Therapeutic potentials of Crataegus azarolus var. eu- azarolus Maire leaves and its isolated compounds

Background Hyperglycemia is a complicated condition accompanied with high incidence of infection and dyslipidemia. This study aimed to explore the phyto-constituents of Crataegus azarolus var. eu- azarolus Maire leaves, and to evaluate the therapeutic potentials particularly antimicrobial, antihyperglycemic and antihyperlipidemic of the extract and the isolated compound (3β-O-acetyl ursolic acid). Methods Total phenolics and flavonoidal contents were measured by RP-HPLC analysis. Free radicals scavenging activity of different extraction solvents was tested in-vitro on DPPH free radicals. The antimicrobial activity of the ethanolic extract and its fractions as well as the isolated compounds were evaluated in-vitro on variable microorganisms. Animal models were used to evaluate the antihyperglycemic and antihyperlipidemic activities of the ethanolic extract along with the isolated compound (3β-O acetyl ursolic acid). Results RP- HPLC analysis of the phenolics revealed high content of rutin, salicylic and ellagic acids. Six compounds belonging to triterpenes and phenolics were isolated from chloroform and n-butanol fractions namely: ursolic acid, 3β-O-acetyl ursolic acid, ellagic acid, quercetin 3-O-β methyl ether, rutin and apigenin7-O-rutinoside. Ethanolic extract showed the highest DPPH radical scavenger activity compared to other solvents. Ethanolic extract, hexane fraction, ursolic acid, 3β-O acetyl ursolic acid and quercetin 3-O-methyl ether showed variable antimicrobial activity against E. coli, P. aeruginosa, S. aureus, and C. albicans. Administration of the ethanolic extract or 3β-O acetyl ursolic acid orally to the mice reduced blood glucose significantly in a time- and dose-dependent manner. Ethanolic extract significantly reduced LDL-C, VLDL-C, TC and TG and increased HDL-C in rats. Ethanolic extract and 3β-O acetyl ursolic acid reduced in-vitro activity of pancreatic lipase. Conclusion This study reveals that Crataegus azarolus var. eu- azarolus Maire has the efficiency to control hyperglycemia with its associated complications. This study is the first to evaluate antihyperglycemic and antihyperlipidemic potentials of 3β-O acetyl ursolic acid. Electronic supplementary material The online version of this article (doi:10.1186/s12906-017-1729-9) contains supplementary material, which is available to authorized users.


Background
Dyslipidemia and increased susceptibility to infections are two typical complications of diabetes mellitus. High glucose levels are highly associated with immune system impairment, particularly on neutrophils [1]. Hyperglycemia reduces the phagocytic activity and ability of neutrophils to form extracellular traps to kill bacteria [1]. On the other hand, hyperglycemia due to both insulin deficiency and insulin resistance significantly affect the lipid metabolic pathways [2]. Diabetic patients usually experience various cardiovascular complications of which dyslipidemia represents a main risk factor [2].
There has been a noticeable increase in the use of both traditional home remedies and herbal medicine instead of relying on conventional treatments [3]. This has caused traditional medicine to become of worldwide importance, with medicinal and economic effects [4].
Crataegus azarolus is indigenous to the Mediterranean Basin. Crataegus azarolus var. eu-azarolus Maire is a low, dense, spiny tree with a beautiful inflorescence up to 6 m tall and with orange fruits [5]. Phytochemical investigation of the plant was performed mainly on the flowers. Antioxidant activity and phenolic composition of the flowers extract were studied [6]. No available literatures concerning the pharmacological activity and the phytochemical constituents of the leaves were found.
This study aimed to explore the phytochemical composition of Crataegus azarolus var. eu-azarolus Maire leaves' extract, assess its acute toxicity and investigate the free radical scavenging and therapeutic potentials particularly antimicrobial, antihyperglycemic and antihyperlipidemic activities.

