European Journal of Medicinal Plants 31(11): 24-37, 2020; Article no.EJMP.58956 ISSN: 2231-0894, NLM ID: 101583475 Characterization of Polyphenols, Flavonoids and Their Anti-microbial Activity in the Fruits of Vangueria madagascariensis J. F. Gmel Peter K. Njenga1*, Samuel M. Mugo2 and Ting Zhou2 1 Department of Botany, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya. 2 Department of Physical Sciences- Chemistry, Grant MacEwan University, Edmonton, Alberta, Canada. Authors’ contributions This work was carried out in collaboration among all authors. The research idea was conceived by authors PKN and SMM. Author SMM sourced for the research funds. Authors PKN and SMM designed the experimental plan. Author PKN identified and collected the plant samples. Author TZ undertook experimental work under the guidance of author SMM. All authors were involved in the drafting of the manuscript. All the authors read and approved the final manuscript. Article Information DOI: 10.9734/EJMP/2020/v31i1130296 Editor(s): (1) Dr. Naseem A. Qureshi, National Center of Complementary and Alternative Medicine, Saudi Arabia. (2) Prof. Marcello Iriti, University of Milan, Italy. Reviewers: (1) Mukesh K. Berwal, ICAR-Central Institute for Arid Horticulture, Bikaner, India. (2) Dalia Mahmood Jamil, Al-Nahrain University, Iraq. Complete Peer review History: http://www.sdiarticle4.com/review-history/58956 Original Research Article Received 12 May 2020 Accepted 18 July 2020 Published 05 August 2020 ABSTRACT Aim: The study aimed to characterize phenolic acids, flavonoids, and determine their antimicrobial activities in fruits of Vangueria madagascariensis (Tamarind of Indies). Study Design: The design of the study included picking of Vangueria madagascariensis fruits from Jomo Kenyatta University of Agriculture and Technology (JKUAT) botanical garden and analysis for their antimicrobial activities at the Botany department research laboratory, JKUAT. Characterization of phenolic acids and flavonoids were conducted at MacEwan University Canada. Place and Duration: JKUAT, Kenya and MacEwan University, Edmonton, Alberta Canada between June 2013 and June 2016. Methodology: Phenolic acids and flavonoids from Tamarind of Indies were determined by highperformance liquid chromatography coupled with photodiode array detection and electrospray _____________________________________________________________________________________________________ *Corresponding author: E-mail: pknjenga@jkuat.ac.ke; Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 ionization tandem mass spectrometry (HPLC-DAD-ESI-MSN). The antimicrobial assay was determined using the disk diffusion method. Results: Based on the retention time, the UV spectrum, and the tandem MS behavior, the results revealed a profile composed of 25 phenolic compounds. Some of the identified phenolic compounds included: 3-caffeoylquinic acid, 5-caffeoylquinic acid, 4-caffeoylquinic acid, 4-feruloyl quinic acid, quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin, 3,4-di-caffeoylquinic acid, 4, 5-di-caffeoylquinic acid, kaempferol, diosmetin, caffeic acid, epicatechin, kaempferol 3-Oglucoside. The fruit extracts had a probable presence of quercetin 3-O-6’-malonylglucoside, ikarisoside C, epimedin C, unknown epigallocatechin-3-gallate and quercetin conjugate derivatives. Furthermore, the fruit extracts from Vangueria madagascariensis showed appreciable antimicrobial properties against human pathogen strains. Strong antimicrobial activity was observed for Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. The Vangueria madagascariensis was found to be highly potent against Escherichia coli and Bacillus subtilis even at low concentrations of 0.1 mg/mL. Conclusion: The research findings may suggest value of the use of Vangueria madagascariensis fruits as a rich source of antioxidants with therapeutic and nutraceutical value. Keywords: Phenolic acids; flavonoids; antioxidants; microbial activity; Vangueria madagascariensis; mass spectrometry. 