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Effective Purification of Ginsenosides from Cultured Wild Ginseng Roots, Red Ginseng, and White Ginseng with Macroporous Resins

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This study was aimed (i) to develop an effective method for the purification of ginsenosides for industrial use and (ii) to compare the distribution of ginsenosides in cultured wild ginseng roots (adventitious root culture of Panax ginseng) with those of red ginseng (steamed ginseng) and white ginseng (air-dried ginseng). The crude extracts of cultured wild ginseng roots, red ginseng, and white ginseng were obtained by using a 75% ethanol extraction combined with ultrasonication. This was followed sequentially by AB-8 macroporous adsorption chromatography, Amberlite IRA 900 Cl anion-exchange chromatography, and Amberlite XAD16 adsorption chromatography for further purification. The contents of total ginsenosides were increased from 4.1%, 12.1%, and 11.3% in the crude extracts of cultured wild ginseng roots, red ginseng, and white ginseng to 79.4%, 71.7%, and 72.5% in the final products, respectively. HPLC analysis demonstrated that ginsenosides in cultured wild ginseng roots were distributed in a different ratio compared with red ginseng and white ginseng.
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J. Microbiol. Biotechnol. (2008), 18(11), 1789–1791
doi: 10.4014/jmb.0800.192
First published online 8 July 2008
Effective Purification of Ginsenosides from Cultured Wild Ginseng Roots,
Red Ginseng, and White Ginseng with Macroporous Resins
Li, Huayue, Jae-Hwa Lee, and Jong-Myung Ha*
Department of Pharmaceutical Engineering, College of Medical Life Science, Silla University, Busan 617-736, Korea
Received: March 9, 2008 / Accepted: April 22, 2008
This study was aimed (i) to develop an effective method
for the purification of ginsenosides for industrial use and
(ii) to compare the distribution of ginsenosides in cultured
wild ginseng roots (adventitious root culture of Panax
ginseng) with those of red ginseng (steamed ginseng) and
white ginseng (air-dried ginseng). The crude extracts of
cultured wild ginseng roots, red ginseng, and white ginseng
were obtained by using a 75% ethanol extraction combined
with ultrasonication. This was followed sequentially by
AB-8 macroporous adsorption chromatography, Amberlite
IRA 900 Cl anion-exchange chromatography, and Amberlite
XAD16 adsorption chromatography for further purification.
The contents of total ginsenosides were increased from 4.1%,
12.1%, and 11.3% in the crude extracts of cultured wild
ginseng roots, red ginseng, and white ginseng to 79.4%,
71.7%, and 72.5% in the final products, respectively. HPLC
analysis demonstrated that ginsenosides in cultured wild
ginseng roots were distributed in a different ratio compared
with red ginseng and white ginseng.
Keywords: Ginsenoside, purification, macroporous resin, HPLC,
cultured wild ginseng roots
Ginseng (Panax ginseng, C.A. Meyer) has been used as a
tonic, antifatigue, sedative, and antigastric ulcer drug for
thousands of years. Recently, many studies have suggested
that its pharmacological effects are mainly due to ginseng
saponins [5, 14]. However, it takes several years to cultivate
ginseng in fields and also needs very sophisticated care
because its growth conditions (i.e., soil, climate, and
pathogenesis) are very difficult to control. For these reasons,
low yields and high costs hamper efforts to meet the
demand of increasing markets. In general, cultured wild
ginseng roots (adventitious root culture of Panax ginseng)
are easily obtained by using a plant cell culture technique
for the production of ginseng and its active ingredient,
ginsenosides, rather than natural cultivation. With the cell
culture technique, fastidious and complicated conditions for
the production of ginseng and ginsenosides can be overcome
and optimized. Although many attempts have been made to
isolate ginseng saponins [8, 9], these were good only
for separation of each single ginsenoside from the total
ginsenosides products. Prior to the process, large amounts
of total pure ginsenosides should be obtained for its latter
separating process and bioassay. Generally, separation of
the ginsenosides was usually performed by using organic
solvents [12], which are not suitable for use in food or
medicine. There is an alternative purification method to
use adsorbents such as silica [6] and Diaion HP 20 [7, 11],
but this method also needs to use toxic organic solvents for
the extraction of crude ginsenosides. In addition, the
absorbents have poor specificity to select ginsenosides with
low efficiency. Thus, a selective and high-yield purification
method for ginsenosides needs to be developed. In this
study, three different macroporous resins (AB-8, IRA 900
Cl, and XAD 16) were used to separate total ginsenosides
from cultured wild ginseng roots (CWG, adventitious root
culture of Panax ginseng), red ginseng (RG, steamed ginseng)
*Corresponding author
Phone: 82-51-999-5467; Fax: 82-51-999-5636;
E-mail: jmha@silla.ac.kr
Fig. 1. Structures of ginsenosides from Panax ginseng.
