Abstract
Objectives
Phytosterols obtained from medicinal plants are well known for their anti-diabetic, anti-cardiovascular, anti-cancer, and anti-microbial properties. Strychnos innocua (a member of the Loganiaceae family) grows in several African nations and is frequently used for medicinal purposes.
Methods
The chromatographic separation of S. innocua (root bark) ethyl acetate extract resulted in the isolation of campesterol (1) and β-sitosterol (2).
Results
The structures of 1 and 2 were confirmed by mass spectrometry, nuclear magnetic resonance (1D and 2D NMR), and literature data. This is a novel report of campesterol and β-sitosterol from S. innocua. Docking studies revealed that the binding affinities of 1 with the binding sites of Staphylococcus aureus pyruvate carboxylase (PDB: 3HO8) and Pseudomonas aeruginosa virulence factor regulator (PDB: 2OZ6) were −7.8 and −7.9 kcal/mol, respectively. Furthermore, 2 had binding affinities of −7.6 and −7.7 kcal/mol with binding sites of S. aureus and P. aeruginosa, respectively, whereas ciprofloxacin (a standard drug) had binding affinities of −6.6 and −8.7 kcal/mol.
Conclusion
This study indicated that S. innocua root bark is rich in campesterol and β-sitosterol. In silico molecular docking demonstrated that the compounds interact well with the binding sites of S. aureus and P. aeruginosa.
Keywords: β-sitosterol, Campesterol, Docking, Isolation, Strychnos innocua
المخلص
أهداف البحث
تشتهر فيتوسترولس التي يتم الحصول عليها من النباتات الطبية بخصائصها المضادة للسكري والقلب والأوعية الدموية والسرطان والمضادة للميكروبات. ينمو الإسطركن الشوكي (عضو في عائلة اللوغانيات) في العديد من الدول الأفريقية وغالبا ما يستخدم للأغراض الطبية.
طرق البحث
أدى الفصل الكروماتوجرافي لمستخلص أسيتات الإيثيل من الإسطركن الشوكي (لحاء الجذر) إلى عزل كامبيسترول (1) وبيتا سيتوستيرول (2).
النتائج
تم تأكيد هياكلها عن طريق قياس الطيف الكتلي والرنين المغناطيسي النووي وبيانات الأدب. هذا هو تقرير جديد عن كامبيسترولوبيتاسيتوستيرول من النبات. كشفت دراسات الالتحام أن ارتباطات الارتباط 1 مع مواقع الربط للمكورات العنقودية الذهبية بيروفاتالكربوكسيلاز ومنظمعامل الفوعة الزائفة الزنجارية كانت -7.8 و -7.9 كيلو كالوري /مول،على التوالي. علاوة علىذلك،يحتوي 2 على ارتباطات ملزمة تبلغ -7.6 و -7.7 كيلو كالوري / مول مع مواقع الربط ل المكورات العنقودية الذهبيةوالزائفةالزنجارية،علىالتوالي،بينما يحتوي سيبروفلوكساسين (دواء قياسي) على ارتباطات ملزمة -6.6 و -8.7 كيلو كالوري / مول.
الاستنتاجات
أشارت هذه الدراسة إلى أن لحاء جذر الإسطركن الشوكي يحتوي على وجود غني من كامبيسترول وبيتا سيتوستيرول، وأن تحقيقات الالتحام الجزيئي في سيليكو أظهرت أن المركبات تتفاعل بشكل جيد مع مواقع ارتباط الزائفة الزنجارية والمكورات العنقودية الذهبية.
الكلمات المفتاحية: عزل, الإرسا الجزيئي, كامبيسترول, بيتا سيتوستيرول, الإسطركن الشوكي
Introduction
Compounds derived from plants have enormous potential for the development of new drugs.1 The various parts of plants (leaves, stems, roots, fruit, and flowers) are used in multiple applications, including medicinal purposes.2
In medicinal plants, the phytochemicals and secondary metabolites, such as phytosterols, contain active medicinal components with therapeutic potential.3,4 Campesterol is a naturally occurring plant sterol that has been associated with cholesterol lowering and cancer prevention.5 Plumbago zaylanica contains β-sitosterol, which has antibacterial, antimalarial, antifertility, anti-inflammatory, blood coagulation, wound healing, and anticancer properties.6
Several approaches, such as in vitro, in vivo, and in silico methods, have been used to investigate the antimicrobial activity of natural plant constituents. Docking is one technique that has seen widespread use in the development of antimicrobial medicines.7,8
Strychnos innocua (Figure 1) is a plant of the Loganiaceae family with a straight stem and a height up to 18 m. It has a trunk diameter ranging from 7 to 40 cm, and many branches. Its leaves are normally simple, with a rounded base in rare instances. S. innocua can be found in Cameroon, Malawi, and Nigeria. The root bark of the plant has been reported to cure gonorrhea, and a fresh infusion of the plant's root is used to treat snake bites.9,10 The plant can be harvested in Kaduna State, Nigeria.
Figure 1.
Strychnos innocua branches fruit, and leaves.
The chemical compositions and antimicrobial activities of S. innocua root bark extracts have been investigated.11, 12, 13, 14, 15 However, research on the isolation of phytosterols from S. innocua root bark is lacking. Herein, the phytosterols campesterol (1) (Figure 2) and β-sitosterol (2) (Figure 3) were isolated from the root bark of S. innocua, characterized, and docked to assess their antibacterial activity. This is a novel report of phytosterol compounds from the plant root bark.
