Chemical and pharmacological evaluation of the non-flowering aerial parts of Acacia modesta Wall. cultivated in Egypt

Acacia modesta Wall. (A. modesta), often recognized as Phulai, is belonging to family Fabaceae and sub-family Mimosaceae. A. modesta has many beneficial uses. Leaves, wood, flowers, and gum of A. modesta have been used frequently for multiple therapeutic purposes. The chemical investigation of butanol fraction of A. modesta non-flowering aerial parts yielded Vitexin-2′′-β-D-glucopyranoside and Apigenin-6,8-di-C-β-D-glucopyranoside in a flavone mixture as well as (β-D-glucopyranosyl (1-3)-β-D-glucopyranosyl)-3-β-hydroxy-11-oxo-olean-12-en-28-oic acid) an oleanane-type triterpenoidal saponin. Metabolite profiling via ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) of the ethyl acetate fraction resulted in recognizing of eighteen compounds tentatively compared with previously published data. Quantitative measurement of the overall value of flavonoids of A. modesta was found to be 2.824 μg/100 μg ± 0.01 calculated as quercetin. The acute toxicity study of the ethanol extract proved that the plant under investigation is safe and nontoxic to the male albino mice used. The anti-hyperglycemic activity of the ethanol extract performed on type 2 diabetic rats proved that the most potent dosage was 200 mg/kg b. wt. after 4 and 4 weeks of treatment respectively compared to metformin. Furthermore, evaluation of the hepato-protective activity of the ethanol extract of the plant under investigation showed that the most potent extract was with a dose level of 200 mg/kg b. wt. after 3 and 10 days of continuous treatment compared to silymarin. It can be concluded that A. modesta Wall. cultivated in Egypt could be used as a promising anti-diabetic agent and a hepato-protective agent against hepatocellular damage induced by hepatotoxins.

for various medical applications such as dysentery, leprosy, and cough [5]. Different parts of A. modesta have been previously investigated in several pharmacological activities such as antibacterial, antifungal, antihyperglycemic, analgesic, anti-inflammatory, anti-platelet aggregation, anti-termite, antioxidant, brine shrimp cytotoxicity, hemagglutination, insecticidal, phytotoxic, and spasmolytic activities. Reports on A. modesta aerial parts exposed the presence of flavonoids, alkaloids, terpenoids, and tannins [6]. Tracing the available current literature, there is scarce information on the chemical and pharmacological characters of A. modesta cultivated in Egypt. Therefore, the following work has been planned to examine the main active principles and to screen the biological activities to find out the potential benefits of the plant under investigation.

Collection of plant material
The non-flowering aerial parts of A. modesta have been collected in August 2015 from Giza zoo garden, then identified and authenticated by the taxonomist Dr. Threse Labib, specialist in the central gardening administration, Orman garden, Giza, Egypt.

Isolation and identification
Extraction of aerial parts of A. modesta About 500 g of the air-dried powdered non-flowering aerial parts of A. modesta have been exhaustively extracted with 70% methanol, and filtered and then concentrated using rotary evaporator R-3 (Buchi, A.G., Switzerland). The crude extract (100 g) has been mixed with distilled water (500 ml) and then has been partitioned with the following solvents; n-hexane, methylene chloride, ethyl acetate, and n-butanol saturated with water several times and concentrated to give 3 g, 3 g, 8 g, and 17 g, respectively. TLC profile of the n-butanol fraction prompted to focus on its purification. The dried extract of the n-butanol fraction (17 g) was delivered over a silica gel (60) glass column and eluted with methylene chloride, and the polarity of the column was stepwise increased by gradient addition of methanol. Similar fractions of each 50 ml were collected together. Fractions eluted with solvent strength (methylene chloride: methanol 70:30) offered fraction 1 (1.08 g) which was purified using reversed-phase silica column and partitioned with water, then increasing polarity via addition of methanol gradually. Similar fractions (5 ml) each, eluted with (30% methanol), were concentrated to offer compounds 1 and 2 (50 mg) (Supplementary file: Figures S1-S7). Similar fractions eluted from the main column using polarity (methylene chloride: methanol 60: 40) offered fraction 2 (1.37 g) which has been separated via a sephadex LH-20 glass column, using absolute methanol to offer compound 3 (30 mg) (Supplementary file: Figures S8-S15).

Materials for pharmacological screening Extract preparation
Non-flowering A. modesta aerial parts have been left to dry in the air, and grinded and then extracted with absolute ethanol for hepato-protective and anti-diabetic activities. The extracts were dried using rotary evaporator R-3 (Buchi, A.G., Switzerland).

Animals
Adult male albino Sprague dawley rats (130-150 g) have been used for the hepato-protective and anti-diabetic activities. Mice (25-30 g) have been used for the toxicological study. Both have been brought from the animal house colony belonging to the National Research Centre, Dokki, Giza, Egypt. Experiments and animal procedures have been carried out in compliance with the Ethics Committee of the National Research Centre following the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. After each experiment, animals would be sacrificed by cervical dislocation via light anesthesia with ether.