General
Shimadzu 1700 spectrophotometer was used for UV absorption spectra. Melting points were determined on Electrothermal 9100 equipment. Mass spectra were measured on a Jeol Mass Spectrometer SSQ 7000, Digital DEC 300. NMR spectra were measured in DMSO or CD 3 OD or CDCl 3 ; 1 H-NMR spectra were obtained at 400 MHz and 13 C-NMR spectra at 100 MHz on a JEOL GX-400 spectrometer with the chemical shifts (δ ppm) expressed relative to TMS as internal standard. Precoated silica gel 60 F 254 (Merck, Darmstadt, Germany) was used for the TLC analysis. Vacuum liquid chromatography (VLC) was performed on silica gel 60 GF (Merck, Darmstadt, Germany). Sephadex LH-20 (Sigma-Aldrich, St. Louis, Missouri, United States) and silica gel 100 C 18 -Reversed Phase (Fluka, Switzerland) were also used. Analysis of phenolics was performed on Hewlett Packard HPLC (HP 1050HPLCDADw/Data System) equipped with a Hypersil-ODS (4.6 X 250 mm, 5 μm) column and a UV detector.
concentrations from gallic acid (10-50 µg/mL) were prepared for the standard calibration curve. The absorbance was determined at 750 nm. Results were calculated as mg gallic acid/g dry plant weight equivalent.
Aluminum chloride was used to assess the total flavonoid contents of the different extracts according to the procedure described by Dewanto et al. [20]. Serial dilutions of quercetin were used for preparation of the standard calibration curve. The absorbance was measured at 510 nm. All the experiments were carried out in triplicate.

RP-HPLC analysis
Phenolic composition of C. azarolus var. eu-azarolus leaves was investigated in aliquots (1 g, each) of the methanolic extract of the plant via RP-HPLC [21,22] based on the method previously described by the authors [23]. For the phenolic acids composition, the UV detector was set at 280 nm while for flavonoids composition the UV detector was adjusted at 330 nm. Quantification was based on peak area calculation and was done in triplicate.

Large scale extraction and fractionation
Air-dried powdered leaves of C. azarolus var. eu-azarolus (2.5 Kg) were extracted at room temperature by maceration in ethanol (70%, 10 L × 3). The ethanolic extract was evaporated under reduced pressure at temperature 55°C to provide 280 g residue. 200 g residue were successively fractionated with different solvents according to the polarity viz. n-hexane, chloroform and n-butanol saturated with water; while the remaining residual amount was saved for biological evaluation. Partitioning the ethanolic extract with different solvents yielded 20, 15 and 18 g of dried extractive respectively.

Isolation of the constituents of the chloroform extract
An accurately weighed amount of the chloroform extract (13.0 g) was applied on silica gel 60 GF VLC column (25 X 5 cm). Gradient elution was performed using hexane-chloroform, chloroform-ethyl acetate mixtures and ethyl acetate. Fractions (100 mL each) were gathered and monitored by TLC using different mobile phase (System A, chloroform-methanol 9.5:0.5 and System B, ethyl acetate: formic acid: acetic acid: water 10.0:1.0:1.0:0.5). Spots were located by visualization under UV 365 nm before and after exposure to ammonia vapor and by spraying with p-anisaldehyde at 110°C. Similar fractions were pooled to yield 6 collective fractions (F1-F6). According to the weight of the fraction and the number of spots, fractions F3 and F5 were selected for further isolation.

Isolation of the constituents of the n-butanol extract
The n-butanol residue (15 g) was fractionated by silica gel 60 GF VLC column (30 × 3.5 cm). Mixtures from different solvents were used (chloroform-ethyl acetate, ethyl acetate and ethyl acetate-methanol). Fractions (200 mL each) were gathered and seen by TLC (System B and System C, chloroform: methanol: water 8.5:1.5: 0.1). Spots were located before and after exposure to ammonia vapor by visualization under UV 365 nm and by spraying with p-anisaldehyde at 110°C. Similar fractions were pooled to yield 10 collective fractions (P1-P10). According to the weight of the fractions and the number of spots, fractions P7 and P8 were selected for further isolation.