Glc: glucose; Rha: rhamnose; Ara(f): arabinose in furanose form; Ara(p):
arabinose in pyranose form.
1790 Li et al.
and white ginseng (WG, air-dried ginseng). In addition, ten
ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rh1, Rg1, Rg2, and
Rg3) (Fig. 1) were quantitatively compared among three
kinds of ginseng products by HPLC analysis.
Ten g of each ground sample (CWG, RG, and WG) was
extracted with 200 ml of 75% ethanol in an ultrasonic bath for
120 min at 39oC. Then, the concentrated extracts (40 ml)
were passed through an AB-8 polar column (bed volume,
80 ml) to eliminate water, soluble impurities at the flow
rate of 1 BV (bed volume)/h and the adsorbed ginsenosides
were eluted with 3 BV of 70% ethanol (v/v) at 2 BV/h. The
eluent was applied onto the Amberlite IRA 900 Cl strong base
anion-exchange column at the flow rate of 1 BV/h to remove
the pigments and loaded onto the Amberlite XAD 16 column
to get rid of nonpolar substances. The final resulting eluent
was collected and evaporated to yield dried powders. The
purity of the ginsenosides was estimated by using ginsenoside
Re as a calibration standard [1]. A good linear relationship
was obtained in the range of 0.005 to 0.03 mg/ml, and the
regression equation was: y=30.376x
-
0.0258 (R2=0.9983,
n=5), where y represents the absorbance at 544 nm, x the
concentration of total ginsenosides (mg/ml). Analysis of
single ginsenoside content was performed on an HPLC
system with a reversed-phase column (Zorbax Bonus-RP
4.6 mm×150 mm, 3.5 µm).
It has been known that macroporous resins are widely
used in medicine manufacturing and in extraction of active
ingredients in natural plants such as vitexin [2], vanillin [15],
arabinogalactan [4], scutellarin [3], flavone compounds
[10], etc. As the macroporous resin has a lot of advantages
of nontoxicity, good specificity, easy operation, low cost,
and easy regeneration of resin, it can be a powerful method
for industrial use instead of toxic organic solvents. Hence,
here we also tried to develop the purification method to
obtain high levels of yield and purity of ginsenosides from
cultured wild ginseng roots, red ginseng, and white ginseng.
As is shown in Table 1, 3.12±0.14 g, 3.72±0.37 g, and
3.50±0.22 g of crude ethanol extracts were obtained from each
10 g of CWG, RG, and WG, respectively. However, the total
ginsenosides content of each crude extract was only 4.0%,
12.1%, and 11.3%. After purification with the three
macroporous resins, the purity of the ginsenosides increased
to 79.4%, 71.7%, and 72.5% for CWG, RG, and WG,
respectively, indicating that the purification steps used in
this study significantly improved the purity of the
ginsenosides in each product.
