The First Comprehensive Chemical Profiling of Vachellia gummifera (Willd.) Kyal. & Boatwr., a Plant with Medicinal Value

Vachellia gummifera (Willd.) Kyal. & Boatwr. is a medicinal plant endemic to Morocco that has no documented studies on its chemical composition. In this study, the chemical composition of the water/methanol (4 : 1) extracts of air‐dried leaf and stem samples of Moroccan V. gummifera was determined using UHPLC‐MS and NMR. In total, over 100 metabolites were identified in our study. Pinitol was the major compound in both the leaf and stem extracts, being significantly more abundant in the former. Asparagine and 3‐hydroxyheteroendrin were the second most abundant compounds in the stem and leaf extracts, respectively, though both compounds were present in each tissue. The other compounds included flavonoids based on quercetin, and phenolic derivatives. Eucomic acid, only identified in the stems and was the major aromatic compound distinguishing the leaf and stem profiles. Quercetin 3‐O‐(6′′‐O‐malonyl)‐β‐D‐glucopyranoside was identified as the major flavonoid in the leaves but was also present in the stems. Other malonylated derivatives that were all flavonol glycosides based on myricetin, kaempferol, and isorhamnetin in addition to quercetin were also identified. This is the first report of eucomic acid and malonylated compounds in Vachellia species. This report provides valuable insights into the chemotaxonomic significance of the Vachellia genus.


Introduction
Vachellia gummifera (Willd.)Kyal.& Boatwr.(Basionym: Acacia gummifera Willd.) is a thorny flowering plant belonging to the Fabaceae family. [1] In Morocco, the plant's various parts are used in traditional medicine to treat different ailments.For instance, the aerial parts are used to treat bronchitis and cough, [4] decoctions from its roots for type 2 diabetes, [5] poultice of the leaves for wounds and the powder of the bark for measles. [3]Its extracts also showed in vitro nematicidal activity against Meloidogyne ssp. [6]V. gummifera is an important forage plant for Moroccan Dorcas gazelles [7] and its powder biomass can be used as a biosorbent for removing lead and cadmium from polluted water. [8] To the best of our knowledge, there exists no reported studies on the chemical composition of V. gummifera.Mouhajir et al., (2001)  [11] used electronic spin resonance spectroscopy, a method that detects phenolics with free ortho-or para-dihydroxy groups to study compounds in the plant.However, they did not detect any compounds in their study despite phenolics being some of the most abundant secondary metabolites in plants. [12]ince this plant has reported apparent beneficial effects as elaborated above, it is important to assess its phytochemical composition which might provide valuable insights to understand the chemistry responsible for the effects.In line with the above, the objective of this study was to characterise the phytochemical profile of the polar extracts of V. gummifera.The leaves and stems were investigated in this study as they make up the major biomass on the plant and are prominently used in traditional medicine and other applications.This is the first report on the characterisation of this plant's metabolome.

