MATERIALS AND METHODS

Plant material and extraction: Fresh leaves of Z. spina-christi were collected from different locations of Jordan valley. Prior to the extraction, the leaves were washed with sterile water to remove any associated debris and dried at room temperature. After that, the leaves were powdered with a mechanical grinder and stored in an air tight container. In order to extract the crude bioactives, the powdered leaves were extracted by maceration with seven different common solvents (like water, methanol, ethanol, acetone, chloroform, ethyl acetate and n-hexane) at 20% (w/v) concentration for 2 days at the room temperature with occasional shaking. The mixtures were filtered through Whatman no: 4 and then membrane filter (0.45 um). The filtrates were concentrated in rotary evaporator (Buchi, Switzerland) at 40°C and the crude concentrated extracts were calculated for their extractive value. Then, transferred to brown colored sample vial and stored at -20°C for further analysis²⁰.

Quantitative phytochemical analysis
Determination of total flavonoid content (TFC): Aluminium chloride colorimetric method was used for flavonoids determination. Different plant extracts in methanol (mg mL^–1) were separately mixed with 1.5 mL of ethanol, 0.1 mL of 10% aluminium chloride, 0.1 mL of 1 M sodium acetate and 2.8 mL of distilled water. It was kept at room temperature for 30 min. The absorbance of the reaction mixture was measured at 415 nm with double beam UV spectrophotometer. Quercetin was used as the standard for a calibration curve and the results were expressed as mg of quercetin equivalents per gram of extract dry weight (mg QE g^–1 DW)²¹.

Determination of tannin content: The tannins were determined by Folin-ciocalteu method. About 0.1 mL of each sample of extract was added to a volumetric flask (10 mL) containing 7.5 mL of distilled water and 0.5 mL of Folin-ciocalteuphenol reagent, 1 mL of 35% Na₂CO₃ solution and dilute to 10 mL with distilled water. The mixtures were shaken well and kept at room temperature for 30 min. A set of reference standard solutions of gallic acid (20, 40, 60, 80 and 100 μg mL^–1) were prepared in the same manner as described earlier. Absorbance for tests and standard solutions were measured against the blank at 725 nm with an UV/Visible spectrophotometer. The tannin content was expressed in terms of mg of gallic acid equivalent/g of extract dry weight (mg GAE g^–1 DW)²².

Determination total alkaloid content: About 5 g of dry powdered leaves of Z. spina-christi was weighed into a 250 mL beaker and 200 mL of 10% acetic acid in ethanol was added, covered and allowed to stand for 48 h. After filtration, the extract was concentrated using a water bath to 1/4th of the original volume. Concentrated ammonium hydroxide was added in drops to the extract until the precipitation was completed. The whole solution was allowed to settle and the precipitate was collected and washed with dilute NH₄OH and then filtered. The residue is the alkaloid, which was dried and weighed²³.

Heavy metals analysis: The concentration of heavy metals including lead (Pb), copper (Cu), cadmium (Cd), nickel (Ni) and cobalt (Co) were measured in the leaves of Z. spina-christi. For this purpose, plant sample was dried in oven at 72°C for 48 h. Subsequently, the plant dry matter was powdered and extracted according to the method described by Kovacs et al.²⁴. Two grams of powdered sample was mixed with 10 mL of 65% HNO₃ and remained overnight at room temperature. Then, the digest was heated at 85°C to evaporate acid. One mL of 30% H₂O₂ was added; consequently, the digest was filtered and diluted with deionized water to 50 mL. Heavy metals concentration in leaves was determined using inductive coupled plasma (ICP-OES) (Optima 2000, PerkinElmer, USA). Appropriate working standard solutions were prepared for each element.

Statistical analysis: The one-way analysis of variance (ANOVA) and Tukey’s test at the significance level p<0.05 were conducted using SPSS statistical software (version 10). Data were reported as mean±standard deviations. All experiments were performed in three different sets each in triplicates (n = 3).

RESULTS

Yield of extracts: The yield percentage of different Z. spina-christi leave extracts are shown in Fig. 1. Yields range from 0.81-46.2%. The highest significant extractive yield (p<0.05) was obtained from water extract (46.2%), followed by acetone (24.3%), then chloroform (24.2%), methanol (21.4%) ethanol (20.3%), ethyl acetate (15.0%) and n-hexane (0.81%).

Total flavonoid content: The concentration of flavonoids in various plant extracts of Z. spina-christi leaves was determined using spectrophotometric method with aluminum chloride. The content of flavonoids was expressed in terms of quercetin equivalent, the standard curve Eq.:

y = 0.015x+0.005

Fig. 1:	Extract yield percentage of different extracts of Z. spina christi
	Values are the mean average of three replications for each solvent±standard deviation. Columns not sharing the same superscript letter are significantly different p<0.05

Fig. 2:	Total flavonoid content of different extracts of Z. spina christi expressed as mg of quercetin equivalent/g of dry extract (mg QE g–1 DW)
	Values are the mean average of three replications for each solvent±standard deviation. Columns not sharing the same superscript letter are significantly different p<0.05

Fig. 3:	Total tannin content of different extracts of Z. spina christi expressed as mg of gallic acid equivalent/g of dry extract
	Values are the mean average of three replications for each solvent±standard deviation. Columns not sharing the same superscript letter are significantly different p<0.05

Table 1:	Total concentration of alkaloid in dry leaves of Z. spina christi
Value is the mean average of three replications for each solvent±standard deviation

Table 2:	Concentration of heavy metals in dry leaves of Z. spina christi

(R² = 0.999) (Fig. 2). Ethyl acetate extract has significantly the highest total flavonoid content (145.3±2.3 mg QE g^–1 DW of extract) (p<0.05), followed by methanol (120.0±4.9), then ethanol (109.0±2.0), n-hexane (71.3±6.4), acetone (31.3±1.2), chloroform (23.9±0.05) and water (13.0±2.0). The flavonoid contents of the rest of the solvents are also significantly different (p<0.05) except for chloroform and acetone extracts.

