Quality Assessment and Origin Identi cation on Four Species of Curcumae Radix by HPLC Assay and Fingerprint Analysis
Yu-Ling Ho HungKuang University Chi-Ren Liao Central Flagship Pharmacy Chun-Pin Kao Hsin Sheng College of Medical Care and Management Kun-Chang Wu China Medical University Shang-Chih Lai Tzu Chi University Yuan-Shiun Chang ( [email protected] ) China Medical University College of Chinese Medicine https://orcid.org/0000-0002-8149-2638
Research Article
Keywords: Curcumae Radix, HPLC, assay, ngerprint, curcumin, desmethoxycurcumin, bisdesmethoxycurcumin, germacrone, curdione
Posted Date: September 1st, 2021
DOI: https://doi.org/10.21203/rs.3.rs-838863/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1 Quality assessment and origin identification on four
2 species of Curcumae Radix by HPLC assay and
3 fingerprint analysis
4
5 Yu-Ling Ho 1, Chi-Ren Liao2, Chun-Pin Kao3, Kun-Chang 6 Wu4, Shang-Chih Lai5 and Yuan-Shiun Chang6, 7*, 7 8 Affiliation 9 1Department of Nursing, Hungkuang University, Taichung 43302, TAIWAN 10 2Central Flagship Pharmacy, 402, Wu Chuan Road, North Section, Taichung 40446, 11 TAIWAN 12 3Department of Nursing, Hsin Sheng College of Medical Care and Management, 13 Tao-Yuan 32544, TAIWAN 14 4Department of Pharmacy, College of Pharmacy, China Medical University, Taichung 15 40402, TAIWAN 16 5Department of Post-baccalaureate Chinese Medicine, Tzu Chi University, Hualien 17 97004, TAIWAN 18 6Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, 19 College of Chinese Medicine, China Medical University, Taichung 40402, TAIWAN 20 7Chinese Crude Drug Pharmacy, China Medical University Hospital, Taichung 40402, 21 TAIWAN 22 23 24 *Corresponding author 25 Prof. Yuan Shiun CHANG 26 Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, 27 College of Chinese Medicine, China Medical University, Taichung 40402, TAIWAN 28 E-mail: [email protected] 29 Phone: +886-4-2203-0380 30 FAX: +886-4-2208-3362 31 32 33 Abstract 34
35 Background:
36 Curcumae Radix is a multi-source Chinese medicine called Yujin. It is derived
37 from the root tubers of four Curcuma species including Curcuma wenyujin, C.
38 kwangsiensis, C. phaeocaulis and C. longa. The identification of the root tubers of the
39 four Curcuma species often caused confusions.
40 Methods:
41 In this study, we developed HPLC/PDA methods to differentiate these four
42 species of Curcumae Radix by HPLC assay and fingerprint analysis. The methods
43 developed were also validated with their precision, repeatability and accuracy.
44 Results:
45 Curdione could only be detected in C. wenyujin. For C. kwangsiensis, C.
46 phaeocaulis, if the relative peak area value of peak 1 and germacrone peak of the
47 samples was more than 1.024, it could be identified as C. phaeocaulis of Curcumae
48 Radix. Curcumin, desmethoxycurcumin and bisdesmethoxycurcumin could only be
49 detected in C. longa. We also developed a simple method to determine curcumin,
50 desmethoxycurcumin and bisdesmethoxycurcumin in C. longa and germacrone in the
51 other three species of Curcumae Radix.
52 Conclusions:
53 Because of the definite difference of main ingredients between curcumin-free
54 group (C. wenyujin, C. kwangsiensis and C. phaeocaulis) and curcumin containing C.
55 longa from this study, four Curcuma species of Curcumae Radix in the pharmacopeia
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56 should be separated into two monographs, with curcumin-free and germacrone
57 containing species as one and curcumin containing C. longa as the second
58 monograph.
59 The HPLC/PDA methods of HPLC assay and fingerprint analysis developed in
60 this study could be used for the quality control of these four species of Curcumae
61 Radix.
62
63 Keywords: Curcumae Radix, HPLC, assay, fingerprint, curcumin,
64 desmethoxycurcumin, bisdesmethoxycurcumin, germacrone, curdione
65
66 Background
67 Curcuma spp. (Zingiberaceae) is one of the famous condiments in food. There
68 are more than 50 Curcuma species used as herbal medicines and food in the world
69 [1-4]. Around 12 Curcuma species are distributed in China and some of them are used
70 as food and herbal medicines [5-7]. The rhizomes of Curcuma spp. are branched
71 and often with tuber bearing roots under the rhizomes [1] (Figure 1).
72 In TCM, the rhizomes and root tubers of Curcuma wenyujin Y. H. Chen et C.
73 Ling, Curcuma kwangsiensis S. G. Lee et C. F. Liang, Curcuma phaeocaulis Val. and
74 Curcuma longa L. were used separately as different herb items. The root tubers of
75 four Curcuma species including C. wenyujin, C. kwangsiensis, C. phaeocaulis and C.
76 longa designated as Curcumae Radix are called Yujin. The rhizome of three Curcuma
77 species including C. wenyujin, C. kwangsiensis and C. phaeocaulis designated as
78 Curcumae Rhizoma are called Ezhu whilst the rhizome of C. longa known as turmeric
79 designated as Curcumae Longae Rhizoma is called Jianghuang [8-10] (Table 1). The
3
80 turmeric has been used for a long time in many Asian countries as a condiment, dye
81 and traditional medicine [11]. The description of the rhizomes and the root tubers of
82 these Curcuma species often confused the readers. Figure 1 showed the connection of
83 rhizomes and root tubers within a Curcuma species.
84
85 Table 1 The names of different medicinal parts of Curcuma species for TCM use
Source Root tuber Rhizome C. wenyujin Curcumae Rhizoma C. kwangsiensis (Ezhu) C. phaeocaulis Curcumae Radix (Yujin) Curcumae Longae Rhizoma C. longa (Jianghuang ; Turmeric) 86
87 Curcumae Radix, Yujin , the root tubers of four Curcuma species, C. wenyujin
88 (Wenyujin), C. kwangsiensis (Guiyujin), C. phaeocaulis (Lusiyujin) and C. longa
89 (Huangsiyujin) were recorded in both Chinese Pharmacopoeia (2020 edition) [8] and
90 Taiwan Herbal Pharmacopeia (III), 2018 [9]. In TCM, Curcumae Radix is used to
91 activate qi for resolving stagnation, clearing heart fire and cooling blood and
92 normalizing gallbladder to cure jaundice. It is used to treat illnesses like jaundice,
93 cholelithiasis caused by dampness-heat of liver and gallbladder and so on [10]. It is
94 also used to treat gastric cancer [12], diabetes [13], hepatitis [14], improve blood
95 circulation and remove blood stasis [15] in previous studies.