1. INTRODUCTION nuts, seeds, leaves, roots, and barks. Natural antioxidants can be classified loosely as phenolic acids, flavonoids and flavonoid polymers, with the latter being highly diverse subgroup generally present as glycosylated conjugates [2,3,4]. Flavonoids can be further classified into 7 classes as shown in Fig. 1. Vangueria madagascariensis is a species that is native to Madagascar. It belongs to the plant family Rubiaceae. It is an evergreen multistemmed tree and may reach a height of 15 meters [1]. It has been naturalized in many African countries like Kenya, Angola, Tanzania, and Madagascar. The fruits are rounded, green and are often in bunches of 5-6. On ripening fruits converted to yellowish-brown in color. Flowering takes place in the rainy season, while fruit ripening occurs during the dry season. Fruit normally takes 6-8 months from flower fertilization to ripening, depending on locality. The ripe juicy fruits are collected from the tree, peeled and the pulp is eaten fresh. It has a mealy taste like Irish potatoes and is eaten as a snack. The fruit is also used as a flavoring agent in beer. The pleasant-smelling flowers of Vangueria madagascariensis attract bees and are therefore a suitable honey source. A decoction of roots is used as a remedy for various intestinal worms. An infusion of the bark can also be used for treating malaria. On the other hand, numerous classes of phenolic acids have been studied, with chlorogenic acids being the most prevalent and widely distributed in plants. About 30 chlorogenic acids have been determined especially in coffee [5,6,7,8]. A few examples of representative chlorogenic structures are shown in Fig. 2. The public interest towards natural antioxidants (phenolic compounds) has largely been due to various scientific research reports on their values such as antiallergenic, antiartherogenic, antiinflammatory, antimicrobial, antidiabetic, cancer chemopreventive agents (especially anthocyanidins), antiviral activities, antithrombotic, cardioprotective, antimutagenic, vasodilatory, insulin secretion ability, and neuroprotective effects. A classic example of anecdotal evidence of the benefits of phenolics, particularly the anthocyanins (main class of flavonoids in red wine) is the so-called "French Paradox", which refers to the lower incidents of coronary atherosclerosis in the French population compared to the Western populations, though the former consume a diet that contains more fat. Over the last decade, there has been continued research and public interest on natural antioxidants which are largely phenolic compounds. These molecular entities are secondary metabolites synthesized by the plants as protective and defense agents in response to different stress factors, e.g. UV rays, presence of xenobiotics pollutants or to protect themselves from parasitic weeds. Natural antioxidants occur in all parts of plants such as fruits, vegetables, 25 Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 Fig. 1.. Classification of general flavonoids Fig. 2. A few examples of representative chlorogenic structures 26 Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 2.2 Methods The main characteristic of the antioxidants that largely explain their health benefits enumerated above is their inherent ability to scavenge highly reactive free radicals and reactive oxygen species (e.g. superoxides, peroxides, and hydroxyl radicals). These species have been identified as major contributors to the oxidation of cellular entities (nucleic acids, proteins, DNA, lipids, and biomembranes) leading to tissue damage and the initiation of many diseases. With the rapid increase in the number of these cases to an epidemic level, the public has accepted the promise of natural antioxidants. Research output on antioxidants has been enormous, but there remain many unanswered questions on bioavailability, viability, and in general validity of clinical and epidemiological benefits of ingestion of phenolic compounds. Ripe fruits of Tamarind of Indies were picked from trees growing at Jomo Kenyatta University of Agriculture and Technology (Juja, Kenya) compound. The fruits were crushed and the macerated