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A simple and simultaneous identification method for aloe, catechu and gambir by high performance liquid chromatography
Accepted Manuscript Title: A simple and simultaneous identification method for aloe, catechu and gambir by high performance liquid chromatography Author: Yan Zhao Young Ho Kim Wonjae Lee Young Keun Lee Kyung Tae Kim Jong Seong Kang PII: DOI: Reference:
S0731-7085(15)30125-4 http://dx.doi.org/doi:10.1016/j.jpba.2015.08.027 PBA 10227
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
11-5-2015 17-8-2015 19-8-2015
Please cite this article as: Yan Zhao, Young Ho Kim, Wonjae Lee, Young Keun Lee, Kyung Tae Kim, Jong Seong Kang, A simple and simultaneous identification method for aloe, catechu and gambir by high performance liquid chromatography, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.08.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A simple and simultaneous identification method for aloe, catechu and gambir by high performance liquid chromatography Yan Zhaoa, Kyung Tae Kima, Young Ho Kima, Wonjae Leeb, Young Keun Leec, Jong Seong Kanga,* [email protected] a
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
b
College of Pharmacy, Chosun National University, Gwangju, 501-759, Korea
c
Daejeon Health Sciences College, Daejeon, 300-711, Korea
*
Corresponding author. Tel./fax: +82-42-821-5928/+82-42-823-6566.
Graphical Abstract
2.50 OH
2.00
OH
A U
Aloe
O
1.50 OH
1.00
OH
H O
0.50
OH HO
OH
0.00 5.00
10.00
15.00
20.00 Minutes
25.00
30.00
35.00
0.80
A U
0.60
Catechu
0.40
0.20
0.00 10.00
15.00
20.00 Minutes
25.00
30.00
35.00
5.00
10.00
15.00
20.00 Minutes
25.00
30.00
35.00
1.00 A U
Gambir
5.00
0.50
0.00
Highlights · This analysis revealed that aloe, catechu and gambir are mislabeled in the market. · A simultaneous analytical method was developed to discriminate three materials. · We only use (+)-catechin, (-)-epicatechin and aloin as marker compounds. · New content criteria are made by ratio of (+)-catechin and (-)-epicatechin content. · New content criteria discriminate clearly catechu and gambir.
Abstract An effective and rapid method was developed for the simultaneous identification of aloe, catechu and gambir by high performance liquid chromatography-diode array detector (HPLCDAD). Identification of three maker compounds presented in three medicinal materials was performed on high performance liquid chromatography-mass spectrometry (HPLC-MS). Under the optimal HPLC chromatographic conditions, sixty-two samples were processed on an Optimapak C18 column using a solvent system of acetonitrile (from 10% to 35%) and 0.1% phosphoric acid solution (from 90% to 65%) at a total flow rate of 1.0 mL/min and detected at 270 nm. All calibration curves exhibited good linear relationship (r2 > 0.9992). The relative standard deviation values of intra-day and inter-day precision were less than 1% and 2%, respectively. The recoveries of three analytes ranged from 99.48 to 100.97% with low RSDs (< 2%). For the first time, this study demonstrates that the processed aloe, catechu and gambir are sold in local material markets in China and Korea without their correct identification. It indicates the existent of high potential medicinal risk by misuse of three medicinal materials. The developed HPLC method can be applied to prevent unexpected biological activity due to misapplication of medicinal materials. Keywords: Aloe; Catechu; Gambir; Identification; Quantification.
1. Introduction In ancient Asian countries, herbal medicines that are difficult to store for long periods are traditionally dried, thus extending their storage period and making them easier to transport. Processed herbal drugs have several advantages compared to raw materials, including less oxidation, greater stability and increased clinical efficacy. Among the processed herbal medicinal materials, aloe, catechu and gambir are widely used in Korea and China [1]. Aloe is the dried, concentrated substance obtained from the juice of the leaf of Aloe barbadensis Miller [2]. A. barbadensis (family Asphodelaceae) is a very short-stemmed succulent plant and widely used in the diet, cosmetic, and pharmaceutical industries [3]. The rind of the A. barbadensis leaves secretes a yellow exudate from cells abundant in aloin [4]. Aloin extracted from natural sources is a mixture of two diastereomers, termed aloin A and B, which have similar bioactivities [5]. Also, as an important secondary metabolite, aloin has been used as a maker for the quality control of A. barbadensis in the pharmacopeias of several countries, due not only to its extremely high content but also its various pharmacological activities [6-7]. Catechu is the dried water extract of the peeled branches and stems of Acacia catechu Willd [2]. A. catechu, a species of Fabaceae (Leguminosae), is a deciduous thorny tree found in China, India and countries around the Indian Ocean. Catechu has been used for a variety of applications including as a food additive, astringent, tanning agent and dye. It is also a traditional medicinal material due to its anthelminthic and antipruritic properties, and is also used to improve digestion and treat skin disorders [8]. Gambir is the dried aqueous extract of the leaves and young twigs of Uncaria gambir Roxburgh. U. gambir belongs to the family Rubiaceae, found in India, Sri Lanka, Indonesia and Malaysia. Gambir is a traditional medicinal material used as an astringent and tonic [9-10]. A. catechu and U. gambir contain (+)-catechin and (-)-epicatechin as their main secondary metabolites [8-10]. (+)-Catechin and (-)-epicatechin are bioactive constituents [8] generally present as
ubiquitous constituents of vascular plants such as A. catechu and U. gambir [12]. Though the shapes of the original plants are different, they are often misidentified because they are not easily distinguished when processed as shown in Fig. 1. The chemical structures of (+)catechin, (-)-epicatechin and aloin were shown in Fig. 2. However, the misuse of these three medicinal materials would cause unexpected results as aloe is usually used as a stimulant laxative for treating constipation and bowel movements, while catechu and gambir are used in traditional medicine for sore throats and diarrhea [8]. In addition, the content of (+)catechin and (-)-epicatechin in catechu and gambir is variable [8-10]. Therefore the identification of aloe, catechu and gambir is necessary to avoid misapplication and to achieve the correct bioactivity. Until now, there has not been an effective, single analytical method to identify and differentiate aloe, catechu and gambir. This study aims to develop a simultaneous HPLC method to identify these medicinal plants using a single analytical method.