Quantitative estimation of flavonoid content
A spectrophotometric method using aluminum chloride was followed for total flavonoid content estimation based on the measurement of the intensity of the color developed when flavonoids complexed with aluminum chloride, at λ max 415 nm using the standard quercetin (Sigma-Aldrich chemicals, Co., St. Louis, MO, USA). The assay was done in triplicate. The calibration curve was prepared using quercetin solution at a concentration of 5 to 100 μg/ml in methanol [7].

Metabolite profiling via UPLC-ESI-MS
The sample solution of the ethyl acetate fraction of A. modesta (100 μg/mL) non-flowering aerial parts was prepared, the chromatographic separation was conducted on an Acquity UPLC system (Waters) equipped with a reversed-phase BEH C18 column (50 × 2.1 mm, particle size 1.7 μm; Waters), and the analysis was carried out using a binary elution system. Mass spectra were detected between m/z 100-1000 in negative and positive ionization modes on a XEVO TQD triple quadrupole mass spectrometer (Waters Corporation, Milford, USA) [8]. Compounds were recognized tentatively by analyzing their mass data using the Maslynx 4.1 software and making a comparison between their retention time (RT) and mass spectrum with previously reported data.

Acute toxic activity
A preliminary experiment was done to determine the minimal dose that kills all animals (LD 100 ) and the minimal dose that fails to kill any animal. Several doses at equal logarithmic intervals were chosen in between 2 doses; each dose was injected in a group of 10 animals by subcutaneous injection total of forty mice. The mice were then observed for 24 h, and symptoms of toxicity and mortality rates were recorded and LD 50 was calculated [9].
Anti-hyperglycemic activity Induction of hyperglycemia Type 2 diabetes mellitus has been induced via alloxan injection [10]. Then, the measurement of the blood glucose levels was tested after 72 h to ensure hyperglycemia [11].

Induction of liver damage
Liver damage has been ensured by injection of toxic carbon tetrachloride (CCl 4 ) dissolved via liquid paraffin (5 ml/kg of 25%) via intra-peritoneal route, and blood samples were withdrawn for the biochemical study [12,13]. Serum aspartate amino-transferase (AST), alanine amino-transferase (ALT) [14], and serum alkaline phosphatase (ALP) [15] were isolated and then analyzed. The data have been analyzed using Student's t test [16].

Phytochemistry
Phytochemical investigation of the butanol fraction of A. modesta non-flowering aerial parts has yielded three compounds. The chemical structures of the three compounds have been characterized using elemental analysis, 1 H & 13 C NMR correlating with the existing literature data [17].

Quantitative estimation of flavonoid content
The total flavonoid content of A. modesta Stock was found to be 2.824 μg/100 μg ± 0.01 calculated as quercetin.

UPLC-ESI-MS
Tentative identification of the ethyl acetate fraction of A. modesta non-flowering aerial part metabolites has led to the identification of eighteen compounds which were distributed into two major categories: flavonoids and phenolic acids. The compounds were analyzed depending on their molecular weight, mass fragmentation, and compared with previously revealed data to the extent of our knowledge. It is important to mention that this is the first study for evaluating any fraction of A. modesta nonflowering aerial parts via UPLC-ESI-MS analysis (Fig. 2).

Acute toxicity study (LD 50 )
It was found that the median lethal dose of ethanol extract of A. modesta non-flowering aerial parts (LD 50 ) is 7.1 g/kg b. wt., so it is possible to conclude that the LD 50 of alcoholic extract of A. modesta is safe and nontoxic as LD 50 greater than 50 mg/kg b. wt. is nontoxic [19].

Anti-hyperglycemic activity
Results revealed that the ethanol extract of A. modesta showed a potent anti-diabetic activity at the two tested doses. From the examination of the two dose levels at 100 mg/kg b. wt. and at 200 mg/kg b. wt., respectively, of  Figure 2 shows two chromatograms obtained by ultraperformance liquid chromatography-electrospray-mass spectrometry of the ethyl acetate fraction of A. modesta non-flowering aerial parts; the one from the left shows the total ion chromatogram at the positive mode and the chromatogram at the right shows the total ion chromatogram at the negative mode  . 3).

Hepato-protective activity
From the examination of the two doses 100 and 200 mg/ kg b. wt., respectively, of the ethanol extract of A. modesta non-flowering aerial parts cultivated in Egypt in comparison with silymarin as a standard hepatoprotective drug, blood samples were collected at zerotime, 1 week before CCl 4 injection, 3 days, and then 10 days after CCl 4 injection. Results showed a substantial increase in serum levels of ALP, ALT, and AST in nontreated animals, i.e., control (3 days and then 10 days after CCl 4 injection). On the other side, the pretreated animals with the two tested doses respectively revealed a great decrease in the previously mentioned enzymes.

