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ORIGINAL ARTICLE
Year : 2013  |  Volume : 9  |  Issue : 36  |  Page : 38-43  

The ginsenosides and carbohydrate profiles of ginseng cultivated under mountainous forest


College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, China

Date of Submission27-Jul-2012
Date of Acceptance28-Aug-2013
Date of Web Publication07-Sep-2013

Correspondence Address:
De-qiang Dou
77 of life one Road, DD Port, Dalian 116600
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1296.117862

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   Abstract 

Background: Ginseng cultivated under mountainous forest, called "Lin-Xia-Shan-Shen" (LXSS) in China's Pharmacopoeia. In recent years, it has been quickly propelled to plant at a large scale. Objective: To study the profiles of ginsenosides and carbohydrate profiles of LXSS. Materials and Methods: The contents of ginsenosides and carbohydrates, such as soluble sugar, polysaccharide, pectin, and starch in LXSS, were determined. All the above components were profiled, and the correlations between them were analyzed. Results: The results indicated that the contents of total ginsenoside, protopanaxadiol, protopanaxatriol, Rg 1 , Re, Rb 1 , Rc, Rb 2 , Rd, starch, and pectin were negatively correlated with the growing years within 17 years. Among them, the content of starch was positively correlated with that of pectin. The total ginsenosides was positively correlated with starch and pectin, which cannot be found in garden ginseng, maybe resulting of fertilizer and other manual intervention in process of cultivation of garden ginseng. Discussion and Conclusions: The accumulation of ginsenosides and carbohydrate, especially starch and pectin, was different in garden ginseng and LXSS. This research may provide the scientific basis for germplasm evaluation, the cultivation and utilization of ginseng cultivated under mountainous forest.

Keywords: Carbohydrate, ginseng, ginsenoside, pectin, polysaccharide, starch


How to cite this article:
Zhang Jk, Gao R, Dou Dq, Kang Tg. The ginsenosides and carbohydrate profiles of ginseng cultivated under mountainous forest. Phcog Mag 2013;9, Suppl S1:38-43

How to cite this URL:
Zhang Jk, Gao R, Dou Dq, Kang Tg. The ginsenosides and carbohydrate profiles of ginseng cultivated under mountainous forest. Phcog Mag [serial online] 2013 [cited 2019 Jul 20];9, Suppl S1:38-43. Available from: http://www.phcog.com/text.asp?2013/9/36/38/117862


   Introduction Top


Ginseng, the dried roots of Panax ginseng C.A. Meyer, has been held in high esteem in traditional Chinese medicine for 400 years. It belongs to the family of Araliaceae and is a well-known medicinal plant worldwide. Traditionally, only wild ginseng was applied clinically in Traditional Chinese Medicine( TCM) However as the extinction of wild resources, the garden ginseng emerged 400 years ago. Now garden ginseng is the preponderant resource of medicinal ginseng materials, but the development of garden ginseng cultivation accompanies the deforestation and severe damage to the ecological balance as well. In recent decades, the ginseng cultivation under mountain forest has been spread at a large scale so as to preserve forest and imitate the growing conditions of wild ginseng. It was formally called "Lin-Xia-Shan-Shen0" (LXSS) in China's Pharmacopoeia and named consuetudinary "Zi-Hai," meaning that the LXSS was cultivated directly by seeds and disseminated everywhere and grew naturally. Since the formulation of natural forest protection policy in 1998, the cultivation of garden ginseng shrunken and the cultivation of LXSS increased rapidly. However, the chemical study was rarely concerned. Previously, the bioactivities and hemolytic interaction of ginsenosides were studied, [1-4] and the SOP for cultivation and fingerprint of LXSS were proposed. [5],[6] The monthly accumulating profile of ginsenosides in LXSS and the optimal harvest month of LXSS were discussed. [7] More recently, the ginsenosides in garden ginseng and LXSS was compared. [7] Ginsenosides and pectin are medicinal components, and the soluble sugar, starch, and polysaccharide are the reflection of germplasm, such as resistance, genetic character, etc. Content of starch is relevant to the texture of LXSS. In continuation, the ginsenosides and carbohydrates of LXSS were studied. This paper dealt with the determination and correlations of the aforementioned components of LXSS.