2. Materials and methods 2.1. Chemicals and reagents HPLC-grade methanol and acetonitrile were purchased from Burdick & Jackson (NJ, USA). Phosphoric acid and formic acid were analytical grade (Sigma-Aldrich, MO, USA). Water was purified using the Milli-Q system (Sinhan, Korea). For standards, (+)-catechin was isolated from catechu (Sample 13K0052) and identified by Professor Young Ho Kim at the College of Pharmacy in Chungnam National University. The purity of (+)-catechin was higher than 98%. (-)-Epicatechin and aloin were analytical grade, purchased from SigmaAldrich (MO, USA).
2.2. Preparation of sample and standard solutions
Thirty-seven commercial aloe samples, 21 commercial catechu samples and four commercial gambir samples were obtained from China and Korea. Powdered samples (aloe sample 14K0020, catechu sample 13K0052 and gambir sample 14I0051, 0.3 g each) were extracted in 30 mL methanol by sonication for 60 min at room temperature. Standards were prepared by mixing (+)-catechin, (-)-epicatechin and aloin in methanol to a concentration of 0.500 mg/mL, and diluted twice to prepare solutions of 0.250 mg/mL and 0.125 mg/mL. These solutions were stored at 4 °C. All solutions were filtered through a 0.2 μm syringe filter before analysis.
2.3. HPLC analyses HPLC analyses were performed using a LC-10AD series HPLC system (Shimadzu, Kyoto, Japan) with a column oven and a diode array detector (DAD). An Optimapak C18 column (250 x 4.6 mm, 5 μm, RStech, Seoul, Korea) was used at 30 °C for separation. The mobile phase consisted of 0.1% phosphoric acid solution (A) and 100% acetonitrile (B). Samples were eluted at a flow rate of 1 mL/min.
2.4. LC-MS analyses LC–MS analyses were performed using a ProminenceTM UFLC system (Shimadzu, Kyoto, Japan) linked to a LCMS–2020 system (Shimadzu, Kyoto, Japan) in positive mode. LCsolution software was used to control the instruments for data acquisition and processing. The HPLC analysis was performed using an Optimapak C18 column (150 x 2.1 mm, 5 μm, Waters, MA, USA). The mobile phase was 0.1% formic acid solution (A) and 100% acetonitrile (B) from 25% B to 70% B over an 80 min period. Samples were eluted at a flow rate of 0.3 mL/min and a column temperature of 30 °C. The LC–MS was operated with a nebulizing gas flow rate of 10 L/min, CDL temperature of 250 °C, heat block temperature of
200 °C, detector voltage of 1.50 kV and a CDL voltage of 15.0 V.
2.5. Method validation Linearity was examined with standard solutions using five different concentrations by plotting the integrated peak area for each component against its corresponding solution concentration. Precision was expressed as intra-day and inter-day relative standard deviations. Intra-day variability was evaluated by analyzing standards five times in a single day and inter-day variability was verified for five consecutive days. Repeatability, expressed as RSD, was calculated based on the retention and migration times for six repetitive injections. The stability of the sample solutions was tested at 0, 2, 4, 8, 12 and 24 h after preparation and expressed by RSD. Recovery was determined by adding three different concentration levels (120%，100% and 80%) of standard solution into samples in which the content of the three analytes were known. Samples were analyzed in triplicate at each level. The recoveries were calculated using the following equation: recovery (%) = (amount found − amount original)/amount spiked × 100%.
3. Results and Discussion 3.1. Optimization of HPLC-DAD conditions To establish an optimal chromatographic condition, various mobile phase compositions (methanol–water or acetonitrile–water), types of acid (phosphoric acid, formic acid and acetic acid) and detection wavelengths were employed. Retention times of marker compounds were shorter using acetonitrile–water compared to methanol-water, so acetonitrile–water was chosen as the mobile phase. The addition of 0.1% of each acid in the mobile phase decreased peak tailing significantly, and phosphoric acid yielded the best results. By verifying DAD spectra with HPLC chromatograms, the optimal detection wavelength of the three compounds
was determined to be 270 nm. HPLC chromatograms of a standard mixture and the three medicinal materials using optimized HPLC conditions are shown in Fig. 3.
3.2. LC-MS data of marker compounds in the three medicinal materials (+)-Catechin, (-)-epicatechin and aloin were detected in the positive mode (Table 1). The three peaks in the chromatogram predicted to represent the marker compounds were verified by interpretation of retention time, UV absorbance band, source materials, molecular formulae and characteristic MS data acquired from the LC-MS. All characteristics of each peak matched the standard completely. Therefore, the three peaks were confirmed as (+)catechin, (-)-epicatechin and aloin.
