
==== Front
J Ginseng Res
J Ginseng Res
Journal of Ginseng Research
1226-8453
2093-4947
Elsevier

S1226-8453(24)00088-5
10.1016/j.jgr.2024.05.004
Research Article
Monoclonal antibody-based enzyme-linked immunosorbent assay for quantification of majonoside R2 as an authentication marker for Nngoc Linh and Lai Chau ginsengs
Chaingam Jiranan a1
Van Huy Le b1
Noguchi Kanta a
Nuntawong Poomraphie a
Vimolmangkang Sornkanok cd
Yodsurang Varalee ef
Yusakul Gorawit g
Morimoto Satoshi a
Sakamoto Seiichi s.sakamoto@phar.kyushu-u.ac.jp
a⁎
a Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan
b Department of Biochemistry and Medicinal Chemistry, Research Institute for Biotechnology and Environment, Nong Lam University, Ho Chi Minh City, Viet Nam
c Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
d Center of Excellence in Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand
e Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
f Preclinical Toxicity and Efficacy, Assessment of Medicines and Chemicals Research Unit, Chulalongkorn University, Bangkok, Thailand
g School of Pharmacy, Walailak University, Nakhon Si Thammarat, Thailand
⁎ Corresponding author. Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan s.sakamoto@phar.kyushu-u.ac.jp
1 These authors contributed equally to this work.

23 5 2024
9 2024
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© 2024 The Korean Society of Ginseng. Publishing services by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Background

Recent years have witnessed increasing interest in the high amount of ocotillol-type saponin in Panax vietnamensis, particularly in relation to majonoside R2 (MR2). This unique 3%–5% MR2 content impart Ngoc Linh and Lai Chau ginsengs with unique pharmacological activities. However, in the commercial domain, unauthentic species have infiltrated and significantly hindered access to the authentic, efficacious variety. Thus, suitable analytical techniques for distinguishing authentic Vietnamese ginseng species from others is becoming increasingly crucial. Therefore, MR2 is attracting considerable attention as a target requiring effective management measures.

Methods

An enzyme-linked immunosorbent assay (ELISA) was developed by producing monoclonal antibodies against MR2 (mAb 16E11). The method was thoroughly validated, and the potential of the immunoassay was confirmed by high-performance liquid chromatography with ultraviolet spectroscopy. Furthermore, ELISA was applied to the assessment of the MR2 concentrations of various Panax spp., including Korean, American, and Japanese ginsengs.

Results and conclusions

An icELISA using mAb 16E11 exhibited linearity between 3.91 and 250 ng/mL of MR2, with detection and quantification limits of 1.53 and 2.50 − 46.6 ng/mL, respectively. Based on this study, the developed icELISA using mAb 16E11 could be a valuable tool for analyzing MR2 level to distinguish authentic Ngoc Linh and Lai Chau ginsengs from unauthentic ones. Furthermore, the analysis of the samples demonstrated that Ngoc Linh and Lai Chau ginsengs exhibit a notably higher MR2 value than all other Panax spp. Thus, MR2 might be their ideal marker compound, and various bioactivities of this species should be explored.

Graphical abstract

A workflow on the production and development of anti-MR2 monoclonal antibody for the determination of MR2 compounds and the application on the distinguishing of authentic Vietnamese ginseng.

Image 1

Keywords

Enzyme-linked immunosorbent assay
Majonoside R2
Monoclonal antibody
Ngoc Linh ginseng
Vietnamese ginseng
==== Body
pmc1 Introduction

Panax species, commonly known as ginseng, is a member of the Araliaceae family. Panax spp. has been the focus of scientific research for over a century owing to its immunomodulatory properties [1] and a broad range of physiological functions, including its potential as a cardiovascular and central nervous system stabilizer [[2], [3], [4], [5]]. More than 14 species have been identified worldwide with various medicinal applications [6]. Among six known species, common species are known as Korean ginseng (P. ginseng; PG); the others are American ginseng (P. quinquefolius); Chinese ginseng (P. notoginseng); Japanese ginseng, also known as bamboo ginseng (P. japonicum); Vietnamese ginseng (P. vietnamensis); and Himalayan ginseng (P. pseudoginseng) [1].

Among the three recognized ginseng species in Vietnam [7], Panax vietnamensis Ha et Grushv. represents an invaluable and endemic species [8]. This ginseng was discovered in the Ngoc Linh Mountain in 1973, well known as “Ngoc Linh ginseng” [8], and the authenticity of its root was officially protected by the Geographical Indication “Ngoc Linh” as Registration Certificate No. 00049 [[9], [10], [11]]. The ginseng species was further enriched with the discovery of the two subspecies, P. vietnamensis var. fuscidiscus from southern Yunnan, China in 2003 [12] and P. vietnamensis var. langbianensis from Lang Bian Mountain, Vietnam in 2016 [13]. Put differently, the species are divided into three types, namely P. vietnamensis Ha et Grushv. or “Ngoc Linh ginseng,” P. vietnamensis var. fuscidiscus, and P. vietnamensis var. langbianensis [7]. Additionally, P. vietnamensis var. fuscidiscus was also found to be naturally distributed in Lai Chau Province, Vietnam and became well known as Lai Chau ginseng [7,14]. The fresh root of Lai Chau ginseng produced in Lai Chau Province was ensured its authenticity by the trademark “Sam Lai Chau or Lai Chau ginseng” in 2022 [15].