Under the chromatographic conditions used in this study,
all ten calibration curves exhibited good linear regressions
(data not shown). Standard and representative chromatograms
of purified ginsenosides products for CWG, RG, and WG
were compared to be shown in Figs. 2A
-
2D. CWG contained
similar ginsenoside types with RG and WG, but they were
distributed in different ratios compared with the other two
ginseng products (Figs. 2B
-
2D). Some unknown small
Fig. 2. HPLC chromatograms of (A) mixed standards (Rb , Rb ,
Rc, Rd, Re, Rf, Rh , Rg , Rg , and Rg ); purified ginsenosides
products of (B) CWG, (C) WG, and (D) RG. HPLC was performed
on a Zorbax Bonus-RP column (4.6 mm×150 mm, 3.5 µm).
The binary gradient elution system consisted of water (A) and acetonitrile (B)
and separation was achieved using the following gradient: 0
-
30 min, 20% B;
30
-
48 min, 20
-
40% B; 48
-
60 min, 40
-
45% B; 60
-
72 min, 45
-
55% B. The
column temperature was kept constant at 35 C. The flow-rate was 1 ml/min
and injection volume was 10 µl for the standard solution (0.5 mg/ml) and 20 µl
for the samples (1 mg/ml). The UV detection wavelength was set at 203 nm. 1:
Rg ; 2: Re; 3: Rf; 4: Rb ; 5: Rc; 6: Rb ; 7: Rg ; 8: Rh ; 9: Rd; 10: Rg .
Tabl e 1 . Yield and purity of total ginsenosides from 10 g of
ground ginseng (CWG: cultured wild ginseng roots; RG: red
ginseng; WG: white ginseng).
Yield (g) Purity (%)
Crude ethanol extracts CWG 3.12±0.14 04.0±0.8
RG 3.72±0.37 12.1±1.2
WG 3.50±0.22 11.3±0.9
Purified ginsenosides CWG 0.14±0.05 79.4±1.3
products RG 0.46±0.04 71.7±0.9
WG 0.39±0.02 72.5±1.5
EFFECTIVE PURIFICATION OF GINSENOSIDES WITH MACROPOROUS RESINS 1791
peaks observed in Figs. 2B
-
2D indicated that there are
still some uninvestigated minor ginsenosides containing in
each ginsenosides product. As shown in Table 2, the
content of protopanaxadiol-type saponins (Rb1, Rb2, Rc, Rd,
Rg3) was higher than that of protopanaxatriol-type ones (Re,
Rf, Rg1, Rg2, Rh1) in CWG and RG, whereas the contents
were opposite in the case of WG. This result was inconsistent
with the previous data reported by Wan et al. [13] that WG
contained more protopanaxadiol-type saponins. One of the
possible reasons for such a different result is that ginsengs
cultivated in different areas might accumulate different
ratios of the ginsenosides. The ratio of Rb1/Rg1, two major
ginsenosides representing protopanaxadiol and protopanaxatriol,
for CWG, RG, and WG were 2.90, 1.33, and 0.67,
respectively. The Rb1 content was higher than Rg1 both in
CWG and RG; however, the difference was much more
significant in CWG. The Rg1/Re ratio of CWG, which
contained more Re (95.1±0.4 µg/mg) than Rg1 (54.3±
0.4 µg/mg), was contrary with those of the other two
ginseng products. The Rd content in CWG (74.1±0.9 µg/
mg) was about seven times and twelve times more than
that in RG (10.6±0.8 µg/mg) and WG (6.1±0.5 µg/mg),
respectively. In addition, Rg3, a kind of minor ginsenoside,
was found both in CWG and RG, but not in WG.
In conclusion, the purification of ginsenosides using three
macroporous resins (AB-8, IRA 900 Cl, and XAD 16)
significantly increased the purity of the total ginsenosides.
In addition, the contents of each investigated ginsenoside in
cultured wild ginseng roots, red ginseng, and white ginseng
were quite different. These results assist the development of a
new ginsenosides purification method and provide a possibility
of high-quality but low-price ginsenosides products. Further
study on how to improve the efficiency of purification of
ginsenosides, and on the relations between ginsenosides
distribution and their biological activities, should be undertaken.
