|Ahead of print
|Simultaneous quantitative analysis of five components in Angelica sinensis and Angelica acutiloba acclimatized growing in vietnam by high-performance liquid chromatography with photodiode array detector
Thi Minh Tam Pham1, Thi Thu Cuc Le2, Van Thanh Nguyen3, Viet Hung Tran4
1 Department of Microbiology, Institute of Drug Quality Control, Ho Chi Minh City, Vietnam
2 Department of Microbiology; Department of Instrumental Analysis, Institute of Drug Quality Control, Ho Chi Minh City, Vietnam
3 Department of Instrumental Analysis, Institute of Drug Quality Control, Ho Chi Minh City; Institute of Drug Quality Control, Ho Chi Minh City, Vietnam
4 Department of Microbiology, Institute of Drug Quality Control, Ho Chi Minh City; Faculty of Pharmacy, Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam
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|Date of Submission||16-Mar-2020|
|Date of Acceptance||17-Apr-2020|
|Date of Web Publication||18-Jul-2020|
Background: Chinese Danggui (Angelica sinensis) and Japanese Danggui (Angelica acutiloba) are evaluated as the same in using and critical of quality control in Vietnamese Pharmacopoeia. In Vietnam, Japanese Danggui were acclimatized and cultivated in several regions in recent years. Despite the huge climatic difference between Vietnam and Japan, Japanese Danggui grows very well in Vietnam. However, there are no studies to assess the overall quality of this medicinal herbs. Aims and Objectives: The aim of this study is to develop a method to identify and assay simultaneously 5 component compounds of these crude herbs: chlorogenic acid, ferulic acid, scopoletin, xanthotoxin and ligustilide by high-performance liquid chromatography with photodiode array detector (HPLC-PDA). Materials and Methods: The procedure was optimized by using column Phenomenex Gemini 5 μm PR C18, 15 x 4,6 mm with the detection wavelength set at 321 nm for detector PDA and mobile phase was composed of (A) ACN and (B) aqueous solution 1% of acid acetic using a gradient elution. Analytes were performed at 30°C with a flow rate of 1.0 mL/min. Results: The developped method were fully validated for linearity, accuracy, recovery and met the validation requirements, proved practical applicability for the quality control of these herbs and related products. Conclusion: The method was successfully applied to the quantification of five markers simultaneously from collected samples.
Keywords: Chlorogenic acid, Danggui, ferulic acid, ligustilide, scopoletin, xanthotoxin
|How to cite this URL:|
Tam Pham TM, Cuc Le TT, Nguyen VT, Tran VH. Simultaneous quantitative analysis of five components in Angelica sinensis and Angelica acutiloba acclimatized growing in vietnam by high-performance liquid chromatography with photodiode array detector. World J Tradit Chin Med [Epub ahead of print] [cited 2022 Aug 8]. Available from: https://www.wjtcm.net/preprintarticle.asp?id=290080
| Introduction|| |
Chinese Danggui or formally known as Chinese Angelica (Radix Angelica sinensis [AS]) is the root of AS (Oliv.) Diels, which has been used in China for more than 2000 years. This herb is one of the most famous medicinal plants in Traditional Chinese Medicine (TCM) and Traditional Vietnamese Medicine (TVM). The indication is for the treatment of anemia with dizziness and palpitation; menstrual disorders, amenorrhea, dysmenorrhea; constipation; rheumatic arthralgia; traumatic injuries; carbuncles, boils, and sores. There are four composite formulae of TVM in Vietnam, more than 90 composite formulae of TCM in China, and hundreds of functional food products that are sold worldwide contain AS. This widespread use indicates the importance of AS in traditional medicine. There are three species of Danggui which has been stipulated differently in several Asian countries: the roots of AS (Oliv.) Diels. in the Chinese Pharmacopoeia (ChP), the roots of Angelica gigas Nakai (Cham-Danggui, AG) in the Korean Pharmacopoeia (KP), the roots of Angelica acutiloba Kitagawa (Japanese Danggui, AA) and Angelica acutiloba Kitagawa var. sugiyamae Hikino (Japanese Danggui, AA sugiyamae) in the Japanese Pharmacopoeia (JP), and the roots of AA Kitagawa and AS(Oliv.) Diels in the Pharmacopoeia Vietnamica (PV).