96 Chinese medicine was widely used in the world and the source identification
97 played a key role in the quality control, especially for multiple-origin Chinese
98 medicines, such as Curcumae Radix, Fritillariae Cirrhosae Bulbus, Gentianae
99 Macrophyllae Radix and Asari Radix et Rhizoma, etc [8,9]. Yujin, Curcumae Radix,
100 is one of the multiple-origin Chinese medicines. However, it is difficult to 4
101 authenticate their sources of crude drugs of Curcumae Radix in clinic because of their
102 similar morphological features. Even though a number of methods have been
103 developed on qualitative and quantitative analysis of Curcuma species, including TLC,
104 GC, HPLC and UFLC-Q-TOF-MS, etc [16-20]. However, there was no study to
105 investigate the analytical methods on how to distinguish these four species of
106 Curcumae Radix previously. Therefore, we developed a reliable method to
107 discriminate these four species of Curcumae Radix by HPLC assay and fingerprint
108 analysis in this study.
109 In addition, there was no marker constituents regarding these four species of
110 Curcumae Radix had been stipulated in the current Chinese Pharmacopoeia [8] and
111 Taiwan Herbal Pharmacopeia [9]. Therefore, it is also important to develop a method
112 to evaluate the quality of these four species of Curcumae Radix by HPLC assay and
113 fingerprint analysis. Based on their chemical profiles, the chemical constituents of
114 these four Curcuma species can be divided into two categories. Germacrone, a
115 sesquiterpenoid compound, is an important bioactive component in the three species
116 (C. wenyujin, C. kwangsiensis and C. phaeocaulis) of Curcumae Radix. Germacrone
117 showed extensively biological activities including antitumor [21,22], antimicrobial [23]
118 and antiandrogenic [24] activities. However, germacrone was not found in C. longa of
119 Curcumae Radix [25]. Curcuminoids (curcumin, desmethoxycurcumin and
120 bisdesmethoxycurcumin) are the major bioactive compounds in C. longa [26].
121 Curcuminoids showed general biological activities including antioxidant activities
122 [27], neuroprotective effect [28], improvement of Alzheimer's disease [29] as well as
123 improvement of inflammatory bowel disease, pancreatitis, arthritis and chronic
124 anterior uveitis [30]. In this study, we developed a simple method to determine
125 germacrone in C. wenyujin, C. kwangsiensis and C. phaeocaulis of Curcumae Radix
5
126 and curcumin, desmethoxycurcumin and bisdesmethoxycurcumin in C. longa of
127 Curcumae Radix by HPLC assay. Besides, the difference of germacrone content in the
128 three species (C. wenyujin, C. kwangsiensis and C. phaeocaulis) of Curcumae Radix
129 was also investigated. Furthermore, a feasible method was developed in HPLC
130 fingerprint analysis and displayed the result with common characteristic peaks,
131 relative retention time (RRT) and relative peak area (RPA) in four species of
132 Curcumae Radix samples.
133 In this study, we not only established a scientific platform to discriminate the
134 sources of these four species of Curcumae Radix but also develop an analytical
135 method to evaluate the quality of these four species of Curcumae Radix by a sensitive
136 HPLC–PDA method. The results of this study can be used for the quality control of
137 these four species of Curcumae Radix and can provide reference for the future edition
138 of herbal Pharmacopoeias in the world.
139 Methods
140 Materials and reagents
141 The reference compounds, germacrone, curdione, curcumin, desmethoxycurcumin
142 and bisdesmethoxycurcumin (Figure 2) were purchased from the Shanghai Research
143 Center for Modernization of Traditional Chinese Medicine, minimum 98% purity.
144 HPLC grade acetonitrile and formic acid were purchased from branch of Merck in
145 Taipei, Taiwan. De-ionized water was prepared by an Elix 10/Milli-Q Gradient
146 (Millipore, MA, USA). All other reagents used in this study were of analytical grade.
147 Ten batches of each root tuber of C. wenyujin, C. kwangsiensis and C. phaeocaulis
148 and C. longa were collected from different regions in Mainland China and the herbal
149 markets from Hong Kong. The detailed information of the samples is summarized in
6
150 Table 2A and Table 2B. The plant materials were identified by Prof. Yuan-Shiun
151 CHANG, Department of Chinese Pharmaceutical Sciences and Chinese Medicine
152 Resources, College of Chinese Medicine, China Medical University. All the plant
153 specimens have been deposited in Department of Chinese Pharmaceutical Sciences
154 and Chinese Medicine Resources, College of Chinese Medicine, China Medical
155 University, Taichung, Taiwan.
156
7
157 Table 2A Collection information of C. wenyujin, C. kwangsiensis and C. phaeocaulis 158 of Curcumae Radix and the content of germacrone (mg/kg) on the dried basis Sample ID Source Germacrone (mg/kg) W1 Ruian, Zhejiang 455 W2 Ruian, Zhejiang 395 W3 Ruian, Zhejiang 807 W4 Ruian, Zhejiang 883 W5 Lishui, Zhejiang 888 W6 Wenjou, Zhejiang 740 W7 Leqing, Zhejiang 902 W8 Ruian, Zhejiang 500 W9 Ruian, Zhejiang 330 W10 Anxi, Fujian 353 Mean (W1-W10) 625 SD 240 K1 Local Sample (Guangxi) 390 K2 Local Sample (Guangxi) 326 K3 Local Sample (Yunnan) 388 K4 Local Sample (Guangxi) 130 K5 Ginzhou, Guangxi 140 K6 Nanning, Guangxi 152 K7 Ginzhou, Guangxi 309 K8 Ginzhou, Guangxi 140 K9 Ginzhou, Guangxi 135 K10 Ginzhou, Guangxi 136 Mean (K1-K10) 225 SD 114 P1 Local Sample (Sichuan) 131 P2 Local Sample (Sichuan) 178 P3 Local Sample (Sichuan) 151 P4 Local Sample (Sichuan) 160 P5 Shuangliu, Sichuan 124 P6 Shuangliu, Sichuan 222 P7 Chendu, Sichuan 164 P8 Chendu, Sichuan 217 P9 Chendu, Sichuan 180 P10 Chendu, Sichuan 210 Mean (P1-P10) 173 SD 34 159 Note: Root tuber samples W1-W10, C. wenyujin; K1-K10, C. kwangsiensis; P1-P10, 160 C. phaeocaulis. 161 162 8
163 Table 2B Collection information of C. longa of Curcumae Radix, and the content 164 (mg/kg) of curcumin, desmethoxycurcumin, bisdesmethoxycurcumin and total 165 contents (mg/kg) of curcumin, desmethoxycurcumin, bisdesmethoxycurcumin on the 166 dried basis Sample ID Source C D B Total (mg/kg) L1 Jianwei, Sichuan 2036 517 466 3019 L2 Shuangliu, Sichuan 672 104 52 828 L3 Shuangliu, Sichuan 893 153 95 1141 L4 Chendu, Sichuan 798 137 71 1006 L5 Chendu, Sichuan 553 122 82 757 L6 Chendu, Sichuan 549 128 79 756 L7 Leshan, Sichuan 1794 315 157 2266 L8 Leshan, Sichuan 1787 312 156 2255 L9 Leshan, Sichuan 2060 363 194 2617 L10 Jianwei, Sichuan 698 107 78 883 Mean 1182 225 107 1550 (L1-L10) SD 640 141 49 881 167 Note: Root tuber samples L1-L10, C. longa. C: curcumin; D: desmethoxycurcumin; B: 168 bisdesmethoxycurcumin. 169
170 Sample preparation
171 The root tuber samples of Curcumae Radix were pulverized into small
172 homogeneous particles (ca. 20-mesh). Two different extraction procedures were used
