The changes of ginsenoside patterns in red ginseng processed by organic Acid impregnation pretreatment.

In order to enhance bioactive functionalities of ginseng, an acid impregnation processing was applied as a pre-treatment in producing red ginseng. Acid impregnation studies were conducted, and acids (ascorbic, malic, and citric acid) were selected. The optimal concentration of each acid was investigated in this study in terms of ginsenoside contents. The most concerned ginsenoside, Rg3 was increased by ascorbic, malic, and citric acid pre-treated red ginseng up to 1 M acid concentration. In the case of ascorbic acid pre-treated red ginseng, Rg2 concentration was increased depending on acid concentrations. Citric acid pre-treatment enhanced Rg2, Rg3, and Rh1+Rh2 formation in red ginseng. Therefore, ginsenoside patterns in red ginseng could be changed by acid impregnation pre-treatment depending on acid concentration and acid types. This research is expected to contribute to the development of the ginseng industry via new red ginseng products with selective and intensified functionality.

INTRODUCTION

Ginseng (Panax ginseng Meyer) has been used as a marvelous folk remedy in the East for thousands of years [1,2]. Recent scientific research showed that the major active compounds of ginseng have positive nutraceutical effects on various diseases such as high blood pressure, hyperlipidemia, liver disease, depression, diabetes, cancer, fatigue, stress, and Raynaud's phenomenon, as well as on the improvement of memory, learning, immune system, cardiovascular system, anti-arteriosclerosis and cholesterol metabolism [3-6]. Such nutraceutical effects of ginseng come from ginsenosides, a class of steroid saponins. Saponins are often subdivided into two main classes, the triterpenoid and the steroid saponins by the structure of aglycone (nonsugar moiety). The triterpenoid saponins are further divided into oleanane-triterpenoid saponins in Platycodon grandiflorum and Polygala tenuifolia, and dammarane-triterpenoid saponins in ginseng. Representative dammarane-triterpenoid saponins further subdivided into protopanaxadiol, protopanaxatriol and oleanolic acid by the chemical structure [7-10].

More than forty different ginsenosides have been identified so far, and play an important role in the nutraceutical effects of ginseng. Since the amount of an individual ginsenoside is trace, if the amount of a certain type of ginsenoside can be increased to enhance its specific function, it could be a good option for customers. Especially, ginsenoside Rg3 suppresses cancer metastasis and cancer drug resistance, and functional food such as Rg3-enriched red ginseng will draw public attention and contribute to expanding the ginseng market [11,12].

Since red ginseng produced by conventional manufacturing processes contains a small amount of ginsenoside Rg3, many studies focusing on Rg3 enrichment are being conducted in Korea and other foreign countries. Such functional red ginseng products containing a higher amount of Rg3 have already been released into the market, and the competitive launches of new products is also expected. However, previously developed Rg3 enrichment processing methods tend to destruct other functional ginsenosides. Also, the form of previous Rg3 enriched ginseng products is extract type, not root type and show limitations in developing various products due to excessive heat treatments applied during the extraction of red ginseng [13,14].

This study aimed to enrich Rg3 in red ginseng, without destroying the shape of red ginseng, because it will lead to the further development of various red ginseng products. Since previous studies, which aimed to enrich Rg3 with the acid treatment of red ginseng extracts, did not carefully consider the change of other ginsenosides as well as the change of ginsenoside patterns by various organic acids, this study focused on the change of ginsenoside patterns by impregnating various organic acids in producing red ginseng in order to enrich Rg3. We herein describe the change of ginsenoside patterns after impregnation pretreatments using three different organic acids (selected in our previous study) and an ideal condition for organic acid impregnation [15-17].

MATERIALS AND METHODS

Organic acid impregnation pretreatments

Four to five-year-old fresh ginseng roots were purchased from Anseong Ginseng Cooperative (Anseong, Korea), and kept at -2℃ to 0℃ before use. To infiltrate organic acid into fresh ginseng roots, the impregnation processing was performed as follows. Two cleaned fresh ginseng roots (~140 g) were submerged fully in the flask containing 500 mL of acid solution, and the pressure in the flask was reduced by using an aspirator for 30 min. Three different organic acids (malic acid, ascorbic acid, citric acid; Sigma, St. Louis, MO, USA) were selected from previous studies, and various concentrations of the organic acids (0.1 to 2 M) were employed in the pretreatment process [16-19].

