Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Optimal Conditions for Extracting the Ginsenosides Rg3, Rg5, and Rk1 from Black Ginseng

Myo Jin Kwon, Kui-Jin Kim, Byung Wook Yang, Boo-Yong Lee, Hyoung Chun Kim*, Sung Kwon Ko*

College of Pharmacy, Kangwon National University, Chunchon, Republic of KoreaDepartment of Oriental Medical Food & Nutrition, Semyung University, Jecheon, Republic of Korea

Abstract: Ginseng is known to contain numerous ginsenosides, which exhibit beneficial health effects. In particular, ginsenoside Rg3, Rg5, and Rk1, derived from high temperature-processed ginseng, have higher bioactivities compared to other types of ginsenosides. In this study, we determined the optimal conditions for extraction of the valuable ginsenosides Rg3, Rg5, and Rk1 from black ginseng. Our results revealed that black ginseng extracted with 100°C distilled water for 0.5 hours and 6 hours yields large amounts of Rg3, Rg5, and Rk1 compared with other extraction times. We also found that protopanaxadiol (PPD) and protopanaxatriol (PPT) were also efficiently extracted from black ginseng with 100 °C distilled water at 0.5 hours and 6 hours and then rapidly degraded in a time-dependent manner. Therefore, we posit that short extraction times such as 0.5 hours and 6 hours might be optimal for isolation of the ginsenosides Rg3, Rg5, and Rk1 from black ginseng as opposed to longer extraction periods.

Keywords: black ginseng, ginsenoside, prosapogenin, protopanaxadiol, protopanaxatriol, Rg3, Rg5, Rk1

Citation: Myo Jin Kwon, Kui-Jin Kim, Byung Wook Yang, Boo-Yong Lee, Hyoung Chun Kim, Sung Kwon Ko, Optimal Conditions for Extracting the Ginsenosides Rg3, Rg5, and Rk1 from Black Ginseng. Journal of Food and Nutrition Research, 5 (3). 176-179. January 2017. doi:10.12691/jfnr-5-3-6

1. Introduction

Ginseng radix (Panax ginseng Meyer) is used worldwide in the treatment of certain diseases [1,2,3,4,5]. Shin-Nong-Bon-Cho-Kyung, the oldest oriental medicine reference book, first noted that ginseng can be used as a herbal medicine to strengthen activities of the five internal organs and increase stamina [6]. Ginseng is known to contain a number of ginsenosides that exhibit anti-inflammatory, anti-oxidant, anti-obesity, and anti-cancer effects [7,8,9,10,11].

The majority of commercial ginseng is separated into three different types, fresh ginseng, white ginseng, and red ginseng, depending on the food processing method used, such as clean, dry, or steam with high pressure [12,13]. Moreover, fresh ginseng, white ginseng, and red ginseng contain two classes of ginsenosides, including protopanaxadiol (PPD: Rb1, Rb2, Rb3, Rc, Rd, Rg3, and Rh2) and protopanaxatriol (PPT: Rg1, Rg2, Rh1, Re, and Rf) [14,15,16]. Both PPD and PPT are converted during food processing. In particular, high pressure steaming produces enhanced activity of bio-transformed ginsenosides, such as Rg3 and Rh2, resulting in increased pharmacological effects of red ginseng [12,17].

Recently, several reports have suggested that black ginseng, which refers to high pressure steaming and drying of fresh ginseng, contains not only more of the ginsenosides PPD and PPT than red ginseng does (at least nine times) but also has a high quantity of unique types of ginsenosides, i.e., Rg5 and Rk1 [18,19]. The ginsenosides Rg3, Rg5, and Rk1 in black ginseng appear to be promising bioactive compounds for the prevention of certain types of diseases, including obesity, cancer, and inflammation [20,21,22]. However, no studies have attempted to optimize their isolation from black ginseng. In the this study, we determined the effect of different extraction times under chemical hydrolyzing conditions on the recovery of the ginsenosides Rg3, Rg5, and Rk1 from black ginseng.

2. Materials and Methods

2.1. Materials

Black ginseng (Panax ginseng) was purchased from Ganghwa Cheonto Heuksam (Incheon, South Korea) in September, 2014. Ginsenoside standards were purchased from Chromadex (St. Santa Ana, CA, U.S.A.) and the Ambo Institute (Seoul, Korea). Diethyl ether and n-butanol were purchased from Sigma (St. Louis, MO, USA). Acetonitrile (HPLC grade, >99.9% purity) and distilled water (HPLC grade, >99.9% purity) were purchased from JT Baker Inc. (Phillipsburg, NJ, USA).

