Antihypertensive Effects of Rg3-Enriched Korean Vitamin Ginseng in Spontaneously Hypertensive Rats

Abstract

Ginseng is well known to treat various diseases. Ginsenoside Rg3 exhibits a variety of pharmacological activities including cardiovascular protective effects. Vitamins utilized as supplements have minimal interactions with other drugs making them attractive targets for therapeutics. Here, we prepared Rg3-enriched Korean ginseng catalyzed by vitamin (REKVG) and evaluated its ability to improve hypertension in spontaneously hypertensive rats (SHRs). The ginsenoside content in both Korean Red ginseng (KRG) and REKVG were analyzed using high-performance liquid chromatography (HPLC). Male SHRs and Wistar-Kyoto rats (WKYs) were randomly divided into 6 groups (WKY saline, WKY KRG, WKY REKVG, SHR saline, SHR KRG, and SHR REKVG). KRG and REKVG were orally administered once daily to the rats at a dose of 10 mg/kg for 6 weeks, and blood pressure was measured in live rats using the tail-cuff method. Human umbilical vein endothelial cells were used for the in vitro experiment. HPLC chromatograms revealed that the concentration of ginsenoside Rg3 in REKVG was much higher than that in KRG. The administration of REKVG significantly decreased the systolic blood pressure in SHRs at the end of 6 weeks as compared to KRG. Further, REKVG use resulted in a dose-dependent increase in Akt and endothelial nitric oxide synthase (eNOS) phosphorylation and NO production in endothelial cells. In addition, the administration of REKVG significantly increased Akt and eNOS phosphorylation and increased plasma NO levels in SHRs. We conclude that REKVG effectively lowers the blood pressure in rats and therefore could be considered for use in preventing or improving hypertension.

Ginseng belongs to the Araliaceae family (Panax genus) of herbs, which has been used in traditional Chinese and Korean medicine for many centuries. It is also cultivated and is popular in Japan, the USA, and Canada because it may improve health, longevity, and vitality. Although the plant contains many complex molecules, several bioactive components have been identified and used for numerous pharmacological functions.^1

-4 Positive pharmacological effects of ginseng have been demonstrated in the central nervous, cardiovascular, endocrine, and immune systems.^5,6 Although various forms of ginseng are processed for use, red ginseng is the most widely used. Red ginseng is produced by steaming and drying raw ginseng. It is considered more pharmacologically active than other forms, probably due to the production of various chemical constituents during the steaming process.

Korean red ginseng (KRG), ie, red ginseng specifically of Korean origin, has a wide range of beneficial effects. Apart from its pharmacological effects, KRG also has many physiological benefits including anti-inflammatory, antidiabetic, anticancer, and antioxidant effects.^7
-9 The vascular protective properties of KRG include vasorelaxation and hypotensive effects. KRG prevents endothelial cell damage or dysfunction by increasing nitric oxide (NO) and endothelial nitric oxide synthase (eNOS) levels.¹⁰ These vascular, and consequently, cardiological, effects of KRG may be protective against various cardiovascular diseases such as atherosclerosis, diabetes, and hypertension. Instead of using whole ginseng extract, various studies have focused on the actions of individual ginsenosides against various diseases.^11
-13 Of the various ginsenosides, Rg3, Rb1, Rg1, Re, and Rd are the most frequently studied.¹⁴ Individual ginsenosides have different effects in different tissues. Furthermore, the composition of various ginsenosides in any given ginseng extract affects the therapeutic and pharmacological effects of that extract.^15
-17 Ginsenoside Rg3 is well known for its beneficial effects on numerous diseases, including hypertension, diabetes mellitus, and breast cancer.¹⁸ It also protects the cardiovascular system by enhancing NO production, eNOS activity, and vascular function.¹⁹ In previous studies, we have shown that Rg3 improves vascular function through eNOS activation²⁰ and enhances blood pressure stability in spontaneously hypertensive rats (SHRs).²¹

Hypertension is a major risk factor for a range of cardiovascular diseases. Therefore, the development of new antihypertensive drugs with established efficacy and safety is necessary to improve treatment regimens.²² Various in vitro and in vivo studies that have examined the antihypertensive properties of different ginseng components have shown that ginseng confers beneficial effects on the cardiovascular system through several mechanisms, including by lowering blood pressure.^23,24 Vitamins are nutrients or organic compounds that are essential for supporting the normal physiological functioning, growth, and health of the body.²⁵ One important reason that vitamins are needed is that they play an indirect role in catalysis, in which enzymes speed up chemical reactions.

