Abstract

A simple and intuitive method for optimizing the chemical constituents of Coptis Chinensis Franch. is important to assess its quality and clinical efficacy. An high performance liquid chromatography and ultraviolet spectrophotometry method was developed for the determination of berberine hydrochloride, palmatine chloride, jatrorrhizine hydrochloride, epiberberine, coptisine, columbamine and magnoflorine in various tissues (i.e., phloem, xylem and medulla) and rizhome of C. Chinensis Franch. The transection of rhizome from outside–in includes cork layer, cortex, phloem, cambium, xylem and medulla. Cork layer consists of dead cells, and therefore is not of any research significance. Cortex, phloem and cambium were almost impossible to separate, therefore they were studied as a whole in our experiments. They were collectively referred to as “phloem”. The analytes were separated on a Gemini-NX C18 (250 mm × 4.6 mm, 5 μm) reversed phase column using a gradient elution of acetonitrile–0.03 mol/L ammonium acetate solution (containing 0.1% triethylamine and 0.6% ammonium hydroxide) as the mobile phase at a flow rate of 1.0 mL/min and UV detection at 270 nm. The method allowing the simultaneous quantification of seven major active constituents was optimized and validated for linearity, precision, accuracy, limits of detection (LOD) and quantification. The LOD ranged from 0.102 to 0.651 mg/mL (r ≥ 0.9993). Accuracy, precision and recovery were all within the required limits. The average recovery was between 100.14% and 102.75% and the relative standard deviations were <3.34%. At the same time, the absorbance was determined by ultraviolet spectrophotometry at 345 nm wavelength. Based on contents of the seven constituents and clustering result, this investigation suggests that there are significant differences in the distribution of seven alkaloids in the tissues examined. Furthermore, the total alkaloid content in xylem is relatively lower than that in phloem, medulla and rhizome.

Introduction

Medicinal material Coptis chinensis is the dried rhizome derived from the Ranunculaceae family of plants Coptidis chinensis Franch. or Coptis deltoide C.Y. Cheng et Hsiao or Coptis teeta Wall., respectively called “Weilian,” “Yalian” and “Yunlian,” as listed in Chinese Pharmacopeia (version 2010). This herb has heat clearing, dampness drying, fire draining and detoxification properties (1). Coptis chinensis has been commonly used in Traditional Chinese Medicine (TCM) since ancient times and was commonly recommended for inflammatory diseases by famous physicians like Shizhen Li, Shenwei Tang and Hongjing Tao in the TCM history (2). Historically, Sichuan is the main production area for C. chinensis and the geo-authentic crude drug from it (3, 4). Modern chemical research has shown that alkaloid components in C. chinensis are the main pharmacologically active substances (5). Recent pharmacological studies indicated that the alkaloids from C. chinensis showed antibacterial (6, 7), hypoglycemic, antitumor, immune regulatory, anti-inflammatory (8), anticancer (9) and antiviral (10) activities and it can also be used for the treatment of oxidative stress associated with neurodegenerative disease (1113). In clinical applications, C. chinensis and its chemical constituents were often used in the treatment of infectious diseases, insomnia, mental diseases, arrhythmia, hypertension, diabetes and its complications, cerebrovascular diseases, etc. (10). Traditionally, C. chinensis Franch. with thick and massy rhizome, orange-red phloem, and bright or orange yellow xylem are identified as the superior quality plant for medicinal purposes. In order to clearly understand the significance of these features, we undertook this study. Thus, the content analysis on the alkaloids in various tissues of C. chinensis Franch. is paramount. The amount of alkaloids in C. chinensis Franch. directly influence the efficacy of the treatment and therefore breeding of good varieties of this herb is important. A plethora of research has been conducted on this species of plant; however, the content of pharmacologically active alkaloids in various tissues of this plant has not been investigated (14, 15). In order to reasonably guide people to most effective treatment, optimization of total alkaloid contents in various tissues of C. chinensis Franch. is warranted. In this paper, for the first time, we report the contents of pharmacologically active alkaloids, viz. berberine hydrochloride, palmatine chloride, jatrorrhizine hydrochloride, epiberberine, coptisine, columbamine and magnoflorine in various tissues of C. chinensis Franch. after analyzing them by reversed phase-high performance liquid chromatography (RP-HPLC) and ultraviolet spectrophotometry.

Experiment

Materials and reagents

Seventeen batches of C. chinensis Franch. were collected from various locations in Sichuan. The tissue locations and percent composition of seven major pharmacologically active constituents are listed in Table I. The collected plants were authenticated by experts and the samples met the standard of the Chinese Pharmacopoeia. The voucher samples of our collection were deposited at the herbarium of the Key Laboratory of Resource Science and Chemistry in Chinese Medicine Hubei Province, Hubei University of Chinese Medicine, Wuhan, Hubei, China.

Table I.

Studied Samples and % Content of Seven Active Alkaloids in C. chinensis Franch.

