Bioguided chemical study of Boswellia dalzielii Hutch. (Burseraceae) for antibacterial agents and a new glucopyranoxylmethoxybenzyle

Abstract Stem barks of Boswellia dalzielii are used traditionally for the treatment of various bacterial infections. A bioassay guided fractionation of the MeOH-CH2Cl2 (1/1, v/v) stem barks extract led to the isolation of fourteen compounds 1–14, identified based on spectroscopic data. Dalzienoside (1) is reported here for the first time. The broth microdilution method was used to evaluate the antibacterial activity of the crude extract, fractions and compounds against six bacterial strains. The crude extract exhibited moderate antibacterial activity with MIC of 250 μL/ml; two fractions showed significant activities with MICs ranging from 7.8 to 125 μg/ml, while α-boswellic acid (2), β-boswellic acid (3), acetyl-11-keto-β-boswellic acid (4) from these fractions exhibited strong activities with MIC value of 3.125 µg/mL against Staphylococcus aureus, Salmonella typhi, Enterobacter cloacae, Streptococcus pneumonia and Pseudomonia aeruginosa. This study gives insight into the antibacterial constituents of the stem bark of B. dalzielii and justifies its use in ethnomedicine. Graphical Abstract


Introduction
Microbial diseases are a serious public health problem in Africa.Although a significant number of synthesised drugs exist around the world for the treatment of microbial diseases in modern medicine, in most African countries, poverty has encouraged many communities to focus on herbal medicine (Timo et al. 2013;Gera et al. 2015).The pharmacological and phytochemical studies of plants used in traditional medicine could lead not only to the discovery of new antimicrobial compounds, but also to the valorisation of local plant species through the investigation on their efficacy and safety.Boswellia dalzielii Hutch is a common deciduous tree in the wooded savanna growing up to 12 m high; it is mostly called the 'Frankincense tree' (Younoussa et al. 2014).Its stem bark is pale brown and smooth; the leaves are glabrous green and approximately 20-45cm long (Burkill 1985).This species ranges from the Northern Ivory Coast to Nigeria and into Cameroun and Ubangi-Shari (Eslamieh 2010), and is widely used in Africa as medicinal plant.In Nigeria the gum resin is used with other medicines for the treatment of venereal diseases; the bark is boiled to wash for fever, rheumatism; the roots decoction boiled along with Hibiscus sabdariffa is used for the treatment of syphilis (Nwinyi et al. 2004).Decoction of the aerial parts is taken orally for tuberculosis; decoction of roots bark is applied to treat scabies (Kubmarawa et al. 2013).In Niger the leaves are used in the treatment of bilharziasis and given as an oxytocic to pregnant women (Kafuti et al. 2017).In Cameroon, people use the leaves of B. dalzielii to protect maize, millet and sorghum against weevils' attack (Younoussa et al. 2014).The stem bark is used in folk medicine in Cameroun to treat gonorrhoea, syphilis and diarrhoea.However, in spite of its numerous applications in traditional medical practice in the sub Saharan region, the Cameroun specie is less known and studied compared to its more popular congeners.Previous phytochemical investigations of the genus Boswellia have led to the isolation and characterisation of secondary metabolites belonging mainly to the class of triterpenes (Belsner et al. 2003;Verhoff et al. 2014;Manguro and Wagai 2016;Wang et al. 2016), steroids and their glycosides (Shenvi et al. 2014;Ahmed et al. 2015;Sarada and Geetha 2016), diterpenes (Alemika et al. 2004;Jinqian et al. 2018).There are several in vitro, animal and clinical studies which substantiate the antiarthritic, antiinflammatory, antiulcer, antibacterial, and antidiarrheal activities as well as hepato-protective and analgesic effects of extracts obtained from various species of Boswellia (Al-Harrasi et al. 2019).A mixture of aand b-boswellic acids isolated from Boswellia rivae was found to exhibit activity against Bacillus subtilis with a minimum inhibitory concentration of 7.8 lg/mL and also impressive activities (MIC values of 15.7 lg/ml) against Staphylococcus albus and Staphylococcus aureus (Manguro and Wagai 2016).The antimicrobial activities of boswellic acid molecules against 112 pathogenic bacterial isolates including ATCC strains were also evaluated and Acetyl-11-keto-b-boswellic acid was found to be the most active compound showing MIC range of 2-8 lg/ml against the entire gram positive bacterial pathogens tested (Raja et al. 2011).In this study, a bioassay-guided approach was undertaken to further identify constituents which may contribute to the antimicrobial activity of Boswellia dalzielii stem bark.