Thus, in this study, for the first time, we prepared a Korean vitamin ginseng (REKVG) under various conditions using vitamin C as a catalyst during the steaming process. This REKVG is also enriched with the ginsenoside Rg3 making it more effective. Then, we evaluated its ability to improve hypertension and vascular function in SHRs.

Eleven ginsenosides (Rg1, Re, Rf, Rg2, Rh1, Rb1, Rc, Rb2, Rg3, Rk1, and Rg5) were analyzed by high-performance liquid chromatography (HPLC). HPLC chromatograms of KRG and REKVG are shown in Figure 1. As shown in Table 1, the concentration of ginsenoside Rg3 in REKVG was much higher than that in KRG.

Figure 1

High-performance liquid chromatography chromatograms of (a) Korean red ginseng (KRG) and (b) Rg3-enriched Korean ginseng catalyzed by vitamin (REKVG). (1) Rg1, (2) Re, (3) Rf, (4) Rh1(S), (5) Rh1(R) + Rg2(S), (6) Rg2(R), (7) Rb1, (8) Rc, (9) Rb2, (10) Rg3(S), (11) Rg3(R), (12) Rk1, (13) Rg5.

Table 1

The Saponin Contents in Korean Red Ginseng (KRG) and Rg3-Enriched Korean Ginseng Catalyzed by Vitamin (REKVG) (mg/g).

Ginsenoside (mg/g)	Rg1	Re	Rf	Rg2	Rh1	Rb1	Rc	Rb2	Rg3(S)	Rg3(R)	Rk1	Rg5
KRG	4.9	1.2	0.9	0.6	0.6	8.7	1.1	1.1	0.3	0.4	0.06	0.30
REKVG	ND	ND	1.0	2.3	2.2	0.9	0.2	0.2	9.7	7.7	2.0	4.5

Next, we examined the effects of KRG and REKVG treatment on systolic blood pressure (SBP) in Wistar-Kyoto rats (WKYs) and SHRs over a period of 6 weeks. For this, rats in each group were fed KRG and REKVG orally every day at a dose of 10 mg/kg, and blood pressures were recorded daily. Saline was used as a vehicle. As shown in Figure 2, neither extract caused significant changes in SBP in WKYs (Figure 2(a)), although the rats consistently gained weight every week (Figure 2(c)). In contrast, the administration of REKVG significantly decreased SBP in SHRs at the end of 6 weeks (Figure 2(b)). These effects were seen as early as 2 weeks after the start of treatment. In addition, these rats also consistently gained weight every week (Figure 2(d)).

Figure 2

Changes of systolic blood pressure (a) and weight (c) in Wistar-Kyoto rats (WKYs), systolic blood pressure (b) and weight (d) in spontaneously hypertensive rats (SHRs) after daily administration of Korean red ginseng (KRG) and Rg3-enriched Korean ginseng catalyzed by vitamin (REKVG) by oral feeding at a dose of 10 mg/kg for a period of 6 weeks. Saline was used as a vehicle. *P < 0.05 compared with the control. Data are presented as mean ± standard deviation (n = 9).

Akt-eNOS pathway plays an important role in the regulation of vascular tone and blood pressure in vascular endothelial cells. Under exposure to stimuli such as shear stress or acetylcholine, eNOS constitutively expressed in endothelial cells oxidizes l-arginine to generate l-citrulline and NO. Ginseng is known to enhance eNOS phosphorylation and NO production.

Therefore, to examine the pathway of blood pressure-lowering effect of REKVG, we treated KRG and REKVG in human umbilical vein endothelial cells (HUVECs) at doses of 10-300 µg/mL and incubated the cells for 24 hours, followed by an examination of Akt and eNOS phosphorylation by Western blotting. As shown in Figure 3(b), the administration of REKVG increased Ser-473 phosphorylation of Akt and Ser-1177 phosphorylation of eNOS (Figure 3(d)) in HUVECs dose dependently compared to KRG treatment for the same period of time (Figure 3(a) and (c)). Similarly, Ser-473 phosphorylation of Akt and Ser-1177 phosphorylation of eNOS were considerably increased by REKVG treatment in the aortas of SHRs (Figure 4(b) and (d), respectively) as compared to the WKY aortas (Figure 4(a) and (c)).