3.3. Method validation Linearity was verified using five standard concentrations of each compound. Calibration curves for the three marker compounds were linear (r2 > 0.9992) in the range of 25 to 400 μg/mL (Table 2). LODs and LOQs were 1 and 4 μg/mL for (+)-catechin, 0.5 and 1.4 μg/mL for (-)-epicatechin, and 0.5 and 1 μg/mL for aloin, respectively (Table 2). Table 3 shows the precision of the three marker compounds at three concentrations. The intra-day and inter-day precisions of the three marker compounds were less than 2%. As shown in Table 4, the developed method showed good repeatability base on analyte RSD values (<1.81%). Sample solutions were also stable over 24 h as indicated by a low RSD value (<1.25%). The recovery of the three compounds was 99.48 to 100.97%, indicating that the three marker compounds could be extracted completely from herb material without destruction or denaturation. RSD of recovery was less than 1.70%, demonstrating that the method was precise (Table 5). All validation results of the developed HPLC method in this study were satisfactory and met method validation guidelines in Korea and China [3, 9].
3.4. Chromatographical differentiation among aloe, catechu and gambir Differentiation of aloe from the other two medicinal materials using the chromatogram is straightforward; the existence of aloin indicates that the material is aloe. In contrast, differentiation of catechu and gambir is less simple, because both materials contain (+)catechin and (-)-epicatechin. Generally, gambir has a higher content of the marker compounds than catechu. However, higher (+)-catechin and (-)-epicatechin is not sufficient to differentiate gambir from catechu. Another potential criterion for the differentiation of catechu and gambir is the ratio of (+)-catechin and (-)-epicatechin, as the (-)-epicatechin content of gambir is lower than that of catechu. The ratios calculated for all gambir samples were higher than 9.68 and those for all catechu samples were lower than 4.65. The ratio of (+)-catechin and (-)-epicatechin content thus could be a good criterion for the discrimination of catechu and gambir (Fig. 4).
3.5. Hierarchical clustering analysis To evaluate the method of differentiating the three materials, unsupervised clustering analysis was performed using Ward’s method to visualize differences and/or similarities among samples through linkage distances [14]. Using hierarchical cluster analysis, the collected 59 samples in Korea and China were grouped into cluster I (aloe) and cluster II (others), and cluster II was divided into cluster III (gambir) and cluster IV (catechu) (Fig. 5). This indicated that our results were well adapted to the hierarchical cluster analysis model, allowing the three materials to be discriminated during the same analysis (Fig. 5). The results of hierarchical clustering analysis correspond to those of chromatographical differentiation.
4. Conclusion
This is the first report of a simultaneous analytical method to discriminate aloe, catechu and gambir by HPLC-DAD and LC-MS using (+)-catechin, (-)-epicatechin and aloin as marker compounds. The HPLC-DAD method was validated by linearity, precision, repeatability, stability and recovery. This analysis revealed that aloe, catechu and gambir are mislabeled in the marketplace and that the developed HPLC-DAD method can clearly distinguish aloe, catechu and gambir. This new method will prevent the misuse of the three medicinal materials by misidentification. Furthermore, the newly suggested ratio calculating method will be useful to easily distinguish catechu from gambir.
References [1] Q. F. Gong, Chinese Medicinal Herbs Preparation; China Press of Traditional Chinese Medicine, Beijing, 2007, pp. 25. [2] K. Eshun, Q. He, Aloe Vera: A Valuable Ingredient for the Food, Pharmaceutical and Cosmetic Industries—A Review, J. Crit. Rev. Food. Sci. Nut. 44 (2004) 91-96. [3] The State Commission of Pharmacopoeia, Pharmacopoeia of People’s Republic of China, The Medicine Science and Technology Press of China, Beijing, 2010, Part I, pp. 151152; Part I, pp. 9; Appendix, pp. 29. [4] J. H. Park, S.W. Kwon, New Perspectives on Aloe, Springer, New York, 2006, pp. 19-34. [5] X. L. Cao, D. F. Huang, Y. M. Dong, H. Zhao, Y. Ito, Separation of aloins A and B from Aloe vera exudates by high speed countercurrent chromatography, J. Liq. Chromatogr. Relat. Technol. 30 (2007), 1657-1668. [6] X. F. Wu, W. J. Ding, J. S. Zhong, J. Z. Wan, Z. Y. Xie, Simultaneous qualitative and quantitative determination of phenolic compounds in Aloe barbadensis Mill by liquid chromatography–mass spectrometry-ion trap-time-of-flight and high performance liquid chromatography-diode array detector, J. Pharm. Biomed. Anal. 80 (2013), 94106. [7] X. F. Wu, S. Yin, J. S. Zhong, W. J. Ding, J. Z. Wan, Z. Y. Xie, Mushroom tyrosinase inhibitors from Aloe barbadensis Miller, J. Fitoterapia 83 (2012), 1706-1711. [8] Y. G. Kang, Identificology of Chinese Materia Medica; China Press of Traditional Chinese Medicine, Beijing, 2007, pp. 452-453. [9] Ministry of Food and Drug Safety, Korean Pharmacopoeia，2012, Part Ⅱ, pp. 13001301. [10] Ministry of Health, Labour and Welfare, Japanese Pharmacopoeia，2011, Part I, pp.
1641. [11] K.S. Tong, M. Jain. Kassim, A. Azraa, Adsorption of copper ions from its aqueous solution by a novel biosorbent Uncaria gambir: Equilibrium, Kinetics and Thermodynamic studies, Chem. Eng. J. 170 (2011), 145-153. [12] Napolitano, J. G.; Gödecke, T.; Lankin, D. C.; Jaki, B. U.; McAlpine, J. B.; Chen, S. N.; Pauli, G. F. Orthogonal analytical methods for botanical standardization: Determination of green tea catechins by qNMR and LC-MS/MS, J. Pharm. Biomed. Anal. 93 (2014), 59-67. [13] Taniguchi, S.; Kuroda, K.; Doi, K.; Tanabe, M.; Shibata, T.; Yoshida, T.; Hatano, T.; Revised structures of gambiriins A1, A2, B1, and B2, chalcane-flavan dimers from gambir (Uncaria gambir extract), J. Chem. Pharm. Bull. 55 (2007), 268-272. [14] R. M. Cormack, A review of classification, J. Roy. Stat. Soc. A. 134 (1971), 321-367.