These two authentic ginsengs share high similarities regarding their molecular and morphological characteristics, invaluable bioactive constituents, precious biological activities, and intensive cultivation stages to meet the market demand [7,8,14,16]. Both authentic ginsengs were especially characterized by their extremely high concentrations of invaluable majonoside R2 (MR2), which is a tetracyclic triterpenoid saponin with a furan ring linked to aglycones (Fig. 1). The MR2 contents of authentic ginsengs are often >5 %, whereas those of other adulterants are small or zero [[17], [18], [19], [20], [21], [22], [23], [24]]. More attractively, studies have demonstrated that MR2 exhibits chemoprotective, anticancer, or hepatocytoprotective activities [[17], [18], [19], [20], [21], [22]]; thus, most extant studies on both ginsengs have intensively determined MR2 as a key active component. The investigation of saponin accumulation in P. vietnamensis Ha et Grushv. during its cultivation also focused on MR2 as a major component, and MR2 reached a maximum of ∼5 % at the age of 5 years, indicating an appropriate harvest age [25].Fig. 1 Chemical structure of MR2.

Fig. 1

As characterized with essential chemical and biological properties, “Ngoc Linh ginseng” was recognized as a national product in 2017 and the price of 1 kg of the best fresh root of genuine “Ngoc Linh ginseng” was 3000 US dollars (2017) [21]. However, the commercialization of the two authentic ginsengs in Vietnam is hindered by less valuable adulterants, such as P. stipulealatus Tsai & K.m. Feng, P. vietnamensis var. langbianesis, and P. bipinnatifidus Seem., which are extremely challenging to distinguish with the naked eye.

The two genuine ginsengs can be distinguished from adulterants using their molecular and morphological characteristics [[26], [27], [28]] or by chemical analysis. Biological tools do not evaluate the concentrations of essential chemical compounds, which are low at early ages and accumulate at the later stage of development in the Ngoc Linh ginseng [25]. Thus, quantitative analysis was performed to authenticate the root of authentic Ngoc Linh ginseng based on the total saponin content and three major compounds, including MR2, ginsenoside Rg1, and ginsenoside Rb1 [7,8]. The biological activity and high concentration (>5 %) of MR2 make it unique; thus, it is the most important compound in quantitative analyses for authenticating Ngoc Linh ginseng, with an approximate requirement of 3.6 % MR2 in authentic root at the age of 6 years [[9], [10], [11]]. Additionally, the trademark of the fresh root of Lai Chau ginseng was recently granted in 2022, and the quantitative authentication was achieved by the determination of total saponin content and the unique characterization of MR2, with a minimum requirement of 3 % MR2 in absolute dried material [15]. Hence, MR2 was highlighted as the most crucial molecule and even a unique compound used for the quantitative authentication of the Vietnamese ginseng roots, as well as the presence of prominent biological benefits therein.

The existing general method for detecting MR2 involved employing analytical techniques that leverage chromatographic properties in addition to various detection methods [[29], [30], [31], [32], [33]]. Further, the absence of chromophore characteristics limits the capability of detecting MR2 via simple UV spectroscopy [19]. Therefore, developing a more sensitive approach for distinguishing Ngoc Linh ginseng and Lai Chau ginseng from other ginseng types, as well as ensuring accurate quality control in pharmaceutical and therapeutic applications, are of utmost significance.

An immunoassay is a highly specific technique that is based on antigen–antibody complex reactions. Many small molecules can be determined by immune-specific reactions [34]. Enzyme-linked immunosorbent assay (ELISA) has become increasingly prevalent in the measurement of plant secondary metabolites [[34], [35], [36], [37], [38]] owing to its economical nature, notable effectiveness, and environmentally sustainable process [34].

A MR2-targeting monoclonal antibody was generated in this study. The generated monoclonal antibody 16E11 (mAb 16E11) was characterized by determining its cross-reaction with structure-related compounds and an ELISA using mAb 16E11 was validated for its sensitivity and precision. Thereafter, the developed method was employed to establish ELISA for quantifying the MR2 content as well as distinguishing Ngoc Linh ginseng and Lai Chau ginseng from other species. Moreover, the accuracy of the newly devised method was confirmed through traditional detection using high-performance liquid chromatography using ultraviolet–visible spectroscopy (HPLC/UV–Vis).