material extracted with hexane (5x100 mL, each extraction for 5 hours) at room temperature. All the extracts from the five extractions were pooled, evaporated and preserved for analysis. To extract the phenolics, the fruit residues were extracted (5x100 mL, each extraction for 5 hours) with 80% aqueous acetone (v: v). Acetone was evaporated and the remaining aqueous phase was further extracted by 5x30 mL of ethyl acetate to extract the flavonoids (labeled VAF) while leaving phenolics in the aqueous phase. The pH of the aqueous phase was adjusted to pH 1.5 and then extracted by 5x30 mL of ethyl acetate to extract the phenolic acids (labeled VAP). Although there is no doubt about the importance of antioxidants, there is still a need to profile the presence of these phytochemicals in plants. As a result, a huge research opportunity in the antioxidant profiling of plants from developing countries, especially in Africa, has arisen. Therefore, this work was developed to determine phenolics in the fruits of Vangueria madagascariensis, by high-performance liquid chromatography (HPLC) coupled with diode array (DAD) and electrospray ionization mass spectrometry with tandem MS (HPLC-DAD-ESIMS/MS). 2.2.1 Phenolic acid standards Caffeic acid, ferulic acid, vanillic acid, protocatechuic acid, p-coumaric acid, gallic acid, and chlorogenic acid were used to prepare a 0.1mg/mL standard mixture. UV spectra and MS/MS data were recorded as a reference, which was then used to compare with the plant extract chromatograms and spectra. A mango peel extract that has been extensively characterized by Schieber, et al [9] was used as a standard to help in the structural identification of the phenolic constituents in Vangueria madagascariensis fruit extract. The extract from the Vangueria madagascariensis fruits was also evaluated for antibacterial properties against five human pathogen strains including Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. 2.2.2 Coffee extract preparation Since it was not possible to obtain all the required pure standards for complete identification of phenolic compounds present in Vangueria madagascariensis., the coffee extract was used as a standard. The coffee extract has been extensively characterized by several authors and its wide availability makes it an inexpensive pseudo standard [10,11,12]. 2. MATERIALS AND METHODS 2.1 Materials Acetonitrile, methanol (HPLC grade), formic acid, acetic acid, caffeic acid, ferulic acid, vanillic acid, protocatechuic acid, p-coumaric acid, gallic acid, and chlorogenic acid were purchased from Sigma-Aldrich, Oakville Ontario Canada. C-18 SPE cartridges were obtained from Fisher Scientific, Edmonton, Canada, and 0.22 µm hydrophobic filters were obtained from VWR Edmonton, Canada. 500 mg of ground green robusta coffee beans were bought from a local store and extracted (4 x25 mL, 25 min each) with 70% methanol/30% water (v:v). The extract was then preconcentrated by the removal of methanol using a rotary evaporator (rotavap). The aqueous fraction was filtered through a hydrophilic syringe filter and analyzed by LC-MS. 27 Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 Table 1. HPLC gradient setting for the separation of phenolic acids and flavonoids No. 0 1 2 3 4 5 6 7 8 9 Time 0.00 15.00 30.00 70.00 85.00 95.00 100.00 103.00 105.00 107.00 C% 100.0 70.0 60.0 40.0 20.0 0.0 0.0 0.0 100.0 100.0 D% 0.0 30.0 40.0 60.0 80.0 100.0 100.0 100.0 0.0 0.0 Flow rate (µL/min) 500.0 500.0 500.0 500.0 500.0 500.0 500.0 500.0 500.0 500.0 Table 2. ESI-MS setting for the detection of phenolic acids and flavonoids Polarity Spray voltage (kV) Sheath gas flow rate Auxiliary gas flow rate Sweep gas flow rate Capillary temperature Capillary voltage (V) Tube lens (V) MS/MS isolation width MS/MS collision energy (%) Negative 5.0 50 30 0 270.0 -19.0 -125.0 3.0 35.0 2.3 Apparatus The effluent from DAD detector was then introduced to the ESI interface in the negative n mode for sample ionization and MS detection. Table 2 shows the ESI-MS settings. High purity nitrogen was used as the sheath and auxiliary gas. Structure information of phenolic acids and flavonoids was obtained by tandem MS n spectrometry (MS ) (n = up to 3) through collision-induced dissociation (CID), where argon n gas was used as the collision gas. MS was performed with a relative collision energy setting of 35%. Data acquisition was obtained using Xcalibur software version 2.0 (Thermo, San Jose, CA, USA). 