Figure Captions Fig. 1. The appearances of aloe, catechu and gambir. Fig. 2. Chemical structures of (+)-catechin, (-)-epicatechin and aloin. Fig. 3. HPLC chromatogram of (a) standard mixture, (b) catechu, (c) gambir and (d) aloe; 1: (+)-catechin, 2: (-)-epicatechin, 3: aloin. Fig. 4. (a): Total content of (+)-catechin, (-)-epicatechin and (b): the ratio of (+)-catechin / (-)epicatechin in catechu (white) and gambir (black) samples. Fig. 5. Dendrogram of hierarchical cluster analysis.
Aloe
Catechu Fig. 1.
Gambir
(+)-Catechin
(-)-Epicatechin
Fig. 2.
Aloin
(a) A U
2
1
0.15
3
0.10
0.05
0.00 5.00
(b)
10.00
20.00 Minutes
25.00
30.00
35.00
15.00
20.00 Minutes
25.00
30.00
35.00
15.00
20.00 Minutes
25.00
30.00
35.00
30.00
35.00
1
0.80
2
0.60 A U
15.00
0.40
0.20
0.00 5.00
(c)
10.00
1 A U
1.00
0.50
2
0.00 5.00
10.00
2.50
(d)
3
2.00
A U
1.50
1.00
0.50 0.00 5.00
10.00
15.00
20.00 Minutes
Fig. 3.
25.00
Fig. 4.
Fig. 5.
Tables Table 1. Characterization of compounds in aloe, catechu and gambir by LC-MS. RT (min)
MS
Molecular formula
UV λ (nm)
Identified compound
Material
21.96
291.5 [M+H]+
C15H14O6
212.5, 278.6
(+)-Catechin
Catechu, Gambir
25.13
291.5 [M+H]+
C15H14O6
212.5, 278.6
(-)-Epicatechin
Catechu, Gambir
C21H22O9
193.7, 269.1, 296.4, 354.8
Aloin
Aloe
50.10
+
419.4 [M+H] 441.4 [M+Na]+
Table 2. Calibration curve, LOD and LOQ of marker compounds (n = 5). Analyte
Regression equation Y(area) X(conc. μg/mL)
r2
Linear range (μg/mL)
LOD (μg/mL)
LOQ (μg/mL)
(+)-Catechin
Y = 5847.3X + 4478
0.9994
25-400
1.0
4.0
(-)-Epicatechin
Y = 7107.2X + 6838
0.9994
25-400
0.5
1.4
Aloin
Y = 4222.3X + 4358
0.9992
25-400
0.5
1.0
Table 3. Inter-day and Intra-day precision of marker compounds (n = 6). Intra-day Analyte
(+)-Catechin
(-)-Epicatechin
Aloin a
Inter-day
Conc. (μg/mL)
Detecteda (μg/mL)
Detecteda (μg/mL)
RSD (%)
62.5
62.5±0.34
0.54
62.5
62.6±0.43
0.68
125.0
125.0±0.72
0.58
125.0
125.1±0.49
0.39
250.0
250.0±0.99
0.40
250.0
250.1±1.14
0.46
62.5
62.5±0.34
0.40
62.5
62.6±0.70
1.12
125.0
125.0±0.55
0.44
125.0
125.0±1.03
0.83
250.0
250.0±1.64
0.66
250.0
250.1±0.39
0.16
62.5
62.5±0.26
0.42
62.5
62.8±1.05
1.68
125.0
125.0±0.85
0.68
125.0
126.1±0.99
0.79
250.0
250.0±1.61
0.42
250.0
249.2±4.19
1.68
Data are represented as mean±SD.
RSD (%)
Conc. (μg/mL)
Table 4. Repeatability and stability of marker compounds (n = 6). Repeatability Analyte
(+)-Catechin
(-)-Epicatechin Aloin a
Stability
Material
RT (min)a
Content (mg/g)a
RSD (%)
Catechu
9.61±0.03
145.2±1.67
1.15
Gambir
9.55±0.02
252.3±2.43
Catechu
12.13±0.02
Gambir Aloes
Data are represented as mean±SD.
RT (min)a
Content (mg/g)a
RSD (%)
9.61±0.04
145.5±1.12
0.77
0.96
9.59±0.04
251.6±0.83
0.33
72.8±1.10
1.51
12.13±0.03
73.2±0.68
0.93
12.10±0.02
22.7±0.41
1.81
12.14±0.04
21.7±0.27
1.25
24.13±0.01
447.1±1.31
0.29
24.10±0.02
446.0±1.76
0.39
Table 5. Recovery data of the developed method (n = 3). Analyte
Material
Concentration Original (μg/ml)
Catechu
64.5
(+)-Catechin Gambir
Catechu
33.5
22.4
(-)-Epicatechin Gambir
Aloin a
Aloe
Data are represented as mean±SD.