In Vietnam, Japanese Danggui was acclimatized and cultivated in several regions in recent years. Despite the huge climatic difference between Vietnam and Japan, Japanese Danggui grows very well in Vietnam, and the herb became an alternative choice of local herbalists while food supplement manufacturers also became an exportable item. Jeong et al. admitted that Danggui has been distributed with the same name because of similarities in shapes, although it has different origin species and scientific names. Therefore, it is needed to contribute to the determination of origin species using marker compounds for a suitable use of Danggui. Referring to concerned pharmacopoeia as KP, ChP, JP, and VP, we found that KP described the quantitative assay of nodakenin, decursin, and decursin angelate, while ChP described the quantitative assay of ferulic acid (FA), volatile oil, and extractable content in powdered Chinese Angelica by dilute ethanol (70%) soluble extract method. Other pharmacopoeia have simplified method of analysis of extractable content in powdered Japanese Angelica root by dilute ethanol soluble extract method or extractable content in powdered Chinese Angelica and in powdered Japanese Angelica acclimatized root by dilute ethanol (50%) soluble extract method. Apart from macroscopic and microscopic authentication, DNA sequencing and chemical fingerprint are new and useful tools for the identification of herbal materials. For fingerprint method, chlorogenic acid (CGA), FA, ligustilide, scopoletin, and xanthotoxin appear as favorite markers of many authentic studies in recent years.
The aim of this study is “simultaneous quantitative analysis of five components in AS, AA, and AA acclimatized growing in Vietnam by HPLC/PDA.” Through this work, we expected to establish a simple method of analysis using common chemical markers in Danggui to assess the Radix AA acclimatized growing in Vietnam and its processed products.
| Experimental|| |
Twelve samples corresponding to four AS samples AS1–AS4 purchased from China; three AA samples from AA1 to AA3 purchased in Japan; five AA acclimatized samples from AAV1 to AAV5 grown (harvested) and collected in Vietnam are encoded as shown in [Table 1]. All samples were identified using genetic sequencing methods to determine correctness. The detail taxonomic results based on DNA sequencing are shown in [Table 1].
Powdered Danggui was sieved through a 45-mesh sieve. Accurate moisture content is determined via the gravimetric method.
About 1.0 g of the powder was accurately weighed, 10 mL of methanol was then added, and the sample was sonicated for 60 min at temperature 50°C. The solution was filtered through a 0.45-μm membrane filter, and the filtrate was collected in a conical flask. Repeat three times with fresh methanol. Combine the filtrates and concentrate to 10 mL under a flow of nitrogen gas.
Chemicals and reagents
Methanol (MeOH) and acetonitrile (ACN), for liquid chromatography (high-performance liquid chromatography [HPLC] grade), were purchased from JT Baker; glacial acetic acid (CH3 COOH) and ammonia (NH3) are of pure analytic grade from Merck. Other working standard substances have their purities above 95% including: FA and CGA are from USP; ligustilide (LIG) is from ChromaDex; xanthotoxin (XAN) is from PhytoLab; and scopoletin (SCO) is from Sigma.
Ultra-Fast Liquid Chromatography - PhotoDiode Array detector system (UFLC-PDA) from Shimazdu-Japan; Gemini® 5μm C18 110Š, 150 x 4.6 mm analytical column. Chromatographic conditions: 30°C column temperature, 1 mL/ min flow rate. Injection volume 10 μL, detector PDA set at single wave 321 nm. Mobile phase A: Acetonitril, B: 1% acetic acid. Chromatographic program is shown in [Table 3].
Analytical method validation
The working standards were accurately weighed and then dissolved with methanol to produce stock standard solutions series as shown in [Table 2].
