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

Future Journal of Pharmaceutical Sciences volume 6, Article number: 122 (2020) Cite this article

Abstract

Background

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.

Results

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.

Conclusion

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.

Background

Traditional medicine is used globally and has significant economic benefits in both industrialized and developing economies using natural plants that are rich sources of active components, so knowledge about both the area’s plant diversity and local people’s medicinal uses is of utmost importance. Herbal plants play a key role in medicare and are therefore essential natural resources both for the traditional and innovative medicinal products [1]. Family Fabaceae is one of the fastest growing flowering plant families in the world, the third largest group of plants with 19,400 species, and has been categorized into 730 genera. Family Fabaceae plants supply a reliable and safe therapy for many diseases. Species of this family range from dwarf herbs to tropical rain forest massive trees [2]. Genus Acacia is a heterogeneous collection of ever more than 1000 species, most of which are trees, mainly found in Australia; others are found in South-East Asia [3]. Acacia species were first used in the beginnings of human civilization as traditional medicinal plants and have a really significant economic value [4]. Gum, leaves, flowers, and wood of A. modesta were used 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, anti-hyperglycemic, 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.

Methods

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 λmax 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 (LD100) 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 LD50 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].

Hepato-protective activity

Induction of liver damage

Liver damage has been ensured by injection of toxic carbon tetrachloride (CCl4) 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].

Results

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, 1H &13C NMR correlating with the existing literature data [17].

Vitexin-2′′-β-glucopyranoside (compound 1)

Brownish yellow amorphous powder, Rf: 0.53 (methylene chloride: methanol: distilled water 70:30:3). According to the chromatographic properties, this compound was expected to be an apigenin derivative [17] (Fig. 1).

Fig. 1
figure 1

Isolated compounds of Acacia modesta Wall. non-flowering aerial parts. Figure 1 shows the three compounds isolated from the butanol fraction of A. modesta non-flowering aerial parts, compounds 1 and 2 were isolated in a flavone mixture, while compound 3 was isolated in a pure form

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Apigenin-6,8-di-C-β-D-glucopyranoside (compound 2)

Yellow amorphous powder, Rf: 0.53 (methylene chloride: methanol: distilled water 70:30:3) (Fig. 1).

β-D-glucopyranosyl (1-3)-β-D-glucopyranosyl)-3-β-hydroxy-11-oxo-Olean-12-en-28-oic acid (compound 3)

Amorphous powder

It showed a positive test of Lieberman-Burchard test for terpenoid [18], Rf: 0.8 (methylene chloride: methanol: distilled water 70:30:3). The spectrum of 13C NMR showed 30 carbon signals (Fig. 1).

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 non-flowering aerial parts via UPLC-ESI-MS analysis (Fig. 2).

Fig. 2
figure 2

UPLC-ESI-MS analysis of Acacia modesta Wall. non-flowering aerial parts. Figure 2 shows two chromatograms obtained by ultra-performance 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

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Acute toxicity study (LD50)

It was found that the median lethal dose of ethanol extract of A. modesta non-flowering aerial parts (LD50) is 7.1 g/kg b. wt., so it is possible to conclude that the LD50 of alcoholic extract of A. modesta is safe and nontoxic as LD50 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 the ethanol extract of A. modesta non-flowering aerial parts cultivated in Egypt in AITD (Alloxan-induced type 2 diabetic rats) after 4 and 8 weeks of continuous treatment against standard anti-diabetic drug metformin with dose 100 mg/kg b. wt.: results showed a substantial decrease in blood glucose levels from 259.8 mg/dl in non-treated AITD to 209.1 mg/dl to 186.8 mg/dl in AITD treated with the two tested doses, respectively, compared with metformin (143.6 mg/dl). Furthermore, after 8 weeks of continuous treatment blood glucose levels was lowered from 268.4 mg/dl in non-treated AITD to 153.6 mg/dl to 139.9 mg/dl in AITD treated with the two tested doses, respectively, compared with metformin (85.4 mg/dl) (Supplementary file: Table S1) (Fig. 3).