18.0
77.4
Recovery (%)
RSD (%)
155.0±0.17
100.22
0.12
64.5
129.1±0.08
100.07
0.06
38.7
103.0±0.70
99.48
0.68
Spiked (μg/ml)
Found (μg/ml)a
90.3
46.9
80.4±0.21
100.00
0.26
33.5
67.2±0.29
100.52
0.43
20.1
53.6±0.91
100.00
1.70
31.4
53.8±0.05
100.00
0.10
22.4
44.7±0.08
99.75
0.17
13.4
35.8±0.40
100.00
1.12
25.2
43.2±0.10
100.00
0.22
18.0
36.2±0.13
100.97
0.37
10.8
28.8±0.49
100.00
1.70
108.4
185.8±0.69
100.00
0.37
77.4
154.8±0.79
100.02
0.51
46.4
123.8±2.10
100.00
1.69
S0731-7085(15)30125-4 http://dx.doi.org/doi:10.1016/j.jpba.2015.08.027 PBA 10227
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
11-5-2015 17-8-2015 19-8-2015
Please cite this article as: Yan Zhao, Young Ho Kim, Wonjae Lee, Young Keun Lee, Kyung Tae Kim, Jong Seong Kang, A simple and simultaneous identification method for aloe, catechu and gambir by high performance liquid chromatography, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.08.027 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A simple and simultaneous identification method for aloe, catechu and gambir by high performance liquid chromatography Yan Zhaoa, Kyung Tae Kima, Young Ho Kima, Wonjae Leeb, Young Keun Leec, Jong Seong Kanga,* [email protected] a
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
b
College of Pharmacy, Chosun National University, Gwangju, 501-759, Korea
c
Daejeon Health Sciences College, Daejeon, 300-711, Korea
*
Corresponding author. Tel./fax: +82-42-821-5928/+82-42-823-6566.
Graphical Abstract
2.50 OH
2.00
OH
A U
Aloe
O
1.50 OH
1.00
OH
H O
0.50
OH HO
OH
0.00 5.00
10.00
15.00
20.00 Minutes
25.00
30.00
35.00
0.80
A U
0.60
Catechu
0.40
0.20
0.00 10.00
15.00
20.00 Minutes
25.00
30.00
35.00
5.00
10.00
15.00
20.00 Minutes
25.00
30.00
35.00
1.00 A U
Gambir
5.00
0.50
0.00
Highlights · This analysis revealed that aloe, catechu and gambir are mislabeled in the market. · A simultaneous analytical method was developed to discriminate three materials. · We only use (+)-catechin, (-)-epicatechin and aloin as marker compounds. · New content criteria are made by ratio of (+)-catechin and (-)-epicatechin content. · New content criteria discriminate clearly catechu and gambir.
Abstract An effective and rapid method was developed for the simultaneous identification of aloe, catechu and gambir by high performance liquid chromatography-diode array detector (HPLCDAD). Identification of three maker compounds presented in three medicinal materials was performed on high performance liquid chromatography-mass spectrometry (HPLC-MS). Under the optimal HPLC chromatographic conditions, sixty-two samples were processed on an Optimapak C18 column using a solvent system of acetonitrile (from 10% to 35%) and 0.1% phosphoric acid solution (from 90% to 65%) at a total flow rate of 1.0 mL/min and detected at 270 nm. All calibration curves exhibited good linear relationship (r2 > 0.9992). The relative standard deviation values of intra-day and inter-day precision were less than 1% and 2%, respectively. The recoveries of three analytes ranged from 99.48 to 100.97% with low RSDs (< 2%). For the first time, this study demonstrates that the processed aloe, catechu and gambir are sold in local material markets in China and Korea without their correct identification. It indicates the existent of high potential medicinal risk by misuse of three medicinal materials. The developed HPLC method can be applied to prevent unexpected biological activity due to misapplication of medicinal materials. Keywords: Aloe; Catechu; Gambir; Identification; Quantification.
1. Introduction In ancient Asian countries, herbal medicines that are difficult to store for long periods are traditionally dried, thus extending their storage period and making them easier to transport. Processed herbal drugs have several advantages compared to raw materials, including less oxidation, greater stability and increased clinical efficacy. Among the processed herbal medicinal materials, aloe, catechu and gambir are widely used in Korea and China [1]. Aloe is the dried, concentrated substance obtained from the juice of the leaf of Aloe barbadensis Miller [2]. A. barbadensis (family Asphodelaceae) is a very short-stemmed succulent plant and widely used in the diet, cosmetic, and pharmaceutical industries [3]. The rind of the A. barbadensis leaves secretes a yellow exudate from cells abundant in aloin [4]. Aloin extracted from natural sources is a mixture of two diastereomers, termed aloin A and B, which have similar bioactivities [5]. Also, as an important secondary metabolite, aloin has been used as a maker for the quality control of A. barbadensis in the pharmacopeias of several countries, due not only to its extremely high content but also its various pharmacological activities [6-7]. Catechu is the dried water extract of the peeled branches and stems of Acacia catechu Willd [2]. A. catechu, a species of Fabaceae (Leguminosae), is a deciduous thorny tree found in China, India and countries around the Indian Ocean. Catechu has been used for a variety of applications including as a food additive, astringent, tanning agent and dye. It is also a traditional medicinal material due to its anthelminthic and antipruritic properties, and is also used to improve digestion and treat skin disorders [8]. Gambir is the dried aqueous extract of the leaves and young twigs of Uncaria gambir Roxburgh. U. gambir belongs to the family Rubiaceae, found in India, Sri Lanka, Indonesia and Malaysia. Gambir is a traditional medicinal material used as an astringent and tonic [9-10]. A. catechu and U. gambir contain (+)-catechin and (-)-epicatechin as their main secondary metabolites [8-10]. (+)-Catechin and (-)-epicatechin are bioactive constituents [8] generally present as
ubiquitous constituents of vascular plants such as A. catechu and U. gambir [12]. Though the shapes of the original plants are different, they are often misidentified because they are not easily distinguished when processed as shown in Fig. 1. The chemical structures of (+)catechin, (-)-epicatechin and aloin were shown in Fig. 2. However, the misuse of these three medicinal materials would cause unexpected results as aloe is usually used as a stimulant laxative for treating constipation and bowel movements, while catechu and gambir are used in traditional medicine for sore throats and diarrhea [8]. In addition, the content of (+)catechin and (-)-epicatechin in catechu and gambir is variable [8-10]. Therefore the identification of aloe, catechu and gambir is necessary to avoid misapplication and to achieve the correct bioactivity. Until now, there has not been an effective, single analytical method to identify and differentiate aloe, catechu and gambir. This study aims to develop a simultaneous HPLC method to identify these medicinal plants using a single analytical method.
