Phytochemical, pharmacological and in silico studies on Teucrium stocksianum Bioss

2.1 General experimental procedures

The polarimeter (JASCO DIP-360) was used for optical rotations whereas Melting points (MP) were determined with the aid of Gallenkamp apparatus. Hitach UV-3200 and Shimadzu FTIR-8900 spectrometer were employed for UV and IR spectral measurement respectively. The 1D-NMR spectra (¹H- along with -¹³C) were taken down on Bruker AM-400/500 spectrometers with the presence of deuterated solvents. The determination of 2D-NMR (HMQC, COSY, HMBC, NOESY) peaks with the aid of above same models. For all NMR spectra the ppm chemical shifts (δ) along with coupling in Hz. The Mass spectra (MSEl/MSHREI) were calculated with electron impact (EI) mode on Finnigan MAT-112/ 113 spectrometers. The E. Merck silica gel (size; 70–230 mesh) employed for various used column chromatography techniques. The pre-coated silica gel G-25-UV₂₅₄ plates (E. Merck) used for the purpose of TLC followed by UV detection at 254/366 nm along with spraying with Ce(SO₄)₂ in 10% H₂SO₄ for UV inactive phytochemicals.

2.2 Collection and identification

The Teucrium stocksianum (whole, 12 kg) was collected from, Ziarat Valley, Baluchistan, Pakistan and recognized by Plant Taxonomist, Prof. Dr. RB Tareen, University of Baluchistan, where voucher specimen No. 1310BUH was placed in the herbarium.

2.3 Extraction, fractionation and purification

The shade dried T. stocksianum whole plant (12 kg) was crushed into fine powder and extracted with ethanol (15 L × 4, each 7 days). The obtained combined extract (EtOH) was evaporated at a low pressure to produce greenish residue. It was dissolved further within distilled H₂O (0.5 L) and successively partitioned with n-hexane (7 × 3.0 L; 980 g), dichloromethane (DCM) (6 × 3.0 L; 443 g), ethyl acetate (AcOEt) (4 × 3.0 L; 70 g), n-butanol (7 × 3.0 L; 535 g) and water (907 g), respectively. The ethyl acetate sub-portions (45 g) subjected to CC on silica gel and elution was attained with n-hexane-DCM, DCM, DCM- EtOAC, EtOAC-MeOH. Using the above solvents in order of increasing solvent polarity, 13 sub-portions (A-M) were collected. The sub-portion-A was acquired from n-hexane:DCM (7.0:3.0), chromatographed over silica gel, eluted with n-hexane:DCM (7.5:2.5) to obtain1-dotriacontanol (12). The sub-portion-B was acquired by eluting n-hexane: DCM (6.0:4.0) to achieve binary mixture. The 2H-chromen-2-one (8) and stearic acid (11) was obtained by further CC using solvent system n-hexane: DCM (6.5:3.5 and 6.0:4.0). The n-hexane:DCM (5.0:5.0) elution gave C sub fraction (mixture), further chromatographed over adsorbent silica gel and eluted with n-hexane:DCM (4.5:5.5 and 4.0:6.0) to give benzoic acid (9) and cinnamic acid (7), respectively. The D subfraction was obtained with n-hexane:CH₂Cl₂ (3.0:7.0), was a single compound with lingering of impurities, whose further CC separation affoard salicylic acid (10). The G subfraction affoards ursolic acid (2) after chromatographed over silica gel with eluent as DCM:EtOAc (6.0:4.0). The H subfraction was obtained with CH₂Cl₂:MeOH (9.9:0.1), was a semi pure compound, followed with same system CC afford teucriol (1) 8 mg as pure compound. The I subfraction was obtained with CH₂Cl₂:MeOH (9.5:0.5), further same mobile phase CC was afford pure apigenin (3). The binary mixture J subfraction attained with CH₂Cl₂:MeOH (9.4:0.6), was rechromatographed over silica gel and eluted with CH₂Cl₂:MeOH (9.6:0.4) to obtain compound4. The elution with CH₂Cl₂:MeOH (9.3:0.7) gave K subfraction, was a mixture, rechromatographed over silica gel and eluted with CH₂Cl₂:MeOH (9.4:0.6) to afford luteolin (5). The subfraction was obtained with CH₂Cl₂:MeOH (9.0:1.0), rechromatographed over silica gel, eluted by CH₂Cl₂:MeOH (9.1:0.9) to obtain apigetrin (6).

