Standardization of Diploid Codonopsis laceolata Root Extract as an Anti-Hyperuricemic Source

Abstract

Codonopsis lanceolate exerts various medicinal effects and has been used as a traditional medicine for inflammation, asthma, gastritis, and liver disease. Recently, we reported the xanthine oxidase inhibitory activity of C. lanceolata extract and that lobetyolin, one of the key components, was a xanthine oxidase inhibitor. Lobetyolin showed anti-hyperuricemic activity in vitro and in vivo. In this study, we prepared various types of C. lanceolata extracts for the development of functional materials and natural drugs. We present the optimal analytical approach for the quality control and extraction optimization of C. lanceolata preparations. We established and validated a HPLC analysis for easy separation and quantification of the lobetyolin biomarker. Solvent extracts of C. lanceolata root were prepared and the profiles of the active marker and the optimal extraction methods were evaluated. The 100% ethanolic extract demonstrated the highest lobetyolin content. The validated HPLC method confirmed that lobetyolin was present in C. lanceolata root extracts. We suggest that the anti-hyperuricemic activities of C. lanceolata extract could be attributed to this marker compound. The results proposed that the 100% ethanolic extract could be used for the prevention of hyperurecemia, and that this analytical method and biomarker could be useful for the quality control of C. lanceolata preparations.

1. Introduction

Uric acid is an oxidative product of the purine metabolism pathway catalyzed by xanthine oxidase (XO) [1]. Hyperuricemia is characterized by increased uric acid levels in the blood as a result of XO mediation and inhibition of the renal excretion of uric acid by the body [2]. Gout is caused by the production of insoluble urate, resulting in gouty arthritis [3]. The prevalence of gout ranges from 0.1% to 10% across the world. More than two million people in the United States have gout [4,5]. Two commercial drugs, allopurinol and febuxostat, are used to treat hyperuricemia (HU) and gout [6,7]. However, these drugs have severe side effects that include skin rashes, infections, liver failure, fever, and allergic reactions [8,9,10]. Thus, new XO-inhibitory bioactive sources and plant-based medicines need to be explored as alternative medicines for HU and gout. In a previous study, we isolated the XO inhibitor lobetyolin from an extract of C. lanceolata root [11].

Lobetyolin is generally found in Codonopsis species, such as C. pilosula, C. tubulosa, and C. lanceolata [12]. It has been reported that lobetyolin has anticancer effects [13,14], and lobetyolin analogues have been reported to inhibit mucus secretion in airway epithelial cells [15]. We previously described the anti-hyperuricemic effect of lobetyolin in vitro and in vivo. Lobetyolin was found to inhibit XO activity via a mixed-type mechanism. Additively, oral lobetyolin at 50 mg/kg significantly reduced hepatic XO activity in vivo [11].

Codonopsis lanceolata possesses various medicinal effects and has been used as a traditional medicine for asthma, inflammation, gastritis, and liver disease. Recently, several studies have reported that Codonopsis lanceolata extract shows anti-hypertensive effects, and anti-obesity and anti-cancer activity [16,17,18]. The XO inhibitory activity of C. lanceolata extract was assessed, and lobetyolin was one of the key components as well as a xanthine oxidase inhibitor. Han et al. reported that treatment with dosages of C. lanceolata extract (200 and 400 mg/kg) significantly reduced systolic blood pressure (SBP) in hypertensive rats. C. lanceolata extract tended to increase acetylcholine (Ach)- and sodium nitroprusside (SNP)-induced vascular relaxation in hypertensive rats. Seol et al. suggested the use of several active markers such as lancemaside derivatives using liquid chromatography–mass spectrometry (LC–MS) [19]. Qiao et al. reported the quantitative results of lobetyolin among Codonopsis species [12]. However, Han et al. did not report quantitative results when defining lancemaside derivatives as the main effective biomarkers; as such, there is insufficient evidence for the commercialization of C. lanceolata extract [16].