2 Materials and methods

2.1 Chemicals and reagents

MR2 and related compounds (≥98 % purity), including majonoside R1 (MR1), pseudoginsenoside RT4 (PRT4), vinaginsenoside R1 (VR1), vinaginsenoside R2 (VR2), and vinaginsenoside R11 (VR11), were isolated and identified by nuclear magnetic resonance by Dr. Le Van Huy of the Research Institute for Biotechnology and Environment (Nong Lam University, Ho Chi Minh City, Vietnam). Enriched RPMI-1640 Delbecco's-Ham's F12 (eRDF) and RD-1 were acquired from Kyokuto Pharmaceutical Industrial (Tokyo, Japan). Bovine serum albumin (BSA), ovalbumin (OVA), aminopterin, and polyethylene glycol (PEG) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The employed fetal bovine serum (FBS) was acquired from Gibco (Carlsbad, CA, USA). The chemicals employed in this study, i.e., 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hypoxanthine, were acquired from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The goat anti-mouse IgG H&L (HRP) (ab6789) was acquired from Abcam (Cambridge, Massachusetts, USA). The additional chemicals and reagents employed in the experiments were of analytical grades.

2.2 Animals

Male BALB/c mice weighing 17–22 g were obtained from Charles River Laboratories (Kanagawa, Japan) and were aged 5 weeks before distributed. The mice were subjected to standard treatment, following the guidelines established by the Committee on the Ethics of Animal Experiments at Kyushu University's Graduate School of Pharmaceutical Sciences (reference no. A20-002-3)

2.3 Preparation of Panax spp. root samples

This study comprised the analyses of six Panax roots and one Randia siamensis (R. siamensis) fruit (14), which reportedly exhibit incredibly high triterpenoid saponin contents [39]. Five Panax root samples, namely P. vietnamensis var. fuscidiscus or Lai Chau ginseng (1), P. vietnamensis var. vietnamensis or Ngoc Linh ginseng (2), P. vietnamensis var. langbianesis N.V. Duy et al. (3), P. bipinnatifidus (4), and P. stipuleanatus (5), were collected and identified by Dr. Le Van Huy. Further, P. japonicus (6) and P. ginsengs collected in Japan (PG - 1 (7) root powder were purchased from Uchidawakanyaku Ltd. (Tokyo, Japan). Furthermore, four additional P. ginsengs were acquired from China (PG - 2, (8)); prescribed by a doctor from Korea, Korean white ginseng (PG - 3, (9)); company product from Korea (PG - 4, (10)), and Korean red ginseng (PG - 5, (11)). Two P. quinquefolius species, which originated from Canada (PQ - 1, (12)) and China (PQ - 2, (13)), were utilized. Voucher specimens are at the Herbarium of the Faculty of Pharmaceutical Sciences, Kyushu University (Voucher specimen Nos. P0001–P0013 and R0001). All the plant samples were ground into fine particles and weighed accurately (50 mg) for extraction. Thereafter, they were extracted four times with methanol using an ultrasonic sonicator (500 μL/time). The supernatant solutions were collected after centrifugation for 10 min at 9200 g, transferred into a 2 mL tube, and dried completely at 42 °C. Further, each sample was redissolved with 1 mL of methanol. After complete dissolution, the sample extracts were diluted to suitable concentrations for detection by ELISA and HPLC methods.

2.4 Preparation of the MR2 conjugates

The MR2–BSA immunogen and MR2–OVA immobilized antigen were produced by the reaction of sodium periodate (NaIO4), as detailed in several extant studies [35,38]. In summary, in the context of immunogenic conjugates, a solution of distilled water (1 mL) and NaIO4 (3.02 mg) was added gradually to a solution of MR2 (3.01 mg) in methanol (1 mL), after which the reaction proceeded for 3 h at 25 °C. Subsequently, BSA (6.35 mg) was added to a 50 mM carbonate buffer solution (pH 9.6; 2 mL). Next, the resultant reaction mixture was subjected to continuous stirring at 25 °C for 14 h. The product reaction required dialysis for three days to remove the excess reagents before lyophilization. The conjugates were verified by matrix-assisted laser desorption/ionization combined with time-of-flight mass spectrometry (MALDI-TOF MS) to quantify the hapten quantity [34]. To obtain the MR2–OVA conjugates, similar steps based on the NaIO4 reaction were employed to prepare the coating agent using MR2 (3.03 mg) and OVA (6.13 mg).