2.4 Teucriol (1)

White amorphous, [α]²⁰_D (c 0.3, MeOH): −28.5, UV (CH₃OH) λ_max nm (logε): 202 (4.1), 204 (3.4), 193 (4.2), IR ν_max (KBr) cm⁻¹: 3406 (OH), 3021 (CH₃), 1280 (O-C). ¹H/¹³C NMR (CD₃OD, 300/75 MHz) see Table 1; EI-MS (70 e/v) (rel. Int %) m/z: 190.1 ([M]⁺, 6), 172.1 ([M−H₂O]⁺, 9), 154.1 ([M−2H₂O]⁺, 15), 136.1 (20); HR-EI-MS: m/z 190.1561 [M]⁺ (calcd for C₁₀H₂₂O₃, 190.1569).

2.5 Pharmacological evaluation assays

The ethanol extract, its fractions and new isolate of T. stocksianum were subjected to measure various pharmacological potential through reported protocol and strains were selected based on availability at the time of potential measurement. Antibacterial potential was determined against Streptococcus pneumonia, Staphylococcus aureus, Klebsiella pneumonia and Escherichia coli by agar well diffusion method. In vitro antifungal potential against Fusarium solani, Candida albicans, Candida glabrata, Microsporum Canis and Aspergillus flavus was assessed through agar tube dilution method. Insecticidal activity was appraised against insects viz Tribolium castaneum and Rhyzopertha dominica by impregnated filter paper test. Cytotoxicity, phytotoxicity and DPPH radical scavenging activity were also measured. The detail procedures including computational details (Imran and Irfan 2020, Irfan et al., 2020a,b, Imran et al., 2021) are given in supplementary material.

2.6 Computational details

The optimization of structural geometry for teucriol (1) was achieved by DFT at B3LYP/TZ2P method. Then electronic properties, various descriptors, MEP and Hirshfeld analysis was performed to shed some light on the biological activity nature of newly isolated compound. All these calculations were performed by ADF software (see supporting information).

3.1 Pharmacological evaluation

The in vitro pharmacological evaluation of T. stocksianum Bioss crude extract, all its fractions and new isolate were carried out for antimicrobial (antibacterial and antifungal), cytotoxic, phytotoxic, insecticidal and antioxidant DPPH scanvenging activities. The antibacterial activity results exhibited that crude extract and all its fractions and new isolate showed remarkable antibacterial potentials against all under examined bacteria Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli and Klebsiella pneumonia when compared with the standard ceftriaxone (Table 2). Extract, fractions and pure isolate, except aqueous fraction, showed better potent potential against S. aureus, E. coli and K. pneumonia as compared to standard ceftriaxone, and all showed excellent potential against S. pneumonia but slightly less than the standard. In antifungal activity, crude extract and all fractions, and new isolate showed inactivity against all under investigation fungi Candida albicans, Fusarium solani, Candida glabrata, Aspergillus flavus, and Microsporum canis, further all of them also showed inactivity against Tribolium castaneum and Rhyzopertha dominica insects (Table 2). Crude extract and fractions, and new isolate are also almost inactive in cytotoxicity and phytotoxicity (Table 3). Based on potent antibacterial potential and inactivity in cytotoxicity, extract, fractions and pure isolate can be excellently used against bacterial infections. In DPPH scavenging antioxidant activity, all fractions showed promising potential (Fig. 3). The antioxidant potential was measured at three different concentrations 500 µg, 400 µg and 100 µg, and this potential is linked with concentration i.e. on increasing concentration the potential is also increased. The n-hexane fraction showed the best antioxidant potential which is 35 % at the highest and n-butane fraction showed the lowest antioxidant potential.

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Fig. 3

Antioxidant activity of various fractions of ethanolic extract of T. stocksianum.