Results and Discussion
The UHPLC-MS total ion chromatograms (Figure 1) and the 1 H-NMR spectra (Figure 2) of the leaf and stem polar extracts showed that many of the compound peaks were common to both extracts.However, certain peaks were also observed to be Inspection of the 1 H-NMR spectra of both the leaf and stem crude extracts revealed several differences in the distribution of compounds found in both plant parts.The major peak in both spectra is a singlet at δ H 3.59 (s, 3H) (Figure 2a).Additional signals at δ H 3.64 (t, J = 9.8 Hz, 1H), 3.74 (dd, J = 9.9, 2.8 Hz, 1H), 3.80 (dd, J = 9.8, 2.8 Hz, 1H), 3.98 (m), and another overlapping with the residual methanol signal corresponded to pinitol when compared with the 1 H-NMR data of an authentic standard of the compound.Quantification of the compound in both crude extracts by qNMR revealed that it was significantly more abundant in the leaves with 64.83 � 0.88 mg g À 1 DW compared to the stems where 24.89 � 0.06 mg g À 1 DW was observed (Figure 4).Pinitol has previously been identified in Vachellia species including V. nilotica, [13] V. farnesiana, [14] and V. etbaica Schweinf. [15] This probably explains the reported use of V. gummifera in traditional medicine for treating type 2 diabetes. Therefore, the presence of the compound as the major metabolite in the extracts could be a consequence of the plant material having been sampled from plants growing in Morocco which is an arid area implying that they experienced significant drought and water stress during their growth.
The upfield region of the 1 H-NMR spectra of the crude extracts also showed the presence of various clear signals arising from amino acids (Figure 2b).The identity of these amino acids was confirmed by comparing their multiplet peaks in the crude spectra with authentic standards.The presence of asparagine was confirmed from its characteristic signals at δ H 2.83 (dd, J = 16.9, 8.0 Hz, 1H) and 2.95 (dd, J = 16.9, 4.1 Hz, 1H).Asparagine was significantly more abundant in the stems (13.90 � 1.50 mg g À 1 DW) compared to the leaves (9.41 � 0.20 mg g À 1 DW), and in fact a careful examination of the whole spectrum reveals that it is the second major compound after pinitol in the stem extract.Proline, which was significantly more abundant in the leaves (14.79 � 0.13 mg g À 1 DW) compared to the stems (7.95 � 0.23 mg g À 1 DW) was also confirmed from some of its signals at δ H 2.00 (m, 2H), 2.07 (m, 1H), 2.34 (m, 1H) and 4.10 (dd, J = 8.8, 6.4 Hz, 1H). Additionally, valine with 1.54 � 0.03 mg g À 1 DW in the leaves and 0.65 � 0.01 mg g À 1 DW in the stems was also À ) corresponding to a hexose fragment.Comparison of its 1 H-NMR data to that of a compound previously isolated from V. sieberiana var.woodii confirmed the identity of 9 to be 3-   Naringenin [a] Stems hydroxyheteroendrin. [20] This compound was significantly more abundant in the leaves with 24.40 � 0.65 mg g À 1 DW compared to the stems with just 1.52 � 0.14 mg g À 1 DW.On the other hand, inspection of the aromatic region of the 1 H-NMR spectra of both the leaf and stem extracts revealed two major signals at δ H 7.14 (d, J = 8.4 Hz) and 