Preliminary Phytochemical Screening, Isolation, Characterization, Structural Elucidation and Antibacterial Activities of Leaves Extracts Rhus Vulgaris (Kimmo)

Background : Rhus vulgaris commonly known as sumac, a plant that is known to possess different therapeutic values including antioxidant and antibacterial activities. Medicines from plants contributed largely to human health. The aim of this study was to screen the phytochemical constituents, isolate, elucidate the structure and antibacterial activity of methanol extract from the leaves of Rhus vulgaris . Methods: The methanolic extract of Rhus vulgaris was subjected to column chromatography and eluted with solvent mixture of methanol: chloroform ( 1:8) ratio. The eluted fractions were run in the TLC mobile phase with the different solvent ratio. Based on the TLC profile the fractions with similar Rf values were pooled together. The structure of the isolated compound was characterized based on the spectral data (IR, 1 H NMR, 13 C NMR, and DEPT) and extracts from Rhus vulgaris has been shown to have antibacterial activity were tested against four strains bacteria Streptococcus aureus( gram-positive ) and Escherichia coli, Salmonella typhimurium , and K. pneumoniae ( gram-negative) using Agar well diffusion method. Result: The results showed that the methanol extracts were active against all the tested bacteria. The structure of this compound 1-p-tolyl pentadeca-7,9-dien-1-ol was characterized by means of 1H NMR, 13C NMR, and IR spectral data. Conclusion: Therefore, it is concluded that the use of herbal plants and their recipes are the major source of drugs in a traditional medicinal system to cure different diseases.


Introduction
Plants have been used to treat a wide range of diseases throughout the history of human beings and this practice continues to date. This is mainly because most of these herbals are accessible, affordable and the extracted chemicals have little or no side effects as compared to drugs synthesized in the laboratory. Plants comprise the largest component of the diverse therapeutic elements of traditional health care practices both in humans and animals. The medicinal values of plants are due to the chemical substances that produce a definite physiological action on the human body and are called phytochemicals [1,2].
Natural products as the term imply 'naturally occurring compounds that are the end products of secondary metabolism and they are unique compounds for particular organisms or classes of organisms [3]. Natural products therefore continue to play a crucial role in drug development as they account for almost 50% of new chemical entities in drug discovery and hence providing a starting point for new synthetic drugs [4]. The value of medicinal plants in drug discovery is known to us well and human being have used them for various purposes from the beginning of human history. Traditional folk remedies from plants have always guided scientists to search for new medications in order to maintain and promote healthy life for humans and animals [5].
Sumac is the common name for a genus (Rhus) that contains over 250 individual species of flowering plants in the family Anacardiaceae [6], which occur mainly in the tropics, subtropics and temperate areas of the world. The sumac name is derived from "sumaga", meaning red in Syriac .In general, Rhus species can grow in non-agricultural regions and various species have been used by indigenous people for medicinal and other purposes, suggesting a potential for commercializing the bioactivity of these plants without competing for food production land uses [7].
Previous studies on phytochemical investigation of the stem bark of Rhus vulgaris revealed the presence of secondary metabolites such as tannins, saponins, flavonoids, terpenoids, glycosides, alkaloids and phenol. R. vulgaris methanolic extract (1000 mg/kg) showed greater antiinflammatory activity compared to indomethacin (10 mg/kg), the standard anti-inflammatory drug, with a decrease in inflammation for up to 90 min. The dichloromethane, ethyl acetate and aqueous extracts of R. vulgaris stem bark, root and leaves have exhibited moderate to toxic toxicity against brine shrimp with LC50 values ranging from 3.55 μg/ml to 734.06 μg/ml while cyclophosphamide, the positive control, demonstrated an LC50 value of 15.28 μg/ml [8].
Several reports describe isolations of new biflavonoids on the genus Rhus. Such as agathisflavone, amentoflavone, hinokiflavone, rhus flavanone and succedanea flavone has been sourced from Rhus species and evaluated for activity against a range of pathologically significant viruses. In another study, hinokiflavone was found as the most active among 65 natural flavonoids to inhibit the pro-coagulant activity of adherent human monocytes stimulated by endotoxin and interleukin-1-β in vitro. Other Rhus biflavonoids have also shown cytotoxic and antimalarial activities [10,11].
Further, the paper aims to isolate possible biological active compounds from the leaf of Rhus vulgaris by using various chromatography techniques such as column chromatography and preparative thin layer chromatography. The structure of the active component was elucidated by means of spectroscopic techniques (IR and NMR). Beyond this, the bioactivity of Rhus vulgaris leaves against different pathogens was checked.