Discussion
Genus Acacia belongs to the family Fabaceae. Acacia species were being used in the beginnings of civilization as traditional medicinal herbs which are of considerable medicinal and economic value. Therefore, investigation of non-flowering aerial parts of A. modesta cultivated in Egypt has led to the following findings: The chemical investigation of butanol fraction of A. modesta nonflowering aerial parts yielded three compounds. According to the chromatographic properties, compound 1 was expected to be apigenin derivative [17], confirmed by 1 H NMR spectrum and 13 C NMR spectrum showing typical signals of the apigenin aglycone moieties giving the confirmation of vitexin-2′′-β-glucopyranoside that has been separated for the first time from genus Acacia. In the same spectra of 13 C-NMR, another compound having signal strength close to 1:2 of the first compound, the aglycone moiety of the second compound seems to be  identical to the first one except in carbon number 6 of the aglycone. Comparing the chemical shift of the carbon spectra, compound 2 was confirmed to be apigenin-6,8-di-C-β-D-glucopyranoside (vicenin II), that was separated for the first time from genus Acacia. According to the chromatographic properties, 1 H and 13 C NMR spectrum of compound 3 revealed 30 carbon signals of the aglycone that have been sorted into nine methylenes, nine quaternary carbons, seven methyls, and six methines by DEPT experiments. This data suggested that it is a triterpene of an oleanane-type, the carbonyl function appears at δ 200.1 corresponding to ketone function at C-11. The previous data in addition to the other carbons in the spectra with the 2D-NMR experiments (H-H COSY, HSQC, DEPT, HMBC) came in complete accordance with the previously published data of β-D-glucopyranosyl (1-3)-β-D-glucopyranosyl)-3-βhydroxy-11-oxo-olean-12-en-28-oic acid which was separated for the first time from family Fabaceae. Eighteen compounds were tentatively identified using (UPLC-ESI-MS) analysis of the ethyl acetate fraction compared with previously published references presented in Table 1 and are discussed in the following section:  Figure S21). Compound 6 RT 8.67; it has been tentatively identified as apiin with the molecular Fig. 6 Effect of alcoholic extract of Acacia modesta Wall. non-flowering aerial parts and silymarin on serum alanine amino-transferase ALT level in liver-damaged rats. Figure 6 presents serum alanine amino-transferase ALT level in liver-damaged rats with the collection of blood samples at zero time, 7 days before liver damage with continuous administration of the two doses of A.   Figure S35). Eventually, the ethanol extract of A. modesta Stocks of the two tested doses of 100 mg/kg b. wt. and 200 mg/kg b. wt. decreased the blood sugar level of AITD after 4 weeks by 21.1% and 28%, respectively, when compared to the reference drug metformin (44.5%). Also, the ethanol extract of A. modesta Stocks decreased the blood sugar level of AITD after 8 weeks by 42.1% and 46.1% in the two tested doses, respectively, when compared to the reference drug metformin (67%). It can be concluded that the most potent extracts were A. modesta (200 mg/ kg b. wt.) followed by A. modesta (100 mg/kg b. wt.) after 4 weeks and 8 weeks with a potency 62.9% and 68.8% and a potency 47.6% and 62.8%, respectively, compared to metformin (100 mg/kg b. wt.) which is considered 100% potent. It is important to mention that levels of blood glucose were tested in normal and AITD, given ethanol and ethanol: water (1:1) leaf extracts of A. modesta cultivated in India, at two dose levels of 100 and 300 mg/kg/day [36]. Nevertheless, this is the first report for the anti-diabetic activity of the plant cultivated in Egypt. Regarding the hepato-protective activity, a remarkable reduction in the levels of serum enzymes was observed. After treatment with the two doses of 100 mg/ kg b. wt. and 200 mg/kg b. wt., respectively; the percent of reduction were found to be 42.1%, 56% for AST; 57.7%, 61.2% for ALT; and 58.3%, 65% for ALP compared with the reference drug silymarin 67.8%, 59.6%, and 75.7% for AST, ALT, and ALP, respectively, after 3 days. While after 10 days, percent of reduction was found to be (53.7%, 66% for AST and 64.7%, 67.8% for ALT and 69.4%, 73% for ALP compared with the reference drug silymarin 75.7%, 75.7%, and 90% for AST, ALT, and ALP after treatment with the two tested doses, respectively. It could be concluded that the most potent extract was that of dose level 200 mg/kg b. wt. with percent of potency (82.6%, 102.7%, and 85.9%) for AST, ALT, and ALP, respectively, after 3 days of treatment. While the percent of potency was found to be (88%, 89.6%, and 81.1%) for AST, ALT, and ALP, respectively, after 10 days of treatment compared to the standard drug silymarin (which is considered 100% potent). It is important to mention that the hepato-protective activity of the stem bark of A. modesta was previously investigated using 80% methanolic extract of the Pakistanian cultivated species crude extract [37]. This is the first report for the hepatoprotective activity of A. modesta cultivated in Egypt.

Conclusion
Acacia modesta Wall. cultivated in Egypt revealed the presence of a variety of phytochemical constituents and showed low toxicity profiles with high safety margins and valuable hypoglycemic, hepato-protective activities. This superior activity would be attributed to their high contents of phenolic components and flavonoids. Further investigation is recommended on the total extracts and individual components. Clinical trials should be performed in order to support the above investigation and to facilitate their pharmaceutical formulation.