6.78 (d, J = 8.4 Hz) that were present in the stems but absent in the leaves (Figure 2b).These signals represented the major difference in the aromatic profile of the two extracts.From the isolated fractions, both doublets corresponded to compound 28.This compound eluted at rt 15.50 min, and its MS showed a deprotonated molecular ion ( ).The 1 H-NMR spectrum of the isolated fraction showed two aromatic signals similar to those in the crude extract spectrum at δ H 7.12 (d, J = 8.5 Hz, 2H) and 6.80 (d, J = 8.5 Hz, 2H) indicating a parasubstitution of a phenyloxy, as well as two pairs of aliphatic methylene signals at δ H 2.97 (d, J = 13.8Hz, 1H)/ 2.86 (d, J = 13.8Hz, 1H) and 2.93 (d, J = 16.1 Hz, 1H)/ 2.66 (d, J = 16.1 Hz, 1H). To the best of our knowledge, this is the first time eucomic acid has been reported in any Vachellia or Acacia species.Unlike the spectra of the stem extracts, the aromatic region in the 1 H-NMR spectra of the leaf extracts was dominated by flavonoid related signals, many of which were also present in the spectra from stem extracts, albeit in differing abundances.
Interestingly, inspection of the total ion chromatograms of the leaves and stems showed that the [MÀ H] À peak of eucomic acid (28) at m/z 239.0557 was also the most abundant peak in the stem extract and absent in the leaf extract (Figure 1).Therefore, this compound can be considered as the main aromatic compound differentiating the UHPLC-MS profiles of the leaves and stems of V. gummifera.In addition to 28, the compounds 11, 12, and 13 appeared only in the chromatogram of the stem extract and were not visible in extracts from the leaves (Figure 1).).The 1 H-NMR spectrum of the compound showed a similar aromatic pattern as 28 with signals at δ H 7.13 (d, J = 8.5 Hz, 2H) and 6.78 (d, J = 8.5 Hz, 2H).However, its spectrum showed two aliphatic signals at δ H 4.45 (s, 1H) and 3.02 (m, 2H). Interestingly, the compounds that eluted at rt 7.72 (12) and 7.77 (13) min exhibited similar [MÀ H] À ions, MS/MS fragmentation patterns and 1 H-NMR chemical shifts as compound 11.They might represent two of the three other stereoisomers as expected for a two-stereocenter molecule like piscidic acid.This finding is even more important as stereochemical occurrences of secondary metabolites and their origin in the natural world remain a much discussed topic in natural product chemistry. [25]Recently, piscidic acid was identified as the major compound in the branch extracts of V. nilotica yet a significantly smaller amount was identified in its gum. [26]ompound 94, one of the major abundant peaks in the chromatogram of the stem extract, was found only at trace level in the leaf extract chromatogram.It showed the [MÀ H] À ion at m/z 331.082 corresponding to a compound with the molecular formula C 17 H 16 O 7 .Analysis of its UHPLC-MS and 1 H-NMR data (Table 2) showed that it agreed with the identity of di-O-methyltaxifolin. [27] However, the data was not sufficient for confirmation of the exact methylation positions.c] Putative identification based on UHPLC-MS data.Product ions in bold are the base peaks of the MS/MS spectra.R t is the retention time.