Biological study Microorganisms
Three bacterial strains and one fungus, were kindly provided by Rashid hospital (Dubai-UAE) and were used for the antimicrobial screening. This included one representative of the Gram-positive group (Staphylococcus aureus RMTCC 3161), two representatives of the Gramnegative group (Escherichia coli RMTCC 2682 and Pseudomonas aeruginosa RMTCC 1687) and one fungus (Candida albicans RMTCC 5122). Microorganisms were grown on appropriate media: nutrient agar for S. aureus and P. aeruginosa, MacConkey agar for E. coli and Sabouraud dextrose agar for C. albicans.

Antimicrobial activity
The ethanolic extract, and its fractions, n-hexane, chloroform and n-butanol as well as the isolated compounds of C. azarolus at doses of 375 μg/mL for each extract, and 140 μg/mL for the isolated compounds were subjected to in-vitro qualitative screening, for evaluation of their antimicrobial potentialities. The agar diffusion technique was used [25]. Solubilization of the samples was assisted by sterile DMSO. The effects were compared with gentamicin (30 μg/mL) and antifungal, fluconazole (30 μg/mL). Diameters of zones of inhibition (in mm) were taken as a measure for the growth inhibitory activity against the selected strains.

Experimental animals
Male albino mice (30 ± 5 g) were used for acute toxicity and antihyperglycemic studies. Antihyperlipidemic experiments were performed on Sprague Dawley rats weighing 210 ± 5 g. All animals were kept under standard conditions, fed with regular diet and water supplied ad libitum. Mice were accommodated for 1 week prior to the experiments. All animal investigations were accepted from the Ethical Research Committee of the Dubai Pharmacy College, Dubai UAE and done according to the ethical standards of laboratory animals [26].
Acute oral toxicity study LD 50 was determined by probit test [27]. Mice were divided into five groups (10 each) and they received various oral doses of the ethanolic extract (250, 500, 1000, 2500 and 5000 mg/kg). Later, they were observed over 72 h for any signs of morbidity or abnormal behavior and their death was recorded [27].

Evaluation of antihyperglycemic activity Induction of diabetes in mice
One night before the induction of hyperglycemia, the animals were kept fasted but given water ad libitum. The next morning, animals were injected 150 mg/kg alloxan monohydrate solution in acetate buffer (0.15 M, pH 4.5) intraperitoneally. The animals were observed over a week and their blood glucose values were measured. Mice with blood glucose levels between 180 and 360 mg/dL were assigned diabetic and were used later for further studies [28].

Oral glucose tolerance test
Animals were divided into four groups (n = 6 each). The mice were fasted for 18 h and provided water ad libitum. Each animal serves as its own control, Group I received only glucose at dose of 2 g/kg. Groups II and III received 250 and 500 mg/kg of the ethanolic leaves extract respectively, while group IV received the isolated compound in a dose of 50 mg/kg. All the tested samples were given orally 90 min before the glucose dose (2 g/kg, p.o.). Levels of blood glucose were calculated before and subsequently at 30, 60, 120 and 240 min after the administration of glucose dose. Blood glucose was measured by glucose estimation kit.

Experimental procedure
The diabetic animals were divided into five groups (n = 6 each). Group I kept as control, group II received 5 mg/kg glibenclamide as positive control, groups III and IV received the extract at doses of 250 and 500 mg/kg respectively, and group V received the isolated compound (2) at dose of 50 mg/kg. Levels of the blood glucose were measured pre and post (120 and 240 min) the treatment.

Evaluation of antihyperlipidemic activity Induction of hyperlipidemia in rats
The rats were fed daily by means of gavage tube with cholesterol at 25 mg/kg suspended in coconut oil given at 10 mL/kg daily for 30 days [29].