2.5 Production and purification of MR2–targeting monoclonal antibody

The initial immunization emulsion was prepared by thoroughly mixing a solution of the MR2–BSA conjugates dissolved in phosphate-buffered saline (PBS) using Freund's complete adjuvant (Becton Dickinson Company, MD, USA) in a 1:1 ratio. The subsequent dose was prepared in the same manner as the first dose using Freund's incomplete adjuvant (Becton Dickinson Company, MD, USA) in the same ratio, and an emulsion containing 50 μg of immunogen was delivered intraperitoneally to individual animals 14 days after initial immunization. The next immunization (100 μg of immunogen) was administered without an adjuvant. After the second dose, mouse serum was collected from the tail vein after 5 days of administration to test for the antibody titer and activity against MR2 using indirect ELISA (iELISA) and indirect competitive ELISA (icELISA). Following the fifth immunization, the mice exhibiting the most evident activities and displaying the lowest cross-reactivity (CR; %) toward the structurally related compounds to MR2 were subjected to splenectomy. The splenocyte with SP2/0 (a mouse myeloma cell line) was fused using the well-established PEG technique [40]. Subsequently, the hybridoma cells were grown in an eRDF medium comprising RD-1 and 10 % FBS. Hypoxanthine, aminopterin, and thymidine were included as additives to facilitate the separation of the hybridoma cells from the nonhybridized ones. After two weeks of the culturing process, the presence of antibodies against MR2 in the culture media was assessed by iELISA. The clone-selection process was performed using limiting dilution, followed by antibody titer and activity screening via iELISA and icELISA [41], for the identification of positive clones. Subsequently, the 16E11 clone exhibiting minimal CR (%) was expanded in a nonaminopterin additive to enhance the production of anti-MR2 mAb until a volume of 1 L was achieved. Thereafter, the colony 16E11 cells were transferred to an FBS-devoid medium and cultured for three days before purification.

To purify mAb 16E11 from the hybridoma (16E11)-secreted supernatant, a Protein G Sepharose 4 Fast Flow column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was equilibrated with 10 mM phosphate buffer (PB, pH 7.0) . The supernatant was applied to the column after filtration and pH adjustment (pH 7.0) with a 1 M Tris-HCl (pH 9.0) solution, followed by a washing buffer (10 mM PB, pH 7.0) and an elution buffer (100 mM citrate buffer, pH 3.0). The eluted portion was promptly neutralized using a solution of 1 M Tris-HCl (pH 9.0). Each eluted fraction was determined by iELISA. Subsequently, the fractions containing mAb 16E11 were combined, dialyzed, and allowed to dry to yield the powder.

2.6 Indirect and indirect competitive enzyme-linked immunosorbent assay procedures

Next, mAb 16E11 was characterized using iELISA and icELISA. These methods were deployed to assess the reactivity of the antibody as well as its ability to competitively bind to free MR2. The experimental protocol for iELISA followed previously established methods [[35], [36], [37], [38]] with slight adjustments. Briefly, the MR2–OVA conjugates were immobilized by adding the MR2–OVA conjugates in a 50 mM carbonate buffer (pH 9.6) (2 μg/mL; 100 μL/well) on a 96-well immunoplate. The reaction plate was incubated at 37 °C for 1 h. Next, PBS containing skim milk (5 % (w/v); 300 μL/well) was employed to mitigate non-specific binding to other substances. After 1 h of incubation, 5 % (v/v) methanol (50 μL/well) and various mAb 16E11concentrations in phosphate-buffered saline containing 0.005 % (v/v) Tween 20 (TPBS) (50 μL/well) were mixed and incubated for another 1 h (37 °C), followed by 10,000 times dilution of goat anti-mouse IgG H&L (HRP) (100 μL/well) for 1 h (37 °C) and a substrate solution (0.3 mg/mL ABTS in 100 mM citrate buffer (pH 4.0) supplemented with 0.003 % (v/v) H2O2; 100 μL/well). After 20 min of incubation, the UV absorbance was measured at 405 nm using a microplate reader (Multiskan™ FC Microplate Photometer, Thermo Fisher Scientific, Inc., Waltham, MA, USA). At every step after incubation, the 96-well plate was washed three times with TPBS.

In icELISA, competitive inhibition using a free antigen was evaluated. After the blocking step, various concentrations of MR2 in 5 % (v/v) methanol (50 μL/well) and mAb 16E11 (50 μL/well; 6.25 μg/mL) were mixed well and incubated for 1 h before proceeding to the following step, as previously described.

2.7 Development of indirect competitive enzyme-linked immunosorbent assay for MR2 detection and evaluation to verify antibody performance

To validate the immunoassay, CR (%) of mAb 16E11 was determined by comparing it with those of the other secondary metabolites present in P. vietnamensis var. vietnamensis. By calculating the half-maximal inhibitory concentration (IC50) of MR2 competing with the investigated compounds, CRs (%) was evaluated using the equation below, and the results confirmed the specificity of the method toward MR2 rather than other similar chemicals.CRs(%)=IC50ofMR2IC50ofcandidatecompounds×100.