2. Materials and methods 2.1. Chemicals and reagents HPLC-grade methanol and acetonitrile were purchased from Burdick & Jackson (NJ, USA). Phosphoric acid and formic acid were analytical grade (Sigma-Aldrich, MO, USA). Water was purified using the Milli-Q system (Sinhan, Korea). For standards, (+)-catechin was isolated from catechu (Sample 13K0052) and identified by Professor Young Ho Kim at the College of Pharmacy in Chungnam National University. The purity of (+)-catechin was higher than 98%. (-)-Epicatechin and aloin were analytical grade, purchased from SigmaAldrich (MO, USA).
2.2. Preparation of sample and standard solutions
Thirty-seven commercial aloe samples, 21 commercial catechu samples and four commercial gambir samples were obtained from China and Korea. Powdered samples (aloe sample 14K0020, catechu sample 13K0052 and gambir sample 14I0051, 0.3 g each) were extracted in 30 mL methanol by sonication for 60 min at room temperature. Standards were prepared by mixing (+)-catechin, (-)-epicatechin and aloin in methanol to a concentration of 0.500 mg/mL, and diluted twice to prepare solutions of 0.250 mg/mL and 0.125 mg/mL. These solutions were stored at 4 °C. All solutions were filtered through a 0.2 μm syringe filter before analysis.
2.3. HPLC analyses HPLC analyses were performed using a LC-10AD series HPLC system (Shimadzu, Kyoto, Japan) with a column oven and a diode array detector (DAD). An Optimapak C18 column (250 x 4.6 mm, 5 μm, RStech, Seoul, Korea) was used at 30 °C for separation. The mobile phase consisted of 0.1% phosphoric acid solution (A) and 100% acetonitrile (B). Samples were eluted at a flow rate of 1 mL/min.
2.4. LC-MS analyses LC–MS analyses were performed using a ProminenceTM UFLC system (Shimadzu, Kyoto, Japan) linked to a LCMS–2020 system (Shimadzu, Kyoto, Japan) in positive mode. LCsolution software was used to control the instruments for data acquisition and processing. The HPLC analysis was performed using an Optimapak C18 column (150 x 2.1 mm, 5 μm, Waters, MA, USA). The mobile phase was 0.1% formic acid solution (A) and 100% acetonitrile (B) from 25% B to 70% B over an 80 min period. Samples were eluted at a flow rate of 0.3 mL/min and a column temperature of 30 °C. The LC–MS was operated with a nebulizing gas flow rate of 10 L/min, CDL temperature of 250 °C, heat block temperature of
200 °C, detector voltage of 1.50 kV and a CDL voltage of 15.0 V.
2.5. Method validation Linearity was examined with standard solutions using five different concentrations by plotting the integrated peak area for each component against its corresponding solution concentration. Precision was expressed as intra-day and inter-day relative standard deviations. Intra-day variability was evaluated by analyzing standards five times in a single day and inter-day variability was verified for five consecutive days. Repeatability, expressed as RSD, was calculated based on the retention and migration times for six repetitive injections. The stability of the sample solutions was tested at 0, 2, 4, 8, 12 and 24 h after preparation and expressed by RSD. Recovery was determined by adding three different concentration levels (120%，100% and 80%) of standard solution into samples in which the content of the three analytes were known. Samples were analyzed in triplicate at each level. The recoveries were calculated using the following equation: recovery (%) = (amount found − amount original)/amount spiked × 100%.
3. Results and Discussion 3.1. Optimization of HPLC-DAD conditions To establish an optimal chromatographic condition, various mobile phase compositions (methanol–water or acetonitrile–water), types of acid (phosphoric acid, formic acid and acetic acid) and detection wavelengths were employed. Retention times of marker compounds were shorter using acetonitrile–water compared to methanol-water, so acetonitrile–water was chosen as the mobile phase. The addition of 0.1% of each acid in the mobile phase decreased peak tailing significantly, and phosphoric acid yielded the best results. By verifying DAD spectra with HPLC chromatograms, the optimal detection wavelength of the three compounds
was determined to be 270 nm. HPLC chromatograms of a standard mixture and the three medicinal materials using optimized HPLC conditions are shown in Fig. 3.
3.2. LC-MS data of marker compounds in the three medicinal materials (+)-Catechin, (-)-epicatechin and aloin were detected in the positive mode (Table 1). The three peaks in the chromatogram predicted to represent the marker compounds were verified by interpretation of retention time, UV absorbance band, source materials, molecular formulae and characteristic MS data acquired from the LC-MS. All characteristics of each peak matched the standard completely. Therefore, the three peaks were confirmed as (+)catechin, (-)-epicatechin and aloin.
3.3. Method validation Linearity was verified using five standard concentrations of each compound. Calibration curves for the three marker compounds were linear (r2 > 0.9992) in the range of 25 to 400 μg/mL (Table 2). LODs and LOQs were 1 and 4 μg/mL for (+)-catechin, 0.5 and 1.4 μg/mL for (-)-epicatechin, and 0.5 and 1 μg/mL for aloin, respectively (Table 2). Table 3 shows the precision of the three marker compounds at three concentrations. The intra-day and inter-day precisions of the three marker compounds were less than 2%. As shown in Table 4, the developed method showed good repeatability base on analyte RSD values (<1.81%). Sample solutions were also stable over 24 h as indicated by a low RSD value (<1.25%). The recovery of the three compounds was 99.48 to 100.97%, indicating that the three marker compounds could be extracted completely from herb material without destruction or denaturation. RSD of recovery was less than 1.70%, demonstrating that the method was precise (Table 5). All validation results of the developed HPLC method in this study were satisfactory and met method validation guidelines in Korea and China [3, 9].