2.3.1 HPLC-DAD-ESI-MS separation of coffee extract, mango peel extract, standard mixture, Vangueria m. (phenolic acids and flavonoids extract portions) The characterization of phenolic acids and flavonoids in coffee bean extract, mango peel extract, phenolic acid standard mixture, Vangueria madagascariensis phenolics, and flavonoids extracts (VAF) was subjected to a Thermo LCQ Fleet HPLC-DAD-ESI-MS system. The system consisted of a Surveyor binary pump, a Surveyor autosampler with tray temperature setting at 6ºC, a column oven, a sixport injection valve, a Surveyor DAD detector, electrospray ionization (ESI) source, and an ion trap MS detector. A Varian C18 PAH RPLC column (250 x 3.0 mm I.D., 5 m) (Agilent, Santa Clara, CA, USA) operating at 20ºC with a flow rate of 500.0 µL/min was used to separate phenolic acids and flavonoids. The diode array detector (DAD) had a scan range from 190 nm to 400 nm, with three detection channels at 280, 320, and 380 nm. 2% acetic acid in water (C) and 0.5% acetic acid in water/acetonitrile (50/50, v/v) (D) were used as mobile phases in gradient separation. Table 1 shows the gradient setting during a 107-min run. 2.3.2 Antimicrobial assay test Organisms An equal ratio of VAP and VAF dry extracts were combined, reconstituted in distilled water to make extract solutions of 100, 10, 1, and 0.1 mg/mL. The extract's microbial inhibition activity was tested using the disc diffusion method [13]. The following microorganisms were used as test organisms: Staphylococcus aureus (ATCC 25923), a facultative anaerobic Gram - positive coccal bacterium frequently found as part of the normal skin flora on the skin and nasal passages, Bacillus subtilis (ATCC 6633), a Gram - positive obligate aerobe commonly found in soil), Escherichia coli (ATCC 25922), a 28 Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 3.1 Profiling of Phenolic Acids and Flavonoids from Standard Mixture, Coffee Extract and Mango Peel Extract Gram - negative, rod - shaped bacterium commonly found in the lower intestine of endotherms, Pseudomonas aeruginosa (ATCC 2785), a Gram - negative, aerobic rod - shaped bacteria, and Candida albicans (ATCC 90028), a diploid fungus that grows both as yeast and filamentous cells. 3.1.1 Standard mixture n The ESI-MS results obtained from authentic standards are summarized in Table 3. The MS and tandem MS data were used as the reference for the identification of phenolic acids and flavonoids in VAF and VAP extracts. All the bacteria were obtained from the Botany department laboratory of Jomo Kenyatta University of Agriculture and Technology (JKUAT), Juja, Kenya, and standard isolates were used. The bacteria were maintained at 4 0C on nutrient agar plates. Base plates were prepared by pouring 20 mL Mueller-Hinton agar into sterile Petri dishes and allowed to air dry. 0 Molten Mueller-Hinton agar held at 48 C was inoculated with a broth culture of the test organism and poured over the base plates forming a homogenous top layer. Filter paper discs (Whatman No. 3, 6 mm diameter) were sterilized by autoclaving. Ten μl of each extract (100, 10, 1 and 0.1 mg/mL) were applied per filter paper disc. The discs were air-dried and placed onto the seeded top layer of the MuellerHinton agar plates. Each extract was tested in triplicate. Air-dried Dimethylsulfoxide saturated discs were used as negative controls. Gentamicin antibiotics (30 μg/ml) in distilled water was used as a positive control. The plates were evaluated after incubation at 37°C for 24 hr after which the zones of inhibition were measured. The size of the inhibition zone in (mm) produced by the plant extract and the inhibition zone around the gentamicin reference (mm) was used to express antibacterial activity. Antifungal