Lee et al. investigated the anti-obesity effect of C. lanceolata extract in vivo. C. lanceolata extract decreased fat in the subcutaneous and visceral tissue and exhibited lower serum levels such as low-density lipoprotein (LDL), total cholesterol, and insulin in a high-fat diet-induced mouse model (up to 360 mg/kg). Lee et al. calculated the bioactive materials composing the water extracts of C. lanceolata and found that C. lanceolata extract contained anti-obesity components such as epigallocatechin, phloroglucinol, and gallic acid [17]. In addition, C. lanceolata root extract was successful in clinical trials for hypersensitivity immune response in Korea in 2018 (IRB no 2017-09, Semyung univesity Korean hospital). C. lanceolata root extract was given to allergic rhinitis patients for 8 weeks, and a significant decrease in B cells was confirmed compared to the control group. Researchers have reported a biological effects and active constituent analysis of C. lanceolata, but have not reported any studies establishing the optimal extraction and analysis methods for its development as a functional food source. To maximize its biological activity, extraction optimization is essential, and it is also important to determine the optimal analysis method for biomarkers. Recently, we focused on developing C. lanceolata as a medicinal and functional resource for hyperuricemia and/or gout. To date, no validated analytical approach for the standardization and quality control of C. lanceolata preparations has been reported. We established a validated method using HPLC which could identify and quantify lobetyolin from water/ethanol extracts of C. lanceolata, and investigated the XO inhibitory activity of the extracts.

2. Materials and Methods

2.1. Plant Material

Diploid C. lanceolata root was supplied by Wellphyto Co. (Gwangju, Korea) and deposited in the Mokpo National University (MNUCSS-CL-01). The air-dried, powdered C. lanceolata root (20 g) was boiled twice with hot water at 100 °C for 4 h. C. lanceolata root (20 g) was stirred with 20–100% ethanol (200 mL) at 18 °C for three days. The optimal extraction ratio was 1:5 (powder:solvent, w:v, data not shown). After removing the solvent, the pale-yellow powder was stored at −80 °C. In the form of extracts from health functional foods in Korea, water/ethanol solvents were usually used. Traditionally, plant extracts are hot water extracts or extracted using alcohol to improve their health functions or for therapeutic purposes. In addition, if other organic solvents are used as extraction solvents, there is a risk of toxicity and residue; thus, water and ethanol were used to extract the plant materials. For the development of functional materials in Korea, we performed limited solvent selection and analysis feasibility studies for specific materials.

2.2. Instrumentation and Chromatographic Conditions

All analyses were performed on a Waters Alliance 2695 HPLC system with a photodiode array detector. Operation details are described in

Table 1. Analytical conditions of HPLC for analysis of lobetyolin.

Parameters	Conditions
Column temp	Zorbax extended-C18 (C18, 4.6 mm × 150 mm, 5 µm)/25 °C
Flow rate	1 mL/min
Injection volumn	10 μL
UV detection	280 nm
Run time	30 min
Gradient	Time (min)	% A	% B
	0	10	90
	7	20	80
	8	20	80
	20	25	75
	21	100	0
	25	20	80
	30	20	80

(A) acetonitrile, (B) 0.2% acetic acid.

.

The resolution time of lobetyolin was independently detected in the extract and adjusted for the analysis conditions. For the appropriate wavelength, the wavelength showing the maximum absorption in the UV spectrum of lobetyolin was selected. Method validation was performed in detail according to ICH guidelines.

2.3. Preparation of Standards and Sample Solutions

Lobetyolin (>98%, ChemFaces, Wuhan, China) was dissolved in methanol to obtain a stock solution of 500 μg/mL. The solution was subsequently diluted 2-fold serially to 31.25 μg/mL. The crude extract was dissolved in methanol to obtain a concentration of 5–50 mg/mL.

2.4. Method Validation

For the evaluation of method validation, the following parameters were determined under the analytical conditions for lobetyolin: (1) specificity and linearity, (2) sensitivity, and (3) accuracy, precision, and recovery [20].

(1) Specificity and Linearity: The resolution of the main compounds found in the extract of C. lanceolata root was determined by analyzing the chromatograms of the standard solution and the sample solution. Linearity was analyzed using calibration curves obtained from lobetyolin standards in the concentration range of 31.25–500 μg/mL. The linear regression data were analyzed using Excel^® software.

(2) Sensitivity: The limit of detection (LOD) and quantification (LOQ) were determined from the standard curves of lobetyolin. The LOD was established using the following equation: SDR × 3/S, (SDR: standard deviation of the response, S: slope of the calibration curve). The LOQ was established from the following equation: SDR × 10/S.

(3) Accuracy, Precision, and Recovery: The accuracy and precision were evaluated by the recovery of the lobetyolin at three different concentrations (62.5, 125, and 250 μg/mL lobetyolin). The samples were injected in triplicate for HPLC analysis within one day or over three consecutive days. Intra- and inter-day accuracy was expressed as recovery (%). Intra- and inter-day precision was expressed as the RSD (relative standard deviation). Recovery was calculated based on the data repeated six times at three concentrations (62.5, 125, and 250 μg/mL). Variations were expressed as μg/mL ± standard deviation (SD) and relative standard deviation (RSD).

