Sanhuang tablet (Sanhuang Pian
in Chinese, SHT), comprised of the powder of Rhei Radix et Rhizoma (dried root and rhizome of Rheum palmatum
L., R. tanguticum
Maxim. Ex Balf, or R. officinale
Baill.), berberine hydrochloride, and extracts of Scutellariae Radix (dried root of Scutellaria baicalensis
Georgi) is a commonly used Chinese patented drug [1
]. SHT is widely used in the treatment of constipation, gastroenteritis, dysentery, and vaginitis [2
]. According to the public access database on the website of the China Food and Drug Administration, as of September 2016, SHT, with 210 licenses, is one of the top 25 licensed Chinese patented drugs in China. Meanwhile, Health Canada has licensed four SHT products as natural health products.
Different from conventional Chinese medicine preparations, SHT is not a mixture of herb extracts, but rather a mixture of a single chemical component, two herb extracts, and a powdered raw herb. This formula is derived from Sanhuang Xiexin decoction, which consists of Coptidis Rhizoma (Dried rhizome of Coptis chinensis
Franch, C. deltoidea
C. Y. Cheng et Hsiao, or C. teeta
Wall), Rhei Radix et Rhizoma, and Scutellariae Radix. As recorded in the currently available literature [5
], this formula was first mentioned in Synopsis of Prescriptions of the Golden Chamber
, written in the Eastern Han dynasty (A.D. 25–220) by Zhang Zhong-Jing. Different dosage forms, such as honey bolus and powder, were invented in the Tang dynasty (618–907). The honey bolus was officially recognized in the Song dynasty (960–1279) when the Bureau of Imperial Physicians recorded it in an official formulary, Prescriptions from the Great Peace Imperial Grace Pharmacy
. Its tablet preparation was developed in 1958 when Cotidis Rhizoma was replaced with its main component, berberine hydrochloride, and Scutellariae Radix was replaced with its extract.
According to the chemical profiles, SHT is more complex than Sanhuang Xiexin decoction. Raw, powdered Rhei Radix et Rhizoma contributes a chemical diversity that includes anthraquinones and their glycosides, anthrones and their glycosides, stilbenes, polysaccharides, tannins, and some organic tissues of the plant [8
]. The extract of Scutellariae Radix mainly contains flavonoids [9
], especially when the water extraction and subsequent acid precipitation heightens the content of baicalin [10
]. Furthermore, the berberine hydrochloride that represents Coptidis Rhizoma is supposed to be a single compound. However, it is always accompanied by several analogues due to inherent difficulties in separating single components from a natural product.
SHT has the pharmacological activities of anti-inflammatory, anti-bacterial and purgation [2
]. The purgative, antibacterial and antivirus activities of Rhei Radix et Rhizoma make it become the principal drug of the formulation. Anthrones, such as sennoside, rheidin, and palmidin, are responsible for the purgative activity [12
]. The free-anthraquinone derivatives provide inhibition effect on Helicobacter pylori, which is one of the causes of inflammation and ulcer in stomach and duodenum [13
]. Aloe-emodin has inactivation effects on varicella zoster virus, pseudorabies virus and influenza virus [13
] and an anti-inflammatory effect [14
]. The active components of Scutellariae Radix are flavonoids. Their bacteriostatic, antipyretic and analgesic activities are strongly related to the action of Sanhuang tablet [15
]. Meanwhile, berberine hydrochloride itself is considered as an antibacterial drug effective against Staphylococus aureus
and Enterococcus faecalis
Despite the great advances in analytical tools, the quality control of Chinese medicines, especially in terms of quantitative analysis of the chemical profile, remains unsatisfactory. On the other hand, the successful licensing of botanical drugs, like VEREGEN, by the FDA has highlighted the importance of quantitative analysis of natural complexes like Chinese medicines. In the case of SHT, the published quantitative analysis has assessed no more than ten compounds [17
], missing many important ingredients. For example, sennoside A, a major component responsible for the purgative action of Rhei Radix et Rhizoma [20
] and an assay marker of the herb in the Japanese Pharmacopoeia and Korea Pharmacopoeia [22
], has not been quantified in SHT by the aforementioned studies nor the Chinese Pharmacopoeia. Furthermore, SHT also contains plenty of carbohydrates such as monosaccharides, oligosaccharides, and polysaccharides; however, the analytical methods published to date for the analysis of SHT have not determined either the identity or amount of the carbohydrates. This study aims to establish a more comprehensive analytical method to quantitatively analyze the chemical profile of SHT. Such an approach will be useful not only in determining the quality of various commercial SHT products but also in assessing other, similarly complex, herbal preparations.
3. Experimental Section
3.1. Chemicals and Materials
Commercial SHT products were purchased from various suppliers in mainland China. Details of the 27 samples, including 3 film-coated and 24 sugar-coated tablets, produced by 12 manufacturers are listed in Table 3
. In addition, we prepared our own sample as a control using Rhei Radix et Rhizoma from Gansu, China and Scutellariae Radix from Shandong, China. These herb materials were authenticated by Professor Hu-Biao Chen from the School of Chinese Medicine, Hong Kong Baptist University, China. Voucher specimens of the herbs were deposited at the School of Chinese Medicine, Hong Kong Baptist University, China.
