One-Pot Process for the Production of Ginsenoside Rd by Coupling Enzyme-Assisted Extraction with Selective Enzymolysis

Abstract

One-pot process for the production of ginsenoside Rd by coupling enzyme-assisted extraction with selective enzymolysis was explored in this paper. Several detection methods including HPLC-MS were used to identify and quantify the products in the enzymolysis solution of pectinase. Results showed that ginsenoside Rd was the main component in enzymolysis solution, pectinase specifically hydrolyzes protopanaxadiol (PPD)-type ginsenoside and was a selective enzyme to convert ginsenoside Rb1 to Rd in a way. In addition the influencing factors on the yield of ginsenoside Rb1 and Rd were optimized using L₉(3⁴) orthogonal design data. The enzymolysis conditions for the higher yield of Rd were 52.5 °C, pH 6 and 1 h with a yield of 0.8314 from 50 mg drug material. The controllable transformation hypothesis of the PPD-type ginsenoside was also explored from the perspective of the molecular steric hindrance. Pectinase could be used as an efficient enzyme for one-pot producing ginsenoside Rd.

INTRODUCTION

Panax Quinquefolii Radix (Panax quinquefolius L.) is one of the well-known herbal medicines, used as a tonic, sedative, anti-fatigue and anti-gastric ulcer drug, as well as for its antidiabetic and antitumor activities.^1–4) Some protopanaxadiol (PPD)-type ginseng saponins, such as ginsenoside Rd, Rg3 and CK, as Fig. 1 showed, naturally present at low concentrations, were reported to be the primary bioactive components in Panax Quinquefolii Radix.^5,6) Therefore, separation, transformation and preparation of such ginsenosides would be of great interest to enable their medical applications.^7,8) Ginsenoside Rd was reported to have many pharmacological activities, such as decreasing atherosclerosis risk,⁹⁾ immunological adjuvant functions,¹⁰⁾ improved learning and memory,¹¹⁾ neuroprotection and thrombosis prevention.^12,13) However, the content of ginsenoside Rd is low in natural resoures, so sourcing it for use as a drug is a pressing concern. In addition, ginsenoside Rd is difficult to synthesize because of its complex structure. Fortunately ginsenoside Rb1 can be converted into ginsenoside Rd by hydrolysis because they have similar glucoside structures. In addition, Panax Quinquefolii Radix is rich in ginsenosides, and its drug materials have a high content of ginsenoside Rb1.¹⁴⁾ To our knowledge, many studies focused on conversion of ginsenoside Rb1 to Rd, usually by chemical, microbial or enzymatic transformations. Chemical transformation methods usually have poor selectivity, low efficiency, are environmentally deleterious and involve heating, acid or alkaline hydrolysis that can decrease bioactivity.¹⁵⁾ Microbial transformation is not suitable for industrial applications because of high cost, low selectivity, low yield and production of complex fermentation products that are difficult to purify.^16–19) In addition ginsenosides contents differed markedly, depending on whether liquid or solid fermentation was used. Furthermore, conversion of ginsenoside Rb1 to Rd was usually incomplete, even with solid fermentation.²⁰⁾

Fig. 1. Chemical Structure of PPD-Type Ginseng Saponins

R1-Ginsenoside Rb1, Glc(2→1)glc; Rd, Glc(2→1)glc; Rg3, Glc(2→1)glc; Rh2, glc; CK, H. R2-Ginsenoside Rb1, Glc(6→1)glc; Rd, glc; Rg3, H; Rh2, H; CK, H.

In comparison with chemical or microbial methods, enzymatic transformation has many advantages such as environmental compatibility, mild reaction conditions, high selectivity and controllable reactions. Some reports described enzymatic transformation of ginsenosides, including biotransformation of ginsenoside Rb2 to compound Y,²¹⁾ ginsenoside Rb1 to ginsenoside Rg3 and ginsenoside Rb1 to compound-K.^22–24) In our previous report, it was found that pectinase could hydrolyzes PPD-type ginsenoside and convert ginsenoside Rb1 into ginsenoside Rd in a way.^25,26) In addition, some optimum seeking methods including orthogonal design were often used to increase the yield of target products and to find the optimum operating conditions.^27,28) So far as we know, few reports involved the one-pot process coupling enzyme-assisted extraction and enzymatic transformation of ginsenosides from drug material. Based on L₉(3⁴) orthogonal design, our study examined the efficient one-pot process for producing ginsenoside Rd using enzyme-assisted extraction and enzymatic transformation simultaneously.