Figure 2.
Structure of campesterol (1).
Figure 3.
Structure of β-sitosterol (2).
Materials and Methods
Plant collection
S. innocua was obtained in the wild in Kaduna State, Nigeria. Mr. Namadi Sunusi identified and authenticated the specimen at the Department of Biological Sciences at ABU, Zaria, where V/N-01884 is the herbarium voucher number.
Extraction
The root bark of S. innocua was dried in the shade, then crushed to a fine powder. According to a prior report,11 the powder (i.e., crushed sample, 2 kg) was subjected to extraction with the maceration method with solvents n-hexane (HEX), ethyl acetate (EA), and methanol, with increasing polarity.
General experimental procedure
GC–MS analysis of the isolated compounds was performed on a GC 7890B, MSD 5977A, Agilent Tech instrument. The carrier gas used was helium at a flow rate of 1 mL/min. The sample supernatant (1 μL) was injected into the GC while the temperature of the GC oven was set to rise from 80 °C to 150 °C at a rate of 12 °C/min, then to 270 °C at a rate of 9 °C/min, followed by a 5-min isothermal step at 325 °C. The ion source temperature was set at 230 °C, and the ionization voltage was set to 70 eV. The NMR (1D and 2D) spectra were obtained on a Varian–Vnmrs 400 MHz spectrometer with chloroform (CdCl3). Chemical shifts (δ) are reported in ppm.
The chemicals and reagents used in the investigation were of analytical grade.
Isolation and purification
With several solvent systems, thin layer chromatography (TLC) of ethyl acetate extract revealed many spots. The extract (30 g) was mixed with 60–120 mesh silica gel and dried. After parking (with silica gel and HEX), the dried extract was placed in a column (size, 5 × 60 cm) and eluted with a suitable solvent (HEX:EA) under gradually increasing polarity (HEX 100%, 9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, 1:9, and 100% EA) at a flow rate of 1 drop/sec, thereby yielding 261 collections of 50 mL. To monitor these collections, we used TLC plates pre-coated with spraying reagent (CH3OH:CH3COOH:H2SO4:CH3OC6H4CHO at a ratio of 85:10:5:0.l mL), thus generating 24 fractions (F1–F24). Fractions 8 and 9 were mixed and separated with column chromatography with increasing concentrations of HEX:EA (HEX 100%, HEX:EA, 9:1) to obtain 60 collections of 5 mL each. A pre-coated TLC plate was also used to monitor the collections, thus yielding eight subfractions (FF1–FF8). The FF4 and FF5 subfractions were further combined and separated on a column before being eluted with HEX:EA (9.1), thus yielding three smaller fractions (SF1, SF2, and SF3). On TLC, SF2 revealed one spot representing compound 1 (Rf = 0.41), thus yielding 46 mg. FF2 and FF3 were combined and eluted with HEX:EA (9:1), and four smaller fractions (SF1, SF2, SF3, and SF4) were obtained. On TLC, SF3 revealed one spot, which was used to obtain compound 2 (Rf = 0.18), with a yield of 37 mg.
Molecular docking study
Compounds 1 and 2, as well as ciprofloxacin (standard drug), were docked in silico with target receptors (PDB: 3HO8, and 2OZ6) obtained from the Protein Data Bank (www.rcsb.org). ChemDraw professional 16.0 was used to design its two-dimensional (2D) structure, which was subsequently optimized in three dimensions with Spartan 20v.1.1/2020. The target receptors were created in three dimensions with Discovery Studio Visualizer, saved in PDB file format, and then uploaded to the Pyrx program for docking. To investigate protein–ligand interactions, the docking output was shown in Discovery Studio together with the binding energy.16,17
Results and discussion
Compound 1 (46 mg), in the form of a white powder, had a melting point of 162 °C. The mass spectrum (Figure 4) of 1 at retention time (RT = 10.880 min) showed peaks at an m/z of 400, with molecular ion and fragment ion m/z values of 367, 316, 289, 255, 213, 173, 145, 109, 81, and 43, thus suggesting a molecular formula of C28H48O. The NMR spectra data (Table 1) of 1 was highly similar to that of campesterol in the literature, with 1H NMR (Figure 5) displaying δH for one olefinic methine proton (δH 5.51 H-6), one hydroxyl proton (δH 4.53 OH), six methyl protons (δH 0.83 H-18, 0.67 H-19, 0.81 H-21, 0.77 C-26, 0.80 H-27, and 0.66 C-28), ten methylene protons (δH 1.99 H-1, 1.82 H-2, 1.60 H-4, 1.13 H-7, 1.12 H-11, 1.20 H-12, 2.12 H-15, 1.92 H-16, 2.20 H-22, and 1.08 H-23), and eight methine protons (δH 3.53 H-3, 1.80 H-8, 0.98 H-9, 1.46 H-14, 1.80 H-17, 2.27 H-20, 0.90 H-24, and 1.27 H-25). The 13C NMR (Figure 6) and DEPT revealed 28 carbon signals for six methyl carbons (δC 15.58 C-18, 12.19 C-19, 14.35 C-21, 21.28 C-26, 20.01 C-27, and 15.64 C-28), ten methylene carbons (δC 37.44 C-1, 31.85 C-2, 42.49 C-4, 32.12 C-7, 23.25 C-11, 39.97 C-12, 24.51 C-15, 26.22 C-16, 34.31 C-22, and 34.13 C-23), eight methine carbons (δC 72.03 C-3, 32.09 C-8, 51.44 C-9, 56.96 C-14, 56.23 C-17, 36.35 C-20, 39.25 C-24, and 33.90 C-25), three quaternary carbons (δC 145.43 C-5, 36.71 C-10, and 46.01 C-13), and one olefinic methine carbon (δC 121.94 C-6) (see Table 2).
