Phytochemical Analysis of Acacia auriculiformis Pericarp through Liquid Chromatography-Electrospray Ionization-Mass Spectrometry

: Phytochemicals in Acacia auriculiformis Pericarp Acacia auriculiformis , widely used for afforestation, produces large amount of fruits which has no specific use other than as fuel for cooking by the ethnic people. The aim of our study is to determine chemical constituents with their definite proportion from different solvent extracts of Acacia auriculiformis pericarp at different maturity stages. Qualitative phytochemical analyses of three different solvents extracts viz . methanolic, 80 % ethanolic and aqueous of nine different maturity stages pericarps were screened to identify the presence of secondary metabolites. Quantitative analyses were performed through standard biochemical procedures to obtain the amount of the secondary metabolites. Finally, liquid chromatography-electrospray ionization-tandem mass spectrometry analysis was performed to identify different fractions of the major phytochemical groups. The biochemical assay of the preparation helped to identify saponin, phenols, tannin, flavonoid, alkaloid, steroid, terpenoids, glycosides, cardiac glycosides and reducing sugar as the major groups. Methanolic extract of the 9 th maturity stage pericarp contains the highest amount of saponin and a fairly good amount of phenol, flavonoids, tannin and proanthocynidin. With the help of liquid chromatography-electrospray ionization-tandem mass spectrometry analyses 7 triterpenoid saponins (acaciasides) with their relative percentages, 22 flavonoids, 8 tannins and 1 phenolic rich fraction-galloyl glucose were detected. Among the 7 triterpenoid saponins, acaciasides A and B are worked out as the major components. A detail toxicological study may help to establish it as a substitute of conventionally used piscicides as well for other pharmaceutical purposes.

Acacia auriculiformis (A. auriculiformis), commonly called Northern black wattle and in Bengali as akashmoni, has been used widely in afforestation for its rapid growth rate. It produces large amount of fruits which have no particular utility other than as fuel by the ethnic people and that too causing coughing to the users. So, there is an availability of the fruit in plenty from a small area at a time. Research on chemical component of the fruit is still in its infancy; only 7 compounds have been identified till date [1][2][3] . Limited general pharmacological activities of the fruit ingredients have been established [4][5][6][7][8] . However, it is neither used in medicinal purpose at industrial scale nor explored for other usages. Though the whole plant with fruit is famous as one of the folk medicines in its natural habitat Australia, there is a dearth of information about detailed phytochemical analysis of the fruit. It is a general feature that chemical composition and percentage (%) content of the ingredients of a plant part varies with the plant's habitats, maturity stages and extraction procedures [9][10][11] . At full maturity, the pods are split, the seeds are dispersed and the pericarps are shed off from the plant. It becomes easier to collect the fallen fruit parts lying underneath the plant. Keeping all these considerations in mind a comprehensive study has been apprehended for phytochemical analyses of Acacia pericarp to explore the possibility of its utilization. The present study is thus aimed to analyse the phytochemical contents of A. auriculiformis pericarp at different maturity stages.

Collection of plant material:
A. auriculiformis (A. Cunn. ex Benth.) pericarps of 9 different maturity stages were collected at every 15 d intervals, ranging from small and green to ripen. The samples were collected from Sonajhuri forest (23º41´36´´N 87º40´33´´E), a part of Ballavpur wildlife sanctuary adjacent to Santiniketan, Bolpur, West Bengal, India. The collected pericarps were cleaned, sundried and pulverized separately in powdered form and sieved through uniform mesh no. 40.

Preparation of Acacia pericarp extracts:
Solvent extractions of 9 different stages of pericarp were done with petroleum ether in 60°-80° followed with Chloroform (CHCl 3 ). The aqueous extract were done soaking the pericarp powder in distilled water and subsequently filtered through Whatman filter paper No.1 and evaporated through rotary evaporator. Both alcoholic (methanolic and 80 % ethanolic) extractions were done in soxhlet apparatus at 40°-50° for 4 h and then evaporated through rotary evaporator.