173 to extract germacrone in C. wenyujin, C. kwangsiensis and C. phaeocaulis of
174 Curcumae Radix and curcumin, desmethoxycurcumin and bisdesmethoxycurcumin in
175 C. longa of Curcumae Radix.
176 The first extraction procedure was used for extracting germacrone in C. wenyujin,
177 C. kwangsiensis and C. phaeocaulis of Curcumae Radix. Two point five grams of
178 each powdered sample was carefully weighed into a 50-mL conical flask. Twenty
179 milliliters of 50% methanol was then added into the conical flask and shaken briefly
180 to mix. Each sample was then extracted with 20 mL of 50% methanol in an ultrasonic
181 bath (Delta DC 400H) at a frequency of 40 kHz at 25 ℃ for 30 min. Transfer the
9
182 solution to a 50-mL volumetric flask. Repeat the extraction for two more times each
183 with 15 mL of 50% methanol. Combine the solutions and make up to the mark with
184 50% methanol. The final combined extract was filtered through a 0.45 μm PVDF
185 syringe filter before analysis. An aliquot of 10 μL solution of each sample was used
186 for HPLC analysis.
187 The second extraction procedure was used for extracting curcumin,
188 desmethoxycurcumin and bisdesmethoxycurcumin in C. longa of Curcumae Radix.
189 Zero point five gram of each powdered sample was carefully weighed into a conical
190 flask. Thirty milliliters of 75% ethanol were then added into the 50-mL conical flask
191 and shaken briefly to mix. Each sample was then extracted with 30 mL of 75%
192 ethanol in an ultrasonic bath (Delta DC 400H) at a frequency of 40 kHz at 25 ℃ for
193 30 min. Transfer the solution to a 100-mL round-bottomed flask. Repeat the
194 extraction for one more times with 30 mL of 75% ethanol. Evaporate the solvent to
195 dryness at reduced pressure with a rotary evaporator. Dissolve the residue with 75%
196 ethanol and transfer the solution to a 20-mL volumetric flask. Make up to the mark
197 with 75% ethanol. The final combined extract was filtered through a 0.45 μm PVDF
198 syringe filter before analysis. An aliquot of 10 μL solution of each sample was used
199 for HPLC analysis.
200
201 HPLC condition
202 The HPLC system consisted of a Waters e2695 HPLC system equipped with Waters
203 2998 photodiode array detector (PDA), Waters e2695 separations module and column
204 heater module. Chromatographic separation was performed on an X-Bridge Shield
205 RP18 column (250 mm × 4.6 mm i.d., 5 μm) with an autosampler injection volume of
206 10 μL. The other HPLC parameters were divided into two conditions. The first HPLC
10
207 condition was used for determining germacrone in C. wenyujin, C. kwangsiensis and
208 C. phaeocaulis of Curcumae Radix. For assay, the mobile phase was a mixture of
209 acetonitrile and water (55:45, v/v) and the elution time was about 30 min. The mobile
210 phase of HPLC fingerprint analysis consisted of a mixture of water (A) and
211 acetonitrile (B). The optimized elution conditions were as follow: 0–35 min, 51% A
212 (isocratic), 35–60 min, 51–20% A (gradient). The flow rate for HPLC assay and
213 fingerprint analysis was 1.0 mL/min. Each sample was monitored at 210 nm. An
214 aliquot of 10 μL solution of each sample was used for HPLC analysis. The
215 autosampler and column compartment were maintained at 25 ℃.
216 The second HPLC condition was used for determining curcumin,
217 desmethoxycurcumin and bisdesmethoxycurcumin in C. longa of Curcumae Radix
218 samples. The mobile phase of HPLC assay consisted of acetonitrile: 0.2% formic acid
219 = 55: 45, and each sample was monitored at 430 nm. Besides, the mobile phase of
220 HPLC fingerprint analysis consisted of a mixture of acetonitrile (A) and 0.2% formic
221 acid (B) The optimized elution conditions were as follow: 0–5 min, 40% A (isocratic),
222 5–20 min, 40–47% A (gradient), 20–45 min, 47% A (isocratic), 45–60 min, 47–85%
223 A (gradient), and each sample was monitored at 335 nm. The flow rate for HPLC
224 assay and fingerprint analysis was 1.0 mL/min. An aliquot of 10 μL solution of each
225 sample was used for HPLC analysis. The autosampler and column compartment were
226 maintained at 25 ℃.
227 Results
228 Optimization of extraction method
229 In order to extract germacrone, curcumin, desmethoxycurcumin and
230 bisdesmethoxycurcumin from the herbal samples efficiently, several variable factors
11
231 were compared in this study, including extraction solvent (methanol, ethanol, 75%,
232 50%, 25% methanol and ethanol), extraction time (10, 20, 30, 40, 50, 60 min), and
233 number of extractions. 50% methanol was the best solvent to extract germacrone in
234 the samples of C. wenyujin, C. kwangsiensis and C. phaeocaulis of Curcumae Radix
235 (Table S1, Supporting information). 75% ethanol was the best solvent to extract
236 curcumin, desmethoxycurcumin and bisdesmethoxycurcumin in the samples of C.