Description
Rhus vulgaris is found in all parts of Tanzania; Uganda and Kenya and from Cameroon to Ethiopia and south to Mozambique, Malawi, Zambia and Zimbabwe [16]. It is a shrub or small tree that occasionally reaches 1-9 m; bark smooth, dark brown, branches yellow-red-brown, often densely hairy. Leaves are 3 leaflets, dull green, softly hairy, the central leaflet larger, 4.11 cm long x 2.6.5 cm wide, the two laterals smaller, shortly stalked, edge entire or soft toothed towards the tip, which is blunt or pointed, leaflets dark above, paler below. Flowers are small cream-green-yellow, parts in fives, in terminal loose heads or from upper leaf axils, 5.20 cm long, all densely hairy. Fruits are drupes, with thin flesh, flat and round, red-brown, only 3.5 mm across [17].

Taxonomic Classification
According to the international code of botanical nomenclature, the present taxonomic classification of Rhus vulgaris:

Reagents used
The chemicals and reagents that were used in the study include: Sulphuric acid, acetic acid Molisch's reagent, Millon's reagent, and lead acetate are more relevant to make this experiment.

Apparatus/Equipments
The apparatus and instruments that were used for the study include

General experimental procedures
The isolate was mixed with 200 mg KBr (FT-IR grade) and pressed into a pellet. The sample pellet was placed into the sample holder and FT-IR spectra were recorded in the range 400-4000 cm -1 in FT-IR spectroscopy (Bruker FT-IR Spectrometer, USA).
Nuclear Magnetic Resonance Spectroscopy: The 1 H, 13 C-NMR and 2D NMR spectra of base degradation impurities were recorded in DMSO-d6 solvent on Bruker 400 MHz Avance -III HD NMR spectrometer equipped with broadband observe (BBO) probe. The 1H and 13C chemical shifts are reported on the δ scale in ppm, relative to tetramethyl silane (TMS) as an internal standard. The spectra were set to δ 0.00 ppm in 1 H NMR (TMS) and δ 39.50 ppm in 13 C NMR (DMSO-d6).

Collection of plant:
The fresh and healthy leaves of R. vulgaris were collected from a local farm in the Region of Amara; Central Gondar Zone in Takusa

Preparation of extracts:
Extraction and isolation of organic compounds found in the leaves of Rhus vulgaris were done by using maceration method. The maceration method is more preferable for exhaustive extraction of R.vulgaris leaves to reduce any possibility of thermal decomposition of any thermolabile compounds that may be present [18].

Column Chromatographic
The methanol extract was subjected to silica gel (60-120 mesh ASTM, Merck) glass column chromatography (20-25 mm diameter). Briefly, silica gel (150 g) was mixed with chloroform to form a homogenous suspension/slurry and stirred using a glass-stirring rod to remove bubbles. The silica gel slurry was then poured into a glass column. The sample to load on the column was prepared by dissolving 6g of the extract in 40 ml of methanol. To the solution, 10 g of silica was added and mixed by stirring with a glass rod. The mixture was allowed to dry at room temperature.
The dried silica extract mixture was layered on the column layer bed. The column was first eluted with methanol: chloroform in a ratio of 1:8 as the mobile phase and allowed to run until it reached a consistent flow.

Preliminary Phytochemical Screening
Phytochemical screening was carried out to assess the qualitative chemical composition of crude extracts using commonly employed precipitation and coloration to identify the major natural chemical groups such as alkaloids, phenolic compounds, glycosides, carbohydrates, flavonoids, saponins, terpenoids, anthraquinones, tannins, steroids, amino acids, coumarins, and proteins.
General reactions in these analyses revealed the presence or absence of these compounds in the crude extracts tested. Crude extracts of the plants previously prepared and stored in a refrigerator were used for the phytochemical tests. [19][20][21][22][23].

Antibacterial Activities of the Leaves extracts of Rhus vulgaris
The test organisms that were used to check the antimicrobial activity of the crude extract were Antibacterial activities of R. vulgaris extracts were tested against four strains of bacteria using the agar well diffusion method. The test cultured bacteria were swabbed on the top of the pre-leveled media (about 45-50ml) and allowed to dry for 10 minutes. The sterilized well borer (6mm diameter) was used to bore holes on the plates and 100 mg/ml of each extract (n-hexane, chloroform and methanol) were dissolved by DMSO. Gentamicin disc (30 mcg/disc) will be used as positive antibiotic control. The Petri dishes were then incubated at 37℃ for 24 hrs. After incubation the zone of inhibition was measured and recorded the average inhibition zone in millimeters.