Number Locations Type of tissue and rhizome part Ma Jh Cb Eb Cp Pc Bh Total alkaloid 
Four group, Jinchuan village, Longchi town, EMei city, Sichuan province Phloem 0.899 0.711 1.555 3.035 3.276 3.835 11.158 0.520 
Xylem 1.035 0.148 0.242 0.664 0.912 0.856 4.153 0.156 
Medulla 0.573 0.832 1.799 3.076 2.412 5.181 14.487 0.559 
Rhizome 1.011 0.541 0.801 2.269 2.350 2.589 10.511 0.407 
Five group, Jinchuan village, Longchi town, EMei city, Sichuan province Phloem 0.417 0.440 1.154 3.065 3.389 2.743 10.291 0.491 
Xylem 0.484 0.170 0.253 0.905 1.196 0.841 5.067 0.168 
Medulla 1.073 0.648 1.335 2.650 2.197 4.393 13.605 0.505 
Rhizome 0.804 0.453 0.892 2.563 2.617 2.446 8.960 0.353 
Two group, Jinchuan village, Longchi town, EMei city, Sichuan province Phloem 0.510 1.050 2.082 4.008 4.521 5.076 12.974 0.621 
Xylem 0.347 0.278 0.193 0.921 1.105 0.740 4.237 0.177 
Medulla 0.291 0.822 0.973 3.396 2.448 3.977 14.451 0.538 
Rhizome 0.755 0.506 0.825 2.745 3.225 2.774 11.332 0.473 
Two group, Fuyou village, Longchi town, EMei city, Sichuan province Phloem 0.682 0.466 0.574 3.164 4.274 2.230 11.375 0.471 
Xylem 1.198 0.142 0.118 0.617 1.002 0.568 3.536 0.155 
Medulla 0.659 0.752 0.885 2.622 2.940 3.874 14.525 0.586 
Rhizome 0.705 0.495 0.600 2.580 3.689 1.949 10.080 0.413 
Eight group, Fuyou village, Longchi town, EMei city, Sichuan province Phloem 0.955 0.634 1.376 3.288 3.589 3.736 13.185 0.546 
Xylem 0.575 0.117 0.196 0.584 0.790 0.679 4.042 0.171 
Medulla 0.524 0.853 1.759 2.178 1.967 5.098 14.355 0.566 
Rhizome 0.552 0.679 0.803 1.902 2.705 2.762 8.780 0.389 
Four group, Fuyou village, Longchi town, EMei city, Sichuan province Phloem 0.814 0.449 0.420 1.423 2.257 1.700 9.470 0.364 
Xylem 0.730 0.253 0.230 0.617 0.961 0.970 5.741 0.202 
Medulla 0.533 0.664 0.855 2.158 1.960 3.932 13.907 0.461 
Rhizome 1.006 0.456 0.570 2.225 3.080 2.058 9.129 0.427 
Five group, Fuyou village, Longchi town, EMei city, Sichuan province Phloem 0.596 0.618 1.740 3.351 3.299 4.203 10.647 0.502 
Xylem 0.504 0.118 0.196 0.756 0.820 0.775 3.727 0.140 
Medulla 0.871 0.603 1.649 3.253 1.871 5.138 11.718 0.528 
Rhizome 0.499 0.518 0.781 1.881 2.295 2.309 7.846 0.398 
Three group, Sangou village, Longmenshan Town, Pengzhou city, Sichuan province Phloem 0.543 0.643 1.137 3.267 2.805 3.332 9.769 0.428 
Xylem 0.943 0.257 0.282 0.796 0.929 0.928 4.180 0.167 
Medulla 0.757 0.648 1.091 2.040 1.906 3.884 11.291 0.638 
Rhizome 0.782 0.537 0.481 2.539 2.323 1.900 7.549 0.313 
Four group, Guoping village, Longmenshan town, Pengzhou city, Sichuan province Phloem 0.487 0.664 0.891 3.668 2.941 2.970 8.809 0.447 
Xylem 0.563 0.117 0.140 0.742 0.685 0.506 2.582 0.119 
Medulla 0.365 0.582 0.790 3.007 1.523 3.202 9.102 0.337 
Rhizome 0.461 0.485 0.629 2.531 2.010 2.133 6.637 0.371 
10 Eight group, Wannian village, Huangwan town, EMei city, Sichuan province Phloem 0.578 0.631 0.884 3.102 3.156 2.762 11.077 0.474 
Xylem 0.753 0.331 0.303 1.088 1.110 1.125 4.944 0.196 
Medulla 0.703 0.813 0.745 2.417 2.041 3.357 11.991 0.478 
Rhizome 0.716 0.424 0.535 2.611 2.752 2.183 9.985 0.413 
11 Seven group, Wannian village, Huangwan town, EMei city, Sichuan province Phloem 0.348 0.305 0.802 2.213 3.094 3.092 12.747 0.598 
Xylem 0.244 0.056 0.112 0.465 0.362 0.414 3.972 0.170 
Medulla 0.462 0.318 0.552 2.495 1.469 3.855 14.515 0.565 
Rhizome 0.656 0.533 0.890 3.554 3.090 2.604 9.437 0.460 
12 Two group, Heishan village, Gaomiao town, Hongya county, Sichuan province Phloem 0.447 0.136 0.232 2.049 3.052 1.282 10.539 0.436 
Xylem 0.365 0.058 0.114 0.338 0.435 0.274 2.771 0.133 
Medulla 0.578 0.450 0.715 2.583 2.080 2.836 13.922 0.461 
Rhizome 0.593 0.497 0.804 2.188 2.502 2.415 7.405 0.354 
13 Six group, Jinchuan village, Hongya county, EMei city, Sichuan province Phloem 0.937 0.489 0.731 3.239 3.874 2.385 12.286 0.517 
Xylem 0.868 0.121 0.165 0.637 0.731 0.522 3.381 0.138 
Medulla 0.564 0.545 0.669 2.018 2.444 3.137 12.973 0.485 
Rhizome 0.575 0.483 0.654 2.484 2.830 2.522 10.154 0.433 
14 Three group,Tiantaishan village, Bailu town, Pengzhou city, Sichuan province Phloem 0.322 0.585 1.100 3.398 3.184 2.823 9.706 0.426 
Xylem 0.682 0.206 0.265 0.945 1.027 0.896 4.692 0.196 
Medulla 0.257 0.736 1.423 2.330 1.848 4.436 12.334 0.513 
Rhizome 0.778 0.622 0.896 3.330 2.628 2.770 9.299 0.441 
15 Five group, Xianfeng village, Fandian town, Leshan city, Sichuan province Phloem 0.460 0.752 1.330 2.573 3.055 4.314 12.083 0.516 
Xylem 0.595 0.225 0.230 0.380 0.840 0.837 3.791 0.133 
Medulla 0.292 0.796 0.799 1.657 1.785 3.934 11.639 0.437 
Rhizome 0.454 0.763 1.695 1.768 2.784 3.784 12.129 0.476 
16 Six group, Xianfeng village, Fandian town, Leshan city, Sichuan province Phloem 0.993 0.538 0.868 6.225 4.619 2.915 14.403 0.602 
Xylem 0.739 0.179 0.167 1.767 1.556 0.892 5.698 0.207 
Medulla 0.522 0.766 0.953 4.499 2.794 4.425 14.506 0.595 
Rhizome 0.596 0.553 1.003 2.790 3.248 3.028 11.588 0.478 
17 One group,Tianbao village, Yinghua town, Shifang city, Sichuan province Phloem 0.519 0.403 0.826 4.071 3.013 2.646 8.607 0.411 
Xylem 0.604 0.154 0.213 0.635 0.970 0.772 3.588 0.165 
Medulla 0.343 0.480 0.711 2.828 1.610 3.318 9.150 0.389 
Rhizome 0.506 0.495 0.763 2.355 2.879 2.434 8.617 0.415 