237 longa of Curcumae Radix (Table S2, Supporting information). Three extraction times
238 for germacrone (Figure S1, Supporting information), two extraction times for
239 curcumin, desmethoxycurcumin and bisdesmethoxycurcumin (Figure S2, Supporting
240 information) and 30 minutes for these four reference components of Curcumae Radix
241 samples could be extracted completely (extraction rate more than 99%) (Table S3 and
242 S4, Supporting information).
243
244 Optimization of chromatographic conditions
245 There were two different chromatographic conditions used to determine
246 germacrone in C. wenyujin, C. kwangsiensis and C. phaeocaulis of Curcumae Radix
247 samples and curcumin, desmethoxycurcumin and bisdesmethoxycurcumin in C. longa
248 of Curcumae Radix samples respectively. Moreover, several variable factors including
249 mobile phase system, detection wavelength and column temperatures were controlled
250 in HPLC assay and fingerprint analysis.
251 In order to obtain sharp and symmetrical peaks and smooth baseline during
252 analyzing germacrone in C. wenyujin, C. kwangsiensis and C. phaeocaulis of
253 Curcumae Radix samples, the different mobile phase systems, including
254 methanol–water, acetonitrile–water, acetonitrile–0.1% formic acid and
255 acetonitrile–0.1% phosphoric acid were tested in this study. As a result, good
12
256 resolution, baseline, sharp and symmetrical peaks were obtained with using
257 acetonitrile–water system. Furthermore, the different mobile phase systems were also
258 tested for determining curcumin, desmethoxycurcumin and bisdesmethoxycurcumin
259 in C. longa of Curcumae Radix samples, including methanol–water,
260 acetonitrile–water, acetonitrile–0.1% formic acid, acetonitrile–0.2% formic acid and
261 acetonitrile–0.1% phosphoric acid. The results showed acetonitrile–0.2% formic acid
262 was the best mobile phase system used to determine curcumin, desmethoxycurcumin
263 and bisdesmethoxycurcumin in C. longa of Curcumae Radix samples.
264 For a better resolution on analyzing germacrone in the samples in C. wenyujin, C.
265 kwangsiensis and C. phaeocaulis of Curcumae Radix and curcumin,
266 desmethoxycurcumin and bisdesmethoxycurcumin in the samples in C. longa of
267 Curcumae Radix samples, several columns (Waters X-Bridge Shield RP18, Waters
268 XTerra RP18 and Grace Alltech Alltima C18) were screened before Waters X-Bridge
269 Shield RP18 (250 mm × 4.6 mm i.d., 5 μm) was finally selected as the column for
270 HPLC analysis.
271 The detection wavelength was monitored at 210 nm for HPLC assay and
272 fingerprint analysis for C. wenyujin, C. kwangsiensis and C. phaeocaulis of Curcumae
273 Radix because of the maximum UV absorption of germacrone. For C. longa of
274 Curcumae Radix samples, the detection wavelengths were monitored at 430 nm in
275 HPLC assay and 335 nm in HPLC fingerprint analysis for determining curcumin,
276 desmethoxycurcumin and bisdesmethoxycurcumin respectively. The samples were
277 monitored at 430 nm for HPLC assay due to the maximum UV absorption of
278 curcumin, desmethoxycurcumin and bisdesmethoxycurcumin. However, the samples
279 were monitored at 335 nm for HPLC fingerprint analysis in view of showing more
280 characteristic peaks in HPLC fingerprint chromatogram. Different column
13
281 temperatures at 25, 30 and 35 ℃ were also carried out in this study. The column
282 compartment at 25 ℃ was selected for determining germacrone in the samples of C.
283 wenyujin, C. kwangsiensis and C. phaeocaulis of Curcumae Radix and curcumin,
284 desmethoxycurcumin and bisdesmethoxycurcumin in the samples of C. longa of
285 Curcumae Radix.
286
287 Validation of the HPLC procedure
288 The stock solutions containing germacrone in 50% methanol as one group. The
289 stock solutions containing curcumin, desmethoxycurcumin and
290 bisdesmethoxycurcumin in 75% ethanol as another group. Two groups of stock
291 solutions were prepared and diluted to five appropriate concentrations for the
292 construction of calibration curves. Five concentrations of each analyte were injected
293 in triplicate. The calibration curves were constructed by plotting the peak areas under
294 the curve versus the concentration (mg/L) of the analytes, and the R square of each
295 standard curve was more than 0.999 (Table 3).
296 The limit of detection (LOD) and limit of quantitation (LOQ) of each standard
297 was determined by injecting a series of diluted standard solutions until the
298 signal-to-noise (S/N) ratio was 3 for LOD and 10 for LOQ respectively (Table 3). The
299 calibration curves of each pure standard analytes showed good linearity in relatively
300 wide dynamic ranges (Table 3).
301
302
14
303 Table 3 Regression equations, LOD and LOQ of germacrone, curcumin,
304 desmethoxycurcumin and bisdesmethoxycurcumin
Marker Calibration curvea Concentrations r2 LODb LOQc (mg/L) (mg/L) (mg/L) Germacrone y = 26573x - 5884.2 2-100 0.9999 0.2650 0.8830 Curcumin y = 76046x – 4472.6 5-200 1.0000 0.1108 0.3725 Desmethoxycurcumin y = 78242x + 8556.9 2-80 1.0000 0.1159 0.3844 Bisdesmethoxycurcumin y = 78884x + 53740 2-80 0.9999 0.0973 0.2732
305 a y is the peak area in UV chromatograms, x is the concentration (mg/L) of the 306 analyte. 307 b LOD refers to the limit of detection, s/n = 3. 308 c LOQ refers to the limit of quantitation, s/n = 10. 309
310 Table 4 Results of precision, repeatability, recovery of germacrone, curcumin,
311 desmethoxycurcumin and bisdesmethoxycurcumin
Precision RSD (%) Intra-day Repeatability RSD (%) Recovery (%) Marker Inter-day RSD RSD (n = 5) (n = 5) (n = 9) (n = 5) 2.094 (W1) 96.185 ± 1.698 Germacrone 1.282 2.632 2.372 (K1) 96.949 ± 2.690 1.307 (P1) 101.953 ± 2.068 Curcumin 0.480 0.281 4.155 99.920 ± 1.020 Desmethoxycurcumin 0.368 0.365 2.811 104.422 ± 4.550 Bisdesmethoxycurcumin 0.710 0.656 3.949 100.501 ± 3.411 312 Note: Samples W1, C. wenyujin; K1, C. kwangsiensis; P1, C. phaeocaulis. 313
314 The HPLC procedure was also validated with its precision, repeatability, and
315 accuracy. Intra- and inter-day variations were chosen to perform the precision of the
316 procedure. The reference markers including germacrone (20 mg/L), curcumin (50
317 mg/L), desmethoxycurcumin (20 mg/L) and bisdesmethoxycurcumin (20 mg/L) were
318 analyzed five times within one day. While for inter-day variability, the markers were
319 examined in triplicate for three consecutive days respectively (Table 4). The RSD
15
320 (relative standard deviations) values of intra- and inter-day of each marker which
321 were less than 3% (Table 4). Repeatability was evaluated by extracting and analyzing
322 five replicates of the same batch of sample (W1 of C. wenyujin, K1 of C.