MATERIALS AND METHODS

Materials

Acetonitrile and methanol were HPLC grade (Tedia Company, Inc., U.S.A.). Water used in the mobile phase was double distilled. Panax Quinquefolii Radix samples from a local drugstore in Jinan (Shandong, China) were powdered in a mill and filtered through a 40-mesh sieve. Pectinase (60000 units/mL, Jinan, China) was used. Standard ginsenoside Rg1, Re, Rb1 and Rd samples were purchased from National Institute for Food and Drug Control (Beijing, China). Phosphoric acid, n-butanol and ethanol were from Tianjin Bodi Chemical Ltd. (Tianjin, China).

Apparatus

The analysis was performed on a Shimadzu HPLC equipped with LC-20 A pumps, an auto-injector and a SPD-20 A UV-VIS detector. LC-MS was performed on an Agilent 6520 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS system (G6520A), with a dual-nebulizer electrospray ionization (ESI) source. Mass Hunter Workstation and Qualitative Analysis by Agilent Technologies Inc. were used for qualitative analysis.

Chromatographic Conditions

A Venusil XBP C₁₈ column (250 × 4.6 mm i.d., 5 µm, theoretical plate number >5000) was used to separate ginsenosides eluted with a binary solvent consisting of acetonitrile (A) and 0.1% phosphoric acid water solution (B) with a gradient elution: 0.01–25 min, 19–20% A; 25.01–60 min, 20–40% A; 60.01–80 min, 40.1–100% A. Other conditions were as follows, flow rate of the mobile phase, 1.0 mL min⁻¹; column temperature, 30 °C; detection absorbance, 203 nm; injection volume, 20 µL.

HPLC-MS Conditions

An Acquity BEH C₁₈ column (100 × 2.1 mm i.d., 1.7 µm) was used to separate ginsenosides in a binary solvent consisting of acetonitrile (A) and 0.1% formic acid water solution (B) with a gradient elution: 0.01–25 min, 19–20% A; 25.01–60 min, 20–40% A; 60.01–80 min, 40.1–100% A. Other conditions were as follows, flow rate of the mobile phase, 0.2 mL min⁻¹; column temperature, 40 °C; drying gas flow rate, 5 L min⁻¹; cone gas temperature, 325 °C; nebulizer pressure, 15 psi; capillary voltage 4000 V.

Enzymolysis Procedure

About 50 mg drug power made from Panax Quinquefolii Radix (24-mesh) was weighed and placed in a 50 mL round-bottom flask. Water (25 mL) containing a known amount of enzyme (about 6.0 mg) was then added, the pH was adjusted with 0.12 mol L⁻¹ HCl solution and the mixture incubated for indicated time periods and temperatures in a water bath. After enzymolysis, each mixture was extracted with an equal volume of water-saturated n-butanol, shaken and filtered. Then, 25 mL of each filtrate was evaporated to dryness. The residues were each dissolved in 5 mL 50% methanol, filtered and the filtrates retained for analysis.

Experimental Design

An L₉(3⁴) orthogonal test design with blank column was used to optimize the enzymolysis conditions since the existence of blank column in orthogonal table could be used for error estimation and the possible interactions between factors.²⁸⁾ Based on the previous experiment results,²⁵⁾ especially the fact that pectinase was usually used within 40–60 °C, the proper value of the factors was determined. The parameters, including reaction temperature (42.5, 47.5, 52.5 °C), pH (6.0, 4.0, 2.0) and time (1.0, 2.0, 3.0 h), were selected as variables and coded as A, B, and C, respectively on our previous research. In addition the blank column in orthogonal design was coded as D.

Statistical Analysis

All assays were performed in triplicate and results presented as means ± standard deviations.

