Research Progress on Quality Control Methods for Xiaochaihu Preparations

: Xiaochaihu (XCH) is a classic Chinese medicine formula. XCH tablet, XCH granule, XCH capsule, and XCH effervescent tablet are included in the Chinese Pharmacopoeia. In this review, the formula and quality standards of XCH preparations at home and abroad were compared. The differences in manufacturing process of XCH preparations are discussed. The progress of research on the qualitative identiﬁcation, quantitative detection and ﬁngerprint chromatogram/speciﬁc chromatogram of XCH preparations was reviewed. The characteristic components of Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine and Jujubae Fructus was rarely analyzed for XCH preparations. It is suggested that the speciﬁcity of drug quality detection methods should be improved. Considering drug safety and drug efﬁcacy, it is suggested to set the upper and lower limits of the content of saikosaponins. The standards for heavy metals and other limited items for XCH preparations are also suggested to be set.


Introduction to XCH Formula
Xiaochaihu (XCH) formula, which was created by Zhang Zhongjing in the East Han Dynasty, is capable of inducing sweat to dispel heat, channeling the liver, regulating the spleen, soothing the stomach [1], etc. Traditionally, the recipe is composed of Bupleuri Radix, Scutellariae Radix, Ginseng Radix Et Rhizoma (Ginseng Radix), Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle (Glycyrrhizae Radix), Zingiberis Rhizoma Recens, Jujubae Fructus, and Pinelliae Rhizoma [2]. According to the principle of JUN-CHEN-ZUO-SHI (emperor-minister-assistant-courier in English), in this formula, Bupleuri Radix is JUN, Scutellariae Radix is CHEN, Glycyrrhizae Radix is SHI, and the others are ZUO. Modern research has verified that XCH has anti-inflammatory [3] and antitumor [4] functions and regulates the endocrine system [5]. Clinically, the formula is applied to treat various diseases of the respiratory system [6], digestive system [7], urogenital system [8], immune system [9], circulatory system [10], etc. The mechanism of XCH acting on the human body can be preliminarily explored by means of liquid chromatography-mass spectrometry, network pharmacology, and animal experiments. For fever, the widest application of XCH, potential antipyretic mechanism includes the reduction of inflammation level, inhibition of endogenous pyrogen and COX-2 [11]. Some active ingredients of XCH including quercetin, baicalein, and hanbaicalein can significantly inhibit the growth of hepatocellular carcinoma and induce apoptosis of hepatocellular carcinoma cells [12]. In recent years, many novel applications have been reported, including the prevention and treatment of methicillinresistant Staphylococcus aureus [13], syncytial virus, and adenovirus [14], as well as the inhibition of influenza A virus [15], etc. For the period from 2000 to 2020, an overall trend of a steady rise in the numbers of publications in the field of XCH could be found. In the

Differences in XCH Preparation Methods
There are different manufacturing processes for preparing XCH [29][30][31][32] preparations. Manufacturing processes included in the Chinese Pharmacopoeia [17] are shown in Figures 1-4. For Codonopsis Radix, Glycyrrhizae Radix, Bupleuri Radix, Scutellariae Radix, and Jujubae Fructus, the plants are extracted with water decoction. Jiangbanxia and Zingiberis Rhizoma Recens are extracted with ethanol solution with percolation. Compared with water decoction process, the percolation process is time consuming and solvent consuming. However, the volatilization or degradation of active components can be effectively decreased with the percolation process because it is operated at a low temperature. It has been reported that gingerols are easily degraded at a high temperature [33]. Therefore, it is reasonable to extract active components from Jiangbanxia and Zingiberis Rhizoma Recens with a percolation process [34]. Gingerol and other components in Zingiberis Rhizoma Recens have low solubility in water [35]. Therefore, ethanol solution is generally used as the percolation solvent [36].
In the production process for XCH capsules and XCH tablets, part of Codonopsis Radix and Glycyrrhizae Radix are directly crushed and added, which is significantly different from the process of XCH effervescent tablets and XCH granules. Codonopsis Radix and Glycyrrhizae Radix powder can play a role similar as excipients [37]. The excipients in XCH preparations vary depending on the formulation forms.
Apart from the manufacturing processes included in the Chinese Pharmacopoeia mentioned above, there are several other manufacturing processes for different XCH preparations, such as XCH sustained release tablets [38], nano XCH preparations [39], and others [40,41].