Red ginseng manufacturing process

Red ginseng was manufactured as described by Chang et al. [19]. The fresh ginseng roots pretreated with organic acid were steamed at 96℃ to 98℃ using the autoclave(MG-6845; Mega Science, Seoul, Korea) in normal pressure for 2.5 h. After cooling to room temperature, the ginseng was dried at 70℃ for 24 h and at 50℃ for 72 h in Air Flow Oven (WFO-601SD; Eyela, Tokyo, Japan).

Determination of the content of crude saponin

Each 2 g of red ginseng powder was suspended in 100 mL of 80% methanol. Extraction was performed at 80℃ for 3 h in water bath, and repeated two times. The extract was concentrated at aspiration pressure, and dissolved in 20 mL of distilled water. The extract solution in 250 mL separation funnel was washed with 20 mL of ethyl ether, and extracted with 20 mL of water-saturated butanol three times. Extracts in water-saturated butanol containing crude saponin were collected in a separation funnel, and washed with 60 mL of distilled water two times. The layer of butanol was concentrated in an evaporator, and dissolved in 2 mL of methanol. The final methanol extract was then filtered through a Whatman 0.45 μm filter (Whatman, Maidstone, England) and used as a source for determining the content of crude saponin.

Determination of total saponin content

Crude saponin in 1 mg of red ginseng powder was dissolved in 1 mL of methanol and filtered using 0.45 μm microfilter kit (Whatman). One hundred microliter of crude saponin solution was mixed with 0.3 mL of 8% vanillin and 4 mL of 75℃ sulfuric acid, and incubated at 60℃ for 10 min in water bath. The absorbance of the mixture at 545 nm was measured. Standard curves were obtained by using 0.2 to 1.0 mg/mL of ginsenoside Re (Wako, Osaka, Japan) as a reference standard for colorimetric analysis.

Analysis of ginsenoside composition

Crude saponin in 1 mg of red ginseng powder was dissolved in 1 mL of methanol. After filtering crude saponin, solution was analyzed using HPLC (PU-980; Jasco, Tokyo, Japan). Analytic standard solution was prepared using 12 ginsenosides (Rg1, Re, Rf, Rb1, Rg2, Rh1, Rc, Rb2, Rb3, Rd, Rg2, Rh2; Fleton Reference Substance Co., Chengdu, China), and the concentration of each individual ginsenoside in standard solution was 0.1 mg/mL. Quantification of individual ginsenoside was performed in the following conditions. As a HPLC column, Lichrosorb-NH2 (5 μm, 250×4.6 mm; Merck, Darmstadt, Germany) was used. The mobile phase was a solution A (acetonitrile/water/isopropanol:80/5/15) and solution B (acetonitrile/water/isopropanol:80/20/15). Thirty percent to one hundred percent (0 to 20 min), 100% (20 to 55 min) and 100% to 30% (55 to 65 min) of B solution were employed as a gradient system. Mobile phase flow speed was 1.0 mL/min and the detector was ELSD 2000 (Alltech, Deerfield, IL, USA). Analysis was performed at 92℃ and nitrogen gas (2 L/min) was used as nebulizing gas [20].

RESULTS AND DISCUSSION

Contents of crude and total saponin after acid pretreatment

The change of saponin contents in red ginseng by acid pretreatment was summarized in Table 1. The content of crude and total sapoin in red ginseng was 3.43% and 1.91%, respectively. Pretreatment of citric acid and ascorbic acid did not produce significant change in crude and total saponin content in red ginseng. Different concentrations of citric acid and ascorbic acid in pretreatment also did not change the crude and total saponin content.

Table 1.

The changes of saponin contents in red ginseng by acid pre-treatment

Acid treatment	Concentration (M)	Crude saponin (%)	Total saponin (%)
Control		3.43	1.91
Ascorbic acid	1	3.17	1.60
	0.5	3.02	1.63
	0.1	3.23	1.58
Malic acid	2	2.78	1.34
	1	2.90	1.41
	0.5	2.88	1.44
	0.1	3.37	1.63
Citric acid	2	3.59	1.90
	1	3.24	1.92
	0.5	3.55	1.81
	0.1	3.53	1.91

However, malic acid pretreatment slightly decreased the content of crude and total samponin in red ginseng. Such decrease was already observed in our previous study. It seems that more soluble saponin was released after acid solution was permeated into fresh ginseng roots. The amount of soluble saponin release showed a positive correlation with the concentration of malic acid.