Annotation of Other Compounds
Generally, diverse classes of compounds were isolated and identified from both the leaf and stem extracts.Most of the compounds identified were flavonoids (constituting over half of the characterised compounds) and phenolic acid derivatives.Smaller numbers of other phenyl derivatives, organic acids, amino acids, alkyl glycosides, terpene derivatives and fatty acids were also identified.

Flavonoids
The flavonoid composition of both extracts consisted mainly of glycosylated derivatives of various aglycones from multiple classes, with the majority being mono-or di-glycosylated compounds.Some tri-glycosylated derivatives as well as individual aglycones were also identified.Most compounds were flavonol derivatives mainly based on quercetin, but flavanol, flavone, flavanone and flavanonol derivatives were also identified.

Flavonol Derivatives
The majority of the flavonol derivatives identified were quercetin glucosides.From the MS/MS fragmentation patterns, most of these were monodesmodic quercetin-3-O-glucosyl derivatives as shown by the presence of the base peak ion [M-2H-sugars] À at m/z 300 compared to the ion [MÀ HÀ sugars] À at m/z 301. [28]This was confirmed from the 1 H-NMR resonances of both meta-coupled protons, H-6 and H-8, of most flavonoids in the region of 6.35 and 6.60 ppm compared to 6.60 and 6.90 ppm for the same protons in 7-O-glucosylated flavonoids.Some of the derivatives (33 and 41) included bidesmodic glucosides with substitution in both 3-and 7-positions of quercetin.Five malonylated glycosides of quercetin (41, 73, 76, 78 and 84) were identified, as revealed by the neutral loss of m/ z 44 followed by another of m/z 42 in their MS/MS spectra corresponding to a decarboxylation and loss of CH 2 CO, respectively.These losses are characteristic for compounds bearing a malonyl group.However, the NMR signal of the malonyl methylene protons was not observed in the spectra of all the compounds due to an overlap with the residual methanol solvent signal as was similarly observed by Kazuma  et al., (2003). [29]Thus, the presence of the malonyl moiety was Quercetin derivatives have been reported from Vachellia and Acacia species, [10,[30][31] but as far as we know, no malonylated compounds based on quercetin or any other aglycone skeletons have been reported in any species belonging to the two genera before.Both the type of extract studied, and the methodology employed could be responsible for the annotation of malonylated flavonoids as encountered here when compared to common methodology and extracts of most reports in the literature.Quercetin and its derivatives are known to have anti-inflammatory activity and their abundance in both extracts could potentially explain the use of the plant in traditional medicine for relieving symptoms of cough, bronchitis, and measles. [9]ompounds 67, 79, 85 and 89 all showed a base peak ion,      315.0511 resulting from the loss of m/z 308 corresponding to a combined loss of a rhamnose and a hexose.The sugar sequence could not be defined as the compound showed unresolvable 1 H-NMR data and a putative identification of isorhamnetin 3-O-rhamnosyl hexoside was given.Likewise, the mass spectrum of compound 67 showed its [MÀ H] À ion at m/z 769.2185, higher by 146 Da from that of compound 79 indicating the presence of an additional rhamnose in 67.
Without clear 1 H-NMR signals, as in 79, to confirm the sugar moieties, 67 was putatively identified as isorhamnetin 3-Odirhamnosyl hexoside.Similarly, compound 89 showed a fragment at m/z 315.0510 corresponding to two successive neutral losses of m/z 86 and m/z 162 for malonyl and hexose moieties, respectively.This was hence putatively identified as isorhamnetin 3-O-malonyl hexoside, a compound that is described in Vachellia and Acacia species for the first time.The above mentioned isorhamnetin derivatives were identified only in the stem extract except for compound 89 which was identified in both the leaves and stems.Generally, isorhamnetin derivatives appear to have a rare occurrence in these genera according to the available studies on their phytochemical profiles.
Compounds 51-54 and 59 all showed a major fragment ion [M-2H-sugars] À at m/z 316 in their MS/MS indicating the presence of a myricetin aglycone that is 3-O-glycosylated.Compounds 52 and 53 showed the fragment ions [MÀ HÀ 162] À and [MÀ HÀ 308] À that are characteristic of flavonoid O-hexosides and O-rutinosides, respectively, and were indeed confirmed to be myricetin 3-O-β-D-glucopyranoside (52) and myricetin 3-Orutinoside (53) by comparison with authentic standards.Compound 54 showed the same fragmentation pattern as 52 but with unresolvable 1 H-NMR data, it was putatively identified as myricetin 3-O-hexoside.Compound 59 was confirmed to be myricetin 3-O-(6''-O-malonyl)-β-D-glucopyranoside by inspecting its 1 H-NMR spectrum (Table 2).Compound 51 showed successive neutral losses of m/z 86 and m/z 162 and its molecular formula was consistent with the putative identification as a myricetin 3-O-malonyl hexoside.Two kaempferol derivatives, 81 and 88 were isolated from only the leaves and confirmed by 1 H-NMR data (Table 2), with 88 being malonylated.These malonylated myricetin and kaempferol derivatives are also reported for the first time in Vachellia and Acacia species.

Flavanol Derivatives
Seven flavanol (18, 19, 21, 22, 35, 44 and 48) derivatives of catechins including their oligomers and esters with gallic acid were identified as within the extracts.Catechin (21), epicatechin (35) and procyanidin B3 (18) were confirmed by comparison of their profiles with authentic standards.The MS/MS pattern of compound 19 showed two major product ions at m/z 125.0251 (C 6 H 5 O 3 À ) and 179.0355 (C 9 H 7 O 4 À ) consistent with the retro Diels-Alder fragmentation of (epi)gallocatechin.Compound 44 showed gallic acid and (epi)gallocatechin fragments in its MS/ MS pattern and was confirmed by 1 H-NMR data (Table 2 Compound 48 was also putatively identified as a B-type proanthocyanidin based on its MS/MS pattern.Though not many were identified in this study, condensed tannins are some of the best-known compounds in Acacias. 10 Catechin, epicatechin and their oligomers were identified only in the stem extracts while their galloyl derivatives were identified in both the leaf and stem extracts.These flavanol derivatives are common constituents in Vachellia species and have been identified in different plant parts including the leaves of V. tortilis, [32] the leaves, bark, flowers and pods of V. nilotica, [33] and the leaves of V. karroo and V. xanthophloea. [30]These compounds are known to have antioxidant and anti-inflammatory activities. [9]