activity was performed on Candida albicans. In vitro activity was performed as described for bacterial assays except that Sabourands dextrose agar was used for antifungal bioactivity testing and incubation was done at 30°C for 24 hr. 3.1.2 Coffee extract Coffee beans have been extensively studied and have been found to contain a huge amount of phenolic acids especially chlorogenic acid derivatives. Analysis of coffee extract by HPLCDAD, the UV chromatogram at 320 nm is shown in Fig. 3a. The chromatogram peaks were identified by elucidation of diagnostic daughter ions emanating from HPLC-MSn (n = up to 3) and comparison with literature. As shown in Table 4, numerous chlorogenic acid derivatives were identified, including caffeoylquinic acid (CQA), feruloyl quinic acid (FCQA), and dicaffeoyl quinic acid (diCQA). The isomerization of CQA has been reported with 3 isomers of the quinic acid in three different positions on the caffeoyl backbone: (3-CQA), (4-CQA), and (5-CQA). To identify the three possible CQA isomers, MS/MS fragmentation and detection were conducted. 4CQA was verified by the MS peak at m/z 173, while other CQA isomers did not have this peak. 3- and 5-caffeoyl quinic acid were identified based on the parent ion peak at m/z 2 191 and MS ions at m/z 179. As reported in the literature the intensity of m/z 179 was higher in 3-CQA than that in 5-CQA [10,12]. 3. RESULTS AND DISCUSSION A series of dicaffeoyl quinic acid (diCQA) isomers were also identified based on MS/MS 2 data. A MS peak at m/z 173 was detected based on the MS parent peak at m/z 515, which illustrated the presence of 3, 4-di CQA or 4, 5-di CQA. This m/z 173 peak is 4-CQA, thus 3,4-di CQA loses the caffeoyl moiety at position 3, while 4,5-di CQA loses the caffeoyl moiety at position 5. These two dicaffeoyl quinic acid isomers were further distinguished by the peak intensity at m/z 335, which is more intense in 3,4-di CQA than that in 4,5-di CQA [11,12]. Using LC-ESI-MS/MS, the profile of phenolic compounds in the fruits of Vangueria madagascariensis. was characterized and most of the major compounds present were tentatively identified. The identification was based on the comparison of mass spectrometry data with that of authentic standards, coffee extracts, and mango peel extracts. Some of the compounds were also tentatively identified from the published MSn data in the literature, which will be explained in detail. 29 Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 n Table 3. ESI-MS results of authentic standard mixture - [M-H] (m/z) 169 153 353 167 179 163 193 289 289 311 MS/MS (m/z) 125/81/79 109 191 152/123/108 135 119/93 178/149/134 245/137 245/206 179/149 Identity Gallic acid Protocatechuic acid 3-caffeoylquinic acid Vanillic acid Caffeic acid p-Coumaric acid Ferulic acid Catechin Epicatechin Caftaric acid Table 4. Identification of phenolic acids in the coffee extract - 1 2 3 4 5 6 7 8 [M-H] (m/z) 353 353 353 367 367 367 515 515 MS/MS (m/z) 191 191 173 193/173 191 191/149 353/191 353/173 Identity 3-Caffeoylqunic acid 5-Caffeoylqunic acid 4-Caffeoylqunic acid 3-Feruloylquinic acid 5-Feruloylquinic acid 4-Feruloylquinic acid 3,4-Dicaffeoylqunic acid 4,5-Dicaffeoylqunic acid Table 5. Identification of flavonoids in mango peels extract Peak number 1 2 3 4 5 6 7 [M-H]- (m/z) 463 463 433 433 433 447 447 MS/MS (m/z) 301 301/179 301 301/300 301 301 285/284/255 Similarly, three isomers of feruloyl quinic acid were distinguished by tandem MS. The parent ion of FQA at m/z 367 was fragmented and the MS/MS data were compared. Only 3-FQA can form a daughter ion at m/z 193. 