared to controls, and under heat stress increased worm survival was higher by 9% [39]. The combined action of carnosic acid and carnosol against ROS and lipid radicals makes this diterpenoid tandem an effective antioxidant defense. In the Ames test, carnosol was found to have significant antioxidant and anti-mutagenic activity comparable to ascorbic acid [40]. In a micronucleus test, it was found that carnosol is even more effective than ascorbic acid in protection against gamma radiation [41]. Carnosol protects cells from eco-toxicants [42].
Diterpenes and diterpenoids are reported for antiviral, antibacterial, antiparasitic, antifungal, and antiprotozoal action. Some terpenoids are toxic to microorganisms and insects and play an important role in plant protection [43,44]. Extract of Stevia and its glycosides (for example, steviol), in addition to their value as sweeteners, has a therapeutic effect against cystic fibrosis. Carnosic acid and carnosol, isosteviol, andrographolide, and dehydroabietic acid have several important protective properties, including antituberculosis and antiseptic, and can be used in the treatment of colds, showing antibacterial and antiviral activity [45,46].
González-Cofrade et al. identified 19 diterpenoids with anti-inflammatory activity. For their anti-inflammatory activities against NLRP3 inflammasome activation, the authors assessed a series of dehydrohispanolone derivatives. IL-1β secretion was substantially inhibited by four derivatives, being the most active (IC50 = 18.7 and 13.8 µM, respectively). Analysis of the expression of IL-1β and caspase-1 showed that these four diterpenoids were selective inflammasome NLRP3 inhibitors, confirming the anti-inflammatory properties of hispanolone derivatives shown earlier [48].
The efficacy of seven predominant wine terpenoids (i.e., alpha-pinene, limonene, myrcene, geraniol, linalool, nerol, and terpineol) against foodborne pathogenic bacteria was determined by Chung-Yi Wang et al., and their antioxidant activities were observed. Against foodborne pathogenic bacteria, antibacterial activities were observed. The MIC 50 and MBC values were 0.420-1.598 and 0.673-3.432 mg/mL for Escherichia coli, Salmonella enterica, and Staphylococcus aureus, respectively. The fastest free radical scavenging DPPH (IC50 value = 12.57 ± 0.18 mg/mL) and the highest reducing strength (L-ascorbic acid equivalent 213.7 ± 5.27 µg/mL) were shown by the terpenoid alpha-pinene. New possible sources of natural antibacterial and antioxidant agents for use in the food industry may be the seven predominant terpenoids in wines that were found in the recorded study [50].
Antibacterial and antioxidant cassane diterpenoids from Caesalpinia benthamiana have been reported by Rita Akosua Dickson et al. Using the microdilution assay method and the DPPH spectrophotometric and TBA lipid peroxidation assays, the antibacterial and antioxidant activities of the isolated cassane diterpenoids were evaluated. For both Staphylococcus aureus and Micrococcus flavus, Benthaminin 1 was identified as the more active antibacterial compound with MIC values of 47.8 microM. The more active antioxidant compound was found to be benthaminin 2 and showed IC 50 values of 42.7 microM and 74.2 microM for DPPH and TBA assays, respectively. Deoxycaesaldekarin C possessed both antibacterial and antioxidant activities [51].
Diterpeniod, abietane 7 alpha-acetoxy-6β-hydroxyroyleanone (AHR), obtained from plant extracts, has been stated to be an attractive lead for drug production due to its established antimicrobial properties by Carlos E et al. [52].
The compliance of the biological effects of terpenoids with the parameters of geroprotectors, including primary criteria, was evaluated by Ekaterina Proshkina et al. Among the different terpenoid groups, the number of substances showing the greatest compliance with both primary and secondary geroprotector criteria was found. Terpenoids are therefore an underrated source of potential geroprotectors that can have an effective impact on ageing and age-related disease mechanisms [53].

Conclusions
The bioassays guided isolation from ethyl acetate soluble fraction of the root material of the plant resulted two new compounds (1, 2) along with three already reported compounds (3)(4)(5). The structures of the compounds 1-5 were elucidated with the help of 1D and 2D NMR techniques. The compounds 1-5 were found active against different microorganisms and exhibited antioxidative properties. The medicinal efficacy of these compounds will, however, be explored through more in vivo studies.