Optimization of Solvent Systems for the Extraction of Vitexin as the Major Bioactive Flavonoid in Prosopis farcta

Prosopis farcta, a plant belongs to the mimosoideae, is characterized by a very wide spectrum of various bioactive and medical constituents. Vitexin, the marker flavonoid found in Prosopis, has potent and broad antitumour efficacy in preclinical models. Many studies had been done for the isolation of flavonoids (vitexin) by completely different chromatographically methodology. During this study, vitexin was isolated from Prosopis farcta by 6 different extraction methods in which parameters as the type, concentration and pH of the extracting solvents considered. Among different solvent systems used, methanol-water (40%, containing acetic acid 0.5%) was found to be the best solvent generating the highest yield (0.554 mg∙g −1 DW) from Prosopis leaves. The present work suggests an efficient method for estimation the greatest content of vitexin analyzed by HPLC technique and introduces Prosopis farcta as a suitable source of this flavonoid with several pharmacological properties.

For example, it may be used in the treatment of inflammation, measles, diabetes [2] [3] and also reduce cardiac or chest pain [1]. Antitumour activity [4], antioxidant capacity [5] and antimicrobial activity [6] have been reported for Prosopis species.
The medicinal value of these plants can be observed from the chemical agents they possess which may alter certain physiologic actions in the human body. The most important of these bioactive constituents of Prosopis species are L-arabinose, Lectin, alkaloids, flavonoids and phenolic compounds [7] [8]. Recently, flavonoids have attracted interest due to the discovery of their pharmacological activities [9] [10]. Vitexin, a nature-derived lignin compound (Figure 1), is found in Prosopis farcta [8]. It has been shown to have broad anti-tumor activity, activate caspases and induce cell apoptosis [11]. Additionally, vitexin inhibits an analgesic effect in a variety of inflammatory pain models by preventing the decrease of reduced glutathione levels and modulating cytokine production [12].
Extraction is the first crucial step in preparation of active ingredients from plant materials. Optimizing the extraction process with high yield may be the result of a synergistic combination of fundamental factors as extraction technique and efficacy of solvent extraction [13]. HPLC method as the main choice for fingerprinting study, has gained increasing importance for the analysis of plant extracts [14]. The efficacy of solvent extraction is influenced by many parameters such as the type of solvent, solvent concentration, time, temperature, pH and liquid-to-solid ratio [15]. Thus, the main objectives of this research were aimed 1) to introduce the best extraction method to provide for the maximum yield of vitexin from P. farcta and 2) to report this species of Prosopis as a considerable source of antinociceptive and antioxidant flavonoid.

Plant Materials and Growth Conditions
Seeds of Prosopis farcta were collected in western Ilam province, IRAN. The healthy seeds of uniform size were selected and scarified with 98% sulfuric acid for 13 min and surface sterilized with 2% sodium hypochlorite solution, followed by repeated washings with distilled water, and germinated by placing in a Petri dish with two layers of water-saturated filter paper. Germinated seedlings with 20 mm-long roots were then transferred into plastic containers with 2.5 dm 3 of Hoagland nutrient solution (pH 6.8

Method 1: 85% Ethanol Extraction
The extraction was carried out using 2 g of powdered leaves with 30 ml of 85% ethanol in an ultrasonic extraction device for 30 min, repeated twice. The extract was collected and filtered, the filtrate was dried at 50˚C in a rotary evaporator. The dried extract was dissolved in the methanol-acetonitrile-water (40:15:45, v/v/v) containing 1.0% acetic acid. After filtering through a filter paper and a 0.45 µm membrane filter (Millipore), the extract was injected directly into the HPLC [16].

Method 2: 70% Methanol Extraction
The extraction was carried out using 2 g of fine powder with 20 mL of 70% methanol. This process involved 2 min of vigorous vortexing, 30 min of sonication and incubation with shaking either for 3 h at room temperature. After the mixture had been centrifuged for 10 min at 1900 g, the supernatant was taken and filtered through a 0.45 µm membrane filter. After partitioning with 20 mL of n-hexane to remove lipids, 1 mL aliquots of the extract were vacuum dried for 2 h at room temperature. The dried aliquot was then resuspended in 10% methanol, and used for HPLC [17].

Method 3: 40% Acidic Methanol Extraction
The dried leaves (2 g) transferred to a tube with 15 mL of 40% aqueous methanol, containing 0.5% acetic acid. After shaking for 4 h, samples were centrifuged (12 min, 13,000 rpm) and the supernatant was used for HPLC [18].