Statistical Analysis: The data were analyzed using Excel^® software.

3. Results and Discussion

3.1. Method Validation

3.1.1. Limit of Detection (LOD) and Limit of Quantification (LOQ)

The basic profiles of the standard and sample chromatograms are shown in Figure 1. The LOD describes the lowest amount of marker in a sample that can be detected. The LOQ describes the lowest amount of marker that can be quantified with fittable precision and accuracy. The LOD was 1.5 μg/mL for lobetyolin, and the corresponding LOQ was 4.54 μg/mL (

Table 2. HPLC data for the validation parameters.

Product	Validation Parameters
	Linearity Regression Plot	Accuracy		LOQ (μg/mL)	LOD (μg/mL)
		Mean Recovery (%)	RSD (%)
Lobetyolin	y = 7849.5X − 26,403	102.22	1.45	4.547	1.501

).

3.1.2. Linearity

The calibration curve for lobetyolin was linear over a concentration range of 31.25–500 μg/mL. The calibration curve exhibited good linear regression (r² > 0.999; Table 2).

3.1.3. Precision and Accuracy

The results of the intra/inter-day precision are shown in

Table 3. Intra-day and inter-day precision and accuracy.

Lobetyolin	Intraday (n = 3)
Concentration (μg/mL)	Detected (μg/mL, mean ± S.D)	R.S.D. (%)	Recovery (%)
62.5	61.76 ± 2.47	1.23	98.82
125	125.305 ± 1.33	1.09	100.24
250	258.539 ± 4.38	1.72	103.42
Lobetyolin	Interday (n = 3)
Concentration (μg/mL)	Detected (μg/mL, mean ± S.D)	R.S.D. (%)	Recovery (%)
62.5	63.22 ± 0.77	1.28	101.16
125	128.03 ± 1.70	1.36	102.43
250	257.69 ± 4.32	1.70	103.08

. The presented method was precise, as the RSD was below <2.5% for intra-day and inter-day precision, which were under the limit suggested by the International Conference on Harmonization (ICH) guidelines [21]. The recovery percentages were in the range of 98.8–103.4%. These results confirmed that the analytical method showed good accuracy (Table 3).

3.1.4. Repeatability

Table 4. Results of recovery test (n = 6).

Analyte	Added (μg/mL)	Recovery (μg/mL)	SD	RSD (%) a
Lobetyolin	62.5	64.87	3.36	0.63
	125	128.93	4.43	0.84
	250	264.86	4.85	0.56

^a RSD: relative standard deviation.

shows the repeatability test. The analytical method was precise; the RSD values were under 1.0%. These results confirmed that the developed method showed good repeatability.

3.2. Contents of Lobetyolin from C. lanceolata Extracts

C. lanceolata were extracted with water and ethanol to find the best extraction solvent conditions (hot water, 20, 40, 60, 80 and 100% ethanol (v/v)). The validated method was practical for analyzing the hot water and ethanolic extracts. The average contents (%wt) of lobetyolin are presented in

Table 5. Content of lobetyolin (LBT) in water/ethanolic extracts from C. lanceolata. Data is expressed as mean ± SD (n = 3).

Extract	Conc (% w/w) ± SD
Hot water	0.17 ± 0.001
20% EtOH	0.17 ± 0.001
40% EtOH	0.21 ± 0.01
60% EtOH	0.38 ± 0.03
80% EtOH	0.38 ± 0.06
100% EtOH	1.90 ± 0.01

. The 100% ethanolic extract showed the best lobetyolin content compared to those of the 0% to 80% ethanolic extracts. There has been no previous report on extraction optimization or analysis validity of lobetyolin from diploid C. lanceolata.

4. Conclusions

In the present study, C. lanceolata root extracts were prepared, and their lobetyolin contents and optimum extraction conditions were determined. The 100% ethanolic extract showed the highest lobetyolin content. The developed HPLC method was validated and applied to identify lobetyolin, which was found to be a xanthine oxidase inhibitor in C. lanceolata root extracts [11]. As far as we know, this is the first study to report a validated analytical approach for the standardization and optimization of diploid C. lanceolata extract preparations. Further investigations are warranted to confirm the biological study of diploid C. lanceolata root extract and the safety of the plant.