323 kwangsiensis, P1 of C. phaeocaulis and L1 of C. longa) with the established method.
324 The method exhibited good repeatability with RSD of germacrone (2.094% of W1,
325 2.372% of K1 and 1.307% of P1), curcumin (4.155%), desmethoxycurcumin (2.811%)
326 and bisdesmethoxycurcumin (3.949%) of samples were less than 5%, which
327 demonstrated this method possessing good repeatability (Table 4). Recovery test was
328 used to evaluate the accuracy for this method. The known amounts of germacrone,
329 (100% of the known amounts) was added to approximate 2.5 g of the sample W1, K1
330 and P1, and curcumin, desmethoxycurcumin and bisdesmethoxycurcumin (100% of
331 the known amounts) were added to 0.5 g of the sample L1, and then extracted and
332 analyzed as described above. The average recoveries were calculated by the formula:
333 recovery (%) = [(spike concentration － original concentration) / adding
334 concentration)]× 100%. The recoveries of germacrone (96.185 ± 1.698% of W1,
335 96.949 ± 2.690% of K1 and 101.953 ±2.068% of P1), curcumin (99.92 ± 1.020%),
336 desmethoxycurcumin (104.422 ± 4.550%) and bisdesmethoxycurcumin (100.501 ±
337 3.411%) of samples were within the range of 90 to 110 % (Table 4).
338
339 Quantitation of germacrone, curcumin, desmethoxycurcumin and
340 bisdesmethoxycurcumin in Curcumae Radix samples
341 The HPLC assay method described above was used to determine germacrone in the
342 samples of C. wenyujin, C. kwangsiensis and C. phaeocaulis of Curcumae Radix and
343 curcumin, desmethoxycurcumin and bisdesmethoxycurcumin in the samples of C.
344 longa of Curcumae Radix. Ten batches of each four species of Curcumae Radix, a
16
345 total of 40 samples were collected and analyzed in this study. The typical HPLC assay
346 chromatograms of the three species (C. wenyujin, C. kwangsiensis and C. phaeocaulis)
347 of Curcumae Radix were shown in Figure 3A. In addition, the typical HPLC assay
348 chromatogram of C. longa of Curcumae Radix was shown in Figure 3B. The contents
349 (mg/kg) of germacrone, curcumin, desmethoxycurcumin and bisdesmethoxycurcumin
350 in samples of Curcumae Radix were shown in Table 2A and 2B. The germacrone
351 contents of C. wenyujin, C. kwangsiensis and C. phaeocaulis species of Curcumae
352 Radix samples varied significantly. The samples of C. wenyujin contained the highest
353 amounts of germacrone (625 ± 240 mg/kg), which was about 2.8-fold higher than C.
354 kwangsiensis (225 ± 114 mg/kg) and 3.6-fold higher than of C. phaeocaulis (173 ± 34
355 mg/kg) (mean ± SD, n=3). The contents of curcumin, desmethoxycurcumin,
356 bisdesmethoxycurcumin of C. longa of Curcumae Radix were 1182 ± 640 mg/kg, 225
357 ± 141 mg/kg and 107 ± 49 mg/kg respectively (mean ± SD).
358
359 HPLC fingerprint analysis of Curcumae Radix samples
360 The HPLC fingerprint analysis for 40 batches of Curcumae Radix samples were
361 developed in this study. The typical HPLC fingerprint chromatograms of 40 batches
362 of four species of Curcumae Radix samples were shown in Figure 4A and 4B. There
363 were four common characteristic peaks which were marked in the HPLC fingerprint
364 chromatograms of C. wenyujin, C. kwangsiensis and C. phaeocaulis of Curcumae
365 Radix samples (Figure 5A). The average peak retention time of four characteristic
366 peaks of 30 batches of samples were shown as peak 1 (RT = 19.635 ± 0.068 min, RSD
367 = 0.346%), peak 2 (RT = 23.921 ± 0.078 min, RSD = 0.326%), peak 3 (germacrone,
368 RT = 31.470 ± 0.116 min, RSD = 0.369%) and peak 4 (RT = 54.134 ± 0.064 min,
369 RSD = 0.118%) respectively (mean ± SD, n=3). For C. longa of Curcumae Radix
17
370 samples, a total four common characteristic peaks had been marked in the HPLC
371 fingerprint chromatograms to determine curcumin, desmethoxycurcumin and
372 bisdesmethoxycurcumin (Figure 5B). The average peak retention time of four
373 characteristic peaks were shown as peak 1 (RT = 23.226 ± 0.037 min, RSD = 0.159%),
374 peak 2 (curcumin, RT = 29.631 ± 0.068 min, RSD = 0.229%), peak 3
375 (desmethoxycurcumin, RT = 33.560 ± 0.081 min, RSD = 0.241%) and peak 4
376 (bisdesmethoxycurcumin, RT = 37.983 ± 0.111 min, RSD = 0.292%) respectively
377 (mean ± SD, n=3).
378 The relative retention time (RRT) of common characteristic peaks was shown in
379 this study. The RRT was calculated by the formula: RRT = retention time of selected
380 peak / retention time of marker peak. Germacrone (peak 3) was assigned as the
381 marker peak in the samples of C. wenyujin, C. kwangsiensis and C. phaeocaulis of
382 Curcumae Radix. The RRTs of other three selected peaks (peak 1, 2 and 4) of these
383 three species of Curcumae Radix were shown in Table 5A. For C. longa of Curcumae
384 Radix, curcumin (peak 2) was assigned as the marker peak. The RRT of other three
385 selected peaks (peak 1, 3 and 4) of four common characteristic peaks were 0.784 ±
386 0.001, 1.133 ± 0.001 and 1.282 ± 0.001 respectively (mean ± SD) (Table 5B). The
387 results showed a good reproducibility in retention time of common characteristic
388 peaks of HPLC fingerprint analysis.