The spiked recovery assay was performed to investigate the matrix effect on the assay by spiking 6.25 %, 12.50 %, 25 %, 50 %, and 75 % (w/w) of free MR2 standard compounds into a P. vietnamensis var. vietnamensis extract exhibiting a MR2 content of 36.9 ± 4.82 mg/g dry weight. The recovery percentage was calculated from the detected and spiked amounts of MR2 at different concentrations using the equation below. The acceptable range of the recovery rate was 80%–120 %, as previously described [42].Recovery(%)=Detectedamount−36.9spikedamount×100

2.8 Determination of the MR2 content of plant samples using icELISA

The plant samples were analyzed by icELISA. Antigen coating, i.e., the MR2–OVA conjugates (4 μg/mL), was applied to a 96-well plate (100 μL/well) and allowed to adhere for 1 h. Thereafter, nonspecific binding was prevented by blocking with PBS containing 5 % (w/v) skim milk. Subsequently, the diluted sample extracts or standard MR2 (50 μL/well) were incubated in the presence of 6.25 μg/mL of mAb 16E11 (50 μL/well). After an incubation period of 1 h, anti-mouse IgG with horseradish peroxidase (HRP), 1:10000, was introduced (100 μL/well). Subsequently, ABTS in a 100 mM citrate buffer, 0.003 % (v/v) H2O2, was added (100 μL/well). After further incubation for 20 min, the UV absorbance was measured at a wavelength of 405 nm. Thereafter, a standard curve was generated for MR2, and the concentrations of MR2 in the extracts were determined from the standard curve–derived range.

2.9 Determination of the MR2 content of plant samples using HPLC-UV

To confirm the MR2 immunoassay-detection approach, HPLC-UV using the SPD-20A UV–Vis detector (Shimadzu Co., Kyoto Japan) was employed to quantify the concentrations of MR2 in the samples. HPLC pump was PU-4580 with DG-4580 as degassing unit (Jasco Co., Tokyo, Japan). The chromatographic analysis was performed using a COSMOSIL Packed column 5C18-AR-II with an inner diameter and length of 4.6 × 150 mm, respectively. The adopted HPLC-UV condition was slightly modified, following extant research findings [19]. In summary, the mobile phase was 27 % (v/v) acetonitrile isocratic, and MR2 detection was performed using a UV detector set at 196 nm. The flow rate was left constant at 1.0 mL/min, whereas the injection volume was 20 μL. The analyses were performed in triplicate. All samples were filtered through a 0.45 μM Millipore® filter, and all mobile phases were degassed before use.

3 Results and discussion

3.1 Production of MR2–targeting monoclonal antibodies

Conjugation was achieved through the NaIO4 reaction between MR2 and the carrier protein, BSA. The immunogenic probability of the MR2–BSA conjugates was investigated by assessing the hapten number. The molecular weights of BSA and the MR2–BSA conjugates, as determined from the MALDI-TOF MS (BRUKER Autoflex III) spectra, were 66,382 and 68,943 Da, respectively (Supplementary Fig. 1). Based on the molecular weight of MR2 (∼787 Da), the analysis revealed that the MR2–BSA conjugates contained approximately three MR2 molecules. The extant studies revealed that a higher number of hapten molecules (ranging from 15 to 30) corresponds to an increased potential to function as an effective immunogen [34]. Conversely, a reduced number of hapten molecules corresponds to eliciting a more specific immunological response. Sakamoto et al. [35] studied the efficacy of mAb against harringtonine (HT), observing that the hapten number of the HT–BSA conjugates exhibited a hapten number of two molecules, as prepared by the NaIO4 reaction. This finding demonstrated a notable level of specificity and sensitivity in relation to the low hapten number. Krittanai et al. [36] and Kitisripanya et al. [38] developed conjugates that gained seven and three hapten molecules, respectively, resulting in a highly specific antibody production.

After the fourth immunization using the MR2–BSA conjugates, the antibody from the mouse serum attained 80 % inhibition. Following the administration of the fifth boost dose, the mouse splenocyte was prepared and fused with the SP2/0 cell to produce a hybridoma. The limiting-dilution technique was employed to obtain a pure, single clone. Based on its superior sensitivity and selectivity toward MR2, the 16E11 clone was selected, and the antibody produced by 16E11 (mAb 16E11) was further purified after being derived from the second clone by limiting dilution. The fraction with a >1.0 absorbance was collected, mixed, and dialyzed against distilled water for 10 days. Subsequently, the resulting solution was lyophilized, resulting in a powdered product (216 mg).

Next, the isotype of the purified mAb 16E11 was identified using an IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Diagnostics, Mannheim, Germany). Based on the result, mAb 16E11 was classified as an IgG1 subclass with a kappa light chain antibody.

3.2 Optimization and validation of indirect competitive ELISA

The reactivity of mAb 16E11 was investigated by iELISA and graphically represented as a sigmoidal curve (Fig. 2A). The graph revealed the optimal concentration required for the implementation of icELISA. Thus, the concentration of mAb 16E11 was established at 6.25 μg/mL based on an absorbance spectrum of ∼1.0.Fig. 2 Reactivity of mAb 16E11 in ELISA. A: The reactivity of mAb 16E11 against MR-2 OVA (4 μg/mL) determined by iELISA. B: The reactivity of mAb 16E11 (6.25 μg/mL) against free MR2 (0.39–250 μg/mL) determined by icELISA. A: the absorbance at 405 nm from the well with free MR2 A0: the absorbance at 405 nm from the control well where control was 5 % (v/v) methanol.