Fig. 3
figure 3

Effect of the alcoholic extracts of Acacia modesta Wall. non-flowering aerial parts and metformin on blood glucose level in male albino rats. Figure 3 presents blood glucose level in male albino rats after treatment with two doses of A. modesta non-flowering aerial parts (100 and 200 mg/kg b. wt.) for 4 weeks and 8 weeks against standard drug metformin with the dose level of 100 mg/kg b. wt

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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 hepato-protective drug, blood samples were collected at zero-time, 1 week before CCl4 injection, 3 days, and then 10 days after CCl4 injection. Results showed a substantial increase in serum levels of ALP, ALT, and AST in non-treated animals, i.e., control (3 days and then 10 days after CCl4 injection). On the other side, the pretreated animals with the two tested doses respectively revealed a great decrease in the previously mentioned enzymes. Serum ALP, ALT, and AST levels in rats treated with the dose of 200 mg/kg b. wt. were (24.2, 21.2 KAU), (61.3, 51.9 u/l), and (65.9, 53.9 u/l) after 3 days and 10 days, respectively. This significant reduction could be comparable to that of silymarin in which the serum AST, ALT, and ALP levels were (48.2, 39.2 u/l) and (63.8, 39.1 u/l) and (16.8, 7.9 KAU) after 3 days and 10 days, respectively (Supplementary file: Table S2-S4) (Figs. 4, 5, and 6).

Fig. 4
figure 4

Effect of alcoholic extract of Acacia modesta Wall. non-flowering aerial parts and silymarin on serum aspartate amino-transferase AST level in liver damaged rats. Figure 4 presents serum aspartate amino-transferase AST level in the 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. modesta non-flowering aerial parts (100 and 200 mg/kg b. wt.) and then 3 days and 10 days after induction of liver damage with continuous administration of the two doses of A. modesta non-flowering aerial parts (100 and 200 mg/kg b. wt.) showing a great reduction of serum levels of AST after treatment with the two doses of A. modesta against standard drug silymarin with the dose level of 25 mg/kg b. wt

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Fig. 5
figure 5

Effect of alcoholic extract of Acacia modesta Wall. non-flowering aerial parts and silymarin on serum alkaline phosphatase enzymes ALP level in liver-damaged rats. Figure 5 presents serum alkaline phosphatase enzymes ALP 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. modesta non-flowering aerial parts (100 and 200 mg/kg b. wt.) and then 3 days and 10 days after induction of liver damage with continuous administration of the two doses of A. modesta non-flowering aerial parts (100 and 200 mg/kg b. wt.) showing a great reduction of serum levels of ALP after treatment with the two doses of A. modesta against standard drug silymarin with the dose level of 25 mg/kg b. wt

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Fig. 6
figure 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. modesta non-flowering aerial parts (100 and 200 mg/kg b. wt.) and then 3 days and 10 days after induction of liver damage with continuous administration of the two doses of A. modesta non-flowering aerial parts (100 and 200 mg/kg b. wt.) showing a great reduction of serum levels of ALT after treatment with the two doses of A. modesta against standard drug silymarin with the dose level of 25 mg/kg b. wt

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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 non-flowering aerial parts yielded three compounds. According to the chromatographic properties, compound 1 was expected to be apigenin derivative [17], confirmed by 1H NMR spectrum and 13C 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 13C-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, 1H and 13C 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-β-hydroxy-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:

Table 1 Identification of the metabolites of the ethyl acetate fraction of Acacia modesta Wall. non-flowering aerial parts
Full size table