389
18
390 Table 5A 391 Relative retention time (RRT) and relative peak area (RPA) of the HPLC fingerprint 392 analysis of C. wenyujin, C. kwangsiensis and C. phaeocaulis of Curcumae Radix
Sample ID Peak 1 Peak 2 Peak 3 Peak 4 (Germacrone) RRT RPA RRT RPA RRT RPA RRT RPA W1 0.622 0.017 0.760 0.154 1.000 1.000 1.724 0.054 W2 0.620 0.015 0.761 0.147 1.000 1.000 1.724 0.098 W3 0.621 0.027 0.760 0.150 1.000 1.000 1.722 0.067 W4 0.621 0.027 0.760 0.156 1.000 1.000 1.718 0.166 W5 0.621 0.028 0.760 0.155 1.000 1.000 1.724 0.170 W6 0.621 0.025 0.760 0.154 1.000 1.000 1.716 0.088 W7 0.621 0.030 0.760 0.141 1.000 1.000 1.720 0.095 W8 0.622 0.018 0.761 0.088 1.000 1.000 1.727 0.102 W9 0.624 0.960 0.760 0.141 1.000 1.000 1.714 0.092 W10 0.625 0.961 0.760 0.101 1.000 1.000 1.720 0.298 Mean 0.622 0.211 0.760 0.139 1.000 1.000 1.721 0.123 SD 0.002 0.395 0.000 0.024 N/A N/A 0.004 0.072 K1 0.625 0.565 0.759 0.080 1.000 1.000 1.720 0.204 K2 0.625 0.107 0.760 0.393 1.000 1.000 1.721 0.356 K3 0.624 0.174 0.760 0.102 1.000 1.000 1.718 0.289 K4 0.625 0.140 0.761 0.113 1.000 1.000 1.720 0.292 K5 0.625 0.208 0.761 0.142 1.000 1.000 1.721 0.308 K6 0.625 0.100 0.760 0.246 1.000 1.000 1.722 0.157 K7 0.625 0.098 0.761 0.100 1.000 1.000 1.723 0.158 K8 0.624 0.135 0.759 0.230 1.000 1.000 1.720 0.161 K9 0.625 0.130 0.760 0.261 1.000 1.000 1.718 0.144 K10 0.625 0.111 0.760 0.271 1.000 1.000 1.720 0.148 Mean 0.625 0.177 0.760 0.194 1.000 1.000 1.720 0.222 SD 0.000 0.141 0.000 0.102 N/A N/A 0.002 0.081 P1 0.624 1.870 0.759 0.220 1.000 1.000 1.714 0.246 P2 0.625 2.148 0.760 0.265 1.000 1.000 1.722 0.264 P3 0.625 1.820 0.760 0.191 1.000 1.000 1.714 0.216 P4 0.625 2.201 0.759 0.218 1.000 1.000 1.713 0.242 P5 0.625 3.274 0.760 0.208 1.000 1.000 1.724 0.267 P6 0.625 3.734 0.761 0.290 1.000 1.000 1.723 0.365 P7 0.625 3.367 0.760 0.188 1.000 1.000 1.720 0.239 P8 0.626 4.025 0.761 0.283 1.000 1.000 1.727 0.337 P9 0.625 3.761 0.760 0.284 1.000 1.000 1.721 0.287 P10 0.625 4.859 0.761 0.247 1.000 1.000 1.718 0.182 Mean 0.625 3.106 0.760 0.239 1.000 1.000 1.719 0.264 SD 0.000 1.041 0.001 0.039 N/A N/A 0.005 0.054 Mean (Total) 0.624 1.165 0.760 0.191 1.000 1.000 1.720 0.203 SD 0.002 1.530 0.001 0.075 N/A N/A 0.004 0.090 393 Note: Root tuber samples W1-W10, C. wenyujin; K1-K10, C. kwangsiensis; P1-P10, 394 C. phaeocaulis. 395 19
396 Table 5B 397 Relative retention time (RRT) and relative peak area (RPA) of the HPLC fingerprint 398 analysis of C. longa of Curcumae Radix
Sample ID Peak 1 Peak 2 Peak 3 Peak 4 (C) (D) (B) RRT RPA RRT RPA RRT RPA RRT RPA L1 0.784 0.280 1.000 1.000 1.133 0.202 1.281 0.115 L2 0.784 0.239 1.000 1.000 1.132 0.294 1.282 0.266 L3 0.783 0.437 1.000 1.000 1.134 0.201 1.284 0.050 L4 0.782 0.342 1.000 1.000 1.133 0.226 1.282 0.117 L5 0.783 0.493 1.000 1.000 1.133 0.213 1.284 0.082 L6 0.784 0.272 1.000 1.000 1.133 0.717 1.283 0.456 L7 0.785 0.266 1.000 1.000 1.132 0.696 1.281 0.524 L8 0.785 0.303 1.000 1.000 1.132 0.215 1.281 0.102 L9 0.784 0.288 1.000 1.000 1.132 0.217 1.282 0.102 L10 0.785 0.256 1.000 1.000 1.132 0.214 1.280 0.106 Mean 0.784 0.318 1.000 1.000 1.133 0.319 1.282 0.192 SD 0.001 0.084 N/A N/A 0.001 0.206 0.001 0.168 399 Note: Root tuber samples L1-L10, C. longa. (C): curcumin; (D): desmethoxycurcumin; 400 (B): bisdesmethoxycurcumin. 401
402 The relative peak area (RPA) of common characteristic peaks was also shown in
403 this study. The RPA value was calculated by the formula: RPA= peak area of selected
404 peak / peak area of mark peak (curcumin for C. longa and germacrone for the other
405 three species of Curcumae Radix). The result revealed a significant difference for RPA
406 value of peak 1 in C. kwangsiensis and C. phaeocaulis samples (Table 5A). The RPA
407 value range of peak 1 in 10 batches of C. kwangsiensis samples was 0.098 to 0.565,
408 and the RPA value range of peak 1 in 10 batches of C. phaeocaulis samples was 1.820
409 to 4.859 (Table 5A).
410 Curdione is the reference component to distinguish these three species (C.
411 wenyujin, C. kwangsiensis and C. phaeocaulis) of Curcumae Radix in HPLC
412 fingerprint analysis. The result showed that curdione could only be detected in C.
413 wenyujin among these three species of Curcumae Radix. The retention time of 20
414 curdione peak of standard and C. wenyujin samples was eluted about 13.2 to 13.3 min
415 (Figure 4A and 5A).
416
417 Discussions
418 There were total four species (C. wenyujin, C. kwangsiensis, C. phaeocaulis and C.
419 longa) recorded in Chinese Pharmacopoeia [8] and Taiwan Herbal Pharmacopeia (III)
420 [9]. However, it is difficult to distinguish their origins of raw materials of Curcumae
421 Radix in the markets because of their similar morphological features. There is no
422 study elucidating how to authenticate these four species of Curcumae Radix by HPLC
423 assay and fingerprint analysis before. We developed a simple and reliable method to
424 discriminate these four species of Curcumae Radix in this study.