   Materials and Methods Top


Materials and reagents

Samples of LXSS were collected in the county of Kuandian, Liaoning province and were identified as Panax ginseng C. A. Meyer by Professor Ting-guo Kang and Bing Wang. The growing years of LXSS were determined by the number of rhizome nodes together with the record of collection areas. Samples of Garden ginseng were purchased from Shenyang Ginseng & Antler Market. The voucher specimens were deposited in College of Pharmacy, Liaoning University of Traditional Chinese Medicine. Sulfuric acid, anthrone, ethyl acetate, acetic acid, sodium hydroxide, iodine, ethanol, anhydrous ethanol, potassium iodide, soluble starch, glucose, and ethanol were analytical grade and purchased from Tianjin Kermel Chemical Reagent Co., Ltd. Pectinase was analytical grade and purchased from Shanghai Hualanchem Co., Ltd. d-Galacturonic acid with a purity of 97% was purchased from Sigma Aldrich Fluka Co. Deionized water was supplied by Xinshengyuan Company.

Instruments

Agilent 1100 HPLC instrument (Agilent Technologies, Inc.), AT-130 column oven (Dalian Zhonghuida Scientific Instrument Co., Ltd.), FW80 high-speed grinder (Tianjin Taisite Instrument Co., Ltd.), HITACHI UV-3010 spectrophotometer (HITACHI Group Ltd.), Sartorius CP225 analytical balance (Sartorius Co., Ltd.), and FW80 high-speed grinder (Tianjin Taisite Instrument Co., Ltd.) were employed during the process of chemical analysis.

Sample preparation

Preparation of samples for ginsenosides analysis

Ginsenosides were extracted and analyzed by methods of Zhou. [7] Then, 0.5 g of the powder of ginseng was weighed accurately into a 250 mL round-bottomed flask and refluxed by methanol two times, 1 h for each time. The solvent was evaporated and the residue was dissolved by methanol to a constant volume, and then the solution was filtered through a suitable membrane (0.45 μm) before injection. Contents of ginsenosides with authentic samples such as Rg 1 , Re, Rf, Rb 1 , Rc, Rb 2 , and Rd were also dissolved in methanol and determined by an external standard method.

The chemical analysis was achieved on an Agilent 1100 HPLC instrument with Agilent C 18 column (150 × 4.6 mm, 5 μm), column temperature at 30 °C, and flow rate at 1.0 mL min -1 . The ginsenosides were separated in 70 min by the mobile phase consisted of acetonitrile (A) and 0.1% phosphoric acid (W) with the gradients as follows: 0-30 min, A:W from 19:81 to 29:71; 30-50 min, A:W from 29:71 to 32:68; 50-70 min, A:W from 32:68 to 51:49. While the determinations of ginsenosides Rg1 and Re were achieved by isocratic elution with A:W (20:80) within 30 min. Components with retention time between Re and Rf in the first condition were separated by gradient elution as follows: 0-30 min, A:W 20:80; 30-38 min, A:W from 20:80 to 21:79; 38-41 min, A:W from 21:79 to 21.5:78.5; 41-57 min, A:B from 21.5:78.5 to 28:72 [Figure 1] and [Figure 2].
Figure 1: HPLC chromatogram of seven standard substances

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Figure 2: HPLC chromatogram of samples

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Preparation of samples for soluble sugars analysis

Soluble sugars were determined by the anthrone-H 2 SO 4 method. [8] Fifty milligrams of the powdered sample were weighed accurately into a 10 mL centrifugal tube, and the mouth was covered with a plug. The samples were extracted with 10 mL of 80% ethanol at 80 °C in a water bath for 30 min with intermittent mixing. After cooling to room temperature, the samples were centrifuged at 4000g for 20 min and the supernatant was transferred into a 25 mL volumetric flask. The sediment was extracted and centrifuged as mentioned above again, then collected all the supernatant liquids and diluted it to 25.0 mL with water. Five milliliters of supernatant liquids were pipetted into a 50 mL volumetric flask and was made up to the mark line with water. Two milliliters of diluted solution was pipetted into a tube. Then, 0.5 mL of 2% anthrone-ethyl acetate solution was added and 5.0 mL of 98% H 2 SO 4 was directed into the middle of the solution with care. The contents were mixed, and the tube was heated in a boiling water bath for 1 min. After cooling, the absorbance of the sample at 630 nm was measured against a reagent blank as the reference. Standard curves were obtained by using d-glucose as a reference standard for colorimetric analyses. The soluble sugar concentration was cauculated by D-glucose.