Method 4: 94% Ethanol Extraction
2 g of powdered leaves were incubated with 15 ml of 94% ethanol in a petri dish for 1 h. The extract was evaporated to 500 µl and transferred into a centrifuge tube. The petri dish was subsequently washed three times with 2 ml of methanol and the extract and washings were combined and evaporated. The extracts were then dissolved into 100 µl of methanol. After adding 100 µl H 2 O, the extracts were centrifuged in eppendorf tubes at 14,000 × g for 3 min. The supernatants were used for HPLC analyses [19].

HPLC Analysis
The

Statistical Treatment
All data were analyzed using MSTAT-C software. Duncan's multiple range tests were used to measure statistical differences between treatment methods and controls. P = 0.05 or =0.01 was considered significantly different.

Results and Discussion
Vitexin peaks were identified in the extracts by comparing the retention time with that obtained from the standard solution (Figure 2), by spiking the standard solution into the sample solution and by a comparison of its UV spectra with that of the standard (Figure 3). The retention time of vitexin peak in the extract of six methods varied from 22.05 to 22.40, compared to 22.25 min in case of vitexin standard. Our study showed the absorption maxima of vitexin obtained from Prosopis leaves ranged as 350 > 254 > 280 respectively. Also, UV spectra of vitexin between 210 and 400 nm is shown in Figure 3. Sample preparation is the most critical point for a successful and reliable analysis. Many sample preparation methods have been developed to extract phenolic compounds from this wide range of samples [23]. We compared the effectiveness of the different reported methods for the extraction of flavonoid vitexin from Prosopis leaves [16]- [21]. As can be seen in Figure 5, the best results were obtained by using method 3, 0.554 mg•g −1 , followed by the method 2 and 5, 0.498 and 0.463 mg•g −1 , respectively. Numerous reports indicate that the amount of phenolic compounds varies with the conditions during extraction. For example, 80% methanol and acetonitrile/HCl solution were used to extract isoflavones from soybeans and soyfood respectively [24] [25]. Other Studies showed the optimized extraction solvent for phenolic compounds is to be 70% ethanol or 58% acetonitrile [26] [27].
The present study divides different extraction methods into three solvent systems: methanol, ethanol and acetonitrile. Methanol system extracted higher amounts of vitexin than ethanol and then acetonitril systems. Moreover, the ratio of water in the solvent system played an important role in the amount of the phenolic compounds extracted [25]. [28] reported that the highest extraction of phenolic compounds from plant material with methanol-water was achieved during the first stage of extraction. As shown in Figure 5, for vitexin extraction, methanol concentration of 40% (method 3), is better than 70% and 100% (methods 2, 5). It appears that alcohol mixed with up to 50% water gives the best blend for vitexin extraction. As shown in Figure 5, no significant differences were found when using extraction methods 1 and 4 while their dried extracts (prepared for injection) was dissolved in different solvent systems (methanolacetonitrile-water (40:15:45, v/v/v) and waret-H 2 O (50:50) respectively). It may show the fact that solvent systems used to the first stage of extraction are relatively important than ones in consecutive procedures [28]. Moreover, it is important to take account the structural diversity of polyphenols because these compounds may have multiple hydroxyl groups that can be conjugated to sugars, acids or alkyl groups [29]. Hydrolysis of flavonoids, frequently used to remove the sugar moieties from glycosides, may be acidic, basic, or enzymatic [23]. Numerous papers have been cited that the glycosides of flavones and flavonols were hydrolysed by refluxing in 1 -2 M HCl in 50% MeOH-H 2 O [23].
Probably, the other effective factor in the selection of method 3 as the best one S. Zafari, M. Sharifi American Journal of Plant Sciences can be acidic pH of the extracting solvents or in other words acidic hydrolysis of vitexin by acetic acid.

Conclusion
Taken as a whole, our results show that the extraction method [methanol/H 2 O (40%) containing acetic acid] combined with determination by HPLC is an accurate method to extract more amount of vitexin in plant material such as 21-day old Prosopis seedlings. Additionally, the amount of vitexin extracted using this method in this work (0.554 mg/g DW) suggests that Prosopis farcta constitutes a promising biotechnological system for producing this anti-tumor agent. When comparing our vitexin production with others previously reported [30] [31], we can infer that Prosopis cultures should be optimized to be competitive at an industrial level.