Figure 4.
Mass spectrum of campesterol (1).
Table 1.
NMR (400 MHz, CDCl3) data for campesterol (1).
| Campesterol |
Literature data18,19 |
|||||
|---|---|---|---|---|---|---|
| Position | 1H (ppm) | 13C (ppm) | DEPT | 1H (ppm) | 13C (ppm) | DEPT |
| C-1 | 1.99 (m, 2H) | 37.44 | CH2 | 1.55 (m, 2H) | 37.30 | CH2 |
| C-2 | 1.82 (m, 2H) | 31.85 | CH2 | 1.52 (m, 2H) | 28.90 | CH2 |
| C-3 | 3.53 (m, 1H) | 72.03 | CH | 3.40 (m, 1H) | 71.90 | CH |
| C-4 | 1.60 (m, 2H) | 42.49 | CH2 | 1.40 (m, 2H) | 42.30 | CH2 |
| C-5 | 145.43 | C | 142.40 | C | ||
| C-6 | 5.51 (m, 1H) | 121.94 | CH | 5.31 (m, 1H) | 121.90 | CH |
| C-7 | 1.13 (m, 2H) | 32.12 | CH2 | 1.33 (m, 2H) | 31.80 | CH2 |
| C-8 | 1.80 (m, 1H) | 32.09 | CH | 1.73 (m, 1H) | 31.00 | CH |
| C-9 | 0.98 (m, 1H) | 51.44 | CH | 51.20 | CH | |
| C-10 | 36.71 | C | 36.50 | C | ||
| C-11 | 1.12 (m, 2H) | 23.25 | CH2 | 1.13 (m, 2H) | 21.10 | CH2 |
| C-12 | 1.20 (m, 2H) | 39.97 | CH2 | 1.21 (m, 2H) | 39.80 | CH2 |
| C-13 | 46.01 | C | 43.10 | C | ||
| C-14 | 1.46 (m, 1H) | 56.96 | CH | 1.83 (m, 1H) | 56.90 | CH |
| C-15 | 2.12 (m, 2H) | 24.51 | CH2 | 21.80 | CH2 | |
| C-16 | 1.92 (m, 1H) | 26.22 | CH2 | 1.92 (m, 1H) | 25.00 | CH2 |
| C-17 | 1.80 (m, 1H) | 56.23 | CH | 1.73 (m, 1H) | 56.10 | CH |
| C-18 | 0.83 (s, 3H) | 15.58 | CH3 | 1.10 (s, 3H) | 19.80 | CH3 |
| C-19 | 0.67 (s, 3H) | 12.19 | CH3 | 0.73 (s, 3H) | 12.20 | CH3 |
| C-20 | 2.27 (m, 2H) | 36.35 | CH | 2.17 (m, 1H) | 32.50 | CH |
| C-21 | 0.81 (d, 3H) | 14.35 | CH3 | 0.81 (d, 3H) | 19.10 | CH3 |
| C-22 | 2.20 (m, 2H) | 34.31 | CH2 | 34.50 | CH2 | |
| C-23 | 1.08 (m, 2H) | 34.13 | CH2 | 1.20 (m, 2H) | 30.30 | CH2 |
| C-24 | 0.90 (m, 1H) | 39.25 | CH | 1.08 (m, 2H) | 42.40 | CH |
| C-25 | 1.27 (m, 1H) | 33.90 | CH | 1.77 (m, 1H) | 36.10 | CH |
| C-26 | 0.77 (d, 3H) | 21.28 | CH3 | 0.83 (d, 3H) | 21.20 | CH3 |
| C-27 | 0.80 (d, 3H) | 20.01 | CH3 | 0.79 (d, 3H) | 19.10 | CH3 |
| C-28 | 0.66 (d, 3H) | 15.64 | CH3 | 0.70 (d, 3H) | 15.39 | CH3 |
| OH | 4.53 (s, 1H) | |||||
Figure 5.
1H NMR spectrum of campesterol (1).
Figure 6.
13C NMR spectrum of campesterol (1).
Table 2.