Ginsenoside composition after acid pretreatment

To investigate the effect of acid concentration on ginsenoside compositions in red ginseng, different concentrations of malic acid (0.1 M, 0.5 M, 1 M, 2 M) were employed in pretreatment, and the result was summarized in Table 2.

Table 2.

Ginsenosides content depending on malic acid concentrations (mg/g)

Ginsenosides	Control	Malic acid concentration (M)
		0.1	0.5	1	2
Rh1+Rh2	0.018	0.010	0.027	0.049	0.011
Rg2	0.043	0.065	0.092	0.080	0.020
Rg1	0.037	0.039	0.020	0.019	0.029
Rg3	0.011	0.053	0.018	0.185	0.111
Rf	0.035	0.055	0.050	0.027	0.003
Rd	0.040	0.029	0.010	0.004	0.015
Re	0.296	0.215	0.175	0.208	0.111
Rb1	0.144	0.205	0.016	0.097	0.081
Rb2	0.049	0.055	0.028	0.019	0.043

Pretreatment of up to 0.5 M of malic acid did not change the content of Rg3 in red ginseng. However when 1 M malic acid was used for the pretreatment, the content of Rg3 (0.185 mg/g) was increased 10 times. Since Rg3 has nutraceutical functions of anti-metastasis of cancer cells, blood vessel relaxation, and anti-thrombosis, we may expect stronger anti-cancer effects from red ginseng produced by the pretreatment of 1 M malic acid. Ginsenoside Rg3 was produced by removing glucose-glucose from Rb1 and arabinose-glucose from Rb2, respectively (Figs. 1 and 2). Since increased content of Rg3 by malic acid pretreatment was accompanied with decreased Rb1 content in red ginseng, Rb1 is a source for Rg3 production.

Fig. 1.

The chemical pathway of protopanaxadiol ginsenosides.

Fig. 2.

The chemical pathway of protopanaxatriol ginsenosides.

The pattern of ginsenoside composition in red ginseng was summarized in Table 3. The content of Rg3 became slightly increased by the pretreatment of up to 0.5 M ascorbic acid, but when 1 M ascorbic acid was used in the pretreatment, the content of Rg3 increased more. That is, the increase of Rg3 content was proportional to the concentration level of ascorbic acident of Rg2 was also increased in proportion to ascorbic acid concentration, but the amount of other individual ginsenosides in red ginseng did not change by ascorbic acid pretreatment. On the contrary of the change by malic acid pretreatment, the pretreatment of ascorbic acid did not lead to a significant decrease in protopanaxadiol ginsenosides of Rb1 and Rb2, but also resulted in only a slight increase in Rg3. Interestingly, the content of protopanaxatriol ginsenoside Re was decreased by ascorbic acid pretreatment. By the ascorbic acid pretreatment, glucose was separated from a hydroxyl group on the 20th carbon of Re, and as the result of this chemical reaction Rg2 was produced. The increased content of Rg2 by ascorbic acid pretreatment was shown in Table 4. The functionality of ginsenoside Rg2 has been reported in cardiovascular diseases, improving memory and anti-cancer. Therefore, the red ginseng pretreated with ascorbic acid would be useful for individuals suffering from these diseases.

Table 3.

Ginsenoside contents depending on ascorbic acid concentrations (mg/g)

Ginsenosides	Control	Ascorbic acid concentration (M)
		0.1	0.5	1
Rh1+Rh2	0.018	0.008	0.020	0.046
Rg2	0.043	0.099	0.140	0.138
Rg1	0.037	0.022	0.033	0.041
Rg3	0.011	0.010	0.046	0.078
Rf	0.035	0.043	0.048	0.070
Rd	0.040	0.027	0.019	0.017
Re	0.296	0.198	0.142	0.127
Rb1	0.144	0.103	0.149	0.193
Rb2	0.049	0.040	0.054	0.069

Table 4.