In order to contribute to the chemotaxonomy knowledge of this plant, non-active fractions (Hexane-EtOAc 8/2 (14.6 g), EtOAc (13 g) and EtOAc-MeOH 8/2 (72 g) were also subjected to chromatographic separations to give 4-methoxy-1-(methoxymethyl)-2-O-a-D-Glucopyranosyl benzene (dalzienoside) (1), Friedelin (6), Lupeol ( 7), b-Sitosterol (8), Stigmasterol (9) (Mahato and Kundu 1994), Angolensin (10) (Harbone 1988)  Dalzienoside (1) was isolated from the EtOAc/MeOH (8/2, v/v) fraction as brown oil soluble in acetone.Its high resolution mass spectra (HR-ESI, Supplementary material Figure S1) shows the pseudo molecular ion [M þ Na] þ at m/z 353.1203 for (C 15 H 22 O 8 Na), with five degrees of unsaturation.The 1 H NMR spectrum (Supplementary material Figure S2) shows the presence of one trisubstituted benzene ring with an ABX system at ẟ H 7.23 (1H, d, J ¼ 8.4 Hz), 6.84 (1H, d, J ¼ 2.4 Hz) and 6.60 (1H, dd, J ¼ 8.4 and 2.4 Hz).Two methoxy signals at d H 3.30 (3H, s), 3.79 (3H, s), two protons of a hydroxymethylene group at 4.60 (d, J ¼ 11.8 Hz, 1H) and 4.35 (d, J ¼ 11.8 Hz, 1H), one proton at ẟ H 4.86 (1H, d, J ¼ 2.3) attributed to the anomeric proton of a sugar unit.The analysis of the coupling pattern and chemical shifts (two protons of a hydroxymethylene group at 3.90 m and 3.71) allowed us to suggest the presence of an a-D-glucopyranoside moiety in the molecule (Gonz alez-Cortazar et al. 2019).The analysis of the 13 C decoupled NMR (Supplementary material Figure S3) and DEPT (Supplementary material Figure S4) spectra revealed the presence of two methyls, two methylenes, eight methines and three quaternary carbons.The four methine signals between d C 71.4 and 78.0 were attributed to the tertiary oxygenated carbon of the glucose unit, and the signal at d C 103.6 to its anomeric carbon, whereas the signal at d C 70.0 was attributed to a benzylic methyleneoxy carbon.The positioning of the established groups on the benzene ring was done by using the HMBC spectrum (Supplementary material Table S2, Figures S5 and S7).Based on this evidence compound 1 was concluded to be methoxy [2-O-a-D-glucopyranoxyl-4-methoxybenzyle] namely dalzienoside.The spectroscopic data of 1 were similar to 12 (Gonz alez-Cortazar et al. 2019), the main difference being the additional methoxy group bearing by the benzylic carbon in the structure of 1, instead of a hydroxyl group in 12.The chemical shifts of all carbons and protons of 1 (Supplementary material Table S2) were attributed using 1 H, 13 C, DEPT, HMBC, COSY (Supplementary material Figure S6), and HSQC (Supplementary material Figure S8).

General experimental procedures
The different masses of extracts, fractions and pure compounds were measured using the electronic scales Cobos model D-6000-SX and Ohaus pioneer.High resolution mass spectra were obtained with a QTOF Compact Spectrometer (Bruker, Germany) equipped with a HRESI and a HRAPCI sources. 1 D NMR spectra ( 1 H NMR, 500 MHz, 300 MHz; 13 C NMR and DEPT 135; 125 MHz, 75 MHz) and 2 D NMR spectra (HMQC, HSQC, HMBC, COSY and NOESY) were measured on Bruker TopSpin 3.0 spectrometers equipped with cryoprobe, with TMS as an internal reference.Chemical shifts are reported in d (ppm) using TMS as internal standard, and coupling constants (J) are measured in Hz.Column chromatography was carried out using silica gel 60 (230-400 mesh ASTM, Merck) and Sephadex LH-20 (St. Louis, MO, USA).Thin layer chromatography (TLC) was performed on Merck precoated silica gel 60 F254 aluminium foil, and spots were detected using UV light (254 nm) and (365 nm), and spraying with 10% H 2 SO 4 in EtOH followed by heating at about 110 C.

Plant material
The stem barks of Boswellia dalzielii were collected at Moutourma in the far North Region of Cameroon.The plant was identified by Doctor Souar e Konssala, a Botanist at the University of Maroua and then confirmed at the Cameroon National Herbarium in Yaound e where a voucher specimen N 64939/HNC is kept.

Antibacterial activity
Antibacterial activities assays were conducted on a total of six bacterial strains, two from American type culture collection, Escherichia coli ATCC25322 and Streptococcus pneumoniae ATCC491619, one from BEI resources namely Pseudomonia aeruginosa HM801 and finally three clinical isolate strains from Laboratory collection namely Salmonela typhi (CPC and CHU), Enterobacter cloacae (CPC) and Staphylococcus aureus (CPC).The bacterial strains were maintained on agar slant at 4 C and subcultured on a fresh appropriate agar plates 24 h prior to any antibacterial test.The minimal inhibitory concentration (MIC) of extracts, fractions and compounds were assessed using the broth microdilution method as described by Eloff (1998), with slight modifications.The tests were performed in duplicates.Each test sample was dissolved in dimethylsulphoxide (DMSO) to give a stock solution.In all wells of a 96 wells microplate, 100 lL of sterile culture broth (MHB) were introduced.Then, 100 lL of each mother solution (2000 lg/mL) was added to the first well, and then distributed to all other wells according to a geometric progression of reason 2, with final concentrations varying from 3.8 to 500 lg/mL and from 0.15 to 500 lg/mL for ciprofloxacin (reference antibiotic) used as positive control.Then, 100 lL of the liquid culture medium (MHB) inoculated with the test germ (2 Â 10 6 CFU/mL) were introduced into the wells to obtain a final concentration of 10 6 CFU/mL.The negative controls consist of wells containing only the culture medium on the one hand, and wells containing a mixture of culture broth and germ on the other hand.The micro culture plates were covered and incubated at 37 C for 18 hours.20 lL of Resazurin were then introduced into all wells and the micro-plates were incubated again at 37 C for 30 minutes (Mativandlela et al. 2006).The colour shift from blue to pink was observed.The MIC was the lowest concentration of the extract that did not permit any visible growth compared to the control wells.The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the sample that prevents bacterial growth.The antibacterial activity of a sample of plant-isolated compound is strong, moderate or weak if the MIC of the plants is 10 lg/mL, 10 < MIC 100 lg/mL or > 100 lg/ mL, respectively.Whereas, the activity of plant extracts is classified as significant if (MIC < 100 mg/mL), moderate if (100 < MIC 625 mg/mL) or weak if (MIC > 625 mg/ mL) (Kuete et al. 2011).

Conclusion
The present study showed that, fractions and pure isolated compounds from the dicloromethane-methanol (1/1) extract of the stem barks of Boswellia dalzielii possess interesting antibacterial properties against Salmonella typhi, Staphylococcus aureus, Enterobacter cloacae, Streptococcus pneumonia ATCC491619, Pseudomonas aeruginosa HM80.The bio guided studies on the actives fractions have led to the identification of some bioactive molecules in the stem barks of B. dalzielii with good activities; the most active compounds against the bacteria strains tested were a-boswellic acid (2), b-boswellic acid (3) and acetyl-11-keto-boswellic (4).From these results, it can be conclude that fractionation has led to an increase of the activity.The overall phytochemical study of the stem barks of this plant allowed the isolation and characterisation of a total of fourteen compounds, including one previously unreported named dalzienoside and thirteen known compounds.These preliminary results obtained from the in vitro assays are consistent with the uses of the plant to treat microbial diseases in the northern part of Cameroun and are also in agreement with previously reported antibacterial activity of the tested compounds.