INTRODUCTION:

Panax ginseng is a perennial herbaceous plant of the Araliaceae family, growing in the Far East of the Russian Federation, the northeastern provinces of China, and the northern Korean Peninsula. The plant raw materials of Panax ginseng, especially its underground part, are used in officinal medicine in Russia and in traditional Chinese medicine.

Panax ginseng has a number of pharmacological effects, such as cardioprotective, antioxidant, anti-inflammatory, anti-diabetic, hair growth stimulating, anti-ulcerogenic, anti-cancer.^1-5 The medicinal plant raw materials (MPRMs)in Russia are white ginseng roots, which are used as a tonic and adaptogenic medicine. Ginseng medicines are sold in various dosage forms, such as tinctures, capsules, coated tablets. Tinctures are a liquid dosage form for internal use, usually colored alcoholic or aqueous-alcoholic extracts obtained from MPRMs (dried or freshly harvested). This dosage form is highly safe without loss of the target pharmacological effect and finds its use in pharmaceutical practice .^6,7

The chemical composition of ginseng roots is represented by such groups of compounds as ginsenosides, polysaccharides, essential oils, fatty acids, amino acids, and, in smaller quantities, alkaloids, flavonoids, vitamins.⁸ Ginsenosides are triterpene tetracyclic saponins of the dammarane series, which are the main group of biologically active compounds of ginseng roots. Qualitative identification of marker ginsenosides and determination of the ginsenosides amount are a key point of the ginseng roots standardization and medicines based on it.

Chen W. et al. determined the ginsenoside profile of ginseng underground parts using HPLC-QTOF-MS. The total content of ginsenosides is 115.69 – 142.49mg/g. The highest content was found in ginsenosides Rb1, mRb1, Re, and Rg1. TheginsenosidesRc, Rb2, Rb3, Rd, mRc, mRb2, andmRb3 havealsobeenidentified.⁹

The ginsenoside profile of white ginseng roots of Russian and Chinese origin was determined by HPLC-MS. 17 ginsenosides have been detected. The highest contents of ginsenosides are Rb1 (5341.36-5376.48 mkg/kg), Rc (3866.43-4941.39mkg/kg), Re (1644.26-1841.68mkg/kg), Rb3 (1393.07-1457.41mkg/kg), Rb2 (1121.59-1208.55mkg/kg), Rg1 (1066.63–1119.27 mkg/kg), Rf (690.15-1904.14mkg/kg).¹⁰

There are no data on the ginsenoside profile of ginseng root tinctures in the scientific literature. There are data obtained by spectrophotometry on the total content of ginsenosides in terms of ginsenoside Rg1 in tinctures of Russian manufacturing companies (0.42-0.61%).¹¹

In this paper, the authors used TLC and UPLC-MS/MS methods to determine the composition and quantitative content of ginsenosides in white ginseng roots and ginseng tinctures. HPLC-MS/MS and UPLC-MS/MS are advanced chromatographic techniques that are widely used in the physicochemical analysis of chemical compounds.^12-15

The State Pharmacopoeia of the Russian Federation recommends the spectrophotometry method to determine the total content of ginsenosides in terms of ginsenoside Rg1.

Thus, the goal of this work is to determine the ginsenoside profile of white ginseng roots and its tinctures and improve approaches to their standardization.

MATERIALS AND METHODS:

Materials:

The objects of the study were white ginseng roots and tinctures based on them. Ginseng roots were purchased from the state farm "Ginseng" (PrimorskyKrai, Russia).Tinctures produced by "Vifitech" and "Ivanovo Pharmaceutical Factory" were purchased from pharmacies in Moscow.

TLC:

Sample preparation and TLC analysis methods were carried out in accordance with pharmacopoeial monograph - PM.2.5.0013.15 “Ginseng roots”.¹⁶ The tinctures were pre-centrifuged at 5000rpm for 15 minutes. A 10% alcohol solution of sulfuric acid was used as a detection solution, followed by development in UV light at λ=365nm.