Determination of tannin contents: The tannin contents in the examined extracts of Z. spina-christi leaves using the Folin-ciocalteu’s reagent is expressed in terms of gallic acid equivalent, the standard curve Eq.:

y = 0.0.13x+0.23

(R² = 0.981). The values obtained for the concentration of tannin are expressed as mg of GAE g^–1 DW of extract (Fig. 3). The tannin contents in the examined extracts ranging from 17.9±4.4 mg GAE g^–1 DW for water extract to 57.2±5.8 mg GAE g^–1 DW for methanol extract. The highest concentration of tannin was measured in methanol and ethyl acetate extracts. Water and chloroform extract had the least tannin contents.

Total alkaloid content: The result of total alkaloid content in the leaves of Z. spina-christi is shown in Table 1. The alkaloid content was found to be 10.1±0.02 mg g^–1.

Heavy metals analysis: The concentrations of various heavy metals in dry powder leaves are summarized in Table 2.

DISCUSSION

The use of bioactive compounds in different commercial sectors such as pharmaceutical, food and chemical industries signifies the need of the most appropriate and standard method to extract these active components from plant materials²⁵. However, extraction efficiency not only depend on the extraction method but also on the solvent used for extraction^12,26. In this study, Several leaves extracts of Ziziphus spina-christi were obtained by using several solvents like water, methanol, ethanol, acetone, chloroform, ethyl acetate and, n-hexane in order to identify the most appropriate solvent for further extraction and isolation of bioactive compounds such as phytochemical secondary metabolites, from this medicinal plant grown in Jordan.

Results of the present study showed that among all the solvent extracts used, the extract obtained by water showed the highest yield percentage (46.2%), while the n-hexane extract exhibited the lowest percentage (0.81%) (Fig. 1). Findings of the current study are in agreement with previous investigation of Do et al.²⁶, who reported that maximum extract yield from Limnophila aromatica was obtained with water extract. This may be attributable to the higher solubility of proteins and carbohydrates in water. Also, Zielinski and Kozowska²⁷ obtained the highest extract yield with water. However, higher extraction yield does not necessarily imply that it will also have high concentration of the bioactive compounds²⁸.

The total content of flavonoid compounds in different extracts was performed using the aluminium chloride method and expressed as milligrams of quercetin equivalents. In the current study, the TFC values of the extracts are ranging from 13.0±2.0 mg QE g^–1 DW for water extract to 145.3±2.3 mg QE g^–1 DW for ethyl acetate extract. Methanol and ethanol extracts also offered high values of TFC (Fig. 2). Results of the current study are in accordance with a previous investigation on Hibiscus tiliaceus wood, wherein, maximum amount of flavonoid was observed in ethyl acetate extract followed by methanol and other extracts²². While Do et al.²⁶ reported that flavonoid content in the extracts decreased with increasing water content in the aqueous solvent.

The total content of tannins in leaves of Z. spina-christi was determined using total phenol method using Folin-Ciocalteu’s reagent and standard gallic acid. The highest concentrations of tannin were measured in methanol and ethyl acetate extracts, while water and chloroform extracts had the least tannin contents (Table 2). Tambe and Bhambar²² observed similar trends as in the present study regarding extraction efficacy of solvents towards recovering of tannins from Hibiscus cannabinus L. wood.

The total alkaloid content was measured in the powdered leaves of Z. spina-christi and was found to be 10.1±0.02 mg g^–1. Almost similar quantities of alkaloids were obtained from other medicinal plants such as Pteris species using the same extraction method²⁹.

From another aspect, the determination of the heavy metals concentration in plants can provide useful information about the environmental health and safety. The amount of excess metal ions in food chain results in damage to ecosystems and threatens the human health, if these plants be used as food or pharmaceutical products¹⁴. Hence, the World Health Organization (WHO) has stressed that the plants with medicinal and curative function, should be assayed for the presence of heavy metals. Analysis of heavy metal content is necessary for final production and processing of herbal products. According to WHO, the specific limit of heavy metals such as Cu, Pb, Ni, Cd and Co in medicinal plants and food are 10,10, 1.5, 1 and 0.2 ppm, respectively^30,31. In the present study, it is observed that crude powder of Z. spina-christi leaves contains the aforementioned metals in lower concentrations than the permissibility (Table 2).

Previously, several studies revealed that the extraction solvents play an important role in the extraction of important secondary bioactive compounds from the medicinal plants. The yield of extraction depends on the solvent with varying polarity, pH, temperature, extraction time and composition of the sample. Under the same extraction time and temperature, solvent and composition of sample are known as the most important parameters^26,32,33. Since biologically active compounds occur naturally in very small concentrations, the choice of an extraction method and the corresponding suitable solvent is an important step in the drug discovery process^11,12.

CONCLUSION

The solvent effects identified in this study, revealed that the most efficient extraction medium for flavonoids was ethyl acetate. Whereas methanol extraction showed the highest tannins content. The leaves extract has contained appreciable amount of alkaloids, in addition to low concentrations of the heavy metals. Furthermore studies are required to isolate and characterize the bioactive components from Z. spina-christi.

ACKNOWLEDGMENTS

The research was funded by the Deanship of Academic Research at Al al-Bayt University, Al Mafraq, (Project No 7570/2015).