Number Locations Type of tissue and rhizome part Ma Jh Cb Eb Cp Pc Bh Total alkaloid 
Four group, Jinchuan village, Longchi town, EMei city, Sichuan province Phloem 0.899 0.711 1.555 3.035 3.276 3.835 11.158 0.520 
Xylem 1.035 0.148 0.242 0.664 0.912 0.856 4.153 0.156 
Medulla 0.573 0.832 1.799 3.076 2.412 5.181 14.487 0.559 
Rhizome 1.011 0.541 0.801 2.269 2.350 2.589 10.511 0.407 
Five group, Jinchuan village, Longchi town, EMei city, Sichuan province Phloem 0.417 0.440 1.154 3.065 3.389 2.743 10.291 0.491 
Xylem 0.484 0.170 0.253 0.905 1.196 0.841 5.067 0.168 
Medulla 1.073 0.648 1.335 2.650 2.197 4.393 13.605 0.505 
Rhizome 0.804 0.453 0.892 2.563 2.617 2.446 8.960 0.353 
Two group, Jinchuan village, Longchi town, EMei city, Sichuan province Phloem 0.510 1.050 2.082 4.008 4.521 5.076 12.974 0.621 
Xylem 0.347 0.278 0.193 0.921 1.105 0.740 4.237 0.177 
Medulla 0.291 0.822 0.973 3.396 2.448 3.977 14.451 0.538 
Rhizome 0.755 0.506 0.825 2.745 3.225 2.774 11.332 0.473 
Two group, Fuyou village, Longchi town, EMei city, Sichuan province Phloem 0.682 0.466 0.574 3.164 4.274 2.230 11.375 0.471 
Xylem 1.198 0.142 0.118 0.617 1.002 0.568 3.536 0.155 
Medulla 0.659 0.752 0.885 2.622 2.940 3.874 14.525 0.586 
Rhizome 0.705 0.495 0.600 2.580 3.689 1.949 10.080 0.413 
Eight group, Fuyou village, Longchi town, EMei city, Sichuan province Phloem 0.955 0.634 1.376 3.288 3.589 3.736 13.185 0.546 
Xylem 0.575 0.117 0.196 0.584 0.790 0.679 4.042 0.171 
Medulla 0.524 0.853 1.759 2.178 1.967 5.098 14.355 0.566 
Rhizome 0.552 0.679 0.803 1.902 2.705 2.762 8.780 0.389 
Four group, Fuyou village, Longchi town, EMei city, Sichuan province Phloem 0.814 0.449 0.420 1.423 2.257 1.700 9.470 0.364 
Xylem 0.730 0.253 0.230 0.617 0.961 0.970 5.741 0.202 
Medulla 0.533 0.664 0.855 2.158 1.960 3.932 13.907 0.461 
Rhizome 1.006 0.456 0.570 2.225 3.080 2.058 9.129 0.427 
Five group, Fuyou village, Longchi town, EMei city, Sichuan province Phloem 0.596 0.618 1.740 3.351 3.299 4.203 10.647 0.502 
Xylem 0.504 0.118 0.196 0.756 0.820 0.775 3.727 0.140 
Medulla 0.871 0.603 1.649 3.253 1.871 5.138 11.718 0.528 
Rhizome 0.499 0.518 0.781 1.881 2.295 2.309 7.846 0.398 
Three group, Sangou village, Longmenshan Town, Pengzhou city, Sichuan province Phloem 0.543 0.643 1.137 3.267 2.805 3.332 9.769 0.428 
Xylem 0.943 0.257 0.282 0.796 0.929 0.928 4.180 0.167 
Medulla 0.757 0.648 1.091 2.040 1.906 3.884 11.291 0.638 
Rhizome 0.782 0.537 0.481 2.539 2.323 1.900 7.549 0.313 
Four group, Guoping village, Longmenshan town, Pengzhou city, Sichuan province Phloem 0.487 0.664 0.891 3.668 2.941 2.970 8.809 0.447 
Xylem 0.563 0.117 0.140 0.742 0.685 0.506 2.582 0.119 
Medulla 0.365 0.582 0.790 3.007 1.523 3.202 9.102 0.337 
Rhizome 0.461 0.485 0.629 2.531 2.010 2.133 6.637 0.371 
10 Eight group, Wannian village, Huangwan town, EMei city, Sichuan province Phloem 0.578 0.631 0.884 3.102 3.156 2.762 11.077 0.474 
Xylem 0.753 0.331 0.303 1.088 1.110 1.125 4.944 0.196 
Medulla 0.703 0.813 0.745 2.417 2.041 3.357 11.991 0.478 
Rhizome 0.716 0.424 0.535 2.611 2.752 2.183 9.985 0.413 
11 Seven group, Wannian village, Huangwan town, EMei city, Sichuan province Phloem 0.348 0.305 0.802 2.213 3.094 3.092 12.747 0.598 
Xylem 0.244 0.056 0.112 0.465 0.362 0.414 3.972 0.170 
Medulla 0.462 0.318 0.552 2.495 1.469 3.855 14.515 0.565 
Rhizome 0.656 0.533 0.890 3.554 3.090 2.604 9.437 0.460 
12 Two group, Heishan village, Gaomiao town, Hongya county, Sichuan province Phloem 0.447 0.136 0.232 2.049 3.052 1.282 10.539 0.436 
Xylem 0.365 0.058 0.114 0.338 0.435 0.274 2.771 0.133 
Medulla 0.578 0.450 0.715 2.583 2.080 2.836 13.922 0.461 
Rhizome 0.593 0.497 0.804 2.188 2.502 2.415 7.405 0.354 
13 Six group, Jinchuan village, Hongya county, EMei city, Sichuan province Phloem 0.937 0.489 0.731 3.239 3.874 2.385 12.286 0.517 
Xylem 0.868 0.121 0.165 0.637 0.731 0.522 3.381 0.138 
Medulla 0.564 0.545 0.669 2.018 2.444 3.137 12.973 0.485 
Rhizome 0.575 0.483 0.654 2.484 2.830 2.522 10.154 0.433 
14 Three group,Tiantaishan village, Bailu town, Pengzhou city, Sichuan province Phloem 0.322 0.585 1.100 3.398 3.184 2.823 9.706 0.426 
Xylem 0.682 0.206 0.265 0.945 1.027 0.896 4.692 0.196 
Medulla 0.257 0.736 1.423 2.330 1.848 4.436 12.334 0.513 
Rhizome 0.778 0.622 0.896 3.330 2.628 2.770 9.299 0.441 
15 Five group, Xianfeng village, Fandian town, Leshan city, Sichuan province Phloem 0.460 0.752 1.330 2.573 3.055 4.314 12.083 0.516 
Xylem 0.595 0.225 0.230 0.380 0.840 0.837 3.791 0.133 
Medulla 0.292 0.796 0.799 1.657 1.785 3.934 11.639 0.437 
Rhizome 0.454 0.763 1.695 1.768 2.784 3.784 12.129 0.476 
16 Six group, Xianfeng village, Fandian town, Leshan city, Sichuan province Phloem 0.993 0.538 0.868 6.225 4.619 2.915 14.403 0.602 
Xylem 0.739 0.179 0.167 1.767 1.556 0.892 5.698 0.207 
Medulla 0.522 0.766 0.953 4.499 2.794 4.425 14.506 0.595 
Rhizome 0.596 0.553 1.003 2.790 3.248 3.028 11.588 0.478 
17 One group,Tianbao village, Yinghua town, Shifang city, Sichuan province Phloem 0.519 0.403 0.826 4.071 3.013 2.646 8.607 0.411 
Xylem 0.604 0.154 0.213 0.635 0.970 0.772 3.588 0.165 
Medulla 0.343 0.480 0.711 2.828 1.610 3.318 9.150 0.389 
Rhizome 0.506 0.495 0.763 2.355 2.879 2.434 8.617 0.415 