RESULTS AND DISCUSSION

Quantitative Analysis of the Main Constituents of Degradation Solutions

In our previous report, the main ginsenosides in the complex sample matrix, including Rg1, Re and Rb1, were separated effectively by HPLC.²⁵⁾ We therefore analyzed the enzymatic hydrolysis solutions by HPLC, finding that chromatograms had well-resolved peaks corresponding to these ginsenosides. During reactions, the peak corresponding to Rb1 was clearly decreased and a new peak appeared during the same time period. Figure 2 shows the HPLC chromatogram of an enzymatic hydrolysis solution.

Fig. 2. HPLC-UV Chromatograms of Enzymolysis Solution Obtained at the Experimental Conditions Using L₉(3)⁴ Orthogonal Design

1-Ginsenoside Rb1, 2-Ginsenoside Rd.

Effects of Enzyme Addition on Ginsenoside Content

The enzyme solution was added to an aqueous solution of the drug material fine powder. Various volumes of enzyme solution were tested, with results shown in Fig. 3. After hydrolysis for 45 min under specified conditions, samples were analyzed by HPLC. The content of ginsenoside Rd was abruptly increased when 0.5 mL enzyme was used, and remained nearly constant from 0.5 to 4.0 mL enzyme, finally dropping sharply. This indicated that the enzyme concentration was sufficient for enzymolysis under these experimental conditions. In addition, ginsenoside Rd content was decreased, because of further enzymolysis, when the added enzyme exceeded 4.0 mL.

Fig. 3. Effect of the Enzyme Addition on the Content of Ginsenoside Re, Rg1, Rb1 and Rd

Conditions: 50 mg medicinal materials sieved by 40 mesh sieve and the enzymolysis conditions were 45 °C, pH 6 and 1h.

Simultaneously, ginsenoside Rb1 content was decreased with enzyme addition and remained nearly constant at 0.5 to 4.0 mL added enzyme, indicating maximal hydrolysis of ginsenoside Rb1. However, when added enzyme was increased to 5.0 mL, the chromatographic peak corresponding to ginsenoside Rb1 was nearly absent but the peak corresponding to ginsenoside Rd was also decreased, indicating simultaneous hydrolysis of the two ginsenosides. The optimum range for preparing Rd was, therefore, 0.5–4.0 mL enzyme.

The contents of ginsenoside Rg1 and Re were almost constant with added enzyme up to 4.0 mL. This suggested that protopanoxatriol (PPT)-type ginseng saponin was difficult to hydrolyze under these conditions. The enzyme concentration in the reaction mixture is an important factor affecting degree of hydrolysis. The amount of enzyme should be as low as possible for enzymatic preparation of Rd under optimal conditions. In this manner, controllable preparation of Rd should be possible, without subsequent hydrolysis of Rd because of excessive enzyme.

In the fourth and fifth tested reaction conditions, the Rd peak area was relatively low and that of Rb1 was high (Fig. 2). Data variability could have been caused by differences in sample amounts or particle sizes. For example, the Rb1 content, as determined, might be higher than expected because of incomplete enzymolysis caused by the relatively large particle size of the herbal material.

Analysis of Orthogonal Design Data

Reaction temperature, pH and time were optimized using an orthogonal L₉ (3⁴) test design. Results of the orthogonal test are shown in Table 1 and were analyzed by a range analysis method. K and k value for orthogonal design represents the sum and the mean of each factor respectively, and R represents the range difference for each factor by range analysis.

Table 1. The Range Analysis and Variance Analysis of Results of the Orthogonal Experimental Design (n = 2)

Run No.		Influencing factors				Ginsenoside Rd (mg/mL)
		A	B	C	D
1		1	1	1	1	0.6245
2		1	2	2	2	0.3628
3		1	3	3	3	0.3671
4		2	1	2	3	0.3618
5		2	2	3	1	0.3804
6		2	3	1	2	0.6007
7		3	1	3	2	0.8314
8		3	2	1	3	0.6487
9		3	3	2	1	0.7697
Ginsenoside Rd	K1	1.3544	1.8177	1.8739	1.7745
	K2	1.3429	1.3919	1.4943	1.7949
	K3	2.2498	1.7375	1.5789	1.3776
	k1	0.4515	0.6059	0.6246	0.5915
	k3	0.7499	0.5791	0.5263	0.4592
	R	0.3023	0.1419	0.1265	0.1391
Priority		A > B > C
K and k value for orthogonal design represents the sum and the mean of each factor, respectively, and R represents the range difference for each factor by range analysis.
Source		Type III sum of squares	df	Mean square	F	Sig.
Corrected model		0.278a)	6	0.046	264.331	0.004
Intercept		2.719	1	2.719	15533.446	0.000
A		0.180	2	0.090	515.482	0.002
B		0.071	2	0.035	201.893	0.005
C		0.011	2	0.005	30.436	0.032
Error		0.000	2	0.000
Total		2.997	9
Corrected total		0.278	8

a) R Squared = .999 (Adjusted R Squared = .995).