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Tabl e 2 . Contents of ten investigated ginsenosides in three purified ginsenosides products.
Origins Protopanaxadiol (µg/mg) Protopanaxatriol (µg/mg) Rb /Rg
Rb Rb Rc Rd Rg Re Rf Rg Rg Rh
CWG 157.7±0.7 76.7±2.3 78.5±0.5 74.1±0.9
-
095.1±0.4 54.6±7.8 054.3±0.4 8.0±1.7
-
2.90
RG 129.4±1.5 40.0±0.7 58.1±0.2 10.6±0.8
-
059.0±0.9 22.6±5.7 097.4±0.6 4.1±1.2 4.3±1.1 1.33
WG 102.7±1.3 28.3±0.6 50.0±0.5 06.1±0.5
-
106.1±1.7 26.2±2.9 153.1±0.8 2.8±0.9
-
0.67
Contents=mean±SD (n=3).
Too low to be measured.
Not detected.
... Suspension cultures of ginseng roots in bioreactors are the primary alternative method for large-scale production of wild ginseng. Various ginsenosides have been discovered as major compounds of both ginseng and cWGRE (Li et al., 2008). However, active compounds of wild ginseng differ from those of cultured ginseng because of differences in environmental factors (Li and Mazza, 2000). ...
... Previous reports have demonstrated that ginsenosides have antioxidative effects (Lim et al., 1998;Yokozawa et al., 1998;Yokozawa et al., 2004) major components of cWGRE (Li et al., 2008). Results of the present study demonstrate that treatment with cWGRE inhibits generation of ROS in boar sperm cultures. ...
... Although conventional macroporous resins have the advantages in repeated operation, sufficient loading capacity, and high recovery rate, their affinity to ginsenosides is not selective enough to obtain high-purity total ginsenosides by one-step enrichment [24,25]. Combining with other column chromatography or technologies like HSCCC can remarkably improve the purity of ginsenosides in the extract. ...
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Anion exchange resin is a modified version of polar/nonpolar macro porous resins with enhanced selectivity on compounds carrying negative charges. Although it is a promising adsorbent for the purification of natural products, no research regarding its application on ginsenosides has been reported elsewhere. In this paper, we first isolated total ginsenosides from Panax ginseng and investigated their static adsorption/desorption capacity on different resins, among which D301 resin was selected as the optimum one. A pH value of 8 was chosen for the balanced interaction between total ginsenosides and the adsorbents. Adsorption kinetics and isotherms of total ginsenosides on D301 and D101 resins were investigated to differentiate the adsorption characteristics of anion exchange and nonpolar macroporous resins. A D301-based chromatographic method was established to purify total ginsenosides with settings as follows: sample loading and elution speed = 4 bed volume per hour, breakthrough volume = 2 bed volume, elution solvent = 80% aqueous ethanol, elution volume = 8 bed volume. HPLC-QqQ-MS qualitatively and quantitatively analyzed nine individual ginsenosides from the eluates, demonstrating an enrichment factor of 5.3 as well as a recovery rate of 80.9% for the whole of these ginsenosides. The purity of these ginsenosides in Panax ginseng extract increased from 17.07% to 91.19% after the purification. Hence, anion exchange resin D301 proved to be a more comprehensive and efficient adsorbent than the conventional nonpolar macroporous resin for the separation of total ginsenosides from natural sources. Based on the findings in this paper, the enrichment process for total ginsenosides could be well established via one-step column chromatography.
... Furthermore, ginseng demonstrates useful activity on endocrine diseases, cardiovascular diseases and the immune system [45]. During processing, Red ginseng is usually steamed and fermented with skinned ginseng and this alters the composition saponin contained in it when done repeatedly [46]. Red ginseng has been shown to possess anti-cancer, anti-diabetic, anti-obesity and immunomodulatory properties [3,4]. ...