Standard samples of berberine hydrochloride (batch number 110713-201212), palmatine chloride (batch number 110732-201108) and jatrorrhizine hydrochloride (batch number 110733-201108) were bought from China Pharmaceutical Biological Product Assaying Institute. Epiberberine and coptisine were bought from Shanghai Yuanye Biological Technology Co., Ltd (Shanghai, China), while columbamine and magnoflorine were purchased from Chengdu Pufei De Biotech Co., Ltd (Sichuan, China). The purity of all reference substances was higher than 98%. Hydrochloric acid and methanol (both from Guoyao, Shanghai, China) were used for the preparation of samples. HPLC-grade acetonitrile (TEDIA, Company Inc. USA), triethylamine (Guoyao, Shanghai, china) and ammonium hydroxide (Guoyao, Shanghai, china) were used for chromatographic experiments. Filters (Nylon 66, 13 mm, 0.22 μm) were bought from Jinteng Laboratory Equipment Co., Ltd (Tianjing, China). Deionized water was purified by a Milli-Q Integral System (Millipore Corporation, USA).

Apparatus and chromatographic conditions

Agilent 1260 HPLC system, UV-1800 ultraviolet and visible spectrophotometer (Shimadzu Corp.), Milli-Q Integral water purification system (Millipore Corp.), and KQ-250B-type supersonic cleaning device (Kunshan Supersonic Instrument Corp., Ltd) were employed in this investigation. Chromatographic separations were conducted on a Gemini-NX C18 reversed phase column (5μm, 250 × 4.6 mm ID, Phenomenex, USA). The detections were conducted at 270 nm at the flow rate of 1.0 mL/min when the column temperature was maintained at 30°C. The mobile phase was in gradient elution, which consisted of Solvents A (acetonitrile) and B (0.03 mol/L ammonium acetate solution containing 0.1% triethylamine and 0.6% ammonium hydroxide, v/v). The gradient program was optimized and conducted as follows: 0 min–15 min, 10–25% A; 15 min–25 min, 25–27% A; 25 min–40 min, 27–45% A; and 40 min–45 min, 45–90% A. An aliquot of 10 μL of the filtrate was injected into the HPLC for analysis. The 3D plot (wavelength vs. time vs. peak height) of chromatographic run using photodiode array detector of the Rhizoma coptidis samples, and the ultraviolet spectra of standard samples of magnoflorine, jatrorrhizine hydrochloride, columbamine, epiberberine, coptisine, palmatine chloride and berberine hydrochloride were recorded from 200 to 400 nm, and are presented in Figure 1. Considering the UV peak maxima of seven analytes, 270 nm was selected as the detection wavelength. Subsequently, the total alkaloid content determinations were conducted on Shimadzu UV-1800 spectrophotometer (Shimadzu Technologies, Japan), using the corresponding reagent solutions [hydrochloric acid–methanol (v/v = 1:100)] as the blank reference to determine its absorbance at the wavelength 345 nm (16).