Reference standards of (1) loganic acid (Lot no. 131220); (5) scutellarin (130306); (8) apigetrin (130315); (11) baicalin (121128); (12) eriodictyol (130606); (13) quercetin (131120); (14) scutellarein (131219); (15) oroxyloside (121122); (17) wogonoside (131214); (18) baicalein (130119); (19) aloe-emodin (130425); (21) wogonin (140521) and (23) oroxylin A (130323) were provided by Chengdu Preferred Biotechnology Co. Ltd. (Chengdu, China). Reference compounds of (3) daidzin (131214); (4) sennoside B (130409); (6) luteoloside (130418); (7) sennoside A (130306); (9) rhaponticin (140604); (10) daidzein (130715); (16) physcion (140211); (20) rhein (131024); (22) chrysophanol (140219); (24) emodin (140422); (25) coptisine (130628); (26) epiberberine (130621); (27) jatrorrhizine hydrochloride (130318); (28) palmatine hydrochloride (130702) and (29) berberine hydrochloride (140314) were provided by Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Reference marker of (2) caffeic acid (130428) was provided by Chengdu Herbpurify Co., Ltd. (Chengdu, China).
The identities of the reference standards were confirmed by mass spectrometry prior to use. The purities of the reference standards were determined to be greater than 98% by UPLC-DAD analysis based on peak area normalization. Reference substances of (30) d-(−)-fructose (SLBB6798V), (31) d-(+)-glucose (070M03801V) and (32) sucrose (SLBF27618) were supplied by Sigma (S. Louis, MO, USA). Complanatoside A (MUST-13011217, IS1) from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China) and evodiamine (C-0337, IS2) from Hong Kong Jockey Club Institute of Chinese Medicine (Hong Kong, China) were used as internal standards.
HPLC grade acetonitrile, methanol, and formic acid were provided by RCI Labscan Limited (Bangkok, Thailand). HPLC grade ethanol was provided by Merck (Darmstadt, Germany). Hydrochloric acid (37%) was provided by VWR Chemicals (Radnor, PA, USA). Water used was purified with Millipore Milli-Q water purification system (Millipore, Bedford, MA, USA).
3.2. Sample Preparation
For LCMS analysis, 10 tablets from each SHT sample were ground into fine powder and passed through a 60–80 mesh filter. An accurately weighed sample (500 mg) of each powder was then extracted three times under ultrasonication with 10 mL of ethanol-water (70:30, v/v) for 30 min in a sealed 20 mL glass bottle. Due to the varied contents of different analytes in samples, some may be beyond the linear ranges, so the extracts were diluted 10×, 250× and 400× before analysis. For determination of most analytes, the extracts were diluted 10 times; for analytes No. 11, 15 and 24 in some samples, due to their higher contents in SHT, extracts were diluted 250 times; for alkaloid analytes No. 25–29, extracts were diluted 400 times. For HPLC-CAD analysis, 100 mg of the above SHT powder was extracted with 5 mL water in a sealed 20 mL glass bottle in a dry bath at 120 °C for 1.5 h. The solution was filtered, and the residue was extracted again by the same method for another one hour. The solution was filtered and combined with the first. Out of the final solution, 0.5 mL of solution from each sample was then freeze-dried before re-dissolving in 1 mL of acetonitrile–water (80:20, v/v) to prepare the sample solution.
In addition, we prepared the control sample by following the procedures in the Chinese Pharmacopoeia 2015. Briefly, 10 g of Scutellariae Radix was decocted with 100 mL water three times, (1.5 h, 1 h and 40 min, respectively). The decoctions were then combined and filtered. The pH value of the filtrate was adjusted to pH 1–2 by adding hydrochloric acid, and one hour later the solution was filtered again. The obtained precipitate was then washed with water to pH 5–7, heated to dryness, and ground into fine powder to get the extract of Scutellariae Radix. Rhei Radix et Rhizoma coarse powder (10 g) was refluxed with 100 mL of 30% ethanol for three times (1.5 h, 1 h and 40 min, respectively). The extracts were combined and filtered. The filtrate was then concentrated in vacuum to get a thick extract, to which 1.4 g of the dried Scutellariae Radix extract and 10 g of Rhei Radix et Rhizoma fine powder were added to make the control sample. Berberine hydrochloride and excipients were not added because berberine hydrochloride has been an identified chemical and the excipients remain unknown to us.
3.3. Standard Solution Preparation
The non-saccharide small molecule reference markers, as well as two internal standard compounds, were accurately weighted and dissolved in methanol to prepare a stock solution. Reference substances of three saccharides were accurately weighted and dissolved in water to prepare a stock solution. Calibration curves were obtained from standard solutions, which were prepared by appropriate dilution of the mixed standard solutions.