Differences in XCH Preparation Methods
There are different manufacturing processes for preparing XCH [29][30][31][32] preparations. Manufacturing processes included in the Chinese Pharmacopoeia [17] are shown in Figures 1-4. For Codonopsis Radix, Glycyrrhizae Radix, Bupleuri Radix, Scutellariae Radix, and Jujubae Fructus, the plants are extracted with water decoction. Jiangbanxia and Zingiberis Rhizoma Recens are extracted with ethanol solution with percolation. Compared with water decoction process, the percolation process is time consuming and solvent consuming. However, the volatilization or degradation of active components can be effectively decreased with the percolation process because it is operated at a low temperature. It has been reported that gingerols are easily degraded at a high temperature [33]. Therefore, it is reasonable to extract active components from Jiangbanxia and Zingiberis Rhizoma Recens with a percolation process [34]. Gingerol and other components in Zingiberis Rhizoma Recens have low solubility in water [35]. Therefore, ethanol solution is generally used as the percolation solvent [36].
In the production process for XCH capsules and XCH tablets, part of Codonopsis Radix and Glycyrrhizae Radix are directly crushed and added, which is significantly different from the process of XCH effervescent tablets and XCH granules. Codonopsis Radix and Glycyrrhizae Radix powder can play a role similar as excipients [37]. The excipients in XCH preparations vary depending on the formulation forms.       Apart from the manufacturing processes included in the Chinese Pharmacopoeia mentioned above, there are several other manufacturing processes for different XCH preparations, such as XCH sustained release tablets [38], nano XCH preparations [39], and others [40,41].

Indicators for Qualitative Identification
There are two kinds of qualitative methods for the preparations of XCH in production: microscopic identification and thin-layer chromatography (TLC) identification. The identification methods and reference materials from the Chinese Pharmacopoeia and the Japanese Pharmacopoeia are summarized in Table 2.
The method involving the microscopic identification of medicinal materials is suitable for fragmentary medicinal materials or powdered medicinal materials. Raw powdered medicinal materials of Glycyrrhizae Radix and Codonopsis Radix are used in the manufacturing of XCH tablets and XCH capsules, which is suitable to be analyzed with microscopic identification. Both Chinese and Japanese XCH preparations adopt thin-layer identification, but there are obvious differences. First, in Chinese Pharmacopoeia, thin-layer identification uses reference medicinal materials, including Glycyrrhizae Radix, Bupleuri Radix, and Codonopsis Radix. However, thin-layer identification uses only reference substances in Japanese Pharmacopoeia. Second, Japanese thin-layer identification uses more reference substances, including the index components from Bupleuri Radix, Zingiberis Rhizoma Recens, Scutellariae Radix, Glycyrrhizae Radix and Ginseng Radix. In contrast, the use of reference medicinal materials in thin-layer identification can provide more information

Indicators for Qualitative Identification
There are two kinds of qualitative methods for the preparations of XCH in production: microscopic identification and thin-layer chromatography (TLC) identification. The identification methods and reference materials from the Chinese Pharmacopoeia and the Japanese Pharmacopoeia are summarized in Table 2.
The method involving the microscopic identification of medicinal materials is suitable for fragmentary medicinal materials or powdered medicinal materials. Raw powdered medicinal materials of Glycyrrhizae Radix and Codonopsis Radix are used in the manufacturing of XCH tablets and XCH capsules, which is suitable to be analyzed with microscopic identification. Both Chinese and Japanese XCH preparations adopt thin-layer identification, but there are obvious differences. First, in Chinese Pharmacopoeia, thin-layer identification uses reference medicinal materials, including Glycyrrhizae Radix, Bupleuri Radix, and Codonopsis Radix. However, thin-layer identification uses only reference substances in Japanese Pharmacopoeia. Second, Japanese thin-layer identification uses more reference substances, including the index components from Bupleuri Radix, Zingiberis Rhizoma Recens, Scutellariae Radix, Glycyrrhizae Radix and Ginseng Radix. In contrast, the use of reference medicinal materials in thin-layer identification can provide more information than using reference substances, which is conducive to assessing the authenticity of the medicinal materials used. Both Chinese and Japanese XCH preparations quantitatively analyze baicalin contents. Therefore, it seems unnecessary to use baicalin as the reference substance in thin-layer identification.
Silica gel G thin layer plate is mostly widely used in TLC identification. A mixed solvent of ethyl acetate-butanone-formic acid-water is usually used for baicalin identification. A mixed solvent of chloroform-methanol-water is usually used for the identification of Glycyrrhizae Radix.  Table 3 lists the quantitative detection methods for XCH preparations in Chinese Pharmacopoeia and Japan Pharmacopoeia. According to the Chinese Pharmacopoeia, the XCH tablets weight 0.4 g per tablet, the XCH capsules weight 0.4 g per capsule, and the XCH effervescent tablets weight 2.5 g per tablet. The XCH granules have three specifications (10 g/4 g/2.5 g per bag) due to the various preparation methods. The only quantitative determined index component for XCH preparations mentioned in the 2020 edition of the Chinese Pharmacopoeia is baicalin, while the determination of baicalin, saikosaponin B2, and glycyrrhizic acid are required in Japan Pharmacopoeia. Considering that there are seven medicinal materials in the formula for XCH preparations, more index components should be determined to control drug quality. The Chinese Pharmacopoeia specifies a lower limit for baicalin, while the Japanese Pharmacopoeia specifies both the upper and lower limits for the contents of saikosaponin B2, baicalin, and glycyrrhizic acid. Herbal materials of XCH preparations were often decocted before quantitative analysis. Methanol is a common solvent for sample preparation.  Table 4 lists the published works on the quantitative detection of XCH preparations. In such work, the raw materials often went under decoction treatment before they were put into use. In addition, methanol is a common solvent in the procedure. HPLC technology is used to separate the components of XCH preparations. The detectors stated in the literature are mostly ultraviolet detectors, and a few are diode array detectors (DAD) and mass spectrometer detectors. Since the content of a saikosaponin is low, mass spectrometer detectors are used more often to analyze it. In some papers, the method of quantitative analysis of multiple components by a single marker (QAMS) was used, which can reduce the cost of testing. There are more reports on the detection of index components of Bupleuri Radix and Scutellariae Radix, which reflects the emphasis on JUN and CHEN drugs. Some literature has detected gingerol, liquiritin, glycyrrhizic acid, lobetyolin, and other substances, which can help control the contents of chemical components in Zingiberis Rhizoma Recens, Glycyrrhizae Radix, and Codonopsis Radix. However, there are still few detections of index components in Jujubae Fructus and Jiangbanxia. "-" means there is no special instruction that is necessary to be presented.