Experimental procedure
The animals were grouped into five treatment categories (n = 6, each), as following: Group I, kept as control group received daily 1% w/v sodium CMC suspension. Animals in groups II-V were hyperlipidemic and received daily cholesterol (25 mg/kg/day) in oil at 10:00 am. Group II represented the hyperlipidemic group, group III served as positive control and received lovastatin (10 mg/kg/day) at 3:00 pm. Similarly groups IV and V were given the ethanolic leaves extract at doses of 250 and 500 respectively at 3:00 pm. For a period of 30 days, the original and the final body weights and food intake of rats were monitored.
After this period, the fasted rats were sacrificed. Under ether anesthesia, samples of the blood were gathered by cardiac puncture. Lipid profile test was done including TC, HL-C, LDL-C, VLDL-C and TG.
Additionally, cardiac risk indicators were calculated by the "Atherogenic Index" TC: HDL-C ratio and LDL-C: HDL-C ratio.

In-vitro evaluation of the effect of the ethanolic extract and its isolated compound (2) on pancreatic lipase and HMGCoA reductase activities
In-vitro lipase inhibitory effect of the ethanolic leaves extract and the isolated compound (2) was assessed according to the method previously described [30]. The final concentrations of the tested samples of the plant extract and isolated compound were ranged from 50 to 500 μg/mL and 20-200 μg/mL respectively.
The following formula was used to calculate the percentage inhibitory activity (I): Where A: activity without inhibitor; a: negative control in absence of inhibitor; B: activity in presence of inhibitor; and b: negative control in presence of inhibitor.
Orlistat and DMSO were used as positive and negative control respectively and their activities were also tested.
For the in-vitro evaluation of HMG-CoA inhibitory activity, similar concentrations range of the plant extract and isolated compound were used. Pravastatin was used as standard drug with concentrations ranged from 0.1-2.5 μg/mL according to the method previously described [31]. HMG-CoA reductase inhibitory activity was calculated by using the following formula: Inhibitory activity (I %) = (Δ Absorbance control-Δ Absorbance test/Δ Absorbance control) × 100.

Statistical analysis
The results were expressed as mean + S.E.M (Standard Error Mean). Data was analyzed by GraphPad Software version 6.00 (San Diego, CA). One-Way ANOVA followed by Bonferroni's multiple comparison tests against the control was performed. For repeated measures in glucose tolerance test, two-way ANOVA assessed the interactive and independent effects of treatment and time. P values < 0.05 were considered significant. IC 50 values for the DPPH radical scavenging, pancreatic lipase Inhibition and HMG-CoA inhibition assays were determined from the dose-response curves using a linear regression analysis. For invitro evaluation of pancreatic lipase and HMG-CoA inhibition activities, inhibition of less than 40% was considered irrelevant and was selected as a cutoff point.

Spectrophotometric determination of total phenolic and flavonoid contents
Different solvents were used for leaves extraction for the flavonoid and the phenolic contents to select the safest and the most effective extracting solvent as shown in Table 1. Spectrophotometric analysis revealed that ethanol was the best solvent to extract both flavonoids and phenolic acids.