Figure 3

The effect of Korean red ginseng (KRG) and Rg3-enriched Korean ginseng catalyzed by vitamin (REKVG) on phosphorylation of Akt, phosphorylation of endothelial nitric oxide synthase (eNOS) and NO production in human umbilical vein endothelial cells (HUVECs). The phosphorylation of Akt (b) and eNOS (d) were increased dose dependently in HUVECs treated with various concentrations (10-300 ug/mL) of REKVG for 24 hours much more than with a similar dose of KRG treatment (a and c). Protein expression was measured by western blot analysis. β-actin was used as a loading control. P-Akt and P-eNOS expression levels were quantified by densitometric analysis. Metabolites of NO (nitrite and nitrate) were measured in the supernatant of HUVECs treated with various concentrations (10-300 ug/mL) of KRG (e) and REKVG (f) for 24 hours. NO production was increased dose dependently in HUVECs treated with REKVG more than with a similar dose of KRG. *P < 0.05 compared with control cells. Data are presented as mean ± standard deviation (n = 9).

Figure 4

The effect of Korean red ginseng (KRG) and Rg3-enriched Korean ginseng catalyzed by vitamin (REKVG) on phosphorylation of Akt, phosphorylation of endothelial nitric oxide synthase (eNOS) in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) aorta and NO measurement in the plasma from WKYs and SHR. The phosphorylation of Akt was significantly increased in WKY (a) and SHR (b) aortas with the treatment of REKVG more than with KRG treatment. The phosphorylation of eNOS was also significantly increased in WKY (c) and SHR (d) aortas with the treatment of REKVG more than with KRG treatment. Protein expression was measured by western blot analysis. β-actin was used as a loading control. P-Akt and P-eNOS expression levels were quantified by densitometric analysis and presented in the lower panels. Metabolites of NO (nitrite and nitrate) were measured in the plasma of WKY rats (e) and SHR (f). NO production was increased in SHR plasma more than in WKY plasma by REKVG treatment as compared to the KRG treatment. *P < 0.05 compared with the control. Data are presented as mean ± standard deviation (n = 9).

High blood pressure is characterized by eNOS deficiency and decreased NO production, particularly in the endothelium. Therefore, we measured NO production in HUVECs treated with KRG and REKVG at doses of 10-300 µg/mL, followed by 24 hours incubation before measurement of NO production. As presented in Figure 3, NO production showed a dose-dependent increase in REKVG-treated HUVECs (Figure 3(f)) more than KRG-treated HUVECs (Figure 3(e)).

Plasma levels of NO in WKYs and SHRs after administration of KRG and REKVG were also measured. Administration of REKVG (10 mg/kg) for 6 weeks significantly increased NO production in WKYs (Figure 4(e)). Similarly, NO production was markedly increased in SHRs administrated by REKVG (10 mg/kg) (Figure 4(f)). Interestingly, although there was a slight increase in NO production by KRG in the SHRs, there was no significant increase seen in the WKY rats by KRG treatment (Figure 4(e) and (f)). This data indicate that the efficiency of REKVG in stimulating NO production was higher than that of KRG in both groups. These results collectively suggest that REKVG stimulates the Akt-eNOS signaling pathway, in turn, leading to an increase in NO production in SHRs.

The present findings demonstrated that REKVG had antihypertensive effects in SHRs. Administration of REKVG (10 mg/kg) daily for a period of 6 weeks significantly induced phosphorylation of both Akt and eNOS and subsequently increased NO production leading to a significant reduction in SBP in SHRs compared to WKYs. Although KRG administration also reduced SBP in SHRs, the effects of REKVG on lowering blood pressure were much greater. These findings suggest that REKVG has a protective effect against hypertension, possibly by reducing SBP and regulating the NO signal transduction pathway.

Ginseng is used to treat various diseases, including hypertension, thrombosis, hyperlipidemia, cancer, and atherosclerosis.^26
-28 With the increased use of herbs, the interest in using KRG for the prevention of cardiovascular risk factors has grown. In a previous study, rats treated with KRG powder had significantly lower blood pressures than controls over a period of 1-2 months. Furthermore, there were reductions of about 6% and 36% in SBP in a group treated with crude saponin extracted from KRG at doses of 50 and 100 mg/kg body weight in SHRs, respectively.