Fig. 2

Quantitative icELISA was performed under optimized conditions, as previously described [[34], [35], [36], [37], [38]], and Fig. 2B shows the linearity curve. The range of the detectable concentration of MR2 was 3.91–250 ng/mL, with a determination coefficient (R2) of 0.9922. Further, IC50 was 10.8 ng/mL. The limit of detection (LOD) and limit of quantitation (LOQ) were 1.53 and 2.50–46.6 ng/mL, respectively.

Furthermore, CR (%) was determined. The concentrations of the triterpenoid saponins, i.e., ginsenoside Rb1, ginsenoside Rd, ginsenoside Re, and ginsenoside Rg1, were <0.005 % each, and PRT4 exhibited the highest CR (%), followed by MR1 and VR1, with corresponding percentages of 70.7 %, 68.1 %, and 39.3 %, respectively (Table 1). This high CR (%)can be accounted for by the conjugate reaction using the NaIO4 oxidative method. This method involves the oxidative cleavage of the adjacent 1,2-diols present in the sugar moiety, resulting in the formation of imides with the amino group of lysine residues found in the carrier protein [34]. Notably, mAb 16E11 could bind to the monoglucoside structure after the cleavage of the second glucose molecule due to the NaIO4 reaction. This indicated that mAb 16E11 could react with PRT4 and MR1 containing those aforementioned monoglucoside structures. The analysis revealed that the MR2 content was significantly higher than those of PRT4, MR1, and VR1 by ∼80, 40, and 160 times [18], [21], [22], [23], [24], respectively, despite the presence of CR (%) with structurally similar compounds. Derived from the root sample with the greatest concentration of 69.2 mg/g, the estimated quantities of PRT4, MR1, and VR1 from %estimate were 0.85, 1.83, and 0.43 mg, respectively. Compared with HPLC, which yielded 66.8 mg/g of MR2 (Table 2), such an outcome is plausible. Based on its precision, it can be concluded that icELISA using mAb 16E11 would be a good option for MR2 detection in Panax spp., particularly P. vietnamensis.Table 1 Cross-reactivity (CR, %) of mAb 16E11 against structured related compounds.

Table 1Compounds, amount in P. vietnamensis (estimated %w/w)	CR (%)	
MR2	100.00	
MR1	68.06	
PRT4	70.67	
VR1	39.26	
VR2	12.24	
VR11	4.21	
Ginsenoside Rb1	<0.005	
Ginsenoside Rd	<0.005	
Ginsenoside Re	<0.005	
Ginsenoside Rg1	<0.005	

Table 2 Determination of MR2 in plant samples including 13 samples with 6 different types of Panax spp. root and 1 R. siamensis's fruit.

Table 2Sample	Total MR2 Amount (%w/w)	
icELISA	HPLC-UV	
(1)P. vietnamensis var. fuscidiscus	6.92 ± 0.463	6.68 ± 0.274	
(2)P. vietnamensis var. vietnamensis	3.83 ± 0.434	3.80 ± 0.142	
(3)P. vietnamensis var. langbianesis	3.48 × 10−3± 1.91 × 10−4	N.D.	
(4)P. pseudoginseng var. bipinnatifidus	3.47 × 10−4± 2.87 × 10−5	N.D.	
(5)P. stipuleanatus	3.06 × 10−4± 2.68 × 10−5	N.D.	
(6)P. japonicus	2.17 × 10−3± 1.41 × 10−4	N.D.	
(7) PG - 1 (P. ginseng cultivated in Japan)	2.07 × 10−3± 1.43 × 10−4	N.D.	
(8) PG - 2 (P. ginseng cultivated in China)	3.60 × 10−4± 2.22 × 10−5	N.D.	
(9) PG - 3 (Korea White Ginseng prescribed by doctor)	3.88 × 10−4± 1.77 × 10−5	N.D.	
(10) PG - 4 (Korea White Ginseng from company)	1.84 × 10−3± 1.15 × 10−4	N.D.	
(11) PG - 5 (Korea Red Ginseng)	1.02 × 10−3± 1.69 × 10−5	N.D.	
(12) PQ - 1 (P. quinquefolius cultivated in Canada)	1.13 × 10−2± 7.70 × 10−4	N.D.	
(13) PQ - 2 (P. quinquefolius cultivated in China)	8.04 × 10−3± 8.04 × 10−4	N.D.	
(14)R. siamensis's fruit	N.D.	N.D.	
N.D. = Not detected.