Isolation of the constituents of the chloroform and n-butanol extracts
The structure of compound 3 was identified as ellagic acid from its physical properties and different spectroscopic spectra (UV and 1 H-NMR) [35]. 1 H and 13 C-NMR spectra of compound 4 demonstrated a methoxy group at δ H 3.84 and at δ C 58.2 respectively attached at position 3; compound 4 was identified as quercetin 3-O -β methyl ether [36].
The structure of compound 5 was identified as rutin from its physical properties and different spectral data (UV, 1 H-NMR and 13 C-NMR) [37].
The UV λ max (335 nm) of compound 6 suggested that it possesses a flavone substituted skeleton. This was confirmed from the 1 H-NMR spectrum. The occurrence of two doublets signals at δ H 6.51 and 6.86 (J = 2.2 Hz) indicated the presence of two meta protons at C-6 and C-8 of ring A respectively. In addition, 2 doublets appeared at δ The sugar moieties were identified as glucose and rhamnose (TLC of acid hydrolysate, 1 H-NMR and 13 C spectral data). Compound 6 could be identified as apigenin 7-O-β-D-glucopyranosyl (6 → 1)-α-L-rhamnopyranosyl (apigenin 7-O-rutinoside) [38]. Figure 1 is showing the chemical structure of the isolated compounds.
Antioxidant activity DPPH free radical scavenging activity DPPH free radical scavenging effects of the extracts were tested and the results are presented in Fig. 2. Both ethanolic and methanolic extracts showed the highest activity as revealed in Fig. 2. IC 50 's of the leaves extracts were 129.2, 140.1, 164.1 and 262.3 μg/mL for the ethanolic, methanolic, ethyl acetate and acetone respectively. While for ascorbic acid, the IC 50 was calculated to be 34.6 μg/mL.

Biological studies Antimicrobial activity
Results displayed in Table 6 revealed that at the tested concentrations, the ethanolic extract as well its n-hexane fraction and the isolated compounds, ursolic acid, 3β-O acetyl ursolic acid and quercetin 3-O-methyl ether, showed variable antimicrobial activity against all the tested pathogenic strains bacteria and fungus. On the other hand, all the tested samples exhibited variable antibacterial activities with inhibition zones ranging from 18 to 28 mm in diameter against P. aeruginosa.
The isolated compound 2, 3β-O acetyl ursolic acid, demonstrated the highest growth inhibitory activity against all the tested microorganisms, followed by quercetin 3-O-methyl ether and lastly, ursolic acid.  Fig. 1 Compounds isolated from C. azarolus var. eu-azarolus leaves Acute oral toxicity study The LD 50 of the ethanolic extract of C. azarolus var. eu-azarolus Maire was safe up to 5000 mg/kg. During the observation period, no signs of morbidity or behavioral alteration in any animals' groups were noticed.

Evaluation of antihyperglycemic activity
Oral glucose tolerance test Blood glucose levels of normal mice were significantly reduced after receiving the plant extract at different doses in a time-and dosedependent manner as shown in Fig. 3. Both doses of the leaves, as well as the isolated compound, 3β-O acetyl ursolic acid, exhibited significant antihyperglycemic effect. The effect was significant at 30, 120 and 240 min. Noticeable significant decrease in level of glucose was noticed at 30 min with the leaves extract at both doses as well as 3β-O acetyl ursolic acid at dose of 50 mg/kg (p < 0.01). This marked improvement in glucose tolerance was continued over the tested time.
Antihyperglycemic activity of the ethanolic extract and 3β-O acetyl ursolic acid on blood glucose levels in diabetic mice The basal glycaemia was 241.7 ± 1.7 mg/dl for the diabetic control mice. There was no statistical difference between the glycaemic levels of the studied groups at time 0, (p > 0.05). The anti-hyperglycemic activity of the ethanolic extract and the isolated compound (2) on the fasting blood sugar levels of diabetic mice is shown in Fig. 4. In diabetic mice, treatment of C. azarolus var. eu-azarolus leaves extract at dose of 250 and 500 mg/kg significantly lowered the basal level of blood glucose at 120 and 240 min (p < 0.01). A highly significant decrease of the blood glucose level was observed with 3β-O acetyl ursolic acid at the same timing intervals, p < 0.001.