Figure 1.

(A) The 3D plot of chromatographic run of Rhizoma coptidis samples. (B) The UV spectra of magnoflorine (Ma), jatrorrhizine hydrochloride (Jh), columbamine (Cb), epiberberine (Eb), coptisine (Cp), palmatine chloride (Pc) and berberine hydrochloride (Bh) recorded between 200 and 400 nm. (C) Representative HPLC chromatograms of the seven components in standard solution (a) and the sample solution of the rhizome (b), phloem (c),medulla (d) and xylem (e).

Figure 1.

(A) The 3D plot of chromatographic run of Rhizoma coptidis samples. (B) The UV spectra of magnoflorine (Ma), jatrorrhizine hydrochloride (Jh), columbamine (Cb), epiberberine (Eb), coptisine (Cp), palmatine chloride (Pc) and berberine hydrochloride (Bh) recorded between 200 and 400 nm. (C) Representative HPLC chromatograms of the seven components in standard solution (a) and the sample solution of the rhizome (b), phloem (c),medulla (d) and xylem (e).

Preparation of standard solutions

Standard stock solutions of the seven reference standards (magnoflorine, jatrorrhizine hydrochloride, columbamine, epiberberine, coptisine, palmatine chloride and berberine hydrochloride) were prepared by dissolving them in hydrochloric acid–methanol (v/v = 1:100). These were then diluted to seven concentrations for the construction of calibration plots in the ranges of 1.464–29.28, 1.086–21.72 , 2.184–43.68, 6.304–126.1, 5.502–110.0, 5.292–105.8 and 14.54–290.9 µg/mL, respectively. Further dilution with the lowest concentrations in the calibration curves were carried out to provide a series of standard solutions for evaluating the limits of detection (LOD) of the compounds. The stock and working solutions were stored at 4°C.

Sample preparation

All plant samples were over-dried at 50°C. A knife was used to carefully brush away the impurities on the surface of the 17 batches of dried C. chinensis Franch. samples. Subsequently, phloem, xylem and medulla part were carefully separated from each batch and ground into powder. Also, for every batch, one sample of entire rhizome was ground into powder. Approximately 0.100 g of each dried sample was weighed and placed in the 100 mL conical flask to which 50 mL of hydrochloric acid–methanol (v/v = 1:100) was added and the total weight was measured. Ultrasonic extraction (power 200 W, frequency 40 kHz) was performed on this mixture for 30 min at room temperature. The mixture was weighed again and hydrochloric acid–methanol (v/v = 1:100) was added to make up for the weight loss. The mixtures at this stage for all samples were shaken and filtered. All filtrates were collected and were subjected to ultraviolet spectrophotometric analysis. For RP-HPLC analysis, a portion of solution was filtered through a 0.22 µm membrane filter, and 10 µL of the filtrate was injected into the HPLC system.

Method validation

Linearity

Under optimal chromatographic conditions, calibration plot was obtained of each alkaloid by injecting a standard solution with seven different concentrations, and this was performed in triplicate. The calibration plots for each compound were plotted based on the linear regression analysis of the integrated peak areas (Y) versus concentrations (X) of the standard. The regression equation was calculated in the form Y = aX + b, where Y and X are the values of the peak area and sample amount, respectively. The standard solution was diluted with hydrochloric acid–methanol (v/v = 1:100) to provide appropriate concentrations.

LOD and LOQ

The LOD and limit of quantitation (LOQ) of the individual compounds were calculated based on a signal-to-noise (S/N) ratio ≥ 3, using following formulas:  

LOD=3×c(SN),LOQ=c(SN),
where c = concentration. The data are presented in Table II.
Table II.

HPLC and Validation Data for the Calibration Graphs and LOQ of the Seven Alkaloids

Alkaloids Regression equation r Linear rang/μg LOD (mg/mL) LOQ (mg/mL) Precision RSD (%, n = 6) Stability RSD (%, 24 h, n = 6) 
Intraday Interday Instrument precision 
Magnoflorine Y = 437.82X− 1.859 0.9997 0.01464–0.2928 0.377 1.260 1.40 0.92 1.50 1.54 
Jatrorrhizine hydrochloride Y = 2539.1X − 6.1978 0.9995 0.01086–0.2172 0.102 0.340 0.48 0.59 1.09 2.41 
Columbamine Y = 3502.7X − 2.9487 0.9993 0.02184–0.4368 0.218 0.727 0.89 1.20 0.80 1.30 
Epiberberine Y = 2069.2X− 13.695 0.9996 0.06304–1.2608 0.615 2.051 0.64 1.60 0.87 2.29 
Coptisine Y = 2948.5X − 15.462 0.9997 0.05502–1.1004 0.265 0.884 0.97 1.10 0.86 2.27 
Palmatine chloride Y = 3282.9X − 49.549 0.9996 0.05292–1.0584 0.313 1.044 1.80 0.90 1.78 2.21 
Berberine hydrochloride Y = 3275.5X− 52.758 0.9996 0.14544–2.9088 0.651 2.171 1.20 0.87 0.74 1.43 
Alkaloids Regression equation r Linear rang/μg LOD (mg/mL) LOQ (mg/mL) Precision RSD (%, n = 6) Stability RSD (%, 24 h, n = 6) 
Intraday Interday Instrument precision 
Magnoflorine Y = 437.82X− 1.859 0.9997 0.01464–0.2928 0.377 1.260 1.40 0.92 1.50 1.54 
Jatrorrhizine hydrochloride Y = 2539.1X − 6.1978 0.9995 0.01086–0.2172 0.102 0.340 0.48 0.59 1.09 2.41 
Columbamine Y = 3502.7X − 2.9487 0.9993 0.02184–0.4368 0.218 0.727 0.89 1.20 0.80 1.30 
Epiberberine Y = 2069.2X− 13.695 0.9996 0.06304–1.2608 0.615 2.051 0.64 1.60 0.87 2.29 
Coptisine Y = 2948.5X − 15.462 0.9997 0.05502–1.1004 0.265 0.884 0.97 1.10 0.86 2.27 
Palmatine chloride Y = 3282.9X − 49.549 0.9996 0.05292–1.0584 0.313 1.044 1.80 0.90 1.78 2.21 
Berberine hydrochloride Y = 3275.5X− 52.758 0.9996 0.14544–2.9088 0.651 2.171 1.20 0.87 0.74 1.43 