The linear regression equation has the form y = ax + b, where y is the peak area and x is the concentration of substances in the sample. The detection limit (LOD) and the quantitative limit (LOQ) are calculated according to the ICH guidelines. The recovery rate was determined by adding the standard compound to 1 g of powdered Danggui sample at levels of 25%, 50%, and 100% of the active ingredient content in the sample and repeat ultrasonic extraction with 10 mL MeOH for 1 h at each additional level. Repeat 6 times/day for 2 days consecutive. Evaluate the results by comparing the relative standard deviation (RSD) during the day and from the other day and using the t-test (comparing two quantitative average contents between two days).
| Results and Discussion|| |
Optimization of sample preparation conditions
Three extracting solvents methanol, methanol acidified with 1% acetic acid, and methanol alkalized with 0.25% ammonium hydroxide were compared with regard to the content of compounds after ultrasonic water bath for 60 min at 50°C. The extraction efficiency comparison was carried out by one-way analysis of variance (ANOVA), and we selected methanol as the extracting solvent throughout this work.
Optimization of chromatographic conditions
Referring to previous studies, we started with the mobile phase consisting of acetonitrile (A) and acetic acid in water (B) using a gradient program. Three different concentrations of acetic acid 10%, 1%, and 0.1% have been tested, and different ratios of mobile phases have been adjusted. The good separation conditions were selected according to the requirements for obtaining the chromatograms, with a better resolution of the adjacent peaks within a short analysis time. The best condition is shown in [Table 3].
Validation of the method
Each correlation coefficient (r2) was >0.99, as determined by the least square analysis, suggesting a good linearity between the peak area ratio and the compound concentrations. Regression equations, linear intervals, LOD, and LOQ are presented in [Table 4].
|Table 4: Calibration graphs, linear ranges, limit of detection, and limit of quantitation|
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The extraction recovery test was performed by extracting a known amount of the five marker compounds from the Danggui powder samples as previously described by Jeong et al.
A known amount of each compound at three different levels low (25%), medium (50%), and high (100%) was mixed with the sample's powder and was extracted with methanol, as described in “Sample Preparation.” Each level was assessed by three times and the average values are shown in [Table 5].
|Table 5: Average recovery value of marker compounds through standard addition|
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The overall recovery rates of total 45 tests were ranging from 90-115%, of which CGA 91.45%–112.44%; SCO 100.95%–112.44%; FA 95.39%–114.99%; XAN 93.43%–107.73%, and LIG 90.69%–106.38% and every RSD lower than 10%.
Interday accuracy was evaluated from the variability of multiple analysis (n = 6) of quality control samples analyzed on a single analytical run for 2 days. All RSDs were lower than 5%; the highest score is SCO at 3.57% [Table 6].
From the results of recovery and accuracy tests, we concluded that this method manifested good precision and accuracy and suitable for use in this study.
The developed HPLC/DAD method was then applied to the simultaneous determination of the five marker compounds in two species of Danggui collected from Japan, China, and Vietnam.
We note that the pattern recognition is quite different between AS and AA as shown in the chromatogram [Figure 1].
|Figure 1: High-performance liquid chromatograms of (a) standard mixture, (b) Angelica acutiloba, and (c) Angelica sinensis with the corresponding peaks: (1) chlorogenic acid, (2) scopoletin, (3) ferulic acid (4) xanthotoxin, (5) ligustilide|
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The main difference between AS and AA fingerprint is that all three AS samples do not contain SCO and XAN (not detectable – ND).
The results are shown in [Table 7].
We noted that the concentration of FA and LIG in AS samples is much higher (about 10 times) than that of AA samples.
| Conclusion|| |
This was the first report on the simultaneous determination of the major compounds in the two species of Danggui Angelica sinensis and AA and its geographic distinctiveness, the AA acclimatized growing in Vietnam, using HPLC. Validation results showed that the method is suitable for simultaneously quantitative analysis of five mentioned marker compounds. In accordance with previous studies,,, we redetermined that xanthotoxin is only present in Japanese Danggui and is not detectable in Chinese Danggui. This study is an optimized chromatographic method with ultraviolet detection which has established a fingerprint and was designed for the quality control of a new herbal material source, the Radix AA acclimatized growing in Vietnam.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Thi Minh Tam Pham,
Institute of Drug Quality Control, 200 Co Bac Street, District 1, Ho Chi Minh City
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]
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