3.4. UHPLC-Q-TOF-MS Conditions
UHPLC data was collected using an Agilent 1290 Infinity UPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a G4220A binary pump, a column compartment with a thermostat, a G4226A HiP sampler, and a degasser. Separations were conducted over an Acquity UPLC BEH C18 (1.7 µm, 2.1 × 100 mm, Waters, Milford, CT, USA) column at 40 °C with a gradient elution consisting of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile) at a flow rate of 0.35 mL/min. The column was eluted with the following gradient program: 0–14 min, 10%–30% B; 14–22 min, 30%–37% B; 22–28 min, 37%–75% B; 28–31 min, 75%–100% B; 31–34 min, 100% B; 34–34.1 min, 100%–10% B; 34.1–36 min, 10% B. The injection volume was 2 µL.
An Agilent 6540 Q-TOF mass spectrometer (Agilent Technologies) equipped with a jet stream electrospray ionization (ESI) source was used to acquire the MS and MS/MS data in the positive and negative ionization modes. Data acquisition was controlled using MassHunterB.03 software (Agilent Technologies). The operating parameters were set as follows: nebulizing gas (N2) flow rate, 8 L/min; nebulizing gas temperature, 300 °C; jet stream gas flow, 8 L/min; sheath gas temperature, 350 °C; nebulizer, 45 psi; capillary, 3000 V; skimmer, 65 V; Oct RFV, 600 V; fragmentor voltage, 150 V. The peaks with the range of 100–1700 m/z were recorded.
3.5. HPLC-CAD Conditions
An HPLC-CAD method was used to determinate fructose, glucose and sucrose contents. A UltiMate 3000 liquid chromatography system (Dionex, Sunnyvale, CA, USA) equipped with a Dionex Corona Veo RS Charged Aerosol Detector through an Alltech Interface 35900E multichannel interface was used. The chromatographic separations were performed on an Asahipak NH2P-50 4E column (4.6 mm × 250 mm, Shodex, Tokyo, Japan) at a column temperature of 30 °C. The column was eluted with a mixture of water (mobile phase A) and ACN (mobile phase B) at a flow rate of 0.6 mL/min. The elution conditions were as follows: 0–17 min, 78% B; 17–21 min, 78%–62% B; 21–27 min, 62%–60% B, 27–27.1 min, 60%–78% B, followed by ten-minute balance at 78% B. The power function of CAD was 1. The injection volume was 10 µL.
3.6. Method Validation
The two developed UHPLC-Q-TOF-MS and HPLC-CAD methods were evaluated for linearity, sensitivity, precision, stability, and spike recovery. MS data was analyzed with MassHunter Workstation Software Quantitative, version B.06 (Agilent Technologies). HPLC-CAD data was analyzed with Chromeleon® 7 Chromatography Data System, version 188.8.131.5294 (Dionex).
Stock solutions of the mixed standards were diluted to a variety of different concentrations to allow for the construction of calibration curves. At least six concentrations of each reference standard were analyzed in triplicate. The calibration curves were constructed by plotting the peak areas versus the concentrations of the corresponding constituents. The limit of detection (LOD) and limit of quantification (LOQ) values for the optimum conditions were determined at signal-to-noise ratios (S/N) of 3 and 10, respectively. The intra- and inter-day variations were used to evaluate the precision of our newly developed methods. Six independently prepared solutions of SHT were analyzed within 1 day to evaluate the intra-day variability of the optimum method. To evaluate the inter-day variability of this method, we examined the same sample twice a day over 3 consecutive days. For stability, the SHT samples were stored at room temperature and analyzed at 8, 12, 16, 24, 36, 48 h after extraction. Variations were expressed as relative standard deviations (RSDs) of the data, which were calculated using the following formula: RSD (%) = (standard deviation/mean) × 100%. A recovery test was performed to evaluate the accuracy of the optimum method by adding three different concentrations of a standard solution (i.e., low, medium and high) to SHT, which contained known quantities of the target compounds. These samples were then analyzed in parallel using our newly established method. Each experiment was conducted in triplicate at each level. The spike recoveries were calculated using the following equation: Spike recovery (%) = (total amount detected − amount original)/amount spiked × 100%.
A comprehensive and sensitive quality analysis method using UHPLC-Q-TOF-MS and HPLC-CAD was successfully established and validated for quantification of 29 non-saccharide small molecules and three saccharides in commercial SHT products. Up to 57.61% (w/w) of SHT was quantified. For the quantified components, there were 18% flavonoids, 3% anthraquinones, 3% alkaloids, and 76% saccharides which were not well-quantified in other studies. The contents of 32 analytes varied in different samples but were relatively stable between batches of the same manufacturer. There were 22 out of 27 commercial SHT samples failed the Chinese Pharmacopoeia 2015 assays. This dissatisfactory result implicated the uneven qualities of Sanhuang tablets in the market.
However, the current Chinese Pharmacopoeia assays focus on only baicalin, emodin, chrysophanol and berberine hydrochloride. They are not capable of reflecting the quality of SHT as there are many other analogue compounds in the SHT. Moreover, the TLC detection of rhaponticin, the indicator of unauthorized Rhei Radix et Rhizoma, is not sensitive enough. There were 15 samples detected with the presence of rhaponticin using UPLC-Q-TOF-MS, while none was detected with rhaponticin by TLC. These results demonstrate the importance to update the QC methods. This new method provides a more fair and comprehensive quality evaluation of commercial SHT products.