425 We could divide the Curcumae Radix samples into two groups by HPLC assay
426 via their chemical profiles. If a sample was detected with germacrone, it could be
427 classified as C. wenyujin, C. kwangsiensis or C. phaeocaulis of Curcumae Radix. On
428 the other hand, if a sample could not be detected with germacrone and detected with
429 curcumin instead, it should be classified as C. longa of Curcumae Radix. Similarly. If
430 a sample was detected with curcumin, it should be C. longa of Curcumae Radix. In
431 addition, the germacrone content of these three species (C. wenyujin, C. kwangsiensis
432 and C. phaeocaulis) was in decreasing order of C. wenyujin (625 ± 240 mg/kg), C.
433 kwangsiensis (225 ± 114 mg/kg) and C. phaeocaulis (173 ± 34 mg/kg) respectively.
434 These three species (C. wenyujin, C. kwangsiensis and C. phaeocaulis) of Curcumae
435 Radix could be further discriminated by HPLC fingerprint analysis as described
436 below.
437 HPLC fingerprint analysis is one of the popular analytic methods which have
21
438 been applied to control the quality of herbal medicine [31,32]. Even though a few
439 HPLC fingerprint studies had been developed for some single species of Curcumae
440 Radix [33,34], however, there is no study performed previously to distinguish these
441 three species of Curcumae Radix by HPLC fingerprint analysis. Therefore, we
442 developed a reliable method to discriminate these three species (C. wenyujin, C.
443 kwangsiensis and C. phaeocaulis) of Curcumae Radix by HPLC fingerprint analysis
444 in this study.
445 First, we could divide these three Curcuma species (C. wenyujin, C.
446 kwangsiensis and C. phaeocaulis) of Curcumae Radix into two groups via
447 determining curdione by HPLC fingerprint analysis. Curdione, one of the
448 sesquiterpenoid compounds and only abundantly exist in C. wenyujin. This distinctive
449 mark was exhibited in the HPLC chromatogram of HPLC fingerprint analysis in this
450 study (Figure 4A and 5A). The retention time of curdione peak of standard and C.
451 wenyujin samples was about 13.2 to 13.3 min and the maximum UV absorption was
452 about at 210 nm.
453 Secondly, we could further differentiate between C. kwangsiensis and C.
454 phaeocaulis of Curcumae Radix via comparing the RPA (relative peak area) value of
455 HPLC fingerprint analysis. The RPA value range of peak 1 in 10 batches of C.
456 kwangsiensis samples was 0.098 to 0.565 while the RPA value range of peak 1 in 10
457 batches of C. phaeocaulis samples was 1.820 to 4.859. The result showed a significant
458 difference of RPA value of peak 1 in C. kwangsiensis and C. phaeocaulis samples
459 (Table 5A). Furthermore, the standard deviation (SD) measures the scatter of values
460 around their mean. In a normal distribution, the mean ± 2 SD will cover about 95% of
461 all such values. The RPA value of peak 1 in 10 batches of C. phaeocaulis samples was
462 1.820 to 4.859, and the mean ± 2 SD value was 1.024 to 5.188. If the RPA value of
22
463 peak 1 of the samples was less than 1.024, it could be identified as C. kwangsiensis of
464 Curcumae Radix. On the other hand, if the RPA value of peak 1 of the samples was
465 larger than 1.024, it could be identified as C. phaeocaulis of Curcumae Radix.
466
467 Conclusions
468 In conclusion, we had developed a simple method to evaluate these four species
469 of Curcumae Radix in HPLC assay via determining germacrone in C. wenyujin, C.
470 kwangsiensis and C. phaeocaulis of Curcumae Radix and curcumin,
471 desmethoxycurcumin and bisdesmethoxycurcumin in C. longa of Curcumae Radix by
472 HPLC assay. The result in this study indicated that the samples of C. wenyujin
473 contained the highest amounts of germacrone (625 ± 240 mg/kg) which was about
474 2.8-fold higher than C. kwangsiensis (225 ± 114 mg/kg) and 3.6-fold higher than of C.
475 phaeocaulis (173 ± 34 mg/kg) in HPLC assay. Moreover, we also developed a reliable
476 method to discriminate these four species of Curcumae Radix by HPLC assay and
477 fingerprint analysis. It is a simple way to use the difference of the RPA value in HPLC
478 fingerprint analysis to distinguish C. kwangsiensis and C. phaeocaulis of Curcumae
479 Radix.
480 Although Curcumae Radix, Yujin , the root tubers of four Curcuma species are
481 included in both Chinese Pharmacopoeia (2020 edition) [8] and Taiwan Herbal
482 Pharmacopeia (III), 2018 [9], there is still no common marker for the four Curcuma
483 species of Curcumae Radix. Because of the definite difference of main ingredients
484 between curcumin-free group (C. wenyujin, C. kwangsiensis and C. phaeocaulis) and
485 curcumin containing C. longa. In accordance with the result, four Curcuma species of
486 Curcumae Radix in the herbal pharmacopeia should be separated into two
23
487 monographs, with curcumin-free and germacrone containing species as one and
488 curcumin containing C. longa as the second monograph.
489 Finally, the rapid HPLC/PDA method of HPLC assay and fingerprint analysis
490 developed in this study can be used for the quality control of these four species of
491 Curcumae Radix and can provide references for the future edition of herbal
492 Pharmacopoeia in the world.
493
494 Abbreviations
495 TCM: traditional Chinese medicine; TLC: thin layer chromatography; GC: gas
496 chromatography; HPLC: high performance liquid chromatography; PDA: photodiode
497 array detector; UFLC-Q-TOF-MS: ultra-fast liquid
498 chromatography/quadrupole-time-of-flight tandem mass spectrometry; LOD: Limit of
499 detection; LOQ: Limit of quantitation; RRT: relative retention time; RPA: relative
500 peak area.
501 Declarations
502 Ethics approval and consent to participate: Not applicable. 503 504 Consent for publication: Not applicable.
505 Availability of data and materials: The datasets used and/or analyzed during
506 the current study are available from the corresponding author upon request.
507 Competing interests: The authors declare that they have no competing interests.
508 Funding: A grant from HK Chinese Materia Medica Standard Office, Department
509 of Health, Hong Kong SAR Government supported this study. The funding sources 24
510 had no role in the collection, analysis, and interpretation of the data and in writing the
511 manuscript.
512 Authors’ contribution: YLH and YSC designed the study. CRL, CPK and KCW 513 performed the experiments. KCW and SCL contributed to the analysis of data. CRL 514 wrote the first draft. YLH and YSC reviewed and edited the manuscript. YSC 515 supervised the whole work. All authors read and approved the final manuscript.