By max–min difference and variance analyses, we identified the factors most influencing enzymolysis. For ginsenoside Rd, the yields from nine runs ranged from 0.3618 to 0.8314 mg. The effect of enzymolysis conditions on ginsenoside Rd yields were decreasing, in the order A > B > D > C, that is, enzymolysis temperature > pH > time, and there was a certain interaction between the influencing factors, based on the R values. Among these reactions, run 7, conducted at 52.5 °C and pH 4 for 3 h, had the highest yield. Runs 8 and 9 also had higher Rd yields than did the others (p < 0.05). Enzymolysis temperature had the strongest influence of the three variables, with Rd yield increasing with increased temperature. Under normal conditions, enzymatic activities are greater at higher temperatures and the enzymolysis temperatures are usually in the range of 42.5–52.5 °C. We used 52.5 °C as the preferred temperature, based on our previous Rd test data, because pectinase activity might be adversely affected at even higher reaction temperatures. Longer enzymolysis times usually produce higher yields but our findings showed that time had no notable effect on Rd yield. The results also showed that pH effects were negligible, though generally pH is an important parameter in enzymolysis.

The highest Rd yield was expected under conditions described as “A3B1C1,” based on the K values in Table 1. The results of range and ANOVA analyses almost led to the same conclusion. So, enzymolysis was performed in triplicate to validate the optimum conditions identified by orthogonal L₉ (3⁴) test design. The highest yield of 0.8326 mg Rd from 50 mg drug material (data not shown), was obtained with A3B1C1 conditions, slightly higher than that in run 7. At last A3B1C1 conditions were selected as the optimum procedure for preparing Rd for producing more ginsenoside Rd with the conversion ratio (%) of 46.15% as Table 2 showed.

Table 2. Conversion Ratio (%) of the Main Ginsenosides from Panax Quinquefolii Radix by the Pectinase Enzymolysis (n = 2)

Ginsenosides	Amount in drug material (mg)	Amount in production (mg)	Theoretical yield of Rd (mg)	Conversion ratio (%)
Rb1	1.638	0.02562	—	46.15
Rd	0.1733	0.8314	1.426

Conditions: 50 mg medicinal materials sieved by 40 mesh sieve and the enzymolysis conditions were 52.5 °C, pH 6 and 1h.

Identification of the Major Ginsenoside Products by LC-MS/MS

The ginsenoside chromatograms were very different before and after degradation, as Fig. 2 shows. In addition a spot corresponding to ginsenoside Rb1 was increased and, at the same time, that for ginsenoside Rb1 nearly disappeared from TLC chromatograms. HPLC-MS was then used to separate and identify changes in the major ginsenosides in the enzymolysis solutions, with results shown in Table 3. ESI-MS data for four ginsenosides agreed with previously reported results.²⁶⁾ The four clearly changing ginsenosides were identified as Rb1, Rd, Rg3 and Rh2, with retention time of 44.18, 48.43, 54.47, and 63.44 min, respectively. Possibly, hydrolysis was hindered by the saponin moiety of protopanoxadiol because protopanoxadiol was not observed in the total ion chromatogram. Results showed that the enzyme catalyzed specific hydrolysis of glucopyranosides at the C-20 position and that these moieties could be readily dissociated under our experiment conditions.