... Для обнаружения гинзенозидов хроматограммы ФМ-40 и ПС сняты в режиме сканирования отдельных ионов, массы которых соответствовали массам молекулярных ионов гинзенозидов: а) 845-846 (Rg 1 ), б) 945-946 (Rd), в) 1077-1078 (Rc и Rb 2 ), г) 1107-1108 (Rb 1 ). В качестве иллюстрации на рис. 5 приведена хроматограмма препарата ФМ-40, снятая в режиме сканирования указанных ионов [28][29][30][31]. ...
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In this paper, four types of middle‐pressure chromatogram isolated gels are evaluated for adsorption/desorption characteristics of ginsenosides from Panax ginseng. Among them, SP207SS and SP2MGS were selected for dynamic investigations based on their static adsorption/desorption capacity of total ginsenoside. Their adsorption kinetics was better explained by pseudo‐second‐order model and isotherms were preferably fitted to Langmuir model. Dynamic breakthrough experiments indicated an optimum sample loading speed of 4BV/h for either SP207SS or SP2MGS. Desorption speed was determined to be 2BV/h according to desorption amount of total ginsenoside in their effluents. Eight ginsenosides were identified and quantified by HPLC‐TQ‐MS in total ginsenoside extract and different fractions during stepwise dynamic elution. For SP207SS, 27.62% of loaded ginsenosides was detected in 40% ethanol fraction, while 59.12% of them were found in 60% ethanol fraction. As on SP2MGS, the number went to 53.71% and 44.43% respectively. Recovery rate of ginsenosides were calculated to 78.65% for SP207SS and 89.53% for SP2MGS. Intriguingly, content of Rg1 and Re in 40% ethanol fraction from SP207SS became 20.1 and 18.6 times higher than that in total ginsenoside extract by one step elution, which could be leveraged for the facile enrichment of these two ginsenosides from natural sources. This article is protected by copyright. All rights reserved
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Abstract Integrative oncology is being increasingly adopted in mainstream cancer care to strengthen anticancer effects and to control cancerrelated symptoms. The objective of this study is to identify the characteristics of patients with lung cancer treated at an integrative cancer center in Korea and to determine the effects of integrative cancer treatment (ICT) on survival outcome in traditional Korean medicine (TKM). We reviewed medical records for lung cancer patients who visited a single integrative clinical setting, East-West Cancer Center, between January 2014 and December 2015. We classified the patients into groups according to their ICT and whether or not they underwent anticancer traditional Korean Medicine treatment with a multiherbal formula containing Panax notoginseng Radix, Cordyceps militaris, P ginseng C.A.Mey., and Boswellia carterii BIRDWOOD (HangAmDan-B), with a herbal formula containing Rhus verniciflua Stoke, or with cultivated wild ginseng pharmacopuncture. A descriptive analysis of the characteristics and a survival analysis using the Kaplan-Meier curves with log rank test and a Cox proportional hazard model were performed. A total of 91 patients were included, and the majority had advanced-stage cancer. Of those patients, 45.1% were in the mono- TKM group and 39.6% were integrative group. Patients with advanced stage had significantly higher mortality than patients with early stage (crude hazard ratio [HR]: 4.41, 95% confidence interval [CI]: 1.56–12.5; adjusted HR: 6.31, 95% CI: 1.24–32.1). In the unadjusted model, for patients in the integrative group, the mortality rate was reduced by 50% compared to mono-TKM group with statistical significance. After adjusting confounders, the mortality rate of integrative group was reduced by 6% compared to mono- TKM group, suggesting positive effect on survival probability of integrative group. The results suggest that integration of TKM and conventional cancer treatment may have survival benefits in patients with lung cancer. Even though this study has limitations including heterogeneity between treatment groups, the study results suggest that ICT has positive effect on survival probability. To clarify the impacts of ICT for lung cancer and other cancers on survival outcome, further prospective study with a rigorous study design is required in multiclinical setting.
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