Preparation of samples for total polysaccharide analysis

After washed with some 80% ethanol, the sediment obtained from centrifugation mentioned above was homogenized in 15.0 mL of 1 mol/L HCl, and extracted at 80 °C in a water bath for 1 h with vibration occasionally. After cooling to room temperature, 6 mL of 10% NaOH was added for neutralization. After centrifugation at 4000g for 10 min, the supernatant was obtained. When some supernatant was taken for reacting with

iodine-potassium iodide solution without the development of a blue color, 2.0 mL of supernatant was pipetted into a 50 mL volumetric flask and was make up to the mark with water. The following procedure was the same as that of measurement of soluble sugars content.

Preparation of samples for starch analysis

Starch was analyzed by using the iodine colorimetric method. [9] A 100 mg of the powdered sample was accurately weighed into a 10 mL centrifugal tube, 1 mL of anhydrous ethanol was added for wetting, then 9 mL of 1 mol/L NaOH was added, and covered the mouth with a plug. The samples were extracted with at 80 °C in a water bath for 10 min with intermittent mixing and then centrifuged at 4000g for 10 min after cooling to room temperature. Five milliliters of supernatant liquids were pipetted into a 25 mL volumetric flask and was make up to the mark with water. Some diluted solution was pipetted into a 100 mL volumetric flask, added with the corresponding volume of 0.09 mol/L NaOH to make the total volume to 10 mL. The reagents were added in the following order: 30 mL water, l mL of l mol/L acetic acid and l mL iodine solution and was made up to the mark with water. After 10 min, when the color was shown stably, the absorbance of the sample was measured at 580 nm against a reagent blank as the reference. Standard curves were obtained by using soluble starch as a reference standard for colorimetric analyses. Starch concentration was caculated by soluble starch.

Preparation of samples for pectin analysis

First, pectin was enzymatic hydrolyzed as Xu et al. [10] descried and then measured by the 3,5-dinitrosalicylic acid (DNS) method of Miller. [11] Five hundred grams of the powdered sample were accurately weighed into a 50 mL centrifugal tube, then operated as "measurement of soluble sugars content" mentioned above besides the volumes of 80% ethanol were 50 mL, and washed the sediment with some 80% ethanol. After evaporation, the sediment was enzymatic hydrolyzed with 50 mL of 0.1 M citric acid-sodium citrate buffer (pH 4) and pectinase of 1200 u at 50 °C for 6 h. The blank was prepared as described above. Followed with filtration, the filtrate was transferred into a 50 mL volumetric flask and was made up to the mark with the buffer. One milliliter of the filtrate was pipetted into a 25 mL tube with a plug. Then, 3 mL DNS solution was added to each tube and boiled for 5 min. After cooling, the blank was added to make up to the mark, agitated, and the absorbance at 520 nm was measured. Standard curves were obtained by using d-galacturonic acid as a reference standard for colorimetric analyses. Pectin concentration was caculated by D-galacturonic acid.

Statistics

All values are expressed as mean ± SD. The correlation analysis of data was achieved by SPSS.


   Results and Discussion Top


Method validation

The methods were validated by linearity, precision, repeatability, stability, and recovery, and all were up to the demands for HPLC and chromatometry determination.

Accumulation trend of ginsenosides in LXSS

The ginsenosides were determined by the procedure mentioned above. The following regularities could be concluded from the data as shown in [Table 1], [Figure 3], and [Figure 4].
Table 1: Contents of ginsenosides (mg/g) in LXSS with different growing years together with those of garden ginseng (n = 5)


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Figure 3: The accumulation trend of total ginsenoside, ppd, ppt and ppt/ppd of LXSS

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Figure 4: The accumulation trend of ginsenosides of LXSS

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(1) The contents of total and protopanaxadiol (PPD) and protopanaxatriol (PPT) ginsenosides accumulated at the initial growing period (from the sixth to the eighth year) of LXSS, and reached a peak at the eighth year of growth, then fell off in the following years of growth. However, negative correlation between the content of total ginsenosides and growing years was figured out after SPSS analysis with the correlation coefficient (R value) of –0.956 (P < 0.01). The PPT and PPD ginsenosides and growing years were negatively correlated with the growing years with R values of -0.953 (P < 0.01) and -0.955 (P < 0.01). The ratio of PPT and PPD was positively correlated with the growing years with R value of 0.931(P < 0.01).