icELISA exhibited high repeatability and reproducibility, as indicated by the low percentages of relative standard deviation (%RSD). Specifically, the %RSD values for the repeatability (intraday) and reproducibility (interday) were in the 0.92%–3.01 % and 4.25%–7.28 % ranges, respectively (Table 3). Furthermore, when assessing the accuracy, the root extract that contained MR2 was used as the plant matrix. The measured amount of MR2 in the extract was 36.9 ± 4.82 mg/g dry weight. To evaluate the accuracy, the extract was supplemented with MR2 at concentrations of 6.25%–75.0 % of MR2 in the extract. The expected amounts of added MR2 were 39.2, 41.5, 46.1, 55.4, and 64.6 mg, respectively. As presented in Table 4, the outcomes exhibited recovery ranging from 85.3 % to 109 %, which is within the acceptable range of 80–120 % [43].Table 3 Intra-day and inter-day validation to determine precision of ELISA method using mAb 16E11.

Table 3Concentration of MR2 (ng/mL)	RSD%	
Intra-day (n = 6)	Inter-day (n = 3)	
62.5	3.01	5.13	
31.3	2.49	7.28	
15.6	2.65	6.26	
7.81	0.923	5.67	
3.91	2.07	5.53	
1.95	1.83	4.25	

Table 4 Recovery determination using 6.25, 12.5, 25.0 50.0 and 75.0 % spike-level level of root sample of P. vietnamensis containing 36.9 ± 4.82 mg/g.

Table 4Spike-level of MR2% (w/w)	Spike-amount of MR2 (mg/g)	Detected MR2 content (mg/g)	Expected amount (mg/g)	Recovery (%)	
0 %	0	36.9 ± 4.82	–	–	
6.25 %	2.30	39.2 ± 3.04	39.2	99.1	
12.5 %	4.60	41.9 ± 5.67	41.5	109	
25.0 %	9.20	44.7 ± 2.53	46.1	85.3	
50.0 %	18.5	54.4 ± 1.08	55.4	95.1	
75.0 %	27.7	64.3 ± 4.60	64.6	99.4	
Note: The standard MR2 were spiked with the amount of 0, 2.30, 4.60, 9.20, 18.5 and 27.7 mg/g to the plant sample with previously observed for MR2 content. Recovery (%) was calculated from detected MR2 content to determine the correlation between the expected and the analyzed amount of samples from ELISA.

3.3 Determination of MR2 in actual plant samples by icELISA

To assess the efficacy of the proposed methodology, the MR2 contents of 13 root samples of Panax spp. (1－13) and one R. siamensis (14) fruit sample were analyzed. The icELISA results revealed that P. vietnamensis var. fuscidiscus (1) and P. vietnamensis var. vietnamensis (2) exhibited high MR2 concentrations, measuring 6.92 ± 0.463 and 3.83 ± 0.434 % (w/w), respectively. Conversely, P. vietnamensis var. langbianesis (3), commonly referred to as unauthentic Vietnamese ginseng, displayed significantly lower MR2 concentrations, measuring 3.48 × 10−3 ± 1.91 × 10−4 % (w/w). Further, PQ - 1 (12) and PQ - 2 (13) from Canada and China exhibited MR2 contents of approximately 1.13 × 10−2 ± 7.70 × 10−4 % (w/w) and 8.04 × 10−3 ± 8.04 × 10−4 % (w/w), respectively, and they were ranked second among other Panax spp. However, the MR2 quantities were significantly lower (400–600 times) than those of P. vietnamensis var. fuscidiscus (1) and P. vietnamensis var. vietnamensis (2). The other Panax spp. exhibited a relatively diminished MR2 quantity. Most of the P. japonicus (6) as well as PG - 1 (7), PG - 2 (8), PG - 3 (9), PG - 4 (10), and PG - 5 (11) samples exhibited comparable MR2 levels with P. pseudoginseng (4) and P. stipuleanatus (5). An examination of the different origins of PG revealed that the source from Japan (7) exhibited comparable or greater MR2 concentration among the other PGs. Furthermore, a comparison between white and red ginseng from Korea sources (PG - 3 (9), PG - 4 (10) and PG - 5 (11)) revealed that no substantial difference existed between their MR2 contents. In the case of R. siamensis, the plant with high triterpenoid saponins content [39], exhibited undetectable amount of MR2. To the best of the authors’ knowledge, this study could be the first publication that identifies the MR2 concentrations of over 6 Panax spp. Furthermore, compared with the MR2 chemical detected in different ginseng types, it can be implied that MR2 may serve as a marker compound for separating P. vietnamensis (Ngoc Linh and Lai Chau ginsengs) from other varieties. According to the Vietnamese government document for P. vietnamensis authenticity, quantitative examination of fresh Ngoc Linh ginseng root requires 3 compounds: MR2, ginsenoside Rg1, and ginsenoside Rb1. The concentration of each saponin rises with root age, as documented from 4 to 15-year-old [10] (Supplementary Table 1). Thus, MR2 was dominating at 4-, 5-, and 6-year-olds, with concentrations of 2.0 %, 3.0 %, and 3.6 %, respectively, and proportionate to ginsenoside Rg1 and ginsenoside Rb1 by increasing with age in each root [11]. While quantitative examination of fresh Lai Chau ginseng roots requires at least 3 % MR2 in absolute dried material at 5-year-old and total saponin content [15]. Thus, the situation emphasizes importance of MR2 in quantitative analysis. To ensure Vietnamese ginseng quality, quantitative MR2 analysis is the most important official authentication step that classifies the specific specie. The quantitative measurement of unique MR2 using icELISA meets official genuine standards and might be utilized as a quality marker for Ngoc Linh and Lai Chau ginsengs. This analytical development can considerably enhance Vietnamese ginseng production and quality control.

