Preparative enrichment of saponins from fruits of Momordica charantia by D101 macroporous adsorption resins

Summary

Saponins are active compounds in natural products. Many researchers have tried to find the method to determine their concentration in herbs. To establish a simple extraction method for the enrichment of saponins from the fruits of Momordica charantia, we used D101 macroporous adsorption resins with optimal conditions in the concentration of loading sample is 6 mg/mL (based on the weight of the crude drug) and the volume of loading sample is 40 BV (bed volume). The extract was passed through the D101 resin column at a flow rate of 5 BV/h. After adsorption, the adsorbed column was first washed with 4 BV of distilled water, and then desorbed by 80% ethanol at a flow rate of 3 BV/h. The contents of saponin in the product were improved greatly after the separation procedure, by up to 15.20 ± 0.47 %.

1. Introduction

        Today, there are some methods for finding the concentration of active constituents from traditional medicinal plants such as solid-liquid extraction; solvent extraction, column chromatography and the preparative high-speed counter-current chromatography [1]. However, these separation methods have limitations such as low extraction yield and requiring multiple steps. Comparatively, separation methods by macroporous resin are popular as a simple procedure with easy operation, low cost, and high efficiency.

        Momordica charantia (English name: Bitter melon), which belongs to the family of Cucurbitaceae, is widely cultivated as a vegetable crop in Vietnam and tropical Asian countries [2,3]. Previous studies showed that the alcoholic extracts of M. charantia fruits inhibited the increase of serum glucose levels in glucose-loaded rats [3]. The extracts of this plant also exhibited anti-hyperglycemic activity which enhanced insulin sensitivity and lipolysis and suppressed postprandial hyperglycemia in rats [2]. Besides, researchers have indicated that the aqueous extract of its fruits has antitumor activities and inhibits tumor formation in CBA/H mice [2]. Previous chemical studies have displayed that M. charantia contains triterpenoids, saponins, flavonoids, polypeptides, and sterols [2]. Among them, the major chemical constituents are the tetracyclic triterpenoids and their glycosides as cucurbitanes, which are well-known to be responsible for anti-diabetic and hypoglycemia activities. In this work, the method for the preparative enrichment of saponins from the fruits of M. charantia, which used D101 macroporous adsorption resin, is reported

2. Materials and methods

2.1. Materials

       Fruits of Momordica charantia were collected at Tuy Loc commune, Cam Khe district, Phu Tho province. These fruits were sliced, removed cores, and dried at 50 ^oC to dryness, after that grown to powder. The contents of saponin in fruits of this species within a range between 0.32 – 0.37%.

2.2. Chemicals and equipment

       Momordicoside-G standard purchased from ALB Technology (purity ≥ 95%, HPLC); 72% sulfuric acid; 8% vanillin; 96% ethanol (EtOH).

D101 macroporous resin (true density: 1.00-1.07 g/mL; surface area 500-550 m²/g; pore volume 1.18-1.24 mL/g (Made in China). It was submerged in 96% EtOH for 24 hours and washed by distilled water before using it. Glass column (3 x 45 cm), spectrophotometer UV-Vis 1800 (Shimadzu, Japan); magnetic stirrer (Velp Scientifica, Italy; rotary evaporator devices (Buchi, Switzerland).

2.3. Methods

2.3.1. Quantification of total saponins

         The contents of total saponins were determined by the photometric method [4]. In brief, 1 mL of the extracts was mixed with 0.3 mL of 8% (w/v) vanillin solution and 3 mL of 72% (w/v) sulfuric acid. The mixture was mixed and incubated at 60^oC for 15 min and cooled on ice for 10 min. The absorbance was measured at 560 nm.

2.3.2. Preparation of sample solutions

        The powder (500 g) was extracted by 1.000 mL of EtOH : water (80:20, v/v) solution for the ultrasonication (1 h x 3 times). The solutions were combined, transferred to a rotary evaporator device, and concentrated under vacuum to dryness at 60 ^oC. The content of total saponin in the dried extract was 3.70% (w/w). In order to investigate the effect of initial concentration, the adsorption tests were performed by 200 mL of sample solutions at different concentrations (2, 4, and 6 mg extract per mL).

2.3.3. Isolation method

The process of adsorption

       The extract of M. charantia was diluted in water to concentrations (2, 4, 6 mg/mL), and then breakthrough column (3 x 45 cm) has filled D101 macroporous adsorption resin (150 g, dry weight) [the bed volume (BV) of the resin is 300 mL and the packed length of the resin bed is 25 cm] with a flow rate 5 BV/h. The content total saponins were determined by the photometric method in the solution of every 600 mL (3 BV) collected.

The process of desorption

       Static desorption experiments were carried out in Erlenmeyer flask 500 mL, having 15 g (dry weight) of the D101 macroporous adsorption resin, adding 300 ml of the extract (6 mg/mL) with a total saponin concentration of 37.0 mg/mL (C₁), stirring with a magnetic stirrer at a rate of 130 rpm for 2 hours. The contents of the saponin were determined after adsorption (C₂). The D101 macroporous resin was filtered into the Erlenmeyer flask (every 1.5 g). Each Erlenmeyer flask was added 30 mL of solvent EtOH/water from 0, 10, 20, 30, 40, 50, 60, 70, 80, 96% EtOH and shaken in 1 hour. The contents of saponin in desorption solutions were determined (C_GHP). The adsorption rate (D) of the solvent is calculated by the formula:

Where: C₁ and C₂ are the concentration of saponin in the solution before and after the adsorption.

C_GHP is the concentration of saponin in the solution of desorption solution.