(2) At first glance, ginsenosides Rf, Rc, and Rb 2 showed the same accumulating trend with total ginsenosides [Table 2] and [Figure 2], while the contents of Rg 1 , Rb 1 , and Rd fell off from the initial growing period of LXSS to the 17th year of growth. While the contents of Re fell off from the initial growing period of LXSS (from the sixth to the eighth year), then rose and fell off again in the following years of growth. But after SPSS analysis, all of them were negatively correlated with the growing years with R values of –0.961 (Rg 1 , P < 0.01), –0.937 (Re, P < 0.01), –0.981 (Rb 1 , P < 0.01), –0.847 (Rc, P < 0.05), –0.905 (Rb 2 , P < 0.05), and –0.957 (Rd, P < 0.01) while the content of Rf was not correlated with the growing years with R values of –0.772 (P0¡á0.05). The ratio of Rg 1 and Re was positively correlated with the growing years with a R value of 0.919 (P < 0.05).
Table 2: Contents of carbohydrate (%) in LXSS with different growing years together with those of garden ginseng (n = 5)


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Accumulation trend of carbohydrate in LXSS

The soluble sugar, starch, total polysaccharide, and pectin were determined by the procedure mentioned above. The following regularities could be concluded from the data as shown in [Table 2] and [Figure 5].
Figure 5: The accumulation trend of carbohydrate of LXSS

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(1) The content of soluble sugar accumulated at the initial growing period (from the sixth to the twelfth year) of LXSS, and reached a peak at the 12th year of growth, then fell off in the following years of growth. While the content of starch increased, reached the highest level during the eighth year of growth then fell off. While the polysaccharide accumulated continually from the sixth to the tenth year, and reached a peak at the tenth year of growth, then fell off to the 17th year of growth. While the pectin accumulated continually from the sixth to the eighth year, and reached a peak at the eighth year of growth, then fell off a little and showed a tendency toward stabilization in the following years of growth. After SPSS analysis, we found that the content of starch and pectin and growing years were negatively correlated with the growing years with R values of –0.872 (P < 0.05) and –0.872 (P < 0.05). While the content of soluble sugar did not show significant correlation with growing years (P > 0.05), so did the total polysaccharide.

(2) The contents of ginsenosides and pectin are decreased a little with the growing year increasing and the weight of LXSS are increased with the growing year increasing [Table 3] either. Therefore, the total ginsenosides and pectin of the same ginseng kept steady or increased a little during the period of growth.

Compared with the increasing of LXSS's weight, its content of starch fell off sharply. This maybe the reason for the tenacity of adventitious root of LXSS is similar to that of wild ginseng. As the cultivation environment and appearance of LXSS are similar to wild ginseng, the price of LXSS is higher than that of garden ginseng. That is in accordance with the application customs of TCM.
Table 3: Total weight (g) of LXSS with different growing years (n = 5)


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Correlations between ginsenosides, starch, and pectin in LXSS

The contents of total ginsenosides were positively correlated to the starch with R values of 0.889 (P < 0.05). Whereas the content of total ginsenosides were positively correlated to the pectin with R values of 0.967 (P < 0.01). The content of starch was positively correlated to the pectin with R values of 0.906 (P < 0.05). We know that ginsenosides that consist of aglycone and sugar are the main secondary metabolites of ginseng and play an important role in its life cycle. From these results, we could conjecture that sugars may mainly derive from the process of partial enzymatic hydrolysis of starch and pectin, which may explain the high correlation between total ginsenosides, starch, and pectin.

Correlations between ginsenosides, starch, and pectin in garden ginseng

The contents of total ginsenosides were positively correlated to the starch with R values of 0.608 (P¡á0.05). Whereas the contents of total ginsenosides were positively correlated to the pectin with R values of 0.771 (P < 0.05). The contents of starch were positively correlated to the pectin with R values of 0.873 (P < 0.01).