NMR (400 MHz, CDCl3) data for β-sitosterol (2).
| β-sitosterol |
Literature data20,21 |
|||||
|---|---|---|---|---|---|---|
| Position | 1H (ppm) | 13C (ppm) | DEPT | 1H (ppm) | 13C (ppm) | DEPT |
| C-1 | 1.72 (m, 2H) | 38.89 | CH2 | 1.85 (m, 2H) | 37.39 | CH2 |
| C-2 | 1.93 (m, 2H) | 30.27 | CH2 | 1.95 (m, 2H) | 31.76 | CH2 |
| C-3 | 3.57 (m, 1H) | 72.06 | CH | 3.55 (m, 1H) | 71.95 | CH |
| C-4 | 2.32 (m, 2H) | 41.25 | CH2 | 2.38 (m, 2H) | 42.39 | CH2 |
| C-5 | 143.71 | C | 140.85 | C | ||
| C-6 | 5.46 (m, 1H) | 122.21 | CH | 5.37 (m, 1H) | 121.85 | CH2 |
| C-7 | 1.95 (m, 2H) | 32.14 | CH2 | 1.99 (m, 2H) | 32.06 | CH2 |
| C-8 | 2.19 (m, 1H) | 31.88 | CH | 2.00 (m, 1H) | 31.93 | CH |
| C-9 | 1.10 (m, 1H) | 51.46 | CH | 0.94 (m, 1H) | 50.28 | CH |
| C-10 | 37.34 | C | 36.64 | C | ||
| C-11 | 1.19 (m, 2H) | 22.91 | CH2 | 1.02 (m, 2H) | 21.22 | CH2 |
| C-12 | 1.22 (m, 2H) | 40.09 | CH2 | 1.16 (m, 2H) | 39.92 | CH2 |
| C-13 | 43.51 | C | 42.46 | C | ||
| C-14 | 1.15 (m, 1H) | 56.52 | CH | 1.00 (m, 1H) | 56.90 | CH |
| C-15 | 1.53 (m, 2H) | 28.95 | CH2 | 1.58 (m, 2H) | 28.39 | CH2 |
| C-16 | 1.20 (m, 2H) | 28.88 | CH2 | 1.09 (m, 2H) | 28.35 | CH2 |
| C-17 | 1.21 (m, 1H) | 55.00 | CH | 1.12 (m, 1H) | 56.18 | CH |
| C-18 | 0.87 (s, 3H) | 14.31 | CH3 | 0.85 (s, 3H) | 12.12 | CH3 |
| C-19 | 0.78 (s, 3H) | 19.52 | CH3 | 0.82 (s, 3H) | 19.40 | CH3 |
| C-20 | 1.51 (m, 1H) | 33.77 | CH | 1.35 (m, 1H) | 36.29 | CH |
| C-21 | 0.97 (d, 3H) | 20.70 | CH3 | 0.95 (d, 3H) | 18.92 | CH3 |
| C-22 | 1.43 (m, 2H) | 33.20 | CH2 | 1.33 (m, 2H) | 34.07 | CH2 |
| C-23 | 1.22 (m, 2H) | 28.98 | CH2 | 1.16 (m, 2H) | 26.14 | CH2 |
| C-24 | 1.14 (m, 1H) | 45.02 | CH | 0.94 (m, 1H) | 45.99 | CH |
| C-25 | 1.57 (m, 1H) | 29.91 | CH | 1.66 (m, 1H) | 28.91 | CH |
| C-26 | 0.82 (d, 3H) | 22.86 | CH3 | 0.83 (d, 3H) | 21.38 | CH3 |
| C-27 | 0.85 (d, 3H) | 22.81 | CH3 | 0.84 (d, 3H) | 19.18 | CH3 |
| C-28 | 1.39 (m, 2H) | 24.85 | CH2 | 1.25 (m, 2H) | 23.20 | CH2 |
| C-29 | 0.88 (m, 3H) | 14.35 | CH3 | 0.85 (m, 3H) | 12.19 | CH3 |
| OH | 4.82 (s, 1H) | |||||
Compound 2 (37 mg), in the form of a clear crystal, had a melting point of 147 °C. The mass spectrum (Figure 7) of 2 at retention time (RT = 27.486 min) indicated fragment ion peaks at an m/z of 396, representing a H2O loss from the molecular ion peak (m/z 414). Other fragmentation ions included m/z of 381, 342, 303, 255, 213, 173, 145, 109, 81, and 43, thereby suggesting a molecular formula of C29H50O. The NMR spectra data (Table 1) of 2 were highly similar to those in the literature for β-sitosterol, with 1H NMR (Figure 8) displaying δH for six methyl protons (δH 0.87 H-18, 0.78 C-19, 0.97 H-21, 0.82 H-26, 0.85 H-27, and 0.88 H-29), eleven methylene protons (δH 1.72 H-1, 1.93 C-2, 2.32 H-4, 1.95 H-7, 1.19 H-11, 1.22 H-12, 1.53 H-15, 1.20 H-16, 1.43 H-22, 1.22 H-23, and 1.39 H-28), nine methine protons (δH 3.57 H-3, 5.46 H-6, 2.19 H-8, 1.10 H-9, 1.15 H-14, 1.21 H-17, 1.51 H-20, 1.14 C-24, and 1.57 H-25), and one hydroxyl proton (δH 4.82 OH). The 13C NMR (Figure 9) and DEPT displayed 29 carbon signals for six methyl carbons (δC 14.31 C-18, 19.52 C-19, 20.70 C-21, 22.86 C-26, 22.81 C-27, and 14.35 C-29), eleven methylene carbons (δC 38.89 C-1, 30.27 C-2, 41.25 C-4, 32.14 C-7, 22.91 C-11, 40.09 C-12, 28.95 C-15, 28.88 C-16, 33.20 C-22, 28.98 C-23, and 24.85 C-28), eight methine carbons (δC 72.06 C-3, 31.88 C-8, 51.46 C-9, 56.52 C-14, 55.00 C-17, 33.77 C-20, 45.02 C-24, and 29.91 C-25), three quaternary carbons (δC 143.71 C-5, 37.34 C-10, and 43.51 C-13), and one olefinic methine carbon (δC 122.21 C-6).