Rg3 is a naturally occurring ginsenoside with a wide range of beneficial effects for the cardiovascular system. Rg3 enriched KRG plays an important role in decreasing blood pressure and inducing vasodilation associated with the release of NO. A pharmacokinetic study of Rg3 showed that, after oral administration of ginseng, the peak plasma concentration was achieved in 150.0 ± 73.5 hours,²⁹ which means that the oral bioavailability of Rg3 was 2.63%, limiting its beneficial effect. In addition, even when ginseng roots are subjected to steam-heat treatment at 95°C for 3 hours, which strongly increases the amount of Rg3 in normal ginseng, the amount is still usually less than 0.5%.²⁰ Therefore, in this study, to improve the biodistribution of Rg3 in vivo, we used REKVG containing a higher percentage of Rg3 isolated from Panax ginseng compared to KRG. The amount of Rg1, Re, Rf, Rg2, Rh1, Rb1, Rc, Rb2, Rg3, Rk1, and Rg5 was 4.9, 1.2, 0.9, 0.6, 0.6, 8.7, 1.1, 1.1, 0.7, 0.06, and 0.3 (mg/g) in KRG, and ND, ND, 1.0, 2.3, 2.2, 0.9, 0.2, 0.2, 17.4, 2.0, and 4.5 (mg/g) in REKVG. These results show that the concentration of ginsenoside Rg3 in REKVG is ∼25 times greater than in KRG (Table 1). The greater effectiveness of REKVG in reducing blood pressure in SHRs shown in this study may be due to its higher Rg3 content compared to that of KRG. Furthermore, during the steaming process, we soaked dried ginseng in vitamin C water. Vitamin C here functioned as a catalyst and allowed a chemical reaction to occur using less energy and less time than would be required under normal conditions. This is the first time that this particular method of preparing ginseng has been used. It is more economical and less time consuming than traditional methods and thus offers a less expensive alternative for the preparation of Rg3 enriched ginseng. In our previous study, we have shown that Rg3 enriched Korean Red Ginseng (REKRG) has a much higher content of Rg3 (around 300 times higher than KRG) so the effectiveness of REKRG on vascular function is higher as compared to KRG.²⁰ However, the total process of making REKRG consumes a lot of time, which results in a higher cost of producing this kind of ginseng. Therefore, in this study, we used vitamin as a catalyst to speed up the steaming process of ginseng, making it faster and cost-effective. Although we did not compare the antihypertensive effect of REKRG and REKVG, there might be a little difference between these two ginsengs.

The beneficial effects of ginseng on the vascular system may depend on the activation of the PI3K/Akt signal transduction pathway.³⁰ Ginseng extract administration stimulates nongenomic Akt-mediated eNOS activation in SHRs.³¹ In line with this, we found that phosphorylation of Akt, which is upstream of eNOS, was significantly higher in vivo and in vitro in the REKVG-treated group than in the KRG-treated group. In addition, phosphorylation of eNOS and NO production was significantly higher in the SHR group treated with REKVG, similar to the in vitro data. NO is an endothelium-derived relaxing factor that plays an important role in the control of vascular tone and function. Its synthesis by the vascular endothelium is responsible for the vasodilator tone, which is essential for the regulation of blood pressure. A reduction in NO bioavailability contributes to vascular diseases such as hypertension, senility, and atherosclerosis. In the present study, REKVG administration stimulated NO production both in vivo and in vitro.

In conclusion, our data provide experimental evidence suggesting that REKVG suppresses hypertension in rats, at least partly by promoting NO production through upregulation of Akt-mediated eNOS phosphorylation and proves to be a cost-effective alternative to other varieties of Ginsengs.

Experimental

Preparation of REKVG

The BTGin Company Limited, (Daejeon, Korea), kindly provided both KRG and REKVG. The method for the preparation of REKVG was as follows: Fresh Korean ginseng was dried in the oven at 50°C-65°C for 2-5 days until the water content was <30%. This step was carried out because dried ginseng absorbs more vitamin C/water solution than fresh ginseng, which contains water and thus absorbs less than the dried form. Next, the dried ginseng was soaked in a 1% vitamin C/water solution at room temperature for at least 70 hours until the water content was about 70%. This was followed by a heating process that resulted in the structural conversion of the ginsenosides by acid hydrolysis. The swollen ginseng was placed in a sealed chamber to avoid water loss during the heating process, and the chamber was incubated for 48 hours at 85°C. After heating, the shaped ginseng was dried in the oven at 50°C-65°C for 2-5 days until the water content was >20%. Finally, REKVG was extracted in an ethanol/water solution (50% v/v, twice). The ratio of the extraction solution to ginseng was 4:1 (v/w). The extracted solution was concentrated to powder using a rotary vacuum evaporator.