4-FQA and 5FQA were then further identified by the MS2 fragments [12,14]. ESI-MS and tandem MS. Our data show that both the flavonoids and phenolic acids fraction generated very similar phenolic compounds with an exemption of a few, with the HPLC-DAD chromatograms shown in Figs. 3b and 3c respectively. When comparing the identified phenolic acids in both plant fractions, some phenolic compounds were confirmed by the use of both authentic phenolic standards and previously identified mango peel and coffee extract fractions. These included: 3-, 5- and 4-CQA, 4-FQA, quercetin-3O-galactoside, and quercetin 3-O-glucoside. By keenly evaluating the fragmentation patterns and comparing the results with those in the literature, a methoxylated flavonoid, diosmetin, was identified in both VAF and VAP. It has a [M-H] peak at m/z 299, and can be fragmented to [M-HCH3]- at m/z 284 and [M-H-CH3-CO]- at m/z 256 [19]. Kaempferol was also present in both fractions. It has a [M-H]- ion at m/z 285 and Similarly, the mango peel extract (chromatogram not shown) was analyzed by HPLC-DAD-ESI-MS with the summary of identified phenolics based on MS and MS2 data shown in Table 5. Similar identifications have been made by other researchers including [9,15,16,17,18]. 3.2 Identification of Polyphenols Flavonoids in Vangueria m. Identity Quercetin 3-O-galactoside Quercetin 3-O-glucoside Quercetin 3-O-xyloside Quercetin 3-O-arabinopyranoside Quercetin 3-O-arabinoforanoside Quercetin 3-O-rhamnoside Kaempferol 3-O-glucoside and Table 6 and Table 7 show the identification of phenolic acid fraction (VAP) and flavonoids fraction (VAF) in Vangueria madagascariensis together with the parent and daughter ions by 30 Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 resulted in a daughter ion at m/z 242 by the loss of CO2 to form ([M-H-CO2] ). Kaempferol 3-Oglucoside was identified in the phenolic acid fraction as [M-H] at m/z 477. With tandem MS, it loses a hexose moiety (-162) and a daughter ion at m/z 285 was detected [20]. 4,5-diCQA was present in VAP and was not detected in VAF. Caffeic acid was detected in VAP, not in VAF. On the other hand, epicatechin and quercerin were identified only in VAP. Epicatechin has a [M-H]ion at m/z 289 and lost 44 amu (CO2) to form a daughter ion at m/z 245 [21,22]. The fragmentation seemed in accordance of that of ikarisoside C. Several peaks in the flavonoids fraction (VAF) could not be identified. These were compounds with MS peaks at m/z 499 (peak 12), 1219 (peak 13), 463 (peak 15), and 504 (peak 16). As shown in Fig. 3b, the peaks at m/z 1219 and 463 were also detected in VAP, which will be discussed in the VAP section. The m/z 499 peak bore a loss of 36 amu due to the loss of two water molecules, resulting in a MS2 fragment ion at m/z 3 463 (possibly a quercetin derivative) and MS ion of m/z 445 (loss of H2O) and m/z of 433. On the other hand the m/z 504 peak lost 19, 163 and 2 169 amu to give MS fragment ions at m/z of 485, 441 and 435. - There was a [M-H] ion at m/z 549 detected in both fractions. By performing tandem MS, the parent ion resulted in a loss of CO2 to give a fragment ion at m/z 505. MS3 of the m/z 505 daughter ion resulted in fragment ions at m/z 463, 301, 271 and 255 with m/z 301 as the predominant peak, which could be caused by the loss of CH2CO (42 amu), acetyl-glucose moiety (204 amu) and hexose moiety (162 amu). This fragmentation behavior was somewhat following literature data for quercetin 3-O-6”malonylglucoside [23]. For the phenolic acids fraction (VAP), Fig. 3c, the following compounds could not be identified: m/z 765 (peak 7), 381 (peak 10), 779 (peak 14), 1135 (peak 16), 1177 (peak 19), and 1219 (peak 20), 544 (peak 22) and 463 (peak 24), where m/z 1219 and m/z 463 were also detected in VAF. Briefly, m/z 765 (peak 7) ion lost 36 amu to give 2 a MS peak at m/z 729, and further lost 286 amu and 350 amu to give MS3 peaks at m/z 443 and 373. Also, the [M-H]- ion at m/z 821 was detected in both fractions. Fragmentation of this ion resulted in daughter ions at m/z 457 and 623. Further fragmentation of the base peak at m/z 457 resulted in fragmentation ions at m/z 429, 369, and 225 in the MS3 spectrum. From the tandem MS spectra, the following mass fragmentation was observed: 232 amu (either malonyldeoxyhexose or succinyl pentose), 144 amu (loss of dideoxy-OCH3-hexose), 198, 364, 28, 108, 204 amu (acetylhexose), 60. The loss between m/z 821 to 369 could be due to the loss of two rhamnosyl (284 amu) moiety and one hexose moiety (162 amu). From the LIPIDS MAPS database, it would seem that fragmentation pattern