Precision, stability and recovery

Coptis chinensis Franch. was selected as a sample to validate the method. The intraday and interday variations for determining the precision of the developed method were evaluated based on the results of six replicate analyses in a single day and duplicating the experiments over a 3-day period. Instrument precision was determined with standard solutions based on the results of six replicate analyses. Variations of the peak area were taken as the measure of precision and were expressed as percent relative standard deviations (RSD).

Sample stability test was determined with one sample at 0, 2, 4, 6, 8, 12 and 24 h. The solution was stored at room temperature. The stability of the method was expressed as the RSD of the data set.

To determine the accuracy of the developed method, the recovery experiments were performed by adding equimultiple reference substances to the samples as well as extracting and quantifying them according to the established procedure (1).

Results

Optimization of extraction method

In order to extract the active compounds efficiently, two procedures of extraction, ultrasonic and refluxing extraction, were investigated. Upon sample preparation from these two procedures, followed by injection into the HPLC system, and calculating the content of alkaloids by peak areas, the result showed that the ultrasonic extraction method is more conducive for the dissolution of test samples. In order to optimize the ultrasonic extraction process, it was performed using several extraction solvents (ethanol, methanol, hydrochloric acid–methanol (v/v = 1:100)), solvent volumes (50, 70 and 100 mL) and extraction time (30, 40 and 60 min). The optimized conditions are described in the Sample preparation section.

Optimization of the chromatographic conditions

Peak resolution is an extremely important criterion for efficient separation of analytes on an HPLC system within a short analysis time (17). In this study, to obtain better resolution and peak shape, numerous HPLC parameters such as the mobile phase, elution condition, analysis time, chromatographic column and column temperature were examined and their results were compared.

Two mobile phases, namely isocratic elution of 50:50 acetonitrile–0.1% aq. phosphoric acid, acetonitrile–0.05% monopotassium phosphate with 0.4 g sodium dodecyl sulfate added per 100 mL to regulate the pH value of 4.0 in phosphoric acid (version 2010 of the People's Republic of China pharmacopoeia method) and gradient elution of acetonitrile–0.03 mol/L ammonium acetate solution (containing 0.1% triethylamine, 0.6% ammonium hydroxide) were investigated. The 0.1% (v/v) triethylamine and 0.6% ammonium hydroxide was added to the later to improve the peak shape. The results showed that acetonitrile–0.03 mol/L ammonium acetate solution (containing 0.1% triethylamine, 0.6% ammonium hydroxide) as mobile phase provided lower pressure and greater baseline stability. Under these experimental conditions, the retention times of the seven alkaloids of interest were within 45 min of analysis time, and the peak resolution was satisfactory.

In these experiments, three chromatographic columns, viz. Agilent Eclipse XDB C18 (5 μm, 250 × 4.6 mm), Agilent Eclipse SB C18 (5 μm, 250 × 4.6 mm) and Gemini-NX C18 (5 μm, 250 × 4.6 mm) were evaluated for their performance. The first two chromatographic columns allow the pH range of 2–9; however, the Gemini-NX C18 column allows the pH in the range of 2–13. Based on to the degree of separation of peaks in the results, Gemini-NX C18 was selected for the all analyses.

The mobile phase consisted of acetonitrile (A) and 0.03 mol/L ammonium acetate solution (B). Considering that seven test compounds have different chemical structures, properties and absorption spectra, it is difficult to resolve the baseline under isocratic elution. Thus, gradient elution was used in HPLC analysis, and based on the optimization results on these samples following gradient composition was used for all analyses: 0–15 min, 10–25% A; 15–25 min, 25–27% A; 25–40 min, 27–45% A; 40–45 min, 45–90% A.

The column temperature was optimized next. RP-HPLC separations were carried out with gradient elution using acetonitrile–0.03 mol/L ammonium acetate solution (containing 0.1% triethylamine and 0.6% ammonium hydroxide) as mobile phase and using the selected Gemini-NX C18 column at 25, 30 and 35°C column temperatures. Short analysis time, excellent peak resolutions and good peak shapes were obtained under these conditions at the column temperature of 30°C.

As indicated in Figure 1, for a 10 μL sample feed, the best chromatographic condition used Gemini-NX C18 chromatographic column (5 μm, 4.6 × 250 mm) and acetonitrile–0.03 mol/L ammonium acetate solution as mobile phase under gradient elution. The flow velocity and the column temperature were, respectively, maintained at 1.0 mL/min at 30°C.

Under the above-mentioned optimized chromatographic conditions, the degree of separation of each adjacent chromatographic peak were all >1.5, the tailing factor was between 0.95 and 1.05, the theoretical pedaling numbers were all over 8,000, all marker compounds were easily detected and showed adequate absorption.

