Collection of plant
Bersama abyssinica plant materials were collected in Dry season of 2021 at Isongole area, at riverline forest patch with Rauvolffia caffra near pine plantation in Rungwe district, Mbeya region (9o34’60”.9 S 33o62’84 E).The collection of leaves and stem bark was done by Dr. Never Zekeya with the assistance of of an experienced Curator, Mr. Frank .M Mbago with collection No. FMM 4052 from University of Dar es salaam. The plant was identified by Mr. Frank Mbago, and the voucher specimen number ND. Zekeya Nos.01 was deposited in the Herbarium (DSM) of Department of Botany at University of Dar es salaam. After collection, leaves and stem bark were dried under shade in room temperature and then pulverized to obtain small pieces before soaking into different solvents. All methods were performed in accordance with relevant guidelines and regulations, and the plant is recently considered as not of conservation concern.
Study site
Extraction and phytochemical analysis bioassay was conducted at Institute of Traditional Medicine at Muhimbili Institute of Health and Allied Sciences and The Governmnt Chemist Laboratory Authority. Active compounds with high phenolic compound and antioxidant activity were shipped to an advanced lab in Basel University for for antiviral assays against SARS-CoV-2 and prolifilation test.
Chemicals, Standard strains and Culture Media
Extraction Chemicals, Standard strains and Culture Media
Methanol (absolute) was bought from Fluka Chemie GmbH (Sigma-Aldrich®, Zwijndrecht, Netherlands) and Dimethyl sulfoxide (DMSO) was purchased from RFCL Limited, Hayana, India. Dichololomethane, ethyl acetate and Ethanol will be purchased from Loba Chemie Pvt Ltd, Mumbai, India). Coronavirus SARS-CoV-2 –Delta B1 isolate was obtained from Basel University.
Preparation of Plant Materials and Extraction
Leaves and stem bark of B. abyssinica collected from Isongole of Rungwe district in Mbeya, Tanzania were selected based on the current use of herbal medicine named Coviba Dawa that is made from two parts and for conservation purpose, roots were not collected for this study. Leaves and stem bark were separately air dried under shade and then pulverized into fine particles by using electric blender (WESTPOINT M012). Then 100 g of leaves and 100g of stem bark were separately sequentially extracted in 1000ml of petroleum ether, dichloromethane, ethyl acetate and ethanol for 48h twice for each solvent. The respective extracts were filtered through muslin cloth on a plug of glass wool in a glass column and solvents were evaporated in vacuum using a rotary evaporator. Water extacts was obtained by decoction of 100g of stem bark into 1L of water by boiling at 1000C for 10 minutes and infusion of 50g of leaves in stembark decoction. Thereafter, the concoction was filted using muslin cloth followed by lyophilization to obtain dry extract. All extract were stored in the refridgerator at 40C before further use.
Determination of bioactive metabolites
Determination of phenol
Two (2) ml of Iron III chloride solution were added to the 2ml of 100mg/ml of each extracts and fraction, the appearance of deep bluish-green solution indicated the presence of phenolic compounds.
Determination of flavonoid
The presence of flavonoid was determined by addition of 5ml of dilute ammonia solution into 2ml of 100mg/ml of extracts followed by addition of few drops of concentrated Sulphuric acid. Thereafter, a yellow coloration indicated the positive result for the presence of flavonoid compounds.
Test for tannin
Test for tannin was done by boiling 100mg of each extracts/fraction in 2ml of water in a test tube and then filter, this was followed by addition of few drops of 0.1% Ferric chloride solution. A brownish green, blue black coloration indicated the present of tannin compounds.
Determination of saponin
100mg of each extracts/fraction was dissolved into 2ml of distilled water in the test tube and warmed, this was followed by vigorously shaken. The formation of froth for at least a minute indicated the present of Saponin compounds.
Determination for antioxidant
100mg of each sample was dissolved in the 1ml of extractor solvents, filter and divide equally into two different test tubes. This was followed by the addition of 0.5ml of pre prepared from 0.1mM DPPH in one of the test tube and the second test tube as control and the mixture was shaken and allowed to stand for 1 minute. The formation of discoloration in comparison to control indicated the present of antioxidant compounds in the extract.
LC-MS/MS analysis of water extract of bark stem bark and leaves
The selection for this analysis was based on phytochemica screening and the most polar extact with potential pharmacological activity was selected for this aanlysis.Analysis of polar extracts was done by LC-MS/MS (Q-orbitrap-Ultra High Performance Thermofisher Company) at the Government Chemist Laboratory Authority. The extract was re-dried by using Rotavap under reducing pressure with flowing of Nitrogen gas 15psi at 45℃ then the Liquid Chromatography was eluted by mobile phases of ((A)0.1% formic acid in water followed by 0.1% Formic acid in Acetonitrile). The column conditions were 35℃ and 1.9µ of oven temperature and particle size respectively. The coupled MS was scanned in range of 150 – 2000m/z with resolution 140000, AGC Target1e6. The maximum IT setting was 200ms with ionization mode (HESI) collision Energy 45v.
Determination of antiviral and cytotocity activity
All antiviral screenining and cytotoxicity experiments were conducted at Basel University Laboratory according to the previously established Standard operating procedures (SOPs). All infections with live SARS-CoV-2 were strictly performed in the BSL-3 facility of the institute in accordance with the official authorization for work with SARS-CoV-2 by the Federal Government of Switzerland (BAG), permit #A202850/3.
Detemination of antiviral activity
Compounds were pre-diluted in a deep-well plate according to the dilution scheme (top graph below) in a way that afterwards the addition of a volume of 50µL will provide the final test concentration on the cells. Remdesivir (RDV) was included as established and validated activity control. After compound addition, cultures were transferred to the BSL-3 facility. After about 30 minutes of preincubation of cells and compound, 100 pfu of the DELTA strain (BS-01) of SARS-CoV-2 virus were added to each culture well.Subsequently, after an adsorption period of 15-30 minutes, every well was overlayed with low-melting agarose according to the corresponding SOP.Cultures were incubated at 37°C as described below to allow virus-induced plaques to form.After 48 h of incubation at 34°C. As cytopathic changes (CPE) develop within hours, the optimal time of harvest was determined by microscopic inspection, before paraformaldehyde (PFA) was added as fixative. Quantitative plaque formation served as proof for viral replication in the infectivity range (number of plaques = ca. 100 / well). The inhibitory potency was judged as plaque reduction at a given compound concentration. The IC50 for the RDV was at ca. 2.5µM, correlating with the reported activity.The plate on the right is a replicate plate with the same compound concentrations but with no virus added. The plate on the left (Fig. 1) displays the fixed and stained culture plate. Viral plaques were visualized as small white spots. Compound dilutions were from top to bottom, and red lines indicate the respective compound concentration of the 50%-inhibition of plaque formation (IC50).
Detemination of cytotoxicity
All compounds were pre-diluted in DMSO (research grade) to obtain stock concentrations of 20mg/mL. Further dilutions were done in culture medium (DMEM/2%FBS) until the final compound concentration as indicated.Cells were pre-seeded on day -1 as detailed in Fig.1 to allow adherence to the culture plate.
Compound dilution and dispensing was as described in Fig. 1 to obtain serial dilutions of each compound. This was to cover the entire anticipated biological activity range. DMSO concentrations on the cells were always below 0.5% final concentration to ensure full cell viability.A cell viability plate, using identical compound concentrations and cell count but without viral infection was included for each compound as control. The cytotoxicity was also assessed for the compound exposure for 48hrs.