to be following Epimedin C and the ion was tentatively identified as such [24,25]. - The compound with [M-H] ion at m/z 779 (peak 14) lost 322 amu to form a daughter ion at m/z 457 (possibly indicative of an epigallocatechin gallate), and further lost 71 amu, 288 amu and 232 amu (could be due to malonyldeoxyhexose 3 or succiylpentose) to give MS peaks at m/z 386, 225 and 169 (gallate).The m/z 1135 ion (peak 16) resulted from a loss of 36 amu (loss of two 2 water molecules) to give a MS ion at m/z 1099 and further by losing 518 amu and 286 amu, to 3 give MS ions at m/z 813, 581 and 457 (possibly indicative of an epigallocatechin gallate). The m/z 1177 peak (peak 19) on the other hand showed a loss of 36 amu (loss of two H2O molecules) to form a daughter ion at m/z 1141, then further gave two MS3 peaks at m/z 855 and 772 by losing 286 amu and 369 amu. The m/z 1219 ion (peak 20) showed a loss of 36 amu 2 (loss of two H2O molecules) to form a MS peak 3 at m/z 1183, and MS peaks with m/z 855, 813 (predominant), 785, 753, 499 and 457. It is all apparent that peaks 14, 16, and 20 are compounds related in structure with fairly similar characteristic daughter ions especially, m/z 457 and m/z 813. The m/z 457 could be associated with epigallocatechin-3- gallate and as such the 3 peaks could be derivatives of epigallocatechin gallate. Another ion in both the two fractions could not be positively identified as the [M-H] ion at m/z 823. The collisionally induced dissociation (CID) on 2 the parent ion resulted in m/z 763 in the MS spectrum indicating a loss of 60 amu. Further CID of the m/z 763 ion resulted in fragment ions at m/z 719, 617, 573, and 455. The confirmed loss included 44 amu (763-719, CO2), 102 amu (719-617), 146 amu (763-617) deoxyhexose also called rhamnose. Other fragmentations observed included 162 amu (617-455, hexose moiety), 308 amu (763-455, either dehydrated rutinose or coumaroyl glucoside) and 118 amu (573-455). 31 Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 Table 6. Identification of phenolic acids and flavonoids in VAF on C18 column Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 - [M-H] (m/z) 353 353 353 367 463 463 549 515 515 821 285 499 1219 299 927/499/463 504 823 2 MS (m/z) 191 191 191/173 301 301/179 505 353 353/173 623/457 241/199 463 1183 284 485/441/435 763 3 MS (m/z) 463/301/271/255 191 429/369/225 445/433 855/813/785/753/499/457 256/151 719/617/455 UV λmax(nm) 242, 322 242, 322 242, 322 308 254, 344 254, 344 254, 346 248, 326 248, 326 243 256, 344 250, 344 242, 331 252, 266sh, 346 252, 342 274, 330 243, 271, small hump at 326 32 Identity 3-caffeoylquinic acid 5-caffeoylquinic acid 4-caffeoylquinic acid 4-feruloyl quinic acid Quercetin 3-o-galactoside Quercetin 3-o-glucoside Could be quercetin 3-O-6”-malonylglucoside 3,4-di-caffeoylquinic acid 4,5-di-caffeoylquinic acid Could be Epimedin C Kaempferol Unknown Unknown but suggested to be an epigllocatechin-3-gallate derivative Diosmetin Unknown Unknown ikarisoside C Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 Table 7. Identification of phenolic acids and flavonoids in VAP on C18 column Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 - [M-H] (m/z) 353 353 353 179 289 463 765 463 463 381 549 447 No MS peak detected 779 515 1135 821 285 1177 1219 301 544 299 927/499/463 823 MS/MS (m/z) 191 191 191/173 135 245/206 301 729 301 301/179 3 MS (m/z) 443/373 505 285 463/301/255 457 353/173 1099 623/457 242 1141 1183 179/151 386/225/169 284 256 763 719/617/573/455 813/581/457 429/369/225 N/A 855/772 855/813/785/753/499/457 UV λmax(nm) 242, 321 242, 321 242, 321 328 242, 273 308 243, 321 254, 344 254, 344 243, 340sh 244, 328 244, 345sh 244, 334sh 247 248, 326 248 244 256, 344 244, small hump at 334 242, 331 242, 309 242, a 331 hump 243, 266sh, 343 252, 268, 342 max 243, 271, small hump at 326 Bold: Most intense ion 33 Identity 3-caffeoylquinic acid 5-caffeoylquinic acid 4-caffeoylquinic acid Caffeic acid Epicatechin 4-feruloyl quinic acid Unknown Quercetin 3-O-galactoside Quercetin 3-O-glucoside unknown Could be quercetin 3-O-6”-malonylglucoside Kaempferol 