2.2. Optimization Procedure of Black Ginseng Extract

Black ginseng was extracted with 20 times volume of deionized water for 0.5, 1, 3, 9, 12, 24, 48, and/or 72 hours at 100°C, and this process was repeated two times. Homogenized black ginseng extract (BGE) was filtered, concentrated under a vacuum at 60 °C and stored at 4°C until use.

2.3. Ginsenoside Standard and BGE Preparation for HPLC Analysis

Precisely 2 g of each water extract was extracted (diluted) with diethyl ether three times by sonication (Kodo Co. Ltd., 4020P, Korea) after removing lipid soluble materials in the diethyl ether phase. The residue was washed with water-saturated n-butanol three times. Then, the fraction was filtered and concentrated by a vacuum evaporator. All extractions were performed quantitatively. The amount of concentrate was equivalent to that of the crude saponin.

2.4. HPLC analysis of Ginsenoside Content of Black Ginseng Extract

The composition of the ginsenoside was analyzed by HPLC as described previously [23]. The total ginsenoside content and ginsenoside composition of each sample were analyzed three times. Pure ginsenoside standards (99%) were used in this experiment. A Waters 1525 binary HPLC system (Waters, MA, USA), fitted with a Eurospher 100-5 C18 column (Knauer, 3 x 250 mm, Germany) was used for analysis. The mobile phase was a mixture of acetonitrile and distilled water. The content of acetonitrile was sequentially increased from 17% to 25% (25 min), 25% to 40% (50 min), 40% to 60% (105 min), 60% to 100% (110 min) and finally adjusted from 100% to 17% (125 min, lasting for 10 min) again. The operating temperature was set to room temperature, and the flow rate was 0.8 mL/min. The elution profile on a chromatogram was obtained by using a UV/VIS detector at 203 nm (Waters 2487 dual λ absorbance detector, Boston, MA, USA).

2.5. Statistical Analysis

Differences among multiple groups were determined by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test using the SPSS software system (SPSS for Windows, version 20; SPSS, Inc., Chicago, IL). Values with different superscript letters are significantly different, P<0.05.

3. Results and Discussion

3.1. Preparation of Black Ginseng for Extraction

Depending on the extraction conditions, various ginsenosides have been derived from processed ginseng [23]. We therefore extracted black ginseng with 100 °C distilled water at different time intervals, such as 0.5 hours, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, and/or 72 hours.

3.2. Analysis of the Ginsenosides Rg3, Rg5, and Rk1 in Black Ginseng Extract

A number of studies reported that the ginsenosides Rg3, Rg5, and Rk1 are most effective natural chemical compound extracted from ginseng and have multiple pharmaceutical activities [24]. In addition, the ginsenosides Rg3, Rg5, and Rk1 are known to result from high temperature steaming. Therefore, we next analyzed the contents of the ginsenosides Rg3, Rg5, and Rk1 in black ginseng extract (BGE). As shown in Table 1, the highest extraction yield of the ginsenosides Rg3, Rg5, and Rk1 were observed at 0.5 hours in BGE. In particular, BGE-0.5 had approximately 1.79% of Rg3-20s and 0.09% of Rg3-20r. These results are consistent with a previous report, where the maximum extraction level of Rg3 was observed as approximately 1.1% at 6 hours at 90 °C from red ginseng [25], indicating that BGE may contain much more of the ginsenoside Rg3 compared to red ginseng. Interestingly, the ginsenosides Rg3, Rg5, and Rk1 disappeared after 1 hour and were increased by extraction at 100°C for 3 hours. These data indicate that the conversion of ginsenosides may be maximal within 3 hours; then, ginsenosides begin to disappear after 6 hours of extraction. Thus, we established the optimal extraction conditions for isolation of the ginsenosides Rg3, Rg5, and Rk1 from black ginseng.