HPLC Analysis

The powder was dissolved in 70% methanol, and ginsenosides were analyzed by HPLC. HPLC was carried out on liquid chromatography (LC) system equipped with a quaternary gradient pump (Agilent, 1260 Quat Pump VL) and UV detector (Agilent, 1260 VWD). A reversed-phase column (HiQ sil C18 HS, 150 × 4.6 mm, internal diameter 5 µm; KYA Technology Co., Japan) was used for the quantitative determination of ginsenosides Rg3. The mobile phase consisted of water (A) and acetonitrile (B). The separation was achieved using the following gradient program: 0-2 minutes (10% B), 2-8 minutes (12% B), 8-10 minutes (16% B), 10-13 minutes (20% B), 13-22 minutes (21% B), 22-27 minutes (25% B), 27-30 minutes (27% B), 30-33 minutes (28% B), 33-35 minutes (29% B), 35-38 minutes (30% B), 38-42 minutes (32% B), 42-45 minutes (36% B), 45-50 minutes (35% B), 50-80 minutes (40% B), 80-85 minutes (70% B), 85-120 minutes (100% B), and 120-125 minutes (10% B). A flow rate of 1.6 mL/minute was used at the beginning of the experiment until 5 minutes, followed by a constant flow rate of 2.5 mL/minute until the end of the experiment (125 minutes). The column was kept at room temperature and the detection wavelength was set at 203 nm.

Cell Culture

HUVECs were purchased from Clonetics (San Diego, CA, USA) and cultured in endothelial growth medium-2 from Lonza (Walkersville, MD, USA). Subconfluent proliferating HUVECs between passages 2 and 8 were used.

Animals and Blood Pressure Measurements

Animal experiments conducted in this study conform to internationally accepted standards and have been approved by the Animal Care Committee of Chungnam National University. All rat experiments were performed in the animal facility according to institutional guidelines, and the institutional review board of Chungnam National University approved the experimental protocols. Male SHRs aged 10-12 weeks and WKYs weighing 250-320 g were purchased from Central Lab Animal Inc. (Seoul, South Korea). SHRs and WKY were randomly divided into 6 groups (WKY saline, WKY KRG, WKY REKVG, SHR saline, SHR KRG, and SHR REKVG), and blood pressure was measured in live rats using the tail-cuff method. Ginseng (10 mg/kg) was orally administered daily to animals for 6 weeks. Saline was used as a vehicle. The animals were raised under controlled lighting (lights on 6:00 to 18:00 daily), and temperature (24 ± 1°C) and body weight were measured every day.

To measure blood pressure, the rats were restrained in a cylindrical restrainer for 10 minutes to acclimatize them to the apparatus (noninvasive blood pressure system, CODA; Kent Scientific Corporation) before blood pressure recordings were made. Fifteen blood pressure recordings were obtained over a period of 25 minutes and then averaged.

Western Blot Analysis

Anti-phospho-eNOS antibody was purchased from Cell Signaling (Beverly, MA, USA). Anti-NOS3, anti-phospho Akt, and total Akt antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analyses were performed by adding 30 µg cell lysate or 30 µg tissue homogenate (obtained from the aortas of the rats) to sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel loading buffer, followed by boiling and separation by electrophoresis, and then transferred to a nitrocellulose membrane. After incubation in appropriate primary and peroxidase-conjugated secondary antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA), the chemiluminescent signal was developed using Super Signal West Pico or Femto Substrate from Thermo Fisher Scientific (Pierce, Rockford, IL, USA). Blots were imaged and band densities were quantified with a Gel Doc 2000 ChemiDoc system using Quantity One Bio-Rad software (Hercules, CA, USA). Values were normalized to β-actin loading control.

Nitrite and Nitrate Measurements

The NO metabolites nitrite (NO₂ ^–) and nitrate (NO₃ ^–), stable breakdown products of NO, were quantified using a commercially available Nitrate/Nitrite Fluorometric Assay Kit from Cayman Chemicals (Lexington, KY, USA) according to the manufacturer’s instructions. Plasma obtained from the rat blood was deproteinized using a 10 kDa cutoff filter (Microcon YM10 Millipore, Burlington, MA, USA). After subtraction of background fluorescence, values were normalized to obtain the total amount of protein.

Statistical Analysis

All experiments were performed at least 3 times. Statistical analyses were performed using SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation. Statistical significance was determined using analysis of variance followed by a multiple-comparison test with a Bonferroni adjustment; P values < 0.05 were taken to indicate statistical significance.

Footnotes

Acknowledgments

The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see:

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article. This article was financially supported for the research, authorship, and/or publication by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014R1A6A1029617), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (HI17C-1202-010019) and by the research fund of the Chungnam National University Hospital.

ORCID ID

Cuk-Seong Kim

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