3-O-glucoside Unknown Unknown but suggested to be an epigllocatechin-3-gallate derivative 4,5-di-caffeoylquinic acid Unknown but suggested to be an epigllocatechin-3-gallate derivative Could be Epimedin C Kaempferol Unknown Unknown but suggested to be an epigllocatechin-3-gallate derivative Quercetin Unknown Diosmetin unknown quercetin conjugate Ikarisodide C Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 Fig. 3. HPLC UV chromatograms for a) Coffee extract; b) Vangueria madagascariensis flavonoid fraction (VAF); c) Vangueria madagascariensis phenolic acids fraction (VAP) (VAP 34 Njenga et al.; EJMP, 31(11): 24-37, 2020; Article no.EJMP.58956 Table 8. Antimicrobial activity of Vangueria madascariensis fruit extract at different concentrations expressed as inhibition zone calculated in mean diameter around the disc in mm. Any diameter above 6 mm indicates some degree of inhibition Extract concentration (mg/mL) 100 10 1 0.1 c.a p.a s.a E.c B.s Gentamicin 10.5 ± 0.7 9 ± 0.6 7 ± 0.0 6 ± 0.0 12±0.7 10.5±0.7 9.5±0.6 7.5 ± 0.3 10 ±0.8 7.5 ± 0.3 7.5 ± 0.4 7.5 ± 0.4 11.5 ±0.7 9 ± 0.6 10 ± 0.7 10.5 ± 0.8 12 ± 0.9 10.5 ± 0.7 12.5 ± 0.9 12.5 ± 0.9 B.s = 20.6 ± 0.4 E.c=22.0 ± 0.3 s.a =21.0 ± 0.6 p.a = 18.7 ± 0.3 c.a =21.7 ± 0.3 c.a- Candida albicans, p.a- Pseudomonas aerugenosa, s.a- Staphylococcus aureus, E.c- Escherichia coli, B.s Baccilus subtilis 1 Finally, peak 24 showed MS of 927, 499, and 463 with the latter being predominant, which would probably be dehydrated (loss of 2- H2O molecules) derivative of m/z 499. It seems 926, could be a dimer of 463. compounds were identified, but several remained still unidentified. The fruit extracts demonstrated significant antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. The high phenolic content and antimicrobial potency make the fruit a potential functional food. To fully identify all the compounds, it would be necessary to collect the fractions of the extracts after HPLC analysis and carry out NMR studies to elucidate the structures of the unidentified species. The antioxidant activity of the extracts can be further evaluated using well-known chemical assays and facile electroanalytical techniques. 3.3 Antimicrobial Susceptibility Test Results against the Test Organisms The results of antimicrobial activity of the combined VAF and VAP extracts at different concentrations are shown in Table 8. Notably, the Vangueria madagascariensis shows very significant inhibition for all the microorganisms tested at high extract concentration. The inhibition while still strong was least for Candida albicans, and decreased with the dilution of the plant extract. At 0.1 mg/mL the extract barely had any inhibitory properties against Candida albicans. A similar decrease with dilution was observed with Staphylococcus aureus. The inhibition against Pseudomonas aerugenosa was very considerable at 100 mg/mL and slowly decreased with dilution, even at 0.1 mg /mL some inhibition of 7.5 ± 0.3 was observed. For both Escherichia coli and Bacillus subtilis, the extract maintained appreciably high inhibitions even at the lowest concentrations of 0.1 mg/ml. This could attest to the potency of the extract against these microorganisms. The strong microbial inhibitory effects were not unexpected especially after considering the chemical composition of the VAF and VAP elucidated using LC-MS/MS. It is evident in the literature [26,27] flavonoids have significant microbial activity. CONSENT It is not applicable. ETHICAL APPROVAL It is not applicable. ACKNOWLEDGEMENTS This research was funded by Grant MacEwan University Research Scholarly Activity and Creative Achievement fund. 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Harnly. A screening Journal of Botany 2010;76(1):25–29. method for the identification of DOI: 10.1016/j.sajb.2009.06.010 _________________________________________________________________________________ 19. © 2020 Njenga et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Peer-review history: The peer review history for this paper can be accessed here: http://www.sdiarticle4.com/review-history/58956 37