Antihyperlipidemic activity
Body weight Compared to cholesterol induced hyperlipidemic control group (group II), lovastatin treated group showed that the reference has no effect on the body weight (p > 0.05). On the other hand, groups received the ethanolic leaves extract showed a significant reduction in the percentage increment in body weight in day 15 and 30 (p < 0.01) as shown in Table 7.
Effect of the ethanolic extract on serum lipid profile in hyperlipidemic rats In hyperlipidemic model, groups treated with the ethanolic leaves extract and lovastatin showed significant reduction in TC, TG, LDL-C, and VLDL-C levels. In addition, serum HDL-C level was increased as compared to the control group (Table 8). Treated Groups with lovastatin and the leaves ethanolic extract demonstrated remarkable decrease in the "Atherogenic Index" and LDL-C: HDL-C risk ratios.
In-vitro effect of the ethanolic extract and 3β-O acetyl ursolic acid on pancreatic lipase and HMGCoA reductase activities C. azarolus var. eu-azarolus ethanolic extract at concentrations of 50-500 μg/mL reduced the activity of pancreatic lipase in-vitro.

Discussion
Diabetes Mellitus and other hyperglycemic disorders are complicated conditions associated with high prevalence of infection, dyslipidemia, hypertension and renal failure. The aim of this study was to find a standard plant extract that has the potential to control hyperglycemia with its associated complications, and to isolate and identify the active components that are responsible for those activities.
Results of total phenolic and flavonoid contents revealed that ethanol was the best solvent to extract both flavonoid and phenolic acids. Therefore, ethanol had been selected for further investigations. RP-HPLC analysis of the phenolics demonstrated high contents of rutin, salicylic and ellagic acids in the plant. Six compounds belonging to triterpenes and phenolic were isolated from chloroform and n-butanol fractions for the first time from C. azarolus var. eu-azarolus Maire. Ursolic acid is a triterpenoidal compound that finds in medicinal herbs, other plants and foods [39]. Ursolic acid showed anti-inflammatory, hepatoprotective, antihyperlipidemic, anticancer, inhibition of lipid peroxidation and antimicrobial activities [39][40][41][42][43]. Most of the available scientific papers are concerned about the activity of ursolic acid with no data regarding the antihyperglycemic and antihyperlipdimic of its acetate Fig. 4 Effect of ethanolic extract of C. azarolus var. eu-azarolus leaves and 3β-O acetyl ursolic acid on glucose level in diabetic mice test, *p < 0.01, **p < 0.001. Group I is the control, group II: positive control and received glibenclamide, group III and IV were given the plant extract at doses of 250 and 500 mg/kg respectively, and group V was treated with the isolated compound  [44,45]. Therefore, 3-β-O acetyl ursolic acid is assumed to have better pharmacokinetics features than ursolic acid itself. In-vitro DPPH assay was used to evaluate the free radical-scavenging effect of the leaves extracts of different solvents. The ethanolic extract showed the highest DPPH radical scavenger potential. This effect could be attributed to the ursolic acid and the phenolic compounds that were isolated from the ethanolic leaves extract. Ursolic acid, a pentacyclic triterpene, was reported to be a strong oxygen species (ROS) scavenger. Similarly, phenolic compounds including flavonoids particularly, quercetin 3-O -β methyl ether, rutin and apigenin 7-O-β-D-glucopyranosyl (6 → 1)-α-Lrhamnopyranosyl and phenolic acids (ellagic and salicylic acids) have been described to have high antioxidant effects [46][47][48][49]. Hyperglycemia worsens the development of infections and vice versa [50]. Blood glucose of more than 200 mg/dL has been strongly associated with reduced neutrophil activity [51]. Diabetic patients are at higher risk of infections from various microorganisms viz. S. aureus, E. coli, P. aeruginosa and C. albicans [52,53]. The ethanolic leaves extract as well, its fractions and the isolated compounds exhibited noticeable antimicrobial activities against a wide range of microorganisms. This effect could support the immune system to fight against invading microorganism. Antipseudomonal activity of the chloroform extract could be ascribed to its isolated compounds namely, ursolic, 3β-O acetyl ursolic and ellagic acids through individual action or in a synergistic way. Other antimicrobial activities of chloroform fraction could be basically related to the triterpenes content only since ellagic acid appeared to be inactive on the other microorganisms.