516 Acknowledgements: The authors are grateful to Prof. Yu-Cheng Li, Sichuan 517 Institute for Food and Drug Control for collecting Curcumae Radix specimen from the 518 field and herbal markets in China and preparing herbarium specimen of the plant 519 materials.
520
521 References
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627
628
629
630 FIGURE LEGENDS
631 Figure 1 The different medicinal parts of C. phaeocaulis, C. wenyujin, C.
632 kwangsiensis (A) and C. longa (B). The root tubers of these four Curcuma species,
633 Curcumae Radix, were called Yujin. The rhizomes of C. phaeocaulis, C. wenyujin and
634 C. kwangsiensis were called Ezhu. The rhizome of C. longa (turmeric) was called
635 Jianghuang.
636 637 Figure 2 Chemical structures of (i) germacrone, (ii) curdione, (iii)
638 bisdesmethoxycurcumin, (iv) curcumin and (v) desmethoxycurcumin used for HPLC
639 assay and fingerprint analysis of Curcumae Radix.
640 641 Figure 3A The typical HPLC chromatograms (210 nm) of HPLC assay of three
642 Curcuma species (C. wenyujin, C. kwangsiensis and C. phaeocaulis) of Curcumae
643 Radix.
644 A: C. wenyujin; B: C. kwangsiensis; C: C. phaeocaulis; D: marker, germacrone (G).
645 646 Figure 3B The typical HPLC chromatograms (430 nm) of HPLC assay of C. longa of 29
647 Curcumae Radix. A: C. longa; B: Mixture standard of curcumin (C),
648 desmethoxycurcumin (D) and bisdesmethoxycurcumin (B).
649 650 Figure 4A The typical HPLC chromatograms (210 nm) of fingerprint analysis of
651 three Curcuma species (C. wenyujin, C. kwangsiensis and C. phaeocaulis) of
652 Curcumae Radix. A: C. wenyujin; B: C. kwangsiensis; C: C. phaeocaulis; D: marker,
653 germacrone (G); E: marker, curdione (C).
654 655 Figure 4B The typical HPLC chromatograms (335 nm) of HPLC fingerprint analysis
656 of C. longa of Curcumae Radix. A: C. longa; B: Mixture standards of curcumin (C),
657 desmethoxycurcumin (D) and bisdesmethoxycurcumin (B).
658 659 Figure 5A The HPLC fingerprint chromatograms (210 nm) of total 30 batches of
660 three species (C. wenyujin, C. kwangsiensis and C. phaeocaulis) of Curcumae Radix.
661 A: C. wenyujin (W1-W10); B: C. kwangsiensis (K1-K10); C: C. phaeocaulis (P1-P10).
662 Four common characteristic peaks (peak 1–4) were chosen in HPLC fingerprint
663 chromatograms. Peak 3, germacrone (G) was chosen as the marker peak.
664 665 Figure 5B The HPLC fingerprint chromatograms (335 nm) of total 10 batches of C.
666 longa of Curcumae Radix. C. longa (L1-L10). Four common characteristic peaks
667 (peak 1–4) were chosen in HPLC fingerprint chromatograms. Peak 2 (C): curcumin;
668 Peak 3 (D): desmethoxycurcumin; Peak 4 (B): bisdesmethoxycurcumin. Peak 2,
669 curcumin (C) was chosen as the marker peak.
670
671 Figure S1 The chromatograms of three species (C. wenyujin, C. kwangsiensis and C.
672 phaeocaulis) of Curcumae Radix extracted with 50% methanol for four times.
673 30
674 Figure S2 The chromatograms of C. longa of Curcumae Radix extracted with 75%
675 ethanol for three times.
676 (C): Curcumin
677 (D): Desmethoxycurcumin
678 (B): Bisdesmethoxycurcumin
31
Figures
Figure 1
The different medicinal parts of C. phaeocaulis, C. wenyujin, C. kwangsiensis (A) and C. longa (B). The root tubers of these four Curcuma species, Curcumae Radix, were called Yujin. The rhizomes of C. phaeocaulis, C. wenyujin and C. kwangsiensis were called Ezhu. The rhizome of C. longa (turmeric) was called Jianghuang.
Figure 2
Chemical structures of (i) germacrone, (ii) curdione, (iii) bisdesmethoxycurcumin, (iv) curcumin and (v) desmethoxycurcumin used for HPLC assay and ngerprint analysis of Curcumae Radix. Figure 3
A The typical HPLC chromatograms (210 nm) of HPLC assay of three Curcuma species (C. wenyujin, C. kwangsiensis and C. phaeocaulis) of Curcumae Radix. A: C. wenyujin; B: C. kwangsiensis; C: C. phaeocaulis; D: marker, germacrone (G). B The typical HPLC chromatograms (430 nm) of HPLC assay of C. longa of Curcumae Radix. A: C. longa; B: Mixture standard of curcumin (C), desmethoxycurcumin (D) and bisdesmethoxycurcumin (B). Figure 4
A The typical HPLC chromatograms (210 nm) of ngerprint analysis of three Curcuma species (C. wenyujin, C. kwangsiensis and C. phaeocaulis) of Curcumae Radix. A: C. wenyujin; B: C. kwangsiensis; C: C. phaeocaulis; D: marker, germacrone (G); E: marker, curdione (C). B The typical HPLC chromatograms (335 nm) of HPLC ngerprint analysis of C. longa of Curcumae Radix. A: C. longa; B: Mixture standards of curcumin (C), desmethoxycurcumin (D) and bisdesmethoxycurcumin (B). Figure 5
A The HPLC ngerprint chromatograms (210 nm) of total 30 batches of three species (C. wenyujin, C. kwangsiensis and C. phaeocaulis) of Curcumae Radix. A: C. wenyujin (W1-W10); B: C. kwangsiensis (K1- K10); C: C. phaeocaulis (P1-P10). Four common characteristic peaks (peak 1–4) were chosen in HPLC ngerprint chromatograms. Peak 3, germacrone (G) was chosen as the marker peak. B The HPLC ngerprint chromatograms (335 nm) of total 10 batches of C. longa of Curcumae Radix. C. longa (L1- L10). Four common characteristic peaks (peak 1–4) were chosen in HPLC ngerprint chromatograms. Peak 2 (C): curcumin; Peak 3 (D): desmethoxycurcumin; Peak 4 (B): bisdesmethoxycurcumin. Peak 2, curcumin (C) was chosen as the marker peak.
Supplementary Files
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CurcumaeradixmanuscriptSupplementaldocuments08222021.pdf