Table 1. Comparison of effects of different extraction times on yields of the ginsenosides Rg3, Rg5, and Rk1 from black ginseng

3.3. Analysis of the ginsenosides PPD and PPT1 in black ginseng extract

We next evaluated the contents of the ginsenosides PPD and PPT to determine whether the optimal extraction conditions, incubation of black ginseng at 100 °C for 0.5 hours and 3 hours, can suitably isolate the ginsenosides PPD and PPT from black ginseng. As shown in Table 2, ginsenoside-PPD and -PPT yields exhibit similar temperature dependence compared to the ginsenosides Rg3, Rg5, and Rk1. The ginsenosides PPD (Rb1, Rb2, Rc, Rd, and Rh2) and PPT (Rg1, Rg2, Re, Rf, and Rh1) had higher extraction yields in BGE at 0.5 hours and then degraded at 1 and 3 hours. Although the ginsenosides PPD (Rb1, Rb2, Rc, Rd, and Rh2) and PPT (Rg1, Rg2, Re, Rf, and Rh1) were recovered in low quantity from BGE at 6 hours, when the extraction time exceeded 6 hours, decreasing yields of the ginsenosides PPD and PPT were observed. Therefore, the extraction of BGE at 100 °C for 0.5 hours was deemed optimal for isolation of PPD and PPT ginsenosides.

Table 2. Yields of the ginsenosides PPD and PPT in black ginseng aqueous extract (at 100°C) depending on extraction time in 100°C distilled water

4. Conclusion

In this study, we observed that BGE contains multiple ginsenosides, including Rg3 Rg5, Rk1, PPD, and PPT (Figure 1A and 1B). We found that the ginsenosides Rg3, Rg5, and Rk1 were rapidly altered between the 0.5-hour and 72-hour extraction of BGE in distilled water at 100 °C. We found the optimal extraction conditions for the ginsenosides Rg3, Rg5, and Rk1 were from black ginseng treated with distilled water and maintained at a temperature of 100 °C for 0.5 hours. Moreover, the ginsenosides PPD and PPT had high extraction yields after 0.5 hours compared to other extraction times. Our results indicate that longer extractions degraded the bioactive ginsenosides in black ginseng. Therefore, we posit that 100°C aqueous extraction for 0.5 hours is the optimal condition to extract the ginsenosides Rg3, Rg5, Rk1, PPD, and PPT from BGE. Use of these conditions might increase yields of valuable ginsenosides from black ginseng.

Figure 1. High-performance liquid chromatography-UV of black ginseng extract. Black ginseng was extracted with distilled water at 100 °C for 0.5 hour (A) and 6 hours (B)

Acknowledgements

This research was supported by High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs.

Conflicts of Interests

The authors declare no conflicts of interest.