Method validation

The method was validated by the linearity, precision, stability of the results. Regression equations were derived from the external standard method. The calculated results are listed in Table II. R2 in Table II refers to the correlation coefficient of the equation. All the standard compounds showed good linearity in a relatively wide concentration range. The LOD ranged from 0.102 to 0.651 mg/mL.

The RSD was taken as a measure of precision. The data showed that the RSD of interday, interday and instrument precision variabilities ranged from 0.48 to 1.8%, 0.59 to 1.6% and 0.74 to 1.78%, respectively. For stability test, the same sample solution was analyzed for 24 h at room temperature, and the results indicated that seven alkaloids remained stable (RSD ≤2.41%, Table II). Meanwhile, a standard addition test at the same concentration level was performed to determine the recovery of each analyte, in which Sample 1 (Table I) was used as the matrix for quantitative analysis. The average recoveries were calculated by the formula:  

recovery(%)=(amountfoundoriginalspiked)amountspiked×100%.

The results showed that the recoveries ranged from 97.03 to 106.19% within RSD of 3.34% (Table III).

Table III.

Recovery of the Seven Major Components

Alkaloids Original (mg/mL) Spiked (mg/mL) Found (mg/mL) Recovery (%) Mean (%) RSD (n = 6) 
Magnoflorine 0.01804 0.01760 0.03611 102.67 102.00 2.36 
0.01801 0.03589 99.11 
0.01905 0.03810 105.30 
0.01746 0.03547 99.83 
0.01896 0.03775 103.96 
0.01873 0.03698 101.12 
Jatrorrhizine hydrochloride 0.01427 0.01420 0.02870 101.62 100.14 2.31 
0.01501 0.02901 98.21 
0.01279 0.02710 100.31 
0.01389 0.02867 103.67 
0.01431 0.02854 99.72 
0.01512 0.02898 97.29 
Columbamine 0.03119 0.02884 0.06010 100.24 100.22 1.76 
0.03502 0.06582 98.89 
0.02785 0.05998 103.38 
0.02968 0.06083 99.87 
0.03289 0.06428 100.61 
0.03321 0.06385 98.34 
Epiberberine 0.06088 0.07340 0.1321 97.03 101.14 3.03 
0.05801 0.1199 101.74 
0.07523 0.1401 105.30 
0.07318 0.1367 103.61 
0.06321 0.1233 98.75 
0.06096 0.1221 100.43 
Coptisine 0.06572 0.07602 0.1406 98.50 100.80 3.13 
0.07354 0.1369 96.79 
0.06021 0.1293 105.60 
0.06732 0.1329 99.79 
0.06514 0.1327 102.82 
0.06821 0.1348 101.28 
Palmatine chloride 0.07694 0.07196 0.1520 104.31 102.35 2.68 
0.08320 0.1603 100.19 
0.07201 0.1489 99.93 
0.07596 0.1576 106.19 
0.07931 0.1592 103.72 
0.07723 0.1540 99.78 
Berberine hydrochloride 0.2238 0.2190 0.4520 104.20 102.75 3.34 
0.2169 0.4382 98.85 
0.2103 0.4462 105.75 
0.2217 0.4569 105.14 
0.2301 0.4491 97.91 
0.2245 0.4587 104.63 
Alkaloids Original (mg/mL) Spiked (mg/mL) Found (mg/mL) Recovery (%) Mean (%) RSD (n = 6) 
Magnoflorine 0.01804 0.01760 0.03611 102.67 102.00 2.36 
0.01801 0.03589 99.11 
0.01905 0.03810 105.30 
0.01746 0.03547 99.83 
0.01896 0.03775 103.96 
0.01873 0.03698 101.12 
Jatrorrhizine hydrochloride 0.01427 0.01420 0.02870 101.62 100.14 2.31 
0.01501 0.02901 98.21 
0.01279 0.02710 100.31 
0.01389 0.02867 103.67 
0.01431 0.02854 99.72 
0.01512 0.02898 97.29 
Columbamine 0.03119 0.02884 0.06010 100.24 100.22 1.76 
0.03502 0.06582 98.89 
0.02785 0.05998 103.38 
0.02968 0.06083 99.87 
0.03289 0.06428 100.61 
0.03321 0.06385 98.34 
Epiberberine 0.06088 0.07340 0.1321 97.03 101.14 3.03 
0.05801 0.1199 101.74 
0.07523 0.1401 105.30 
0.07318 0.1367 103.61 
0.06321 0.1233 98.75 
0.06096 0.1221 100.43 
Coptisine 0.06572 0.07602 0.1406 98.50 100.80 3.13 
0.07354 0.1369 96.79 
0.06021 0.1293 105.60 
0.06732 0.1329 99.79 
0.06514 0.1327 102.82 
0.06821 0.1348 101.28 
Palmatine chloride 0.07694 0.07196 0.1520 104.31 102.35 2.68 
0.08320 0.1603 100.19 
0.07201 0.1489 99.93 
0.07596 0.1576 106.19 
0.07931 0.1592 103.72 
0.07723 0.1540 99.78 
Berberine hydrochloride 0.2238 0.2190 0.4520 104.20 102.75 3.34 
0.2169 0.4382 98.85 
0.2103 0.4462 105.75 
0.2217 0.4569 105.14 
0.2301 0.4491 97.91 
0.2245 0.4587 104.63 

The results of these tests indicated that the established method was accurate for the determination of the seven alkaloids in C. chinensis Franch. samples.