3.4. Chromatographical differentiation among aloe, catechu and gambir Differentiation of aloe from the other two medicinal materials using the chromatogram is straightforward; the existence of aloin indicates that the material is aloe. In contrast, differentiation of catechu and gambir is less simple, because both materials contain (+)catechin and (-)-epicatechin. Generally, gambir has a higher content of the marker compounds than catechu. However, higher (+)-catechin and (-)-epicatechin is not sufficient to differentiate gambir from catechu. Another potential criterion for the differentiation of catechu and gambir is the ratio of (+)-catechin and (-)-epicatechin, as the (-)-epicatechin content of gambir is lower than that of catechu. The ratios calculated for all gambir samples were higher than 9.68 and those for all catechu samples were lower than 4.65. The ratio of (+)-catechin and (-)-epicatechin content thus could be a good criterion for the discrimination of catechu and gambir (Fig. 4).
3.5. Hierarchical clustering analysis To evaluate the method of differentiating the three materials, unsupervised clustering analysis was performed using Ward’s method to visualize differences and/or similarities among samples through linkage distances [14]. Using hierarchical cluster analysis, the collected 59 samples in Korea and China were grouped into cluster I (aloe) and cluster II (others), and cluster II was divided into cluster III (gambir) and cluster IV (catechu) (Fig. 5). This indicated that our results were well adapted to the hierarchical cluster analysis model, allowing the three materials to be discriminated during the same analysis (Fig. 5). The results of hierarchical clustering analysis correspond to those of chromatographical differentiation.
4. Conclusion
This is the first report of a simultaneous analytical method to discriminate aloe, catechu and gambir by HPLC-DAD and LC-MS using (+)-catechin, (-)-epicatechin and aloin as marker compounds. The HPLC-DAD method was validated by linearity, precision, repeatability, stability and recovery. This analysis revealed that aloe, catechu and gambir are mislabeled in the marketplace and that the developed HPLC-DAD method can clearly distinguish aloe, catechu and gambir. This new method will prevent the misuse of the three medicinal materials by misidentification. Furthermore, the newly suggested ratio calculating method will be useful to easily distinguish catechu from gambir.
References [1] Q. F. Gong, Chinese Medicinal Herbs Preparation; China Press of Traditional Chinese Medicine, Beijing, 2007, pp. 25. [2] K. Eshun, Q. He, Aloe Vera: A Valuable Ingredient for the Food, Pharmaceutical and Cosmetic Industries—A Review, J. Crit. Rev. Food. Sci. Nut. 44 (2004) 91-96. [3] The State Commission of Pharmacopoeia, Pharmacopoeia of People’s Republic of China, The Medicine Science and Technology Press of China, Beijing, 2010, Part I, pp. 151152; Part I, pp. 9; Appendix, pp. 29. [4] J. H. Park, S.W. Kwon, New Perspectives on Aloe, Springer, New York, 2006, pp. 19-34. [5] X. L. Cao, D. F. Huang, Y. M. Dong, H. Zhao, Y. Ito, Separation of aloins A and B from Aloe vera exudates by high speed countercurrent chromatography, J. Liq. Chromatogr. Relat. Technol. 30 (2007), 1657-1668. [6] X. F. Wu, W. J. Ding, J. S. Zhong, J. Z. Wan, Z. Y. Xie, Simultaneous qualitative and quantitative determination of phenolic compounds in Aloe barbadensis Mill by liquid chromatography–mass spectrometry-ion trap-time-of-flight and high performance liquid chromatography-diode array detector, J. Pharm. Biomed. Anal. 80 (2013), 94106. [7] X. F. Wu, S. Yin, J. S. Zhong, W. J. Ding, J. Z. Wan, Z. Y. Xie, Mushroom tyrosinase inhibitors from Aloe barbadensis Miller, J. Fitoterapia 83 (2012), 1706-1711. [8] Y. G. Kang, Identificology of Chinese Materia Medica; China Press of Traditional Chinese Medicine, Beijing, 2007, pp. 452-453. [9] Ministry of Food and Drug Safety, Korean Pharmacopoeia，2012, Part Ⅱ, pp. 13001301. [10] Ministry of Health, Labour and Welfare, Japanese Pharmacopoeia，2011, Part I, pp.
1641. [11] K.S. Tong, M. Jain. Kassim, A. Azraa, Adsorption of copper ions from its aqueous solution by a novel biosorbent Uncaria gambir: Equilibrium, Kinetics and Thermodynamic studies, Chem. Eng. J. 170 (2011), 145-153. [12] Napolitano, J. G.; Gödecke, T.; Lankin, D. C.; Jaki, B. U.; McAlpine, J. B.; Chen, S. N.; Pauli, G. F. Orthogonal analytical methods for botanical standardization: Determination of green tea catechins by qNMR and LC-MS/MS, J. Pharm. Biomed. Anal. 93 (2014), 59-67. [13] Taniguchi, S.; Kuroda, K.; Doi, K.; Tanabe, M.; Shibata, T.; Yoshida, T.; Hatano, T.; Revised structures of gambiriins A1, A2, B1, and B2, chalcane-flavan dimers from gambir (Uncaria gambir extract), J. Chem. Pharm. Bull. 55 (2007), 268-272. [14] R. M. Cormack, A review of classification, J. Roy. Stat. Soc. A. 134 (1971), 321-367.