References

[1]   | Attele AS, Zhou Y-P, Xie J-T, Wu JA, Zhang L, Dey L, et al. Antidiabetic effects of Panax ginseng berry extract and the identification of an effective component. Diabetes. 2002; 51: 1851-8.
[2]   | Sotaniemi EA, Haapakoski E, Rautio A. Ginseng Therapy in Non-Insulin-Dependent Diabetic Patients: Effects on psychophysical performance, glucose homeostasis, serum lipids, serum aminoterminalpropeptide concentration, and body weight. Diabetes care. 1995; 18: 1373-5.
[3]   | Forgo I, Kayasseh L, Staub J. Effect of a standardized ginseng extract on general well-being, reaction time, lung function and gonadal hormones. Die Medizinische Welt. 1981; 32: 751-6.
[4]   | Banerjee U, Izquierdo J. Antistress and antifatigue properties of Panax ginseng: comparison with piracetam. Acta physiologica latino americana. 1981; 32: 277-85.
[5]   | Kim K-J, Yoon K-Y, Hong H-D, Lee B-Y. Role of the Red Ginseng in Defense against the Environmental Heat Stress in Sprague Dawley Rats. Molecules. 2015; 20: 20240-53.
[6]   | Namba T. The encyclopedia of Wakan-Yaku with color pictures. Osaka: Hoikusha. 1980: 1-3.
[7]   | Yayeh T, Jung K-H, Jeong H-Y, Park J-H, Song Y-B, Kwak Y-S, et al. Korean red ginseng saponin fraction downregulates proinflammatory mediators in LPS stimulated RAW264. 7 cells and protects mice against endotoxic shock. Journal of Ginseng Research. 2012; 36: 263-9.
[8]   | Bak MJ, Truong V-L, Ko S-Y, Nguyen XNG, Jun M, Hong S-G, et al. Induction of Nrf2/ARE-mediated cytoprotective genes by red ginseng oil through ASK1&#x2013;MKK4/7&#x2013;JNK and p38 MAPK signaling pathways in HepG2 cells. Journal of Ginseng Research.40: 423-30.
[9]   | Koh E-J, Kim K-J, Choi J, Jeon HJ, Seo M-J, Lee B-Y. Ginsenoside Rg1 suppresses early stage of adipocyte development via activation of C/EBP homologous protein-10 in 3T3-L1 and attenuates fat accumulation in high fat diet-induced obese zebrafish. Journal of Ginseng Research. 41: 23-30.
[10]   | Yu T, Yang Y, Kwak Y-S, Song GG, Kim M-Y, Rhee MH, et al. Ginsenoside Rc from Panax ginseng exerts anti-inflammatory activity by targeting TANK-binding kinase 1/interferon regulatory factor-3 and p38/ATF-2. Journal of Ginseng Research.
[11]   | Park E-H, Kim Y-J, Yamabe N, Park S-H, Kim H-k, Jang H-J, et al. Stereospecific anticancer effects of ginsenoside Rg3 epimers isolated from heat-processed American ginseng on human gastric cancer cell. Journal of ginseng research. 2014; 38: 22-7.
[12]   | Kim WY, Kim JM, Han SB, Lee SK, Kim ND, Park MK, et al. Steaming of ginseng at high temperature enhances biological activity. Journal of Natural Products. 2000; 63: 1702-4.
[13]   | Yun TK. Brief introduction of Panax ginseng CA Meyer. Journal of Korean medical science. 2001; 16: S3.
[14]   | Wan JB, Zhang QW, Ye WC, Wang YT. Quantification and separation of protopanaxatriol and protopanaxadiol type saponins from Panax notoginseng with macroporous resins. Separation and Purification Technology. 2008; 60: 198-205.
[15]   | Jia L, Zhao Y. Current evaluation of the millennium phytomedicine--ginseng (I): etymology, pharmacognosy, phytochemistry, market and regulations. Current medicinal chemistry. 2009; 16: 2475-84.
[16]   | Christensen LP. Ginsenosides chemistry, biosynthesis, analysis, and potential health effects. Advances in food and nutrition research. 2009; 55: 1-99.
[17]   | Park J-B, Kwon SK, Nagar H, Jung S-b, Jeon BH, Kim CS, et al. Rg3-enriched Korean Red Ginseng improves vascular function in spontaneously hypertensive rats. Journal of ginseng research. 2014; 38: 244-50.
[18]   | Sun B-S, Gu L-J, Fang Z-M, Wang C-y, Wang Z, Lee M-R, et al. Simultaneous quantification of 19 ginsenosides in black ginseng developed from Panax ginseng by HPLC–ELSD. Journal of pharmaceutical and biomedical analysis. 2009; 50: 15-22.
[19]   | Jin Y, Kim Y-J, Jeon J-N, Wang C, Min J-W, Noh H-Y, et al. Effect of white, red and black ginseng on physicochemical properties and ginsenosides. Plant Foods for Human Nutrition. 2015; 70: 141-5.
[20]   | Kim S-J, Kim AK. Anti-breast cancer activity of Fine Black ginseng (Panax ginseng Meyer) and ginsenoside Rg5. Journal of ginseng research. 2015; 39: 125-34.
[21]   | Kim A-J, Kang S-J, Lee K-H, Lee M, Ha S-D, Cha Y-S, et al. The chemopreventive potential and anti-inflammatory activities of Korean black ginseng in colon26-M3. 1 carcinoma cells and macrophages. Journal of the Korean Society for Applied Biological Chemistry. 2010; 53: 101-5.
[22]   | Lee MR, Kim BC, Kim R, Oh HI, Kim HK, Choi KJ, et al. Anti-obesity effects of black ginseng extract in high fat diet-fed mice. Journal of ginseng research. 2013;37:308-14.
[23]   | Kim S-J, Kim J-D, Ko S-K. Changes in ginsenoside composition of ginseng berry extracts after a microwave and vinegar process. Journal of ginseng research. 2013; 37: 269.
[24]   | Leung KW, Wong AS-T. Pharmacology of ginsenosides: a literature review. Chinese medicine. 2010; 5: 20.
[25]   | Kim C-S, Choi K-J, Yang J-W, Kim S-B. Effect of preheating condition of raw ginseng on the yield and physical property of Korean Red Ginseng extract. Korean Journal of Medicinal Crop Science. 2000; 8: 146-50.