Quantitative analysis of samples

The established method was applied for the simultaneous determination of seven major active constituents and the total alkaloid contents in the different tissues of C. chinensis Franch. Samples were analyzed in triplicate to determine the mean content (%). The representative HPLC profiles are shown in Figure 1 and the results are summarized in Table I. Table I indicates a significant difference in the contents of the seven major pharmacologically active components in various tissues of C. chinensis Franch. In xylem, the contents (%) of magnoflorine, jatrorrhizine hydrochloride, columbamine, epiberberine, coptisine, palmatine chloride and berberine hydrochloride were in the following ranges: 0.244–1.198%, 0.056–0.278%, 0.112–0.303%, 0.338–1.767%, 0.362–1.556%, 0.274–1.125% and 2.582–5.741%, respectively. In phloem, the percent contents of magnoflorine, jatrorrhizine hydrochloride, columbamine, epiberberine, coptisine, palmatine chloride and berberine hydrochloride were found to be in the following ranges: 0.322–0.993%, 0.305–1.050%, 0.232–2.082%, 1.423–6.225%, 2.805–4.619%, 1.282–5.076% and 8.607–14.506%, respectively. Likewise, in medulla, the contents (%) of magnoflorine, jatrorrhizine hydrochloride, columbamine, epiberberine, coptisine, palmatine chloride and berberine hydrochloride were in the following ranges: 0.257–1.073%, 0.318–0.832%, 0.552–1.799%, 1.657–4.499%, 1.469–2.940%, 2.836–5.181% and 9.102–14.525%, respectively. In rhizome, again the contents (%) of magnoflorine, jatrorrhizine hydrochloride, columbamine, epiberberine, coptisine, palmatine chloride and berberine hydrochloride were in the following ranges: 0.454–1.011%, 0.424–0.763%, 0.481–1.695%, 1.768–3.554%, 2.010–3.689%, 1.900–3.784% and 6.637–12.129%, respectively. At the same time, the total alkaloid contents were determined in different tissues of C. chinensis Franch. by the ultraviolet spectrophotometric method (Y = 59.893X − 0.00871, r = 0.9999). The results are shown in Table I and Figure 2. The content of total alkaloid is presented in terms of berberine hydrochloride content. It can be seen from the experiment involving different tissues of C. chinensis Franch., the content of total alkaloid in phloem and medulla of C. chinensis Franch. was relatively high, while the lowest content was found in the xylem.

Figure 2.

The distribution figure of total alkaloids content of Rhizoma coptidis collected from various locations.

Figure 2.

The distribution figure of total alkaloids content of Rhizoma coptidis collected from various locations.

The results of this study showed that the seven major active alkaloids are distributed in every tissue tested. Additionally, in various tissue of in C. chinensis Franch., the distribution of total alkaloids is relatively regular (Figure 2).

Discussion

The variation in the pharmacologically active components in a plant invariably affects its clinical curative effect. Therefore, in the view of the differences in the distribution of pharmacologically active components in a plant variety, the quality optimization method of the TCM materials is crucial in determining the curative effect. Coptidis chinensis Franch. as the main source of medicinal material C. chinensis, a plenty of research has been conducted on it. Previous studies on C. chinensis Franch. have focused on the chemical compositions (1821), pharmacology (2227), molecular biology (2831) and commodity specification (32). The intrinsic relationship between appearance features and the quality of medicine (in terms of abundance of biologically active components) are generally not reported. This investigation focused on development of an effective and accurate HPLC method for the determination of percent contents of seven major alkaloid constituents (berberine hydrochloride, palmatine chloride, jatrorrhizine hydrochloride, epiberberine, coptisine, columbamine and magnoflorine) in various tissues (i.e., phloem, xylem and medulla) and rizhome of C. chinensis Franch. The optimized method was validated for its linearity, precision, stability and recovery. Additionally, ultraviolet spectrophotometry was employed for determining the content of total alkaloids in various tissues (i.e., phloem, xylem and medulla) and rizhome of C. chinensis Franch. The results show that the average total alkaloid content of C. chinensis Franch. in rizhome, medulla and phloem is 0.413, 0.508 and 0.492, respectively. The average contents of total alkaloids were relatively high, while the lowest content was found in the xylem (0.164). Thus, it can be inferred that the total alkaloids content of the C. Chinensis Franch. depends on the proportion of different tissues types in the whole medicinal materials. Larger the size/amount of medulla and phloem in the C. chinensis Franch. sample, higher the total alkaloids content in it, establishing the superior medicinal value of the sample. On the contrary, larger the size of xylem in the C. chinensis Franch. sample, lower the total alkaloids content in it, signifying poor medicinal value of C. Chinensis Franch. sample. This investigation provided a different perspective for selecting C. chinensis Franch. samples of superior medicinal value, in which medulla and phloem tissues account for higher alkaloids content. This observation about the relationship between the appearance characteristic and intrinsic medicinal value can be applied in quality control of the C. chinensis Franch. medicinal materials.

The analysis of the collected samples of C. chinensis Franch. from different locations exhibited variation in the alkaloid contents presumably due to the variation in geographic environment, cultivation model, hereditary character or other factors, such as growth differences of various tissues. Therefore, establishing good agricultural  cultivation practice to ensure stable quality of TCM is a priority. This study will be useful in guiding fellow researchers working on optimizing the pharmacology and cultivation practice of C. chinensis Franch. as an effective TCM.

Conclusion

An HPLC method was adopted for the detection and quantification of seven types of alkaloids in various tissues (i.e., phloem, xylem and medulla) and rhizome of C. chinensis Franch. for the first time. By comparing the contents of pharmacologically active alkaloids in various tissues and rizhome of C. chinensis Franch., the total alkaloid content was found to be relatively higher in medulla and phloem, while the lowest total alkaloid content was found in the xylem. It implies that the C. chinensis Franch. sample with larger the size of medulla and phloem will be of the superior medicinal value. This discovery relating to the growth and the distribution of phloem, xylem and medulla to judge the medicinal quality of C. Chinensis Franch. will be of value to the growers, traders and end users.

Funding

This work was financially supported by the Key Laboratory of Resource Science and Chemistry in Chinese Medicine Hubei Province, Hubei University of Chinese Medicine, Wuhan, Hubei, China.

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Author notes

These authors contributed equally to this work and should be considered co-first authors.