Flavanonol Derivatives
Besides aromadendrin (83), all the other flavanonol derivatives identified were based on taxifolin including free and methylated aglycones, as well as glycosylated entities.Compounds 62 and 68 both with the [MÀ H] À ion at m/z 303.051 corresponding to a compound with the molecular formula C 15 H 12 O 7 , were identified as taxifolin and epitaxifolin (Table 2) respectively.Compounds 23 and 50 had the same molecular ion and both showed a fragment at m/z 303.05 corresponding to the product ion [MÀ HÀ 162] À indicating that they were both taxifolin hexosides.Compound 23 was confirmed to be taxifolin 7-O-β-Dglucopyranoside by analysis of its 1 H-NMR data ( ) indicating methylation of the compound's B-ring.Compound 61 showed a product ion [MÀ HÀ 162] À at m/z 331.0822 as the major fragment in its MS/MS spectrum in line with a dimethylated taxifolin that has lost a hexose.The 1 H-NMR data confirmed the identity of 61 to be di-O-methyltaxifolin-O-β-D-glucopyranoside (Table 2).All the taxifolin derivatives were identified in the stems except for di-O-methyltaxifolin (94) which also showed minor traces in the UHPLC-MS of the leaves.There are very limited reports of taxifolin in Vachellia species and no methylated taxifolin derivatives seem to have been reported in the genus.

Phenolic Acid Derivatives
The major fragment in the MS/MS spectra of compounds 16, 24, 29 and 38 was at m/z 163.04 (C 9 H 7 O 3 À ) indicating the presence of a coumaric acid moiety in each of these compounds.The observed base peaks in compounds 16 and 29 corresponded to the loss of a hexose [MÀ HÀ 162] À while their individual 1 H-NMR spectra exhibited signals of (E)-and (Z)isomers p-coumaric acid 4-O-β-D-glucopyranoside, respectively (Table 2).The MS/MS of the parent ion [MÀ H] À of compound 38 showed successive losses of a pentose (132 Da) and hexose (162 Da).The identity of the sugars as apiose and glucose as well as the configuration of the p-coumaric acid moiety were determined by inspecting the 1 H-NMR spectrum, confirming the structure of 38 to be (Z)-p-coumaric acid 4-O-(2'-O-β-D-apiofuranosyl)-β-D-glucopyranoside (Table 2).Compound 24 had the same molecular formula and fragmentation pattern as 38 but the NMR of the fraction was dominated by signals of other coeluting compounds and conclusive identification could not be achieved.Therefore, 24 was putatively identified as p-coumaric acid pentosyl hexoside, probably an isomer of 38.The MS/MS of compound 40 showed two successive losses of pentose fragments (132 Da) followed by a hexose fragment (162 Da) to yield a coumaroyl fragment from the [MÀ H] À ion and was thus putatively identified as p-coumaric acid dipentosyl hexoside.Whereas 16 and 40 were only identified in the leaves and 24 only in the stems, 29 and 38 were identified in both the leaves and stems.Compound 36 from the leaf extract showed a major fragment at m/z 223.0609 (C 11 H 11 O 5 À ) from the loss of a hexose from the [MÀ H] À ion.This fragment was identified as sinapic acid by 1 H-NMR inspection and 36 was confirmed to be (Z)sinapic acid O-β-D-glucopyranoside (Table 2).
Compounds 7 and 71 were identified as gallic and salicylic acids, respectively, by comparing their profiles with those of authentic standards.Additionally, the gallic acid derivatives 6 and 15 were identified as β-glucogallin and methyl gallate respectively by 1 H-NMR inspection (Table 2).Methyl gallate was implicated as the major compound responsible for the antiplasmodial activity of the leaf extracts of V. xanthophloea. [37]The major fragment in the MS/MS spectra of compounds 20 and 37 was C 9 H 9 O 5 À (m/z 197.04) coming from syringic acid.From the 1 H-NMR spectrum of the isolated fraction, compound 20 was confirmed to be erigeside C (Table 2) whereas there was insufficient information for the complete assignment of 37 and it could only be putatively identified as a syringic acid derivative.Compound 45 showed a major fragment of C 8 H 7 O 4 À at m/z 167.0353 corresponding to the product ion [MÀ HÀ 162] À and was putatively identified as a vanillic acid hexoside.Whereas 37 and 71 were identified from only the leaves and stems respectively, compounds 6, 7, 15 and 20 were identified in both the leaf and stem extracts.