On the other hand, nbutanol fraction showed inhibition activity against both P. aeruginosa and E. coli. These activities are essentially linked to the phenolic constituents namely, quercetin 3-O-methyl ether, rutin and apigenin 7- For the biological studies, doses were selected depending on the LD 50 value (<1/10). Blood glucose levels of normal and diabetic mice received either ethanolic extract or 3β-O acetyl ursolic acid, were significantly decreased in a time-and dose-dependent manner. It has been previously reported that ursolic acid improves hepatic insulin resistance by stimulating the expression of peroxisome proliferator-activated receptors α (PPARα) [54]. Moreover, ursolic acid is reported to have high α-glucosidase inhibitory activity [55]. Those effects explained the antihyperglycemic activity of ursolic acid on fasting state and glucose tolerance test. Furthermore, ellagic acid is reported to possess antidiabetic action through inhibition of glycogen phosphorylase b enzyme [56]. Polyphenolic compounds, including quercetin 3-O -β methyl ether, rutin and apigenin 7-O-rutinoside are reported to have antihyperglycemic effect [57,58]. Such combination of triterpenes and phenolic compounds could have synergistic antihyperglycemic actions. On the other hand, administration of the ethanolic leaves extract markedly decreased the percentile increment in body weight. Furthermore, the leaves extract significantly reduced the serum TC, TG, LDL-C, VLDL-C and increased HDL-C levels. Pancreatic lipase and HMGCoA reductase were used to explore the possible mechanisms for the antihyperlipidemic action. Both ethanolic extract and 3β-O acetyl ursolic acid reduced in-vitro activity of pancreatic lipase. In contrary, the extract showed moderate inhibition of HMGCoA reductase, while 3-β-O acetyl ursolic acid was unable to inhibit the enzyme activity at the tested doses. Based on that, antihyperlipidemic effect of the ethanolic extract can't be solely contributed to HMGCoA inhibition. Thus, another mechanism could be suggested. Ursolic acid is reported to enhance the binding of PPAR-α to the response element in PPAR-α-responsive genes and modifies the lipid metabolism genes expression [59]. Thereby regulating the transcription of PPAR-α genes involved in lipid metabolism. 12.9 ± 0.4* (−23.7%) 1.9 ± 0.3 0.8 ± 0.05 Group I: Control group received the vehicle, groups II-V were hyperlipidemic and received daily cholesterol (25 mg/kg/day) in oil at 10:00 am. Group II received cholesterol, group III received lovastatin, groups IV and V were given the plant extract at doses of 250 and 500 respectively. # p < 0.01 vs group I, * and ** p < 0.05 and 0.01 vs cholesterol induced hyperlipidemic control group respectively Additionally, it reduces cellular cholesterol and triglyceride levels in hepatocytes, possibly by increasing the uptake and oxidation of fatty acid and by inhibiting their synthesis [59]. Interestingly, the enzymatic assay confirmed the inhibitory activity of the ethanoic extract and 3-β-O acetyl ursolic acid on pancreatic lipase. Hence, the antihyperlipidemic effect of the ethanolic extract and attenuation of body weight gain might be due to its inhibitory action on pancreatic lipase.

Conclusion
The current study demonstrates the efficiency of the leaves extract of C. azarolus var. eu-azarolus Maire in controlling hyperglycemia with its associated complications such as infection and dyslipidemia. This multiple pharmacological profile might be due to the synergistic effect of its bioactive constituents including triterpenes, particularly ursolic acid and its acetyl derivative, and the phenolic compounds particularly, quercetin 3-O -β methyl ether, rutin and apigenin 7-O-rutinoside. This study is unique in the sense that it is the first to evaluate the antihyperglycemic and antihyperlipidemic potentialities of 3β-O acetyl ursolic acid.