Yield of solvent extract and isolation of Leaves of Rhus vulgaris
The dried and powdered Roots (800 g) of Rhus vulgaris subjected to exhaustive extraction successively with n-hexane, chloroform and methanol. The solvent from each extract was recovered under reduced pressure using a rotary evaporator to obtain a n-hexane (4.35g), on the basis of spectroscopic evidence as described in the following section.

Phytochemical screening of the Leaves Extracts of Rhus vulgaris
The phytochemical analysis of each crude extract of Rhus vulgaris revealed the presence of pharmacologically useful classes of secondary metabolites. Alkaloids, Glycosides, Steroids, Anthraquinones, and Carbohydrates were present in all extracts. Phenols, Flavonoids, Tannins, Coumarins, and Proteins were present only in methanol extracts while Terpenoids, Saponins and Amino acid were absent in all the extracts were summarized in Table 3.

Antibacterial Activity of the Crude Extracts against Bacterial Strain
The antibacterial property of Rhus vulgaris extract using different solvents showed varying degree of response towards the selected pathogens ( Table 4). In this work three samples were tested by the above method. The results showed the crude methanol extract was strongly active while the pure n-hexane and chloroform extract was not active at all against the S.typhi and K.pneum pathogen.

Characterization of Compounds
In order to characterize the compounds isolated from the leaves of Rhus vulgaris the RF value, and spectroscopic data of the compounds were utilized.

Partial Characterization of AA1
Compound AA1 was dark green gummy material isolated from the leaves of Rhus vulgaris. It was detected as a pink spot under a UV lamp and became yellow when sprayed with 4% vanillin H2SO4. Its Rf value was 0.70 in Methanol/ Chloroform (1:8).

H-NMR Spectrum
The 1 H-NMR spectrum in Fig.6 shows the peak at δ 1.25-2.07(m) correspond to the protons of the nine methylene groups, which appeared as multiplet due to overlap of signals, the peaks from δ 0.84(t) and δ 2.35(d) indicates the signals of the protons of the methyl groups. The peak at δ 4.50-6.03(t) indicates the protons of the five methine groups and δ 6.99-7.07(d) indicate the protons of the four methine groups.

C-NMR Spectrum
The proton decoupled 13 C-NMR spectrum in Fig.7 showed signals of 17 carbon atoms. But in the structure (AA1) proposed there are 22 carbon atoms. The difference in the number of carbon atoms between the proposed structure and the signals obtained from the 13 C NMR spectrum might be due to the presence of chemically/magnetically equivalent carbon atoms. As indicated in Fig. 9, carbon atoms 4,5& 12, 6 & 11, 8,9,10 &3' 1'&4'and 2'&6' might be chemically equivalent.
The (DEPT) spectrum in Fig.8 showed signals for 11 carbon atoms. Out of these 4 signals indicate the presence of 9 methylene groups and the rest 7 signals for CH and CH3 groups. In DEPT spectrum data are collected in such a way that the resulting signal is either up field (CH & CH3) or downfield (CH2) depending on the number of protons attached. In the proton decoupled 13 C NMR spectrum shown in Fig.7 there are signals for 22 carbon atoms, while in the DEPT spectrum overlap of signals were observed for 20 carbon atoms.
The difference in signals between the two spectra indicated the presence of 2 quaternary carbon atoms that are not normally observed in the DEPT spectrum. The 13C and DEPT chemical shifts of the proposed structure are summarized in Table 5 below.

Conclusion
Qualitative phytochemical screening tests were done on the crude extracts of the leaves of R. vulgaris, and the results showed the presence of Alkaloids, glycosides, steroids, anthraquinones, and carbohydrates. The analyses of the results from bioactivity tests confirm the presence of active compounds which were extracted by methanol that has a high inhibition zone.
AA -Study design, Literature search, data collection, data analysis, data interpretation, writing manuscript, GG -Research supervision, Study design, editing manuscript, DS -Research supervision, editing manuscript. All authors read and approved the final manuscript.

Funding
The funding for this study was availed by University of Gondar, during the financial year BU 2019/2021. The funds for the facilitation of the collection of plant materials and the provision of all the consumables utilized during this study.