Quantitative determination of phytochemicals:
Total Saponin Content (TSC): A volume of 1 ml methanolic extract (1 mg/ml) was added to same volume 8 % vanillin (w/v) and incubated in ice-water bath. After adding 8 ml 77 % (v/v) sulphuric acid, the test tube was placed in hot water bath at 60° for 10 min. After cooled down in ice-water bath for 5 min, absorbance was measured at 544 nm by UVvisible spectrophotometer (Beckman Coulter DU730). Diosgenin standard curve was used. TSC was expressed as mg Diosgenin Equivalents (DE) per gram (g) of extract [16] .

Total Phenolic Content (TPC):
A volume of 1 ml methanolic extract (1 mg/ml) was taken with 9 ml distilled water. Then 1 ml of Folin and phenol reagent was added and mixed thoroughly. After 5 min incubation, 10 ml 7 % Sodium carbonate (Na 2 CO 3 ) solution was added and made the volume up to 25 ml with distilled water. After incubation for 90 min absorbance was recorded at 750 nm. TPC were calculated using gallic acid as standard and expressed as mg Gallic Acid Equivalent (GAE) per g of extract [17] .
Total Tanin Content (TTC): Total tannin was determined with small modification of Folin-Ciocalteu method. A volume of 0.1 ml methanolic extract (1 mg/ ml) was taken with 7.5 ml distilled water and 0.5 ml of Folin-Ciocalteu phenol reagent. Thereafter, 1 ml Na 2 CO 3 solution (35 %) was added and adjusted the volume to 10 ml with distilled water. After incubation for 30 min, absorbance was measured at 725 nm. TTC were calculated using tannic acid as standard and expressed as Tannic Acid Equivalent (TAE) per g of extract [18,19] .

Total Flavonoid Content (TFC):
Aluminium chloride colorimetric assay has been used to quantify TFC. A volume of 1 ml methanolic extract (1 mg/ml) was taken with 4 ml distilled water and 0.3 ml 5 % Sodium nitrite (NaNO 2 ). After 5 min incubation, 0.3 ml 10 % aluminium chloride was added. Thereafter, 2 ml of 1 M Sodium hydroxide (NaOH) was added and total volume was made 10 ml with distilled water. Absorbance was taken at 510 nm. TFC was calculated using quercetin as standard and expressed as mg of Quercetin Equivalents (QE) per g of extract [20] .

Proanthocynidin Content (PC):
Total condensed tannin was evaluated by slightly modified method of Tounsi et al. [21] . In brief, 50 µl of the methanolic solution (1 mg/ml) was mixed with 2 ml of 4 % methanol vanillin solution and 450 µl concentrated sulphuric acid. After 15 min, absorbance was read at 527 nm and results were expressed as mg CE/g of extract using catechin methanol as standard solution.

Total Alkaloid Content (TAC):
A volume of 1 ml water extract (1 mg/ml) was mixed with same amount 2 N Hydrochloric acid (HCl) and filtered. Then transferred to separatory funnel and washed three times with 10 ml CHCl 3 . pH was adjusted to neutral with 0.1 N NaOH. After that 5 ml of Bromocresol Green (BCG) solution and 5 ml of phosphate buffer were added to the solution and shaken. The formed complex was then extracted with 1, 2, 3 and 4 ml CHCl 3 by vigorous shaking. Diluted in 10 ml with CHCl 3 and absorbance was determined at 470 nm. TAC was expressed as mg of Atropine Equivalent (AE)/g of extract using atropine as standard [22] .

Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS) analysis:
An untargeted LC-ESI-MS/High-Performance Liquid Chromatography (HPLC)-MS analysis was performed to identify the saponin fractions and other chemical components. A comparison of relative % of the saponin fractions was also subsequently done from the MS data. This analysis was performed on Waters ACQUITY ® TQD equipped with Sunfire C 18 column of 250×4.6 mm, 5 μm. 'Xevo TQ-S micro' quadrupole ion trap mass spectrometer fitted with high performance spray dual-orthogonal Atmospheric Pressure Ionization (API) sources. Ion transfer optics with Ultraperformance Liquid Chromatography- Tandem

Statistical analysis:
Statistical analyses were done through Analysis of Variance (ANOVA) test with repeated measurements (n=5) using Tukey method in Minitab 17 software.