UPLC-MS/MS:

Determination of the content of ginsenosides in the objects was carried out using the UPLC-MS/MS method on an LCMS - 8045 device: Shimadzu (Shimadzu Corporation, Japan).

Sample Preparation:

The MPRMs (ginseng roots) were pulverized into powder. Sequentially, 0.1g fine powder was accurately weighed and extracted with a 2mL extracting solution (methanol: water=7:3) in an ultrasonication water bath at 50°C for 30 min, then cooled to room temperature. The resulting extraction solution and tinctures were diluted 10 times with methanol and then filtered through a 0.22 μm PTFE micro-filter before UPLC-MS/MS analysis.

Conditions for UPLC-MS/MS Analysis:

The Acquity UPLC Acquity BEH C18 column (100mm × 2.1mm, 1.7μm) (Waters Co. Ltd., MA, USA)at 30℃ was used for component separation and quantitative analysis. The mobile phase was composed of 0.1% aqueous formic acid (A) and acetonitrile (B), and the flow rate was 0.3mL/min. The gradient elution program was as follows: 0.0-1.0min 28% B, 1.0-11.0 min 28-40% B, 11.0-13.0min 40-85% B, 13.0-15.0min 85-95% B, 15.0-17.0min 95% B, 17.0-17.01min 95-28% B, and held at 28% B until 23min.

Multiple reaction monitoring (MRM) optimization results of ginsenosides are presented in the Table 1.

Table 1: MRM optimization results of ginsenosides

Component	RT/min	Ionizationmode	m/z	Q1/V	CE/V	Q3/V
Rb1	8.668	+	1131.9000>365.1000	-32	-55	-25
Rb2	10.534	-	1077.4500>191.1500	32	54	19
Rb3	10.838	-	1077.5000>149.1000	34	55	15
Rc	9.559	-	1077.5000>148.9500	32	55	10
Rd	12.238	-	945.4000>101.0000	24	51	18
Re	1.343	-	945.4000>783.3500	38	38	30
Rf	6.097	-	799.4500>475.2500	24	43	24
Rg1	1.383	-	845.5000>637.3500	26	34	24
S-Rg2	8.485	-	783.3500>475.2500	32	42	24
Ro	10.066	-	955.5000>793.2500	22	49	28
S-Rg3	16.302	-	783.5000>100.9500	24	46	10
Rg5	17.347	-	765.4000>70.9500	24	46	26
R-Rh1	8.452	-	683.5000>44.9500	26	27	16
S-Rh2	17.530	-	667.5000>44.8500	24	23	15
Rk1	17.178	-	765.4500>603.2000	22	31	30
RK3	15.895	-	665.2500>44.9000	34	25	17
F2	15.869	-	829.4500>783.4500	26	22	30
F3	8.402	-	769.5000>148.9500	24	31	10
F4	15.839	-	811.3500>765.4000	24	21	30
CK	17.288	-	667.4500X621.3000	20	20	24

RESULTS AND DISCUSSION:

TLC:

In the chromatograms of the objects, light yellow absorption zones were found at the level of the ginsenoside standard Rg1.Six absorption zones from white to dark yellow were detected in the chromatogram of the objects. The chromatograms of tinctures have more intense adsorption zones than the chromatogram of ginseng roots.In all tested samples, an adsorption zone of the same level (Rf=0,72) and color as the adsorption zone of the standard sample of ginsenoside Rg1 was found.The obtained chromatogram is shown in Figure 1.

Figure 1: TLC chromatogram of ginsenosides.

1 - Standard sample of ginsenoside Rg1; 2 - MPRMs ginseng roots; 3 - Ginseng tincture (“Vifitech” Russia); 4 - Ginseng tincture (“Ivanovo Pharmaceutical Factory” Russia)

UPLC-MS/MS:

Total chromatograms of the objects are presented in Figures 2.

Figure 2: Total chromatograms.

A- Standard;B - MPRMs of white ginseng roots;C - ginseng tincture (“Vifitech” Russia);D - ginseng tincture (“Ivanovo Pharmaceutical Factory” Russia)

The results of identification and quantitative content of ginsenosides are given in Table 2.