Garden ginseng's R values between total ginsenosides, starch, and pectin are lower than in LXSS, which may be relevant to manual intervention in the process of cultivation of garden ginseng.

Comparison of contents of ginsenosides and carbohydrate between LXSS and garden ginseng

The results [Table 4] showed statistically normal distribution and were analyzed by the t-test. We found that though there was no significant difference among soluble sugar, total polysaccharide, and the ratio of PPT and PPD in both LXSS and garden ginseng (P > 0.05), total ginsenosides, PPT, PPD, the ratio of Rg 1 and Re, and pectin of LXSS were significantly higher than that in garden ginseng (P < 0.01). Whereas starch of LXSS was significantly lower than in Garden ginseng (P < 0.01). The growing environment between LXSS and garden ginseng has shown a large difference and in comparison with garden ginseng, fertilizer and other manual interventions are not applied in LXSS. Maybe all about these can explain the difference mentioned above between LXSS and garden ginseng.
Table 4: Significance difference test of the difference of component between LXSS and garden ginseng (X ± S)


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   Conclusion Top


The results indicated that the contents of total ginsenosides, PPD, PPT, Rg 1 , Re, Rb 1 , Rc, Rb 2 , Rd, starch, and pectin were negatively correlated with the growing years, whereas the contents of starch were positively correlated with the pectin. The total ginsenosides were positively correlated with starch and pectin, respectively; however, this cannot be found in garden ginseng, maybe partly because of fertilizer and other manual intervention in process of cultivation of garden ginseng. The accumulation of ginsenosides and carbohydrate, especially starch and pectin varied from garden ginseng and LXSS. This research may provide the scientific basis for germplasm evaluation, the cultivation, and utilization of ginseng cultivated under mountainous forest.

 
   References Top

1.Cheng DR, Fu R, Dou DQ, Kang TG. Hemolytic and its protective action of ginsenosides. Mod Chin Med 2007;9:7-8.  Back to cited text no. 1
    
2.Dou DQ, Chen YJ, Liang LH, Pang FG, Shimizu N, Takeda T. Six new dammarane-type Triterpene Saponins from the Leaves of Panax ginseng. Chem Pharm Bull 2001;49:442-6.  Back to cited text no. 2
[PUBMED]    
3.Dou DQ, Zhang YW, Zhang L, Chen YJ, Yao XS. The inhibitory effects of ginsenosides on protein tyrosine activation induced by hypoxia / reoxygenation in cultured human umbilical vein endothelial cells. Planta Med 2001;67:19-23.  Back to cited text no. 3
[PUBMED]    
4.Qiu YK, Dou DQ, Cai LP, Jiang HP, Kang TG, Yang BY, et al. Dammarane-type saponins from Panax quinquefolium and their inhibition activity on human breast cancer MCF-7 cells. Fitoterapia 2009;80:219-22.  Back to cited text no. 4
[PUBMED]    
5.Jiang HP, Dou DQ, Jing SQ, Liu FY. Study on ginsenoside and fingerprint of Lin-Xia-Shan-Shen by HPLC. Mod Chin Med 2008;10:12-5.  Back to cited text no. 5
    
6.Jiang HP, Liu FY, Dou DQ, Li JH. The SOP for cultivation of Lin- Xia-Shan-Shen. Mod Chin Med 2007;10:34-8.  Back to cited text no. 6
    
7.Zhou ZX, Qu Y, Dou DQ, Liu FY, Huo YS, Jiao FB. The ginsenoside profile of ginseng cultivated under mountainous forest. J Med Plants Res 2011;5:410-9.  Back to cited text no. 7
    
8.Zhang ZL, Qu WQ. Guide to Plant Physiology Experiment. 3rd ed., Beijing: Higher Education Pres; 2003. p. 127-8.  Back to cited text no. 8
    
9.Li BL. Photometric determination of starch content in candy. Chin J Public Health 1994;10:551-1.  Back to cited text no. 9
    
10.Xu ZQ, Chen KB, Cai B, Cheng T. Determination of Pectin in Tobacco with Enzymolysis-Autoanalyzer. Tob Sci Technol 2005;9:26-8.  Back to cited text no. 10
    
11.Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426-8.  Back to cited text no. 11
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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