RESULTS AND DISCUSSION
A. auriculiformis fruit needs 135 d on an average from formation to become fully matured and dried up in vivo. The physical characteristic of the fruit have been recorded at every 15 d intervals of maturity period as shown in Table 1. Analysis for 11 major chemical groups resulted in confirmative presence of 10 groups viz. saponin, phenols, tannin, flavonoid, alkaloid, steroid, terpenoids, glycosides, cardiac glycosides and reducing sugar in all stages of pericarps as shown in Table 2. Among the solvent extracts the methanol extract has been resulted the highest yield as shown in Table 1.
Based on this result only the methanolic extract has been selected for further quantitative analyses. The standard curves for biochemical assays are depicted in the fig.  1. Although the pericarp extracts from all the maturity stages are having similar chemical components, their concentration varied considerably as shown in Table 3. While the chemicals such as phenol, flavonoids, tannin, proanthocynidin and alkaloid were found maximum in 1 st stage pericarps, 2 nd highest amounts were obtained in the 9 th maturity stage. Saponin concentration of the extracts was estimated >39 % in all the maturity stages with slightly higher in the stage nine. The phenolics ranks 2 nd highest in content. Although the first maturity stage contains good amount of secondary metabolites, it estimates a less biomass content in comparison to that of the 9 th maturity stage pericarps. For this reason the methanolic extract of 9 th maturity stage pericarp was selected for LC-ESI-MS analysis.
Tannins of eight different components have been detected first time in the pericarp extract through negative ion ESI-MS spectra ( fig. 2, fig. 3, fig.5 and Table 4). In Ultraviolet (UV) chromatogram, epigallocatechin has shown earlier retention time than that of galloepicatechin. So these are established as isomers. In LC-UV chromatogram procyanidin dimer-II has an earlier retention time, followed by procyanidin dimer-III and procyanidin dimer-I has longer retention time. These three fractions are also considered as isomers. In UV chromatogram catechin has an earlier retention time than that of epicatechin. These are exhibiting isomeric feature. There is no earlier report of tannins from A. auriculiformis but reported from other species of Acacia [28] .
This is for the 1 st time, one phenolic rich fraction-gallyl glucose has also been detected in A. auriculiformis ( fig.  3 and fig. 5) (Table 4). In other Acacia species however, it had been reported earlier [28] .
Proanthocyanin like compounds have been identified as an amalgamation of broad peak on UV chromatogram but no ESI-MS spectra of those has been detected because of their molecular weights beyond the range of 2000 m/z. A small % of proanthocyanidin has been quantified in the methanolic extract (Table 3). To get it in ESI-MS, depolymerization of the compounds is required. Those compounds, not reported earlier from A. auriculiformis, are needed to analyse by Nuclear Magnetic Resonance (NMR) spectroscopy for further detailing.
One triterpenoid saponin (acaciaside) (7) which was previously reported from seed [29] has now been identified from the pericarp of A. auriculiformis ( fig. 3) ( Table  4). Acaciamine and five other saponins (1-6) have also been detected in pericarp, were previously detected by Fast Atom Bombardment-MS (FAB-MS) [1][2][3] . However, in the present study relative % of those compounds has been estimated to come to the inference that among the seven triterpenoid saponin, acaciaside A and acaciaside B are the major components as shown in Table 5.   Other than pharmacological applications [4][5][6][7][8]30] , in aquaculture saponin and tannin containing plant products are used as piscicides for prestocking pond management [31] . Since Acacia pericarp contains good amount of saponin and tannin, it has been recommended very recently [32] as a better a substitute of the conventionally used piscicide Mohua (Bassia latifolia) oil cake which also contains saponin as the principal ingredient [32] .
It can be concluded that highest amount of saponin (41 %) with other phytochemicals are present in the methanolic extract of the 9 th stage pericarps. LC-ESI-MS/MS analyses revealed 7 triterpenoid saponins, 22 flavonoids, 8 tannins and 1 phenolic rich compoundgallyl glucose in this preparation. Among these components acaciaside A is present in maximum concentration, followed by acaciaside B.