Other Phenyl Derivatives
Compounds 30, 34 and 74 were identified from the stems only and were confirmed by 1 H-NMR data inspection to be syringin, kelampayoside A and phloretin-3',5'-di-C-β-glucopyranoside, respectively (Table 2).Compound 31 showed a loss of a hexose in its MS/MS spectrum to yield a fragment at m/z 165.0560 (C 9 H 9 O 3 À ).Its NMR was not clear enough but showed signals at δ H 7.22 (d, J = 8.4 Hz, 2H) and 6.79 (d, J = 8.4 Hz, 2H) pointing to the presence of an aromatic core, and an anomeric signal most likely from the hexose at δ H 5.05 (d, J = 7.6 Hz, 1H).This was hence putatively identified as a phenyl hexoside derivative.Compound 82 appearing in only the stems was putatively identified as the lignan derivative, pinoresinol hexoside by comparing its MS/MS pattern with that reported in literature. [38]her Compounds Two cyanogenic derivatives ( ).The presence of the galloyl moiety was confirmed by the appearance of a signal at δ H 7.21 (s, 2H) in the 1 H-NMR spectrum.Similarly signals for the 3-hydroxy-3-methylbutanenitrile moiety were present at δ H 5.66 (s, 1H), 1.56 (s, 3H) and 1.52 (s, 3H).However, the 1 H-NMR was not clear enough for conclusive identification and 42 was putatively identified as a cyanogenic derivative.The closest structure with similar characteristics found in the literature is linamarin gallate isolated from the Nigerian mistletoe Loranthus micranthus (Linn.). [39]Regardless of the position of the galloyl substituent on the core structure of the nitrile glucoside as shared by both compounds, 42 is potentially a new compound.
An additional alkyl alcohol glucoside (8) with a novel structure in nature was detected in the leaves showing the formate adduct, [M + HCOOHÀ H] À , at m/z 297.1191 corresponding to a compound with the molecular formula C 10 H 20 O 7 .Its 1 H-NMR exhibited resonances typical of a β-glucose in addition to aliphatic singlets at δ H 1.39 (s, 3H) and 1.40 (s, 3H) among other signals.While highlighting the novelty of this compound, additional physical and chemical data is needed for its complete characterisation.Another metabolite (10) whose formate adduct at m/z 295.1034 corresponds to a compound with molecular formula C 10 H 18 O 7 was also isolated.This compound shared some similar MS/MS fragments with 9 including the major fragment at m/z 161.046 (C 6 H 9 O 5 À ) pointing to a closeness in their structures.With no exploitable 1 H-NMR data, 10 was putatively identified as an alkyl glucoside with its structure probably being similar to β-D-glucopyranosyl-2-methylpropanoate isolated from the flowers of Moricandia arvensis [40] or related analogues.

Conclusions
To the best of our knowledge, this paper provides the first characterisation of the chemical profile of V. gummifera, a plant with importance in traditional medicine.Over 100 metabolites have been identified and their occurrence in the leaves and/or stems has been reported.Our methodology led to the characterisation of a vast number of metabolite classes.Several compounds were present in both the leaf and stem extracts albeit differing in their abundances in the two tissues.However, eucomic acid and piscidic acid were some of the main compounds differentiating the leaf and stem profiles, being identified in only the latter.Pinitol, a cyclitol known to be an anti-diabetic agent, was the major compound in both leaves and stems.Other metabolites classes included amino, organic, and phenolic acids, in addition to flavonoid derivatives of flavonols, flavanols, flavones, flavanones and flavanonols.Many compounds described in our study are known plant natural products with valuable biological potential.The structures of the two potentially novel compounds that were isolated could not be confirmed and there is thus need for more elaborate analysis to fully characterise these structures.Additional studies are also necessary to provide an even more in-depth fingerprint of the plant's metabolome for instance the less polar and nonpolar compound profiles as well as the chemical profile of other plant parts such as the fruits (pods and seeds).Furthermore, studies are needed to investigate the bioactivity of V. gummifera extracts or its prominent compounds, and to ascertain which compounds contribute to the reported vernacular uses of the plant.