Compound 1 RT 0.86; it has been tentatively proposed as a caffeic acid ester derivative with [M + H]+ of m/z 295 amu, because of showing similar peaks. The MS fragmentation shows the characteristic fragment ion of caffeic acid at m/z 136 (Supplementary file: Figure S16). Compound 2 RT 6.91; the molecular formula of this compound was found to be C21H20O11 (orientin). With the characteristic base peak at m/z 327 (Supplementary file: Figure S17). Compound 3 RT 7.75; it has been tentatively identified as coumaroyl diferuoyl spermidine, with the molecular formula of C36H41N3O8, with the deprotonated ion [M − H]− at m/z 642.2 (Supplementary file: Figures S18-S19). Compound 4 RT 8.27; with the precursor ion peak of [M − H]− at m/z 626.2. It can be tentatively supposed to be quercetin dihexose, with the characteristic aglycone at m/z 301.1, known for quercetin [(M − H)-2Hexose]− (Supplementary file: Figure S20). Compound 5 RT 8.48; could be tentatively assigned as apigenin-O-pentosyl hexoside with the molecular formula C26H28O14. The precursor ion peak [M + H]+ at m/z 565.2 and the [M + Na]+ ion at m/z 587 (Supplementary file: Figure S21). Compound 6 RT 8.67; it has been tentatively identified as apiin with the molecular formula C26H28O4 and [M + H]+ peak at m/z 565.2 with a molecular weight 564 with the characteristic base peak ion at m/z 433 due to the loss of a pentose unit [(M + H)+ − 132]+ and the [M + Na]+ ion at m/z 588.2 (Supplementary file: Figure S22). Compound 7 RT 8.89; it has been tentatively identified as myricetin-rhamnose malic acid was detected in –ve ESI mode showing a precursor ion peak at m/z 579 having the molecular formula C27H30O13. The base peak ion at m/z 463.1 appeared due to the removal of malic acid [(M − H) − 116]− at m/z 579 to give a myricetin-rhamnose moiety. A characteristic fragment of the aglycone myricetin appeared at m/z 316, also the [(M − H) + K]− ion at m/z 609. (Supplementary file: Figure S23). Compound 8 RT 9.02; exhibited a precursor ion at m/z 951.3 which is assigned for granatin B; (Galloyl-hexahydroxydiphenoyl-di-hexahydroxydiphenoyl-hexoside), hexa-hydroxy-diphenoyl (dehydro-ellagitannins of type III-tannins) (Supplementary file: Figure S24). Compound 9 RT 9.88; it is tentatively assigned as catechin trigallate with the precursor ion peak [M − H]− at m/z 745.3, showing a minor production [(M − H) − 152]− at m/z 593.2 due to removal of a gallic acid unit (Supplementary file: Figure S25). Compound 10 RT 10.20 is tentatively assigned as ellagic acid derivative showing a precursor ion at m/z 799.3 and the base peak ion at m/z 271.1 (Supplementary file: Figure S26). Compound 11 RT 11.80; with the molecular formula C27H28O17 and molecular weight 624, it can be recognized as kaempferol hexose glucuronide with a precursor ion peak [M − H]− at m/z 623.3 and the base peak ion fragment [(M + H)-hexose-Glucuronide]+at m/z 287.1 (Supplementary file: Figure S27-S28). Compound 12 RT 22.59; it has been tentatively identified as tricaffeoyl-quinic acid with the base peak ion at m/z 515 (Supplementary file: Figure S29). Compound 13 RT 22.84; it has been assigned as pentagalloyl hexoside as a hydrolysable tannin with [M + H]+ ion peak at m/z 993 and the base peak ion at m/z 496.4 (Supplementary file: Figure S30). Compound 14 RT 23.46; with the molecular formula of C27H28O18 and molecular weight 640 and the precursor ion peak [M + H]+ at m/z 641.2, it can be recognized tentatively as quercetin hexose glucuronide (Supplementary file: Figure S31). Compound 15 RT 23.69; it has been tentatively identified as an isomer of digalloyl hexose with the ion peak of [M + Na]+ at m/z 507.7 and the [M + K]+ ion at m/z 524.7 (Supplementary file: Figure S32). Compound 16 RT 24.35; it has been assigned as quercetin-tri-O-hexoside with the precursor ion peak [M + K]+ at m/z 827.7 and the base peak ion at m/z 303.1 corresponding to the aglycone quercetin (Supplementary file: Figure S33). Compound 17 RT 24.49 exhibited a precursor ion peak at m/z 623.2 and also an observance of galloyl moiety removal [(M − H) − 152]− m/z 469.4. Thus, this compound was tentatively identified as galloyl-valoneic acid bilactone (Supplementary file: Figure S34). Compound 18 RT 27.56 has been assigned as 7-O-methyl-delphinidin-3-O-(2′′galloyl)-hexoside with the precursor fragment at m/z 631.1 and characteristic ions of m/z 153 [(M + H)-delphinidin-hexose]+ due to loss of delphinidin and hexose moieties (Supplementary file: 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 hepato-protective 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.

Availability of data and materials

Data and materials are available upon request.

Abbreviations

ALP:

Alkaline phosphatase

ALT:

Alanine amino-transferase

AST:

Aspartate amino-transferase

UPLC-ESI-MS:

Ultra-performance liquid chromatography-electrospray ionization - mass spectrometry

+ve ESI:

Positive mode of electrospray ionization

A. modesta :

Acacia modesta

AITD:

Alloxan-induced type 2 diabetic rats

b. wt.:

Body weight

BEH C-18:

Ethylene-bridged hybrid carbon 18 reversed-phase

CCl4 :

Carbon tetrachloride

DEPT:

Distortion-less enhancement by polarization transfer

−ve ESI:

Negative mode of electrospray ionization

ext.:

Extract

H-H cosy:

Proton-proton correlation spectroscopy

HMBC:

Hetero-nuclear multiple quantum coherence

HSQC:

Hetero-nuclear single quantum coherence

LD50 :

Median lethal dose

R f :

Retention factor

RP:

Reversed-phase

RT:

Retention time

TLC:

Thin layer chromatography

References

  1. Ayyanar M, Silambarasan R (2015) An ethnobotanical study of medicinal plants in Palamalai region of eastern Ghats. India. J Ethnopharmacol 172:162–178

    Article  Google Scholar 

  2. Ahmad F, Anwar F, Hira S (2016) Review on medicinal importance of Fabaceae family. Pharmacol Online 3:151–156

    Google Scholar 

  3. Or K, Ward D (2003) Three-way interactions between Acacia, large mammalian herbivores and bruchid beetles. Afr J Ecol 41:257–265

    Article  Google Scholar 

  4. Subhan N (2016) Phytochemical and pharmacological investigations of Australian Acacia: an ethnomedicine-guided bioprospective approach. Charles Sturt University, Dhaka, Bangladesh, p 317 Available from: https://researchoutput.csu.edu.au/en/publications/phytochemical-and-pharmacological-investigations-of-australian-ac-3

  5. Bukhari IA, Khan RA, Gilani AH, Sagheer A, Arshad SS (2010) Analgesic, anti-inflammatory and anti-platelet activities of the methanolic extract of Acacia modesta leaves. Inflammopharmacology 18:187–196

    Article  Google Scholar 

  6. Sarwar W (2016) Pharmacological and phytochemical studies on Acacia modesta wall; a review. JPHYTO 5:160–166

    Google Scholar 

  7. Kalita P, Tapan B, Pal T, Kalita R (2013) Estimation of total flavonoids content (TFC) and antioxidant activities of methanolic whole plant extract of Biophytum sensitivum Linn. JDDT 3:33–37. https://doi.org/10.22270/jddt.v3i4.546 Available from: http://jddtonline.info/index.php/jddt/article/view/546

    Article  CAS  Google Scholar 

  8. Osama S, El Sherei M, Al-Mahdy DA, Bishr M, Salama O (2019) Effect of salicylic acid foliar spraying on growth parameters, γ-pyrones, phenolic content and radical scavenging activity of drought stressed Ammi visnaga L. plant. Ind Crop Prod 134:1–10. https://doi.org/10.1016/j.indcrop.2019.03.035

    Article  CAS  Google Scholar 

  9. Kärber G (1931) Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Archiv f Exper Pathol u Pharmakol 162:480–483. https://doi.org/10.1007/BF01863914

    Article  Google Scholar 

  10. Eliasson SG, Samet TM (1969) Alloxan induced neuropathies lipid changes in nerve and root fragments. Life Sci 8:493–498

    Article  CAS  Google Scholar 

  11. Trinder P (1969) Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem:6–24 Available from: https://journals.sagepub.com/doi/abs/10.1177/000456326900600108

  12. Klassen CD, Plaa GL (1969) Comparison of the biochemical alteration elicted in liver of rats treated with carbon tetrachloride, chloroform 1,1,2-trichloroethane and 1,1,1-trichloroethane. Biochem Pharmacol 18:2019–2022

    Article  Google Scholar 

  13. El-Askary HI, El-Olemy MM, Salama MM, Amer MH (2013) Development and validation of a high-performance liquid chromatography method for standardization of the bioactive ethyl acetate fraction of Alstonia scholaris (Linn.) R. Br. Growing in Egypt. Z Naturforsch C J Biosci 68:376–383

    Article  CAS  Google Scholar 

  14. Thewfweld W (1974) The enzymatic methods for determination of serum AST and ALT. Deutsch Med 99:343–347

    Article  Google Scholar 

  15. Kind PR, King EG (1954) A colorimetric method for the determination of serum alkaline phosphatase. J Clin Pathol 7:322

    Article  CAS  Google Scholar 

  16. Snedecor GW, Cochran WG (1971) Méthodes Statistiques, original title: statistical methods, 6th edn. Iowa State University Press, Ames, p 649

    Google Scholar 

  17. Mabry TJ, Markham KR, Thomas MB (1970) The systematic identification of flavonoids. Springer- Verlag, New York, p 354

    Book  Google Scholar 

  18. Xiong Q, Wilson WK, Pang J (2007) The Liebermann-Burchard reaction: sulfonation, desaturation, and rearrangement of cholesterol in acid. Lipids 42:87–96