Table 3. ESI-MS Data of Ginsenoside Rb1 and Its Secondary Glycosides Determined in Positive Mode

Ginsenoside	tR(min)	Molecular formula	m/z [M + Na]+	MS fragments [M + Na]+
Rb1	44.18	C54H92O23	1113.58	407.36	425.37	587.43	749.48
Rd	48.43	C48H82O18	969.68	407.36	425.37	587.42	835.48
Rg3	54.47	C42H72O13	807.7	407.36	587.43	767.49
Rh2	63.44	C36H62O8	645.43	217.19	369.31	425.37

The Hypothesis of Controllable Transformation of Ginsenoside Rb1 to Ginsenoside Rd

Pectinase hydrolyzes generally pectin (mainly, α-(1→4)-linked D-galacturonic acid polymer). A few reports were involved in pharmacology activities of pectinase-processed Ginseng radix (GINST). According to the reports above, the pectinase is capable for producing minor ginsenosides as final products. In fact some secondary glycosides of ginsenoside Rb1 were observed indeed, including Rd, Rg3 and Rh2.²⁶⁾ Both ginsenosides Rb1 and Rd are PPD-type ginseng saponins. Transformation of ginsenoside Rb1 to ginsenoside Rd involved removal of glucosides at the C-20 position. C-20 is a chiral atom and ginsenoside Rd exists as the S-isomer. Chemical, microbial and enzymatic conversion are all readily achievable, perhaps because of the relatively low spatial restriction at the C-20 glycoside bond. With removal of the first glycosyl group from –glu(6→1)glu and conversion of Rb1 to Rd, spatial restriction is further decreased at the C-20 glucoside bond, enabling further hydrolysis of Rd to Rg3 through removal of the second glycosyl group at C-20. Rg3 can also be further hydrolyzed, removing the glycosyl group at C-3. The process proceeds in phases, closely dependent on reaction time in the presence of a constant enzyme concentration. There are two possible reasons for this. First, steric hindrances at the glucoside bonds C-3 and C-20 are successively decreased. Second, the reaction rate is related to the amounts of substrate and enzyme, as in a second-order reaction. As the concentration of ginsenoside Rb1 is high at the beginning of hydrolysis, it is rapidly hydrolyzed, with the hydrolysis reaction at the first stage. At this time, the concentration of Rd is low, and steric hindrance at the glycoside bond is relatively greater, so Rd is resistant to hydrolysis. As the Rd concentration increases, there is less steric hindrance at the glycoside bond and a decrease of the strong tension at glycoside bond C-20 because of loss of the glyco moiety. In this case, activation energy is decreased and the Rd hydrolysis rate increases, with rapid loss of Rd during the second stage. At the same time, the concentration of Rg3 is relatively low, and the second glycoside bond at C-20 has relatively high steric hindrance, so Rg3 is resistant to hydrolysis. With increased Rg3 concentration, its rate of hydrolysis increases, with rapid loss of Rg3 during the third stage. Thus, the reaction stages progress accordingly.

The enzymolysis rates of ginsenosides were closely related to the concentration of each individual ginsenoside and of the enzyme, with enzymolysis behaving like a bimolecular binding reaction. Only the free enzyme has catalytic activity while bound enzyme does not. This is why the secondary products were stably formed. Thus, a controllable transformation of ginsenoside Rb1 to ginsenoside Rd was possible. In summary, conversion of Rb1 proceeded via at least three phases of hydrolysis. During the first stage, Rd was formed by controlling time of hydrolysis and amount of enzyme. During subsequent stages, enzymolysis was more difficult to control because some ginsenosides, including Rd and Rg3, were simultaneously hydrolyzed.

CONCLUSION

Ginsenoside Rd was produced at high yields through one-pot process by coupling enzyme-assisted extraction with enzymatic transformation in this paper. Under these conditions, the concentration of ginsenoside Rd increased, while that of Rb1 clearly decreased. Conversion of Rb1 occurred in phases, involving at least three stages of hydrolysis. Rd was successfully produced by controlling hydrolysis time and the amount of enzyme during the first stage. Our findings suggested potential applicability of an enzymatic method as a suitable means to prepare ginsenoside Rd through one-pot process. However, further studies are needed to more clearly characterize the reaction of ginsenoside and enzyme, enabling precisely controlled enzymolysis to be achieved.

Acknowledgments

The work was supported by A Project of Shandong Province Higher Educational Science and Technology Program (No. J15LM02) and the Program for Scientific Research Innovation Team in Colleges and Universities of Jinan (No. 2018GXRC006).

Conflict of Interest

The authors declare no conflict of interest.

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