Figure Captions Fig. 1. The appearances of aloe, catechu and gambir. Fig. 2. Chemical structures of (+)-catechin, (-)-epicatechin and aloin. Fig. 3. HPLC chromatogram of (a) standard mixture, (b) catechu, (c) gambir and (d) aloe; 1: (+)-catechin, 2: (-)-epicatechin, 3: aloin. Fig. 4. (a): Total content of (+)-catechin, (-)-epicatechin and (b): the ratio of (+)-catechin / (-)epicatechin in catechu (white) and gambir (black) samples. Fig. 5. Dendrogram of hierarchical cluster analysis.
Aloe
Catechu Fig. 1.
Gambir
(+)-Catechin
(-)-Epicatechin
Fig. 2.
Aloin
(a) A U
2
1
0.15
3
0.10
0.05
0.00 5.00
(b)
10.00
20.00 Minutes
25.00
30.00
35.00
15.00
20.00 Minutes
25.00
30.00
35.00
15.00
20.00 Minutes
25.00
30.00
35.00
30.00
35.00
1
0.80
2
0.60 A U
15.00
0.40
0.20
0.00 5.00
(c)
10.00
1 A U
1.00
0.50
2
0.00 5.00
10.00
2.50
(d)
3
2.00
A U
1.50
1.00
0.50 0.00 5.00
10.00
15.00
20.00 Minutes
Fig. 3.
25.00
Fig. 4.
Fig. 5.
Tables Table 1. Characterization of compounds in aloe, catechu and gambir by LC-MS. RT (min)
MS
Molecular formula
UV λ (nm)
Identified compound
Material
21.96
291.5 [M+H]+
C15H14O6
212.5, 278.6
(+)-Catechin
Catechu, Gambir
25.13
291.5 [M+H]+
C15H14O6
212.5, 278.6
(-)-Epicatechin
Catechu, Gambir
C21H22O9
193.7, 269.1, 296.4, 354.8
Aloin
Aloe
50.10
+
419.4 [M+H] 441.4 [M+Na]+
Table 2. Calibration curve, LOD and LOQ of marker compounds (n = 5). Analyte
Regression equation Y(area) X(conc. μg/mL)
r2
Linear range (μg/mL)
LOD (μg/mL)
LOQ (μg/mL)
(+)-Catechin
Y = 5847.3X + 4478
0.9994
25-400
1.0
4.0
(-)-Epicatechin
Y = 7107.2X + 6838
0.9994
25-400
0.5
1.4
Aloin
Y = 4222.3X + 4358
0.9992
25-400
0.5
1.0
Table 3. Inter-day and Intra-day precision of marker compounds (n = 6). Intra-day Analyte
(+)-Catechin
(-)-Epicatechin
Aloin a
Inter-day
Conc. (μg/mL)
Detecteda (μg/mL)
Detecteda (μg/mL)
RSD (%)
62.5
62.5±0.34
0.54
62.5
62.6±0.43
0.68
125.0
125.0±0.72
0.58
125.0
125.1±0.49
0.39
250.0
250.0±0.99
0.40
250.0
250.1±1.14
0.46
62.5
62.5±0.34
0.40
62.5
62.6±0.70
1.12
125.0
125.0±0.55
0.44
125.0
125.0±1.03
0.83
250.0
250.0±1.64
0.66
250.0
250.1±0.39
0.16
62.5
62.5±0.26
0.42
62.5
62.8±1.05
1.68
125.0
125.0±0.85
0.68
125.0
126.1±0.99
0.79
250.0
250.0±1.61
0.42
250.0
249.2±4.19
1.68
Data are represented as mean±SD.
RSD (%)
Conc. (μg/mL)
Table 4. Repeatability and stability of marker compounds (n = 6). Repeatability Analyte
(+)-Catechin
(-)-Epicatechin Aloin a
Stability
Material
RT (min)a
Content (mg/g)a
RSD (%)
Catechu
9.61±0.03
145.2±1.67
1.15
Gambir
9.55±0.02
252.3±2.43
Catechu
12.13±0.02
Gambir Aloes
Data are represented as mean±SD.
RT (min)a
Content (mg/g)a
RSD (%)
9.61±0.04
145.5±1.12
0.77
0.96
9.59±0.04
251.6±0.83
0.33
72.8±1.10
1.51
12.13±0.03
73.2±0.68
0.93
12.10±0.02
22.7±0.41
1.81
12.14±0.04
21.7±0.27
1.25
24.13±0.01
447.1±1.31
0.29
24.10±0.02
446.0±1.76
0.39
Table 5. Recovery data of the developed method (n = 3). Analyte
Material
Concentration Original (μg/ml)
Catechu
64.5
(+)-Catechin Gambir
Catechu
33.5
22.4
(-)-Epicatechin Gambir
Aloin a
Aloe
Data are represented as mean±SD.
18.0
77.4
Recovery (%)
RSD (%)
155.0±0.17
100.22
0.12
64.5
129.1±0.08
100.07
0.06
38.7
103.0±0.70
99.48
0.68
Spiked (μg/ml)
Found (μg/ml)a
90.3
46.9
80.4±0.21
100.00
0.26
33.5
67.2±0.29
100.52
0.43
20.1
53.6±0.91
100.00
1.70
31.4
53.8±0.05
100.00
0.10
22.4
44.7±0.08
99.75
0.17
13.4
35.8±0.40
100.00
1.12
25.2
43.2±0.10
100.00
0.22
18.0
36.2±0.13
100.97
0.37
10.8
28.8±0.49
100.00
1.70
108.4
185.8±0.69
100.00
0.37
77.4
154.8±0.79
100.02
0.51
46.4
123.8±2.10
100.00
1.69