    Article  CAS  Google Scholar 

  19. Buck WB, Osweiler GD, Van Gelder GA (1976) Iodine in clinical and diagnostic veterinary toxicology, 2nd edn. Kendall/Hunt, Dubuque, pp 69–75

    Google Scholar 

  20. Francescato LN, Debenedetti SL, Schwanz TG, Bassani VL, Henriques AT (2013) Identification of phenolic compounds in Equisetum giganteum by LC–ESI-MS/MS and a new approach to total flavonoid quantification. Talanta 105:192–203

    Article  CAS  Google Scholar 

  21. Krasteva I, Nikolov S (2008) Flavonoids in Astragalus corniculatus. Quim Nova 31:59–60

    Article  CAS  Google Scholar 

  22. Pereira OR, Silva AMS, Domingues MRM, Cardoso SM (2012) Identification of phenolic constituents of Cytisus multiflorus. Food Chem 131:652–659

    Article  CAS  Google Scholar 

  23. Brito A, Ramirez JE, Areche C, Sepúlveda B, Simirgiotis MJ (2014) HPLC-UV-MS profiles of phenolic compounds and antioxidant activity of fruits from three citrus species consumed in northern Chile. Molecules 19:17400–17421

    Article  Google Scholar 

  24. Hanhineva K, Rogachev I, Kokko H, Mintz-Oron S, Venger I, Kärenlampi S, Aharoni A (2008) Non-targeted analysis of spatial metabolite composition in strawberry (Fragaria × ananassa) flowers. Phytochemistry 69:2463–2481

    Article  CAS  Google Scholar 

  25. Truchado P, Vit P, Heard TA, Tomás-Barberán FA, Ferreres F (2015) Determination of interglycosidic linkages in O-glycosyl flavones by high-performance liquid chromatography/photodiode-array detection coupled to electrospray ionization ion trap mass spectrometry. Its application to Tetragonula carbonaria honey from Australia. Rapid Commun Mass Spectrom 29:948–954

    Article  CAS  Google Scholar 

  26. Wojakowska A, Muth D, Narozna D, Madrzak C, Stobiecki M, Kachlicki P (2013) Changes of phenolic secondary metabolite profiles in the reaction of narrow leaf lupin (Lupinus angustifolius) plants to infections with Colletotrichum lupini fungus or treatment with its toxin. Metabolomics 9:575–589

    Article  CAS  Google Scholar 

  27. Wojakowska A, Piasecka A, García-López PM, Zamora-Natera F, Krajewski P, Marczak L, Kachlicki P, Stobiecki M (2013) Structural analysis and profiling of phenolic secondary metabolites of Mexican lupine species using LC–MS techniques. Phytochemistry 92:71–86

    Article  CAS  Google Scholar 

  28. Blunder M, Orthaber A, Bauer R, Bucar F, Kunert O (2017) Efficient identification of flavones, flavanones and their glycosides in routine analysis via off-line combination of sensitive NMR and HPLC experiments. Food Chem 218:600–609

    Article  CAS  Google Scholar 

  29. Abu-Reidah IM, Ali-Shtayeh MS, Jamous RM, Arráez-Román D, Segura-Carretero A (2015) HPLC-DAD-ESI-MS/MS screening of bioactive components from Rhus coriaria L. (sumac) fruits. Food Chem 166:179–191

    Article  CAS  Google Scholar 

  30. Fischer UA, Carle R, Kammerer DR (2011) Identification and quantification of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently produced juices by HPLC-DAD-ESI/MSn. Food Chem 127:807–821

    Article  CAS  Google Scholar 

  31. Mena P, Calani L, Dall’Asta C, Galaverna G, García-Viguera C, Bruni R, Crozier A, Del-Rio D (2012) Rapid and comprehensive evaluation of (poly)-phenolic compounds in pomegranate (Punica granatum L.) juice by UHPLC-MSn. Molecules 17:14821–14840

    Article  CAS  Google Scholar 

  32. Maldini M, Montoro P, Hamed AI, Mahalel UA, Oleszek W, Stochmal A, Piacente S (2011) Strong antioxidant phenolics from Acacia nilotica: profiling by ESI-MS and qualitative-quantitative determination by LC-ESI-MS. J Pharm Biomed Anal 56:228–239

    Article  CAS  Google Scholar 

  33. Llorent-Martínez EJ, Zengin G, Fernández-de Córdova ML, Bender O, Atalay A, Ceylan R, Mollica A, Mocan A, Uysal S, Guler GO, Aktumsek A (2017) Traditionally used lathyrus species: phytochemical composition, antioxidant activity, enzyme inhibitory properties, cytotoxic effects, and in silico studies of Lathyrus czeczottianus and Lathyrus nissolia. Front Pharmacol 8:1–20