Fingerprint and Specific Chromatogram
Fingerprint and specific chromatogram detection methods can reflect the overall characteristics of Chinese medicines and are widely used in drug quality analysis. At present, the application of these two methods represents a significant research progress. Both qualitative identification and quantitative detection can be carried out on the basis of fingerprint and specific chromatograms. A summary of the fingerprint and specific chromatogram detection methods for XCH preparations is shown in Table 5. Compared with the quantitative methods listed in Table 4, fingerprint and specific chromatogram detection can identify more components in chromatographic peaks, therefore providing more information. At present, the most identified components are the saponins in Bupleuri Radix, flavonoids of Scutellariae Radix, gingerol in Zingiberis Rhizoma Recens, liquiritin, and glycyrrhizic acid in Glycyrrhizae Radix, etc. However, the characteristic components of Jujubae Fructus and Jiangbanxia have not been identified.
In some of the research works, a quantitative fingerprint or specific chromatogram of the XCH preparation was obtained. The quantitatively determined components are mainly from Scutellariae Radix, Glycyrrhizae Radix, Bupleuri Radix, and Codonopsis Radix. Wang et al. [68] compared the HPLC spectra of XCH granules at different wavelengths and concluded that spectral analysis at a single ultraviolet absorption wavelength is not suitable for quality detection. Liu et al. used charged aerosol detector (CAD) to analyze saikosaponins [69]. Compared with using evaporative light scattering detector (ELSD), lower detection limit and wider detection range can be realized with CAD.
In some studies, the active substances in Bupleuri Radix such as saikosaponin A and saikosaponin D were not detected in XCH granules, which may be due to the hydrolysis of saikosaponin during decoction [70]. There are also reports that the existing detection methods often add an acid to the mobile phase, and saikosaponin A and saikosaponin D are prone to degrade under acidic conditions, which makes them difficult to detect [71].   [86] "-" means there is no quantitative determined or qualitatively identified component.
In conclusion, a fingerprint/specific chromatogram can be used to characterize multiple chemical component information of XCH preparations. However, fingerprint/specific chromatogram is not included in Chinese Pharmacopoeia. Further development of quality control technology with use of the fingerprint/specific chromatogram is required for XCH preparations.

Improvement in the Specificity of Quality Testing
According to Chinese Pharmacopoeia, Glycyrrhizae Radix, Bupleuri Radix and Codonopsis Radix are used as TLC reference materials, and baicalin is used as a TLC reference substance in qualitative identification. However, less attention has been given to Zingiberis Rhizoma Recens, Jujubae Fructus, and Jiangbanxia. The specific components of Jiangbanxia and Jujubae Fructus are not quantitatively analyzed in literature. Recently, guanosine, uridine, hypoxanthine and several other components were analyzed [87], which do not especially belong to Jiangbanxia, but it still suggests a way to improve the specificity of HPLC detection by detecting these compositions with strong polarity.
It is essential to distinguish the authenticity of Bupleuri Radix. There are 36 species, 17 varieties, and seven forms distributed all over China [88]. Among them, Bupleurum marginatum var. stenophyllum and even poisonous Bupleurum longiradiatum are common varieties that are all easy to mix up [89]. To confirm whether or not Bupleurum marginatum var. stenophyllum had been added, Liu et al. using the retention time and peak area of the specific ion detected in the mass spectrum as standards [90]. Liang et al. tried to establish near-infrared spectrum models to distinguish products of different factories, which provided a practical technology for low-cost and rapid detection [91]. Lai et al. used a polymerase chain reaction (PCR) method based on the site specificity of the Internal Transcribed Spacer (ITS) sequence to identify Bupleurum marginatum var. stenophyllum from Bupleurum chinense DC [92]. These new technologies provide ