Phytochemicals Analysis, In Vitro Antibacterial Activities of Extracts, and Molecular Docking Studies of the Isolated Compounds from Melhania zavattarii Cufod Leaves

Melhania zavattarii Cufod is an endemic plant species to Ethiopia and is used to treat ailments related to kidney infection. The phytochemical composition and biological activity of M. zavattarii have been not reported yet. Therefore, the present work aimed to investigate phytochemical constituents and evaluate the antibacterial activity of different solvents' leaf extracts and analyze the molecular binding capacity of isolated compounds from the chloroform leaf extract of M. zavattarii. Accordingly, preliminary phytochemical screening was tested by using standard procedures and the result indicated that phytosterols and terpenoids as major and others like alkaloids, saponins, flavonoids, tannins, phlobatannin, and coumarins were detected as minor in extracts. Antibacterial activity of the extracts was evaluated using the disk diffusion agar method, and the activities revealed that chloroform extract showed the highest inhibition zones, 12.08 ± 0.38, 14.00 ± 0.50, and 15.58 ± 0.63 mm against Escherichia coli at 50, 75, and 125 mg/mL concentrations, respectively, compared to that of n-hexane and methanol extracts at respective concentrations. Methanol extract showed the highest zone of inhibition 16.42 + 0.52 against Staphylococcus aureus at 125 mg/mL concentration compared to that of n-hexane and chloroform extracts. Two compounds, namely, β-amyrin palmitate (1) and lutein (2) were isolated and identified for the first time from the chloroform leaf extract of M. zavattarii, and structural elucidations of these compounds were accomplished by using spectroscopic methods (IR, UV, and NMR). For the molecular docking study, 1G2A, which is a protein of E. coli and chloramphenicol standard target, was selected. Binding energies of −9.09, −7.05, and −6.87 kcal/mol were calculated for β-amyrin palmitate, lutein, and chloramphenicol, respectively. The drug-likeness property result indicated that both β-amyrin palmitate and lutein violated two rules of Lipinski's rule of five with molecular weight (g/mol) > 500 and LogP > 4.15. In the near future, further phytochemical investigation and biological activity evaluation should be conducted on this plant.


Introduction
Over the past decade, traditional medicinal plants have been closely linked and played important roles in the treatment of human illnesses in the world [1]. According to World Health Organization (WHO) estimation, more than 80% of the total world population in developing countries use mostly plant parts as traditional medicine for their primary health care [2]. Still now, medicinal plants have been receiving significant attention [3] since they possess antimicrobial, antioxidant, antimalarial, anti-infammatory, antiemetic, antidiabetic, antifertility, antiasthmatic, antistress, and anticancer activities [4]. It is well-known that the medicinal values of these plants are due to some bioactive compounds that produce a defnite physiological action on the human body since plants synthesize extremely diverse chemical compounds which have great potential for the development of new pharmaceuticals [5].
Even though nearly 7000 higher plant species and 1000 medicinally valued plants are expected to be found in the fora of Ethiopia [6,7], the scientifc research works done are more on ethnobotanical studies which remained in listing and surveying of many plant species. Te research works done on the phytochemical constituents and biological activities of Ethiopian medicinal plants were minimal and limited to a few plant species. It is therefore very apparent to conduct an extensive scientifc investigation on the phytochemical and antimicrobial activity of untouched medicinal plants of Ethiopia to support their traditional therapeutic uses, which may also lead to a search for some new bioactive drugs. Tere is a need for the screening of more efective, afordable, and readily available antimicrobial substances from local medicinal plants or herbs as pathogenic bacteria are developing resistance to common antibiotics [8].
M. zavattarii (locally known as muka bira in Afan Oromo) is a shrub plant, which belongs to the family Sterculiaceae. It is an endemic plant species to Ethiopia, which is found mainly in the eastern and southern parts of the country. M. zavattarii exhibits simple, hairy, margin serrate leaf, and axillary and solitary fowers. Te leaf of the plant is used to treat ailments related to kidney infection in the eastern part of Ethiopia [9]. To the best of our knowledge, no research work has been reported so far on the chemical constituents and antimicrobial activities of this endemic plant species. Hence, the present study is intended to screen major phytochemical classes, investigate antibacterial activities of the crude extracts, and isolate compounds from the chloroform leaf extract of M. zavattarii. Furthermore, the structures of the isolated compounds were identifed based on spectroscopic analysis using UV-VIS, FT-IR, and NMR and comparison with values reported in the literature. Finally, the molecular binding capacity of the compounds was studied by docking against IG2A protein model.

Plant Materials.
Te leaf of M. zavattarii Cufod (Figure 1) was collected from Harla town and its surrounding areas in December 2021. Harla is found in Dire Dawa administrative city, 515 km far from the capital Addis Ababa, Ethiopia. Identifcation of the plant was previously reported with voucher number AHU127 at the herbarium of Haramaya University, Ethiopia [9]. Te collected leaf was washed and dried under shade at room temperature and pulverized into a fne powder using an electric grinder (Shanghai Jinkle, Scientifc Instrument Co., Ltd., China).

Instrumentation.
Stuart SMP3 melting point apparatus determined the melting point. UV-Vis spectra were recorded on Genesys 2PC UV-Vis scanning spectrometer (200-800 nm) in chloroform. Te IR spectral data were recorded with KBr pellets on Perkin Elmer Bx infrared spectrometer in the range of 400-4000 cm − 1 . Optical rotations were measured on ADP 220 polarimeter at 25°in CHCl 3 . All the NMR analyses were performed on Bruker Advance 400 spectrometer (in CDCl 3 ) at 400.13 MHz for the 1 H and 100.6 MHz for the 13 C and dept-135 spectra. Te residual proton signal of the solvent was used as a reference, and the chemical shift was expressed by δ (ppm).

Preparation of Crude Extracts and their Percentage Yields.
To investigate the efect of solvents on the extraction yields, powder leaf of M. zavattarii (80 g) was extracted separately with n-hexane, chloroform, ethyl acetate, and methanol (600 mL, each) by shaking on an orbital shaker at room temperature for 48 hrs. Te extracts were then fltered using Whatman's No.1 flter paper (Whatman International Ltd., England), and the fltrates were concentrated using a rotary evaporator under reduced temperature and pressure. Te corresponding percentage extract yield (%) was calculated using the following formula [10]: Weight of crude extract Weight of powdered sample × 100. (1)

Phytochemical Screening Test.
Te n-hexane, chloroform, ethyl acetate, and methanol leaf extracts were subjected to various phytochemical screening tests following the standard procedures [11,12] with some modifcations to detect the presence or absence of major phytochemical classes, namely, favonoids, alkaloids, phytosterols, tannins, terpenoids, saponins, free anthraquinone phlobatannin, quinines, and coumarin. Te screening analysis was monitored through the observation of the color change and a precipitate formation after the addition of specifc reagents.
2.6. Antibacterial Activity Test. Tree bacterial species namely, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were obtained from Ethiopian Biodiversity Institute based on their availability and relatedness to ethnomedicinal use of the plants. Antimicrobial assay of the extracts was conducted following the standard Mueller-Hinton agar disc difusion method [8,13,14] for the obtained bacterial pathogen. A stock solution of each crude extract (50, 75, and 125 mg/ml) was prepared in DMSO and paper discs of 6 mm diameter were impregnated with 0.01 ml of the extracts. Commercial chloramphenicol antibiotics discs (5 μg) were used as positive control, and the pure solvent (DMSO) impregnated discs were used as negative control. Te diameters of the zone of inhibition around each disc was measured to the nearest millimeter along two axes (i.e., 90°to each other) by using a transparent ruler, and the means of the two readings were recorded.

Fractionation and Isolation of Compounds.
Te powder (300 g) of M. zavattarii leaf was defatted with n-hexane (2.5 L, 3x) for 48 hrs on an orbital shaker. Te remaining residue, after n-hexane extraction, was then extracted successively with chloroform and methanol solvents for 48 hrs. Te fltered and concentrated solvents by using rotary evaporator under reduced pressure aforded 1.92 g, 8.25 g, and 9.48 g of crude extracts of n-hexane, chloroform, and methanol extracts, respectively. Ten after, all extracts were subjected to the TLC analysis using diferent ratios of nhexane/EtOAc and DCM/EtOAc solvent system and visualizing techniques UV-lamp (254 and 365 nm) and iodine vapor. Based on the phytochemical screening and TLC profle results, the chloroform extract was subjected to silica gel column chromatography (CC) as follows. Part of the chloroform extract (5 g) was dissolved in chloroform, adsorbed on silica gel (35 g), and concentrated and applied on CC silica gel. Te elution was carried out using the eluents n-hexane/EtOAc and DMC/EtOAc with diferent polarity ratios, which resulted in the collection of 90 fractions (15-25 mL). Collected fractions were subjected to TLC examination using diferent ratios of hexane/EtOAc and DMC/EtOAc solvent systems and UV-lamp and iodine vapor as detecting methods. Fractions with similar R f values and TLC profles were combined and resubjected to the TLC analysis. Among them, fraction 9-23 (eluted with 85 : 15 of n-hexane/EtOAc) and fraction 81-88 (eluted with 70 : 30 of DCM/EtOAc) showed a relatively better purity on TLC and fractionated further over silica gel CC as follows.
Fraction 9-23 (1.2 g) was dissolved in chloroform, adsorbed in silica gel (7 g), and concentrated and purifed over silica gel CC which led to the collection of 27 subfractions with the eluent n-hexane/EtOAc of diferent ratios. After TLC examination, subfraction 21-23 (eluted with 9 : 1 of n-hexane/EtOAc) presented a single spot with R f value 0.5 using 19 : 1 n-hexane/EtOAc (19 : 1) as the solvent system and UV-lamp (254 and 365 nm) and iodine vapor which aforded yellow amorphous compound 1 (26 mg). Similarly, fraction 81-88(0.10 g) was further chromatographed over silica gel CC with diferent ratios of DCM/EtOAc resulted in the collection of 33 subfractions. Of which, subfraction 25-27 (eluted with 80 : 20 of DCM/EtOAc) exhibited a single spot (R f value of 0.6) on TLC developed with DCM/EtOAc (4 : 1) and visualized under UV-lamp and iodine vapor which furnished a brick-red crystalline solid compound 2 (23 mg). Te structures of the isolated compounds 1 and 2 were established based on the spectroscopic analysis using UV-Vis, IR, 13 C, and 1 HNMR and comparison with values in the literature.

Molecular Docking Analysis.
Te aim of the docking study was to analyze the binding interaction of the isolated compounds with one of the chloramphenicol's targets, peptide deformylase (PDB ID: 1G2A) [15]. AutoDock 4.2 program [16] was used for the docking study of the compounds 1 and 2 with the peptide deformylase enzyme (PDF) (PDB ID: 1G2A). Water molecules and ligands were deleted from the receptor followed by the addition of polar hydrogens and Kollman partial charges using Autodock tools. A grid box with spacing 0.375Å, size of 60 × 60 × 60 along the X, Y, and Z-axes and Lamarckian genetic algorithm was used. LigPlot [17] was used to elucidate the two-dimensional projection of the ligand-target interactions. Ground state geometry of the ligands, compounds 1 and 2 were optimized using Gaussian 09 program package [18]. Swiss ADME, PreADMET, and ProTox-II property explorer software, also predicted the drug-likeness, ADME and toxicity characteristics of the isolated compounds 1 and 2.

Percentage Extraction Yield.
In the present study, the percentage extraction yield of n-hexane, chloroform, ethyl acetate, and methanol solvents extract of M. zavattarii leaf was determined. Methanol extract recorded the highest percentage extract yield (8.42%) followed by chloroform extract (3.24%) and ethyl acetate extract (1.59%), whereas the least extract yield was obtained from n-hexane (0.95%). Te result indicated that the extraction efciency favors the highly polar solvents.

Phytochemical Composition of Leaf
Extracts. Te presence or absence of favonoids, alkaloids, tannins, terpenoids, saponins, free anthraquinone, phytosterols, quinines, phlobatannin, and coumarin was screened for leaf extracts of M. zavattarii. Te extracts showed the presence and absence of certain phytochemical classes, as summarized in Table 1.

Journal of Tropical Medicine
As shown in Table 1, favonoids, terpenoids, saponins, phytosterols, and coumarin were detected in both chloroform and methanol extracts, whereas alkaloids, free anthraquinones, and quinines were not detected in the extracts. Only alkaloid and phytosterol, and terpenoid and phytosterol were detected in ethyl acetate and n-hexane extracts, respectively. Te phytosterols were moderately detected in all extracts using the Lieberman Bouchard test, whereas the free anthraquinone and quinine were screened negatively in all extracts using the Borntrager's and ammonium hydroxide tests. Te chloroform and methanol extracts, respectively, confrmed a moderate and strong presence of favonoids and saponins. Terpenoids were strongly detected both in the n-hexane and in chloroform extracts, whereas tannins were strongly screened only in the methanol extract.

Antibacterial Activity of Leaf Extracts.
Te disc difusion method was used to determine antibacterial activity of nhexane, chloroform, and methanol crude extracts against E. coli, S. aureus, and P. aeruginosa, and the results are presented in Table 2.
As can be seen from Table 2, P. aeruginosa was found resistant to all types of extracts at all concentrations. Compared to the standard chloramphenicol types of extracts at all concentrations, P. aeruginosa showed lower antibacterial activity. Chloroform extract exhibited higher mean diameter zone inhibition (12.08, 14.00, and 15.58 mm) than hexane (7.67, 11.08, and 14.75 mm) and methanol (7.00, 8.75 and 11.00 mm) against E. coli at concentration of 50, 75, and 125 mg/mL, respectively. On the contrary, n-hexane extract exerted the highest activity (10.33, 12.92, and 14.08 mm of the mean zone of inhibition) against S. aureus than chloroform extract with diameter zone inhibition (7.00, 7.17, and 10.60 mm) and methanol extract (7.25, 11.08, and 16.42 mm), respectively, against S. aureus. Te antibacterial activity of the leaf extracts of M. zavatarii in the present study might be attributed to the phytochemical constituents of the extracts (Table 1). Only terpenoids and phytosterols were detected in n-hexane extracts, but it showed comparable antibacterial activity to chloroform and methanol extracts that had more phytochemical constituents. Te diferences in the antimicrobial activities of the various extracts might be due to various solvents having diferent solubility for diferent phytoconstituents [19]. Te quantity of terpenoids might be the reason of this fnding. In line to this, the composition and quantity of alkaloids, saponins, tannins, and terpenoids were associated with antibacterial activities against Salmonella typhi, Shigella boydii, S. aureus, and Enterococcus faecalis [8].
Furthermore, ten methylene carbon signals at δ C 38. were identifed in the 13 C and DEPT-135 spectra which ftted to the olean-12-en pentacyclic triterpene. All the NMR assignments of compound 1 and values in the literature for a similar structure are presented in Table 3. Accordingly, the obtained NMR data of compound 1 were found closely matched with the values reported in the literature for β-amyrin palmitate [21,22]. Based on this, the structure of compound 1 was established as β-amyrin palmitate ( Figure 2).

Journal of Tropical Medicine
Accordingly, the structure of compound 2 was as lutein (Figure 3).
Lutein (tetraterpenoid class of natural carotenoid) was reported to possess anti-infammatory and antioxidant [29], cytotoxicity [27] antitumorigenic, antiangiogenic, photoprotective, hepatoprotective, neuroprotective, and acroleininduced ototoxicity preventing properties [30,31]. Even though β-amyrin palmitate and Lutein have been isolated from diferent plant parts, this is the frst time a report of isolation of the two compounds from Melhania zavattarii is being made. Te reviewed evidence indicated the plant might exhibit a broad range of pharmacological activities, such as antidepressants, antidiabetes, antioxidants, antiinfammatory, and cytotoxicity.

Molecular Docking Study.
As presented in the previous section (Table 2), the crude chloroform extract of M. zavattari showed the best activity against E. coli, which is close to half the activity of the standard chloramphenicol (inhibition zone 14 mm vs. 26.78 mm). In the present study, the docking study aimed to fnd evidence whether the isolated compounds contribute to the observed antibacterial activity of the crude extracts against E. coli, or not.   It is interesting to note that the standard shows more antibacterial activity despite smaller binding energy relative to the two extracts. Tis might be due to the presence of specifc interactions, which are critical for the antibacterial activity. Further study is required for elucidated specifc interaction present between standard and target so that the observed diference in antibacterial activity will be accounted. From the two-dimensional projections of the interactions, it is visible that the standard shows far greater number of specifc hydrogen bond interaction with the protein residues of the target than the two compounds. A similar situation is encountered in the work of [32] where good antitubercular activity of ligands are observed as a result of specifc interactions with important protein residues despite smaller binding energies.

Pharmacokinetic Properties Study of Isolated Compounds 1 and MB 2.
Te results of the pharmacokinetic properties studies are presented in Tables 6-8 below. Te drug-likeness property results (Table 6) indicated that both the isolated compounds 1 and 2 violated two rules of Lipinski's rule of fve with molecular weight (g/mol) > 500 and LogP > 4.15. Te ADME prediction result (Table 7) revealed that both the isolated compounds, unlike to chloramphenicol, showed a low degree of gastrointestinal absorption and interacted with the P-glycoprotein substrate, whereas similar to the chloramphenicol, both compounds were found as nonpermeable to the blood brain barrier and noninhibitors of all the predicted cytochrome-P enzymes. Tese two isolated compounds were found within the toxicity classes of 4 and 2 with LD 50 (mg/kg) values of 339 and 10, respectively; and both exhibited an immune toxicity property (Table 8).

Conclusion
Te results of the present investigation showed that the highest yield of leaf extracts was achieved by methanol followed by chloroform solvent used for maceration extraction from Melhania zavattarii leaf, which could be preferred for further investigation on additional bioactive phytochemical constituents. Furthermore, methanol and chloroform extracts were enriched with phytosterols, terpenoids, favonoids, saponins, tannins, and coumarin as active phytochemical constituents. It seems that the presence of diverse phytochemicals attributed to the antibacterial activity of methanol and chloroform extracts against E. coli and S aureus. However, n-hexane with only terpenoids and phytosterols major class did show comparable antibacterial activity against E. coli and S aureus. Tus, further investigations on the antibacterial activity of terpenoids and phytosterol separately or in combination are needed. Interestingly, the phytochemical investigation conducted on the chloroform leaf extract of M. zavattarii led to the isolation of two compounds namely, β-amyrin palmitate (compounds 1) and lutein (compounds 2) which are categorized under terpenoids, reported herein for the frst time from this plant. Both β-amyrin palmitate and lutein showed stronger interaction with the 1G2A protein of E. coli than the chloramphenicol's interaction with the 1G2A protein target,