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3.2 Electronic properties

The FMOs of newly isolated derivative including highest occupied (HOMOs/HOMOs-1) and lowest unoccupied molecular orbitals (LUMOs/LUMOs + 1) with employing B3LYP/TZ2P level are presented in Fig. 4. The HOMO-1 orbitals can be established on C14, C15, C16, O32 that is more substituted side, HOMO is at O1, C3, the LUMO is localized on H33, H35, with some charge on O32, O34, C19, C26 whereas LUMO + 1 is localized on H2, O1, C3. The comprehensible intra-molecular charge transfer (ICT) was noted from H → L, H-1 → L and H-1 → L + 1 significantly. The antioxidant talent is also interlinked with HOMO spatial scattering which may illuminating the most possible sites (O1, C3) within newly isolated phytochemical that can be confronted by free radicals ultimately exposed the reactive behavior of this compound as antioxidant which can perform better biological activity. Moreover, better antioxidant drugs might also be effective against COVID19. The energies of FMOs and energy gaps are tabulated in Table 4. The Au and Al work functions (W) are 5.10 and 4.08 eV, accordingly (Imran et al., 2020, Irfan et al., 2020a,b). The injection barrier of the hole / electron (HIE / EIE) is being investigated as (HIE = −W of metal − (E_HOMO) and (EIE =  − E_LUMO − (W of metal) from phytochemicals to Al electrode, i.e., teucriol (1) (4.03 eV = −0.05 − (−4.08)) > ascorbic acid (2.92 eV = −1.16 − (−4.08)) > Quercetin (2.09 eV = −1.99 − (−4.08)) while for Au as teucriol (1) (5.05 eV = −0.05 − (−5.10)) > ascorbic acid (3.94 eV = −1.16 − (−5.10)) > quercetin (3.11 eV = −1.99 − (−5.10)). HIE is estimated as: teucriol (1) (3.42 eV = −4.08 − (−7.50) > ascorbic acid (2.63 eV = −4.08 − (−6.71) > quercetin (1.70 eV = −4.08 − (−5.78) for Al-electrode whilst teucriol (1) (2.40 eV = −5.10 − (−7.50) > ascorbic acid (1.61 eV = −5.10 − (−6.71) > quercetin (0.68 eV = −5.10 − (−5.78) for Au. It can be indicated by a healthy electron injection for Al as a right electrode while for hole Au might be appropriate.

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Fig. 4

FMOs density of teucriol (1) along with numbering scheme (contour value = 0.035).

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Other essential parameters i.e., Global chemical reactivity descriptors (GCRD) are good to apprehend the reactivity. The attained HOMO and LUMO energy values are used to measure various GCRD parameters including chemical hardness (η), chemical potential (µ), softness (S), electronegativity (χ), and electrophilicity index (ω), see Table 4 (computational details within SI). The compound η value is unified (Geerlings et al., 2003, Vektariene et al., 2009) and it show the electron affinity to depart from its electronic surroundings. The obtained η value signify the magnitude of the barricade of electronic cloud for deformation. The available value of ω refers to the.

The multi-color MEP maps are significant to foresee the charged sphere of investigated compound. The MEP surfaces for teucriol (1) along with studies standard drugs are depicted in various shape color segments, see Fig. 5. The red and blue color indicates the most potent components that may be auspicious for nucleophilic and electrophilic attacks. The negative potential was observed on the -O atoms while positive on -H of –OH. The Hirshfeld population evaluation scheme by which we can study molecular density into atomic density helpful to understand the biological activities of the compounds. This approach does no longer require a connection with basis sets or their respective locations but is centered totally on a specific mathematical and physical footing. The benefit of this technique is that, while the molecular deformation density is converged to the real solution, the calculated charges will certainly converge. Earlier it became confirmed that the Hirshfeld charges reveal where electrophilic and nucleophilic attacks will ideally ensue, however the charge quantity correlates with experimental nucleophilicity and electrophilicity. Such correlations are feasible, due to the fact nucleophilicity (electrophilicity) measures the potential of a nucleophile (electrophile) to donate (accept) electrons, so traditional chemical knowledge implies that atomic charges must be direct and trustworthy. The nucleophilicity and electrophilicity are established based on the reacting model. Various categories acquire various reacting styles, so their scales are consequently unlike (Zhou, et al., 2014). Hirshfeld approach was showed appropriate for atomic charges establishment (Guerra et al., 2004). The Hirshfeld charges are demonstrated in Fig. 5. The negative charge oxygen atoms IS −0.235 (O1), −0.215 (O32) and −0.214 (O34) while positive charge on hydrogen atoms 0.157 (H35), 0.155 (H2) and 0.151 (H33). These results showed that O1 might be favorable site for electrophilic while H35 for the nucleophilic attacks.

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Fig. 5

Molecular electrostatic potential surfaces (bottom), Hirshfeld charges (center) Hirshfeld charges color by atoms (top) of teucriol (1).

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