Figure 7.
Mass spectrum of β-sitosterol (2).
Figure 8.
1H NMR spectrum of β-sitosterol (2).
Figure 9.
13C NMR spectrum of β-sitosterol (2).
The ethyl acetate extract demonstrated the presence of steroids in the phytochemical investigation, and further displayed potent antibacterial action against S. aureus, P. aeruginosa, and B. subtilis.13 Campesterol and β-sitosterol were isolated from the extract after chromatographic separation, and their structures (Figure 2, Figure 3) were identified with spectroscopic investigations and comparison with data from the literature.18, 19, 20, 21, 22, 23 These compounds are present in a variety of plant species; their biological activities have been thoroughly investigated, and their pharmaceutical effects have been established. The antifungal activity of campesterol and β-sitosterol obtained from Dendrocalamus asper against some fungal pathogens has been determined, and they have been found to have exceptional antifungal properties.24 These compounds have also been shown to have anti-inflammatory, antibacterial, and anti-tumor effects.25 In general, approximately 250 phytosterols are found in plants, including sitosterol, stigmasterol, campesterol, brassicasterol, ergosterol, and β-sitosterol. They are related to cholesterol and, according to their structural components, have been identified in plant biological membranes.26
The interactions of the compounds with the target receptors (PDB: 3HO8 and 2OZ6) were studied with molecular docking and compared with those of ciprofloxacin (standard drug). Compounds 1 and 2 exhibited considerably greater binding energy (Table 3) toward S. aureus pyruvate carboxylase, 3HO8 (receptor) than ciprofloxacin. Campesterol had a greater binding energy (−7.8 kcal/mol) than β-sitosterol (−7.6 kcal/mol); their interactions with the receptor are shown in Figure 10, Figure 11, respectively. The binding energy of ciprofloxacin was −6.6 kcal/mol, and its interaction with the receptor is represented in Figure 12. These figures also indicate that the target residues involved in interactions with the docked compounds included PHE, PRO, LYS, VAL, PHE, TYR, and GLY, thus emphasizing the relevance of these residues in S. aureus suppression. S. aureus has long been associated with soft tissue infections and skin conditions, such as food poisoning, abscesses, respiratory infections, furuncles, pneumonia, cellulitis, and joint infections.27 Campesterol isolated from Fiscus religiosa shows substantial interactions with binding sites in the crystal structure of the Kelch–Neh2 complex (PDB: 2FLU), thus suggesting that it is a viable competitive agent to counteract Keapl and hence may be used in cancer chemoprevention (28).
Table 3.
Binding energy of isolated compounds/ciprofloxacin with receptor (PDB: 3HO8).
| Ligands | Binding score (kcal/mol) | Protein interaction | Types of interaction | Bond distance Å |
|---|---|---|---|---|
| Campesterol | −7.8 | PHE516 | Alkyl | 4.74 |
| PRO410 | Alkyl | 5.03 | ||
| PRO410 | Alkyl | 5.46 | ||
| PRO410 | Alkyl | 5.16 | ||
| LYS518 | Alkyl | 4.92 | ||
| LYS518 | Alkyl | 5.46 | ||
| LYS518 | Alkyl | 4.23 | ||
| VAL404 | Alkyl | 4.37 | ||
| PHE516 | Pi-alkyl | 5.42 | ||
| TYR400 | Pi-alkyl | 5.37 | ||
| TRY400 | Pi-alkyl | 5.27 | ||
| GLY408 | Carbon hydrogen bond | 2.81 | ||
| β-sitosterol | −7.6 | PRO410 | Alkyl | 4.97 |
| PRO410 | Alkyl | 4.00 | ||
| PRO410 | Alkyl | 4.70 | ||
| LEU926 | Alkyl | 5.03 | ||
| LYS518 | Alkyl | 5.25 | ||
| LYS518 | Alkyl | 4.77 | ||
| LYS518 | Alkyl | 4.14 | ||
| VAL404 | Alkyl | 4.79 | ||
| PHE516 | Pi-alkyl | 4.81 | ||
| PHE409 | Pi-alkyl | 4.68 | ||
| PHE934 | Pi-alkyl | 5.13 | ||
| TYR400 | Pi-alkyl | 5.06 | ||
| TYR400 | Pi-alkyl | 4.61 | ||
| TRY923 | Pi-alkyl | 4.77 | ||
| GLY408 | Carbon hydrogen bond | 2.99 | ||
| Ciprofloxacin | −6.6 | PRO410 | Pi-sigma | 3.70 |
| PHE934 | Pi-alkyl | 5.28 | ||
| PHE409 | Pi-alkyl | 5.12 | ||
| PRO410 | Pi-alkyl | 5.06 | ||
| LYS518 | Alkyl | 4.14 | ||
| PRO410 | Pi-alkyl | 5.15 | ||
| ASN403 | Conventional hydrogen bond | 2.73 |
Figure 10.
2D Interaction of campesterol (1) with crystal structure of S. aureus (PDB: 3HO8).
Figure 11.
2D Interaction of β-sitosterol (2) with crystal structure of S. aureus (PDB: 3HO8).
Figure 12.
2D Interaction of ciprofloxacin with crystal structure of S. aureus (PDB: 3HO8).
Furthermore, compounds 1 and 2 had considerably lower binding energy (Table 4) than ciprofloxacin toward the P. aeruginosa virulence factor regulator 2OZ6 (receptor). Campesterol had a higher binding energy (−7.9 kcal/mol) than β-sitosterol (−7.7 kcal/mol), and their interactions with the receptor are shown in Figure 13, Figure 14, respectively. Ciprofloxacin's binding energy was −8.7 kcal/mol, and Figure 15 displays its interaction with the receptor. The target residues (LEU, ILE, and ARG) were involved in interactions with 1, 2, and ciprofloxacin, thus indicating their importance in P. aeruginosa inhibition. The pathogen is a multidrug-resistant bacterium that causes illness in both plants and animals, such as septic shock, pneumonia, gastrointestinal conditions, and urinary tract infection.29 β-sitosterol has also been discovered in Fiscus religiosa, and has been found to substantially interact with binding sites of the Kelch-Neh2 complex (PDB: 2FLU) crystal structure, thus suggesting that it is a potential competitive drug to counteract Keapl and may be applied in cancer chemoprevention.28
Table 4.
Results of binding energy of isolated compounds/ciprofloxacin with receptor (PDB: 2OZ6).
| Ligands | Binding score (kcal/mol) | Protein interaction | Types of interaction | Bond distance Å |
|---|---|---|---|---|
| Campesterol | −7.9 | ALA77 | Alkyl | 3.79 |
| LEU59 | Alkyl | 4.43 | ||
| ILE44 | Alkyl | 4.22 | ||
| VAL79 | Alkyl | 5.46 | ||
| ARG116 | Alkyl | 5.26 | ||
| ARG116 | Alkyl | 4.05 | ||
| ARG116 | Alkyl | 5.57 | ||
| LEU117 | Alkyl | 4.92 | ||
| LEU68 | Alkyl | 3.82 | ||
| LEU68 | Alkyl | 5.42 | ||
| LEU68 | Alkyl | 4.48 | ||
| LEU68 | Alkyl | 4.48 | ||
| LEU68 | Alkyl | 4.42 | ||
| β-sitosterol | −7.7 | ILE56 | Alkyl | 4.16 |
| ILE44 | Alkyl | 4.44 | ||
| ILE44 | Alkyl | 3.95 | ||
| LEU59 | Alkyl | 4.25 | ||
| ARG116 | Alkyl | 4.20 | ||
| ARG116 | Alkyl | 4.50 | ||
| LEU68 | Alkyl | 4.70 | ||
| LEU68 | Alkyl | 4.84 | ||
| MET113 | Alkyl | 4.74 | ||
| MET113 | Alkyl | 4.32 | ||
| LEU117 | Alkyl | 4.16 | ||
| LEU117 | Alkyl | 5.03 | ||
| LEU117 | Alkyl | 4.25 | ||
| LEU117 | Alkyl | 4.19 | ||
| Ciprofloxacin | −8.7 | GLU57 | Pi-anion | 4.48 |
| ILE44 | Pi-sigma | 3.99 | ||
| ALA77 | Carbon hydrogen bond | 2.52 | ||
| LEU68 | Alkyl | 4.76 | ||
| ALA77 | Alkyl | 5.14 | ||
| ALA77 | Alkyl | 4.72 | ||
| ILE56 | Alkyl | 4.42 | ||
| ALA77 | Alkyl | 4.72 | ||
| ARG116 | Pi-alkyl | 4.84 | ||
| LEU68 | Pi-alkyl | 5.43 | ||
| ILE44 | Pi-alkyl | 4.16 | ||
| THR120 | Conventional hydrogen bond | 2.37 | ||
| GLY66 | 2.37 |
Figure 13.
2D Interaction of campesterol (1) with crystal structure of P. aeruginosa (PDB: 2OZ6).
Figure 14.
2D Interaction of β-sitosterol (2) with crystal structure of P. aeruginosa (PDB: 2OZ6).
Figure 15.
2D Interaction of ciprofloxacin with crystal structure of P. aeruginosa (PDB: 20Z6).
Conclusion
The structures of two compounds (campesterol and β-sitosterol) isolated from S. innocua root bark were characterized with MS and NMR spectroscopy. In the docking study, campesterol and β-sitosterol showed binding energies of −7.8 and −7.7 kcal/mol with the binding site of S. aureus (PDB: 3HO8), values higher than that of ciprofloxacin. Furthermore, the compounds showed binding energies of −7.9 and 7.7 kcal/mol with the P. aeruginosa binding site (PDB: 2OZ6), values slightly lower than that of ciprofloxacin (−8.7 kcal/mol). These findings suggest that the compounds might serve as potential antibacterial agents.
Source of funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflict of interest
The authors have declared no competing interests.
Ethical approval
Not applicable.
Authors contributions
MSS developed the procedure for isolation, AJU performed the experiments/wrote the manuscript, ORI assisted in supervising the experiment, and HI contributed to NMR elucidation. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.
Acknowledgment
The authors appreciate Mr. Silas Ekwuribe in the Chemistry Department of Ahmadu Bello University Zaria–Nigeria for contributions toward the success of this research.
The authors are grateful to Mallam Samaila Mustapha, Mallam Kabir Mohammed, and Mr. Silas Ekwuribe for their valuable assistance.
Footnotes
Peer review under responsibility of Taibah University.
Contributor Information
Ahmed Jibrin Uttu, Email: jibuttu@yahoo.com.
Muhammad Sani Sallau, Email: sskaraye@yahoo.com.
Hamisu Ibrahim, Email: hibrahimbk@yahoo.com.
Ogunkemi Risikat Agbeke Iyun, Email: kemiruthiyun@gmail.com.
References
- 1.Sasidharan S., Chen Y., Saravanan D., Sundram K.M., Latha L.Y. Extraction, isolation and characterization of bioactive compounds from plants extracts. Afr J Tradit Complementary Altern Med. 2011;8(1):1–10. PMID: 22238476. [PMC free article] [PubMed] [Google Scholar]
- 2.Umaru I., Badruddin F.A., Umaru H.A. Extraction, isolation and characterization of new compound and anti-bacterial potentials of the chemical constituents compound from Leptadenia hastata leaf extract. ChemRxiv. 2019 doi: 10.26434/chemrxiv.9782585.v2. [DOI] [Google Scholar]
- 3.Mondal M., Hossain M.S., Das N., Khalipha A.B.R., Sarkar A.P., Islam M.T., et al. Phytochemical screening and evaluation of pharmacological activity of leaf methanol extract of Colocasia affinis Schott. Clin Phytosci. 2019;5(8):1–11. doi: 10.1186/s40816-019-0100-8. [DOI] [Google Scholar]
- 4.Uttu A.J., Sallau M.S., Iyun O.R.A., Ibrahim H. Recent advances in isolation and antimicrobial efficacy of selected Strychnos species: a mini review. J Chem Rev. 2022;4(1):15–24. doi: 10.22034/JCR.2022.314381.1129. [DOI] [Google Scholar]
- 5.Choi J., Lee E., Lee H., Kim K., Ahn K., Shim B., et al. Identification of campesterol from Chrysanthemum coronarium L. and its antiantiogenic activities. Phytother Res. 2007;21(10):954–959. doi: 10.1002/ptr.2189. [DOI] [PubMed] [Google Scholar]
- 6.Jian P., Sharma H.P., Basri F., Baraik B., Kumari S., Pathak C. Pharmacological profiles of ethno-medicinal plant: Plumbago zeylanica I. – a review. Int J Pharmaceut Sci Rev Res. 2014;24(1):157–163. [Google Scholar]
- 7.Nour E.A., Abd E., Khaled E., Maher A.E., Samir A.S., Mostafa M.E. Designed, synthesis, molecular docking and in silico ADMET profile of pyrano[2,3-d]pyrimidine derivatives as antimicrobial and anticancer agents. Bioorg Chem. 2021;115 doi: 10.1016/j.bioorg.2021.105186. [DOI] [PubMed] [Google Scholar]
- 8.Emmanuel I.E., Uttu A.J., Oluwaseye A., Hassan S., Ajala A. A semi-empirical based QSAR study of indole β-diketo acid, diketo acid and carboxamide derivatives as potent HIV-1 agent using quantum chemical descriptors. IOSR J Appl Chem. 2015;8(11i):12–20. doi: 10.9790/5736-081111220. [DOI] [Google Scholar]
- 9.Ruffo C.K., Birnie A., Tengnas B. Regional Land Management Unit; Nairobi: 2002. Edible wild plants of Tanzania. [Google Scholar]
- 10.Orwa C.A., Mutua K.R., Jamnadass R., Anthony S. 2009. Agroforestree database: a tree reference and selection guide version 4.0.http://www.worldagroforestry.org/sites/treedbs/treedatabases.asp Retrieved from. [Google Scholar]
- 11.Ibrahim H., Uttu A.J., Sallau M.S., Iyun O.R.A. Gas chromatography–mass spectrometry (GC–MS) analysis of ethyl acetate root bark extract of Strychnos innocua (Delile) Beni-Suef Univ J Basic Appl Sci. 2021;10:1–8. doi: 10.1186/s43088-021-00156-1. 65. [DOI] [Google Scholar]
- 12.Iyun O.R.A., Uttu A.J., Sallau M.S., Ibrahim H. GC-MS analysis of methanol extract of Strychnos innocua (Delile) root bark. Adv J Chem A. 2022;5(2):104–117. doi: 10.22034/AJCA.2022.322806.1295. [DOI] [Google Scholar]
- 13.Sallau M.S., Uttu A.J., Iyun O.R.A., Ibrahim H. Strychnos innocua (Delile): phytochemical and antimicrobial evaluations of its root bark extracts. Adv J Chem B. 2022;4(2022):17–28. doi: 10.22034/ajcb.2022.323148.1104. [DOI] [Google Scholar]