Figure 1 .
Figure 1.Total ion chromatograms (negative ion mode) of the leaves and stems of V. gummifera extracted with H 2 O:CH 3 OH (4 : 1 v/v).
assigned based only on the UHPLC-MS data.Nevertheless, the attachment of the malonyl group in compounds 41, 73 and 76 was confirmed to be on the 6-position of the sugar from the downfield shift in the chemical shifts of the protons at this position.For instance, a shift from δ H 3.66/3.54ppm in Quercetin 3-O-β-D-glucopyranoside (70) to δ H 4.16/4.08ppm in 76.Quercetin 3-O-(6''-O-malonyl)-β-D-glucopyranoside (76) was suggested to be the major flavonoid in the leaf crude extract and appeared as the most abundant phenolic compound in the chromatogram of the extract.Compound 73, also appeared in spectra from both the leaf and stem extracts and was similar to 76 but possessed a galactose instead of a glucose, with the same malonylation pattern.The compounds 78 and 84 could only be putatively identified as isomers of quercetin 3-Omalonylhexoside as their 1 H-NMR data was not obtained.
[M-2H-sugars] À at m/z 314 (as compared to the ion [MÀ HÀ sugars] À at m/z 315) in their MS/MS indicating the presence of an isorhamnetin aglycone with a 3-O-glycosylation.Compound 85 was confirmed to be isorhamnetin 3-O-β-Dglucopyranoside by comparison with an authentic standard.The MS/MS spectrum of compound 79 showed an ion at m/z

Figure 4 .
Figure 4. Comparison of the quantities of selected compounds from leaves and stems of V. gummifera using the t-test.Bars represent the mean values, and the error bars represent the standard deviation.3HHE-3-Hydroxyheteroendrin.*p < 0.05 and ***p < 0.001.

9 and 42 )
were identified.The MS/ MS of the [MÀ H] À ion at m/z 428.1193 of compound 42, only present in the leaf extract, showed product ions at m/z 401.1093 (C 17 H 21 O 11 À ) for a loss of HCN and at m/z 313.0571 (C 13 H 13 O 9 À ) for the loss of 2,3-dihydroxy-3-methylbutanenitrile as in 9 alongside an additional loss of a galloyl moiety shown by the fragment at m/z 151.0044 (C 7 H 3 O 4 À

Table 1 .
Compound profile of the leaf and stem extracts of V. gummifera in water:methanol (4 : 1).

Table 1 . continued
5 À 0.71 93.0351, 119.0508, 151.0042, 169.0148, 177.0198 Compound 11 eluted at rt 7.64 min and its MS showed an [MÀ H] À ion at m/z 255.0507 corresponding to a compound with the molecular formula C 11 H 12 O 7 .The MS/MS of the parent ion afforded product ions at m/z 165.0562 (C 9 H 9 O 3

Table 2 .
1H-NMR data of selected compounds from the leaves and stems of V. gummifera.
) as epigallocatechin gallate.Based on key fragments in the MS/MS data, including those at m/z 125.0247, 169.0149 (C 7 H 5 O 5

Table 2 )
. Compound 87 showed an [MÀ H] À ion at m/z 317.0667 corresponding to a compound with the molecular formula C 16 H 14 O 7 for a mono-methylated taxifolin.The compound was hence putatively identified as methyltaxifolin.It is worth noting that the MS/MS of the parent ion of 87 afforded the product ions at m/z 152.0121 (C 7 H 4 O 4 À ) and 165.0572 (C 9 H 9 O 3 À