The adsorption capacity is calculated by the formula:

Q_e  =

Where: Qe is the adsorption capacity (mg/g).

C₀ and C_e are the concentration of saponin in the solution before and after the adsorption (mg/ml).

V: is the solution volume (mL) of the adsorption.

m: is the weight of dry resin employed.

After the process of adsorption, the solvent of good desorption is selected for use at a rate of 3 BV/h. The contents of saponin were controlled in leakage volumes.

2.3.4. Statistical analysis

Each experiment was carried out three times. The values were expressed as means ± standard deviation (SD). The data were performed using Microsoft Excel and GraphPad Prism software.

3. Results

3.1. The process of adsorption

As shown in Fig. 1, the contents of saponin were lost similarly at three different concentrations of the extracts. The leakage of saponin of the extracts for adsorption increased slowly from 0-10% and then increased rapidly according to the volume of loading. To ensure that the product obtained high saponin, the adsorption is selected at saponin leakage of less than 10%.

Fig. 1. The kinetic adsorption for the extracts on D101 macroporous resin (the concentration of the extracts for A-2, B-4, C-6 mg/mL)

The content of saponin adsorption in 1 g D101 macroporous adsorption resin is calculated by the original saponin and saponin leakage. As shown in Fig. 2, the adsorption capacities of D101 macroporous resin increased when the concentration of the extracts raises, these results of three concentrations are evaluated statistically significant.

Fig. 1C displayed that by using 30 BV of loading volume, the leaked saponin started to exceed 10%. Therefore, the efficient adsorption is reached by using 30 BV of loading volume.

Fig. 2. The adsorption capacity of the extracts on D101 macroporous resin

3.2. The process of desorption

3.2.1. Effect of EtOH concentration on the ratio of desorption:

The absorption capacity of saponin from D101 macroporous resin was determined EtOH/water solutions (EtOH from 0% to 96%) which had been chosen as a desorption solution for the production cost and safety. The results in Table 1 and Fig. 3 show that the desorption ability increased (16.38 ± 0.53 to 88.53 ± 0.63%) when the concentration of the EtOH solution increased from 0 to 96% until it reached a maximum at 80% EtOH (adsorption rate at 94.10 ± 0.61 %). Therefore, 80% EtOH was selected as the desorption solvent. It can be understood that based on the polarity matching principle, a solute is easy to dissolve in the solvent with similar polarity, namely, a nonpolar solute dissolves in a nonpolar solvent easily, while a polar solute can simply emancipate in a polar solvent. The experimental results indicated that 80% EtOH fits with the polarity of saponins in the extract that leads to favorable desorption of saponins from the resin.

Table 1. The results of desorption on D101 macroporous resin

Fig. 3. The static desorption on D101 macroporous resin

3.2.2. The desorption of solvents:

As surveyed above, 80% EtOH is selected for desorption. The change in the concentration of saponin in the eluting solution by the volume of the solvent used is shown in Fig. 4. Accordingly, most of the saponin is eluted after 2 BV. After 4 BV the saponin concentration is still very low (38.9 mg/mL). The eluted solution is concentrated to obtain the contents of saponin at 15.20 ± 0.47% with 94.72 ± 0.64% adsorption efficiency.

Fig. 4. Desorption kinetics curves of 80% EtOH

      The product of plant extracts always interestingly to rich source of bioactive photochemical and is an important step in manufacturing. Currently, the conventional preparative methods are usually performed from the extracts by means of solid phase extraction, and liquid-liquid extraction. These separation techniques are inefficient, time-consuming, requiring large volumes of toxic organic solvents, and the recoveries are unsatisfactory. Alternatively, the adsorption-desorption process is one of the more efficient methods with a moderate purification effect. Nowadays, this method has been focused on low cost adsorbents due to the technical and economical point of view [5]. The macroporous resins have been widely used for isolation and purification of saponins, alkaloids, flavonoids, peptides, and other chemicals [6]. Liu Z. et al. used macroporous resins ADS-7, ADS-17, ADS-5, NKA-9, AB-8, D101, and X-5 for the separation of steroidal saponins in Paris polyphylla var. yunnanensis. The results demonstrated that D101 resin is the best adsorption and desorption [1]. Zhao Y.N. et al. utilized the combination of D101, D201, and D113 resins for enrichment saponins from Chinese Ginseng water decoction, the estimated contents of the total saponins reached approximately 107% based on the colorimetric method [7]. D101 macroporous resin is popularly used in the production flow sheet and green technology when using ethanol-water as the elution solvent instead of toxic organic solvents. The adsorption balance can be affected by the concentration of the extracts when the adsorption dose is large, moreover, the impurities also decrease the desorption, leading to the effect of the separation of saponins. Therefore, in order to achieve the purity of saponin, the adsorbed concentration must match the adsorption capacity. The aim of this research work is to bring saponin production higher than 10% in the extract and can be applying the domain of industrial manufactory. So, we have optimized partially for the application of D101 macroporous resin in the removal of sugar, mucus, gum, and other compounds to obtain the product with the contents of saponins at 15.20 ± 0.47% higher its contents in the initial extract (3.7%) of M. charantia.

4. Conclusion

In the present study, by using D101 macroporous resins for the preparative enrichment of saponins from fruits of Momordica charantia, the contents of saponin were determined by photometric method. After the process of implementation, the contents of saponin are shown to go up to 15.20 ± 0.47%. This method also has a reasonable price, high separation efficiency, and uncomplicated operation. Therefore, the method can be applied to the large-scale production of saponins in practice for drugs and dietary supplements.

References

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