Ginsenoside contents depending on citric acid concentrations (mg/g)

Ginsenosides	Control	Citric acid concentration (M)
		0.1	0.5	1	2
Rh1+Rh2	0.018	0.079	0.155	0.227	0.037
Rg2	0.043	0.179	0.261	0.265	0.075
Rg1	0.037	0.073	0.022	0.064	0.028
Rg3	0.011	0.093	0.346	0.707	0.303
Rf	0.035	0.087	0.071	0.053	0.049
Rd	0.040	0.042	0.020	0.007	0.002
Re	0.296	0.315	0.149	0.055	0.015
Rb1	0.144	0.147	0.120	0.012	0.024
Rb2	0.049	0.093	0.043	0.025	0.007

The pattern of ginsenoside composition in red ginseng was also changed depending on the concentration level of citric acid used in the pretreatment, and it was summarized in Table 4. Unlike two other organic acids, the pretreatment of citric acid decreased the content of ginsenosides Rd, Re, Rb1, and Rb2 in red ginseng in proportion to the increase of acid concentration. However, the pretreatment of 2 M citric acid significantly increased the Rg3 content (0.303 mg/g), and it was about 30 times higher than that (0.093 mg/g) of 0.1 M citric acid pretreatment. Among the organic acids tested in the present study, citric acid was most effective for increasing Rg3 content in red ginseng. This may be attributable to the change of ginsenoside Rb1 and Rb2 into Rg3. The content of Rh1+Rh2 was also increased up to 0.079 mg/g by the 0.1 M citric acid pretreatment, and such increasing content showed a positive correlation with the concentration of citric acid used in the pretreatment. Rh2 is produced by removing glucose from Rg3 and destructed Rg3 was observed in high citric acid condition. Furthermore, the pretreatment of much higher concentration of citric acid (2 M) rather decreased the content of Rh1+Rh2.

Unlike two other acids (malic acid, ascorbic acid) used in the present study, citric acid pretreatment affected the content of both protopanaxadiol and protopanaxatriol ginsenosides in red ginseng. The content of Rg3 was increased when protopanaxadiol ginsenosides (Rb1 and Rb2) were decreased. Citric acid was more powerful than malic acid in increasing the content of Rg3 in red ginseng (Fig. 3). We may also expect that citric acid pretreatment induced the producing of Rh2, which needs further chemical reaction after Rg3 formation. Unfortunately, our present HPLC analysis could not determine the level of Rh1 (triol) and Rh2 (diol) separately. Therefore, further study has to clarify whether Rh2 is produced from Rg3 after citric acid pretreatment. Pretreatment of citric acid also led to change of protopanaxatriol ginsenosides in red ginseng. For example, by citric acid pretreatment, the content of Re was reduced, whereas the content of Rg2 as well as Rh1+Rh2 produced by Rg2 degradation was increased in red ginseng.

Fig. 3.

(A) The patterns of HPLC chromatogram in red ginseng ginsenosids and (B) red ginseng produced by citric acid impregnation pretreatment.

Efforts were made to change the pattern of ginsenoside composition in ginseng extract, but there has been no report on the change induced by the pretreatment using different types of organic acids. Our overall findings showed that the pretreatment of malic acid and ascorbic acid more selectively affected the production of protopanaxadiol and protopanaxatriol ginsenosides in red ginseng, respectively. Interestingly, when citric acid was used in the pretreatment, the composition of both protopanaxadiol and protopanaxatriol ginsenosides was changed in red ginseng. Because of the trace amount of ginsenoside Rg3 in red ginseng, recent studies have been focused on how to increase Rg3 during the manufacturing process of red ginseng extract. However, such efforts either required special equipment for the production of raw materials, or had shortcomings of destructing other useful saponin components in red ginseng extract. Our present study suggests a simple and economic organic acid impregnation pretreatment method, which can process root shape red ginseng and significantly increase functionalities of red ginseng without any notable destruction of ginsenosides or any deformation of root shape. Time and temperature to optimize organic acid impregnation pretreatment will be established in further studies.

To develop ginseng products in root shape with useful functionalities, our present study focused on the optimization of organic acid pretreatment and the characteristics of new red ginsengs produced by organic acid pretreatment. The content of crude and total saponin in the red ginsengs was not affected by either different organic acid pretreatments or organic acid concentrations. However, all three organic acids (ascorbic acid, malic acid and citric acid) used in the pretreatment drastically increased Rg3 content in proportion to the concentration level of organic acid. But a higher concentration of organic acid (2 M) decreased the Rg3 content in red ginseng. The pretreatment of ascorbic acid increased Rg2 content in proportion to the concentration level, whereas the content of Rg3, Rg2, and Rh1+Rh2 was increased by citric acid pretreatment. The pattern of ginsenoside composition in red ginseng was changed depending on type of organic acid employed in the pretreatment, and 1 M of organic acid pretreatment was found to be a suitable concentration level for Rg3 enriched red ginseng. This study introduces the acid impregnation pretreatment that can increase the content of a certain ginsenoside, and the results of this study will serve as a basis for developing new red ginseng products with enhanced selected functionalities.