Table 2: Results of UPLC-MS/MS analysis of ginsenosides

No	Ginsenoside	MPRMs white ginseng roots	Ginseng tincture (“Vifitech” Russia)	Ginseng tincture (“Ivanovo Pharmaceutical Factory” Russia)
No	Ginsenoside	mg /100 g
1.	Rb1	0.849	99.124	92.545
2.	Rd	0.010	0.806	0.498
3.	S-Rg3	0.001	0.014	0.005
4.	CK	-	0.006	0.006
5.	F2	0.007	0.062	0.032
6.	Rg1	0.179	0.256	0.191
7.	R-Rh1	0.004	0.010	0.004
8.	Rg5	0.0004	0.0004	-
9.	S-Rh2	-	0.001	0.0001
10.	Rc	0.036	0.462	0.282
11.	Rk1	-	0.008	-
12.	F4	0.0002	0.001	0.0003
13.	Rg2	0.008	0.019	0.013
14.	Rb3	0.002	0.062	0.052
15.	Re	0.141	2.274	1.356
16.	Ro	0.158	0.253	0.275
17.	Rf	0.015	0.003	-
18.	Rb2	0.006	0.046	0.017
Total content		1.4166	103.4074	95.2764

A total of 18 ginsenosides were found in the «Vifitech» tincture and 15 ginsenosides in the white ginseng root and tincture of the «Ivanovo Pharmaceutical Factory». The following ginsenosides are present in the greatest quantities in white ginseng roots: Rb1 (0.849mg/100g), Rg1(0.179mg/100g), Ro(0.158mg/100g), Re(0.141 mg/100g), Rc(0.036mg/100g), Rf(0.015mg/100g). In tincture of ginseng (“Vifitech” Russia) 18 ginsenosides were found, of which the highest content is: Rb1(99.124 mg/100g), Re(2.274mg/100g), Rd(0.806mg/100g), Rc (0.462mg/100g), Rg1(0.256mg/100g), Ro(0.253 mg/100g). The tincture of ginseng (“Ivanovo Pharmaceutical Factory” Russia) contains 15 ginsenosides with the maximum value for the following ginsenosides: Rb1(92.545mg/100g), Re(1.356 mg/100g), Rd(0.498mg/100g), Rc(0.282mg/100g), Ro (0.275mg/100g), Rg1(0.191mg/100g).

Thus, ginsenosides Rb1, Rg1, Ro, Re, Rc are represented in all samples in the greatest quantities. The high content of ginsenosides Rb1, Re, Rc, Rg1 is also confirmed by literature data, which allows us to consider these ginsenosides as markers.^9,10

The highest total content of ginsenosides was found in ginseng tincture (“Vifitech” Russia) (103.4074 mg/100g), and the lowest in MPRMs of ginseng roots (1.4166 mg/100g).

CONCLUSION:

The main groups of biologically active substances (ginsenosides) in MPRMs of white ginseng roots and tinctures were determined by TLC. The profile of ginsenosides was determined by UPLC-MS/MS. Among the 18 identified ginsenosides, it seems appropriate to consider the following ginsenosides as marker compounds Rb1, Re, Rc, Rg1. The highest total content of ginsenosides was noted in the tincture of ginseng (“Vifitech” Russia). These analysis methods can be used as a basis for developing regulatory documentation for white ginseng roots and tinctures based on it.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS:

Project mobility program in priority areas for the implementation of projects of the Sechenov University Development Program for 2021-2030 within the framework of the Federal Academic Leadership Program “Priority-2030” .

ABBREVIATIONS:

MPRMs, medicinal plant raw materials; TLC, thin layer chromatography; ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS); high performance liquid chromatography-quadrupole time of flight - mass spectrometry (HPLC-QTOF-MS).

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Received on 06.03.2024 Modified on 25.06.2024

Research J. Pharm. and Tech 2024; 17(9):4299-4303.

DOI: 10.52711/0974-360X.2024.00664