3.4 Determination of MR2 in actual plant samples by HPLC-UV

MR2 was previously isolated from P. japonicus (6), although only 0.1 % was detected [30]. The findings reported here indicate that MR2 was present in the additional Panax spp. in addition to P. vietnamesis. Various types of liquid chromatography with different detection methods have been employed to identify and measure MR2. MR2 has been determined using HPLC-UV. Zhu [19] designed an HPLC system to analyze 11 triterpenoid saponins, including MR2; it exhibited an LOD of 3 μg/mL. However, an assessment of the MR2 concentrations of 3 different types of Panax spp., MR2 was detected in P. vietnamensis at 58.80 mg/g dry weight, whereas it was undetectable in the other two Panax spp. High-resolution MS and multiple-stage tandem MS analyses were employed to identify 5 distinct ginseng varieties. The relative abundance (%) revealed that only the MR2 signal was observed exclusively in the root sample of P. vietnamensis, indicating the significantly high presence of MR2 therein. Tran et al. [18] used LC-electrospray ionization tandem mass spectrometry to measure the presence of MR2 in P. vietnamensis samples obtained from Quang Nam Province, Vietnam. The results revealed that the P. vietnamensis sample contained approximately ∼1.85 % of MR2. HPLC-UV detection and charged aerosol detection (CAD) were employed for the analyses of MR2 and other ocotillol saponins. MR2 could be detected at LOD of 0.0312 and 0.0076 mg/mL using UV detection and CAD, respectively [31]. The assessment of other chromatography procedures confirmed that the developed icELISA approach outperformed others in terms of cost-effectiveness and the absence of any specific skill requirement. The specificity of the antigen–antibody combination as well as the increased sensitivity of this method make it more efficient in MR2 detection for differentiating authentic Ngoc Linh and Lai Chau ginsengs from other Panax spp.

To assess the reliability of the developed method, a modified version of HPLC-UV was employed for MR2 determination. The analysis of MR2 presented a challenge owing to its non-chromophore nature for UV detection. Nonetheless, Zhu et al. [19] established an HPLC approach that employed a UV-detection wavelength of 196 nm to effectively quantify ocotillol saponin, including MR2. After a few adjustments, MR2 was examined with LOD and LOQ of 6.52 and 20.0 μg/mL, respectively. The retention time was between the interval of 3.5 and 3.8 min (Supplementary Fig. 2). Further, calibration curves were established for MR2 at a concentration range of 15.6–500 μg/mL. Regarding the comparison between the existing methods and the newly developed ones, icELISA demonstrated the potential to detect MR2 at the nanogram level compared with HPLC-UV at the microgram level. The antigen–antibody complex approach employed in this study demonstrated an outstanding sensitivity level, enabling the detection of low quantities of MR2 observed in Panax spp. other than Ngoc Linh and Lai Chau ginsengs.

4 Conclusions

Here, an MR2-targeting mAb was generated. Further, mAb 16E11 can be employed for icELISA development, achieving a LOD that is as low as 1.53 ng/mL while maintaining satisfactory accuracy and precision levels. This methodology can be employed for the detection of MR2 in Panax spp. The approach reported herein can be used as a quantitative screening tool for Ngoc Linh and Lai Chau ginsengs, which are officially protected by the Geographical Indication “Ngoc Linh” and the trademark “Lai Chau ginseng”, respectively. This will greatly assist the Vietnamese ginseng industry by facilitating the application potential of MR2-rich products. Advanced ELISA can be employed to distinguish the raw materials of the MR2-rich Ngoc Linh and Lai Chau ginsengs from those derived from different species with low content of MR2, which is necessary for quality control in pharmaceutical and medicinal product applications.

Declaration of competing interest

The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or nonfinancial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Appendix A Supplementary data

The following is the Supplementary data to this article.Multimedia component 1

Multimedia component 1

Acknowledgements

Dr. Le Van Huy would like to thank the financial support from the SAM VIET NHAT company for his work in this study.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.jgr.2024.05.004.
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