    Article  Google Scholar 

  34. Sobeh M, El-Hawary E, Peixoto H, Labib RM, Handoussa H, Swilam N, El-Khatib AA, Sharapov F, Mohamed T, Krstin S, Linscheid MW, Singab AN, Wink M, Ayoub N (2016) Identification of phenolic secondary metabolites from Schotia brachypetala Sond. (Fabaceae) and demonstration of their antioxidant activities in Caenorhabditis elegans. Peer J 4:e2404

    Article  Google Scholar 

  35. Lee JH, Johnson JV, Talcott ST (2005) Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC-ESI-MS. J Agric Food Chem 53:6003–6010

    Article  CAS  Google Scholar 

  36. Jawla S, Kumar Y, Khan MSY (2011) Antimicrobial and anti-hyperglycemic activities of Acacia modesta leaves. Pharmacologyonline 2:331–347 Available from: https://pharmacologyonline.silae.it/files/archives/2011/vol2/036.kumar.pdf

    Google Scholar 

  37. Rahaman M, Chaudhry M (2015) Evaluation of antioxidant and hepato-protective effect of Acacia modesta wall. Against paracetamol induced hepatotoxicity. BJPR 5:336–343

    Article  Google Scholar 

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Acknowledgements

The authors are thankful to prof. Amany Sleem professor of Pharmacology, Department of Pharmacology, National Research Centre, Dokky, Giza, Egypt, who carried out the experimental activities in the pharmacology section.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt, Cairo, Egypt

    Eman Mohamed Salah & Mariam H. Gonaid

  2. Faculty of Pharmacy, Pharmacognosy Department, Helwan University, P.O. Box 11795, Ain-Helwan, Cairo, Egypt

    Reham R. Ibrahim & Hesham S. M. Soliman

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  1. Eman Mohamed Salah
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Contributions

EMS is a research scholar who carried out the practical work, data collection, data interpretation, and analysis and was a major contributor in writing the manuscript. MHG is a professor in Pharmacognosy who designed the work and helped in data interpretation and analysis and critical revision of the article. RRI is a lecturer in Pharmacognosy who helped in practical work and data collection. HSMS is a professor in Pharmacognosy who revised the article and gave the final approval of the manuscript to be published. The authors read and approved the final manuscript.

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Correspondence to Eman Mohamed Salah.

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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 care and use of laboratory animals and approved by the Ethical Committee of Faculty of Pharmacy, Helwan University, Ain-Helwan, Cairo, Egypt, of the protocol numbered (008A-16).

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Supplementary Information

Additional file 1:

Figure S1. 1H-NMR spectral data of the flavone mixture (400 MHz, CD3OD). Figure S2. 13C-NMR total carbons spectral data of the flavone mixture (100 MHz, CD3OD). Figure S3. 13C-NMR total carbons spectral data of the flavone mixture (100 MHz, CD3OD). Figure S4. HMBC-DEPT 90 spectral data of the flavone mixture (CD3OD). Figure S5. HMBC-DEPT 135 spectral data of the flavone mixture (CD3OD). Figure S6. HSQC-DEPT 90 spectral data of the flavone mixture (CD3OD). Figure S7. HSQC-DEPT 135 spectral data of the flavone mixture (CD3OD). Figure S8. 1H-NMR spectral data of compound three (400 MHz, CD3OD). Figure S9. 13C-NMR DEPT 90 spectral data of compound three (100 MHz, CD3OD). Figure S10. 13C-NMR DEPT 135 spectral data of compound three (100 MHz, CD3OD). Figure S11. HMBC-DEPT 90 spectral data of compound three (CD3OD). Figure S12. HMBC-DEPT 135 spectral data of compound three (CD3OD). Figure S13. HSQC-DEPT 90 spectral data of compound three (CD3OD). Figure S14. HSQC-DEPT 135 spectral data of compound three (CD3OD). Figure S15. H-H Cosy spectral data of compound three (CD3OD)

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Salah, E.M., Ibrahim, R.R., Gonaid, M.H. et al. Chemical and pharmacological evaluation of the non-flowering aerial parts of Acacia modesta Wall. cultivated in Egypt. Futur J Pharm Sci 6, 122 (2020). https://doi.org/10.1186/s43094-020-00134-x

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