- 14.Uttu A.J., Sallau M.S., Iyun O.R.A., Ibrahim H. Coumarin and fatty alcohol from root bark of Strychnos innocua (Delile): isolation, characterization and in silico molecular docking studies. Bull Natl Res Cent. 2022;46(174):1–12. doi: 10.1186/s42269-022-00862-5. [DOI] [Google Scholar]
- 15.Uttu A.J., Sallau M.S., Iyun O.R.A., Ibrahim H. Isolation, characterization and in silico molecular docking studies of two terpenoids from Strychnos innocua (Delile) root bark for antibacterial properties. Adv J Chem A. 2022;5(3):241–252. doi: 10.22034/AJCA.2022.344451.1315. [DOI] [Google Scholar]
- 16.Ejeh S., Uzairu A., Shallangwa G.A., Abechi S.E. Computational techniques in designing a series of 1,3,4-trisubstituted pyrazoles as unique hepatitis C virus entry inhibitors. Chem Rev Lett. 2021;4:108–119. ISSN: 2645-4947. [Google Scholar]
- 17.Tukur A., Habila J.D., Ayo R.G., Iyun O.R.A. Design, synthesis, docking studies and antibiotic evaluation (in vitro) of some novel (E)-4-(3-(diphenylamino)phenyl)- 1-(4-methoxyphenyl)-2-methylbut-3-en-1-one and their analogues. Bull Natl Res Cent. 2022;46(60):1–12. doi: 10.1186/s42269-022-00745-9. [DOI] [Google Scholar]
- 18.Musa W.J., Duengo S., Kilo A.K. Campesterol from methanol fraction of Brotowali (Tinospora crispa) stem bark. Atlantis Highlights Chem Pharmaceut Sci. 2019;1:95–97. [Google Scholar]
- 19.Pham X.P., Nhung T.T.T., Trinh H.N., Trung D.M., Giang D.T., Vu B.D., et al. Isolation and structural characterization of compounds from Blumea lacera. Pharmacogn J. 2021;13(4):999–1004. doi: 10.5530/pj.2021.13.129. [DOI] [Google Scholar]
- 20.Aliba M.O., Ndukwe I.G., Ibrahim H. Isolation and characterization of β-sitosterol from methanol extracts of the stem bark of large leaved rock fig (Ficus aburilifolia Mig) J Appl Sci Environ Manag. 2018;22(10):1639–1642. [Google Scholar]
- 21.Erwin U., Pusparohmana W.R., Safitry R.D., Marliana E., Usman E., Kusuma I.W. Isolation and characterization of stigmasterol and β-sitosterol from wood bark extract of Baccaurea macrocarpa Miq. Mull. Arg. Rasayan J Chem. 2020;13(4):2552–2558. doi: 10.31788/RJC.2020.1345652. [DOI] [Google Scholar]
- 22.Jain P.S., Bari S.B. Isolation of lupeol, stigmasterol and campesterol from petroleum ether extract of woody stem of Wrightia tinctoria. Asian J Plant Sci. 2010;9(3):163–167. ISSN: 1682-3974. [Google Scholar]
- 23.Okoro I.S., Tor-Anyiin T.A., Igoli J.O., Noundou X.S., Krause R.W.M. Isolation and characterisation of stigmasterol and β–sitosterol from Anthocleista djalonensis A. Chev. Asian J Chem Sci. 2017;3(4):1–5. ISSN: 2456-7795. [Google Scholar]
- 24.Choi N.H., Jang J.Y., Choi G.J., Choi Y.H., Jang K.S., Nguyen V.T., et al. Antifungal activity of sterols and dipsacus saponins isolated from Dipsacus asper roots against phytopathogenic fungi. Pestic Biochem Physiol. 2017;141:103–108. doi: 10.1016/j.pestbp.2016.12.006. [DOI] [PubMed] [Google Scholar]
- 25.Singh B., Singh J.P., Shevkani K., Singh N., Kaur A. Bioactive constituents in pulses and their health benefits. J Food Sci Technol. 2017;54(4):858–870. doi: 10.1007/s13197-016-2391-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Moreau R.A., Nystrom L., Whitaker B.D., Winkler-Moser J.K., Baer D.J., Gebauer S.K., et al. Phytosterols and their derivatives: structural diversity. Distribution, metabolism, analysis, and health-promoting uses. Prog Lipid Res. 2018;70:35–61. doi: 10.1016/j.plipres.2018.04.001. [DOI] [PubMed] [Google Scholar]
- 27.Dalen R.V., Peschel A., Sorge N.M. Wall teichoic acid in Staphylococcus aureus host interaction. Trends Microbiol. 2020;28(12):985–998. doi: 10.1016/j.tim.2020.05.017. [DOI] [PubMed] [Google Scholar]
- 28.Syahputra H.D., Masfria M., Hasibuan P.A.Z., Iksen I. In silico docking studies of phytosterol compounds selected from Ficus religiosa as potential chemopreventive agent. Rasayan J Chem. 2022;15(2):1080–1084. doi: 10.31788/RJC.2022.1526801. [DOI] [Google Scholar]
- 29.Diggle S., Whiteley M. Microbe profile: Pseudomonas aeruginosa: opportunistic pathogen and lab rat. Microbiology. 2020;166(1):30–33. doi: 10.1099/mic.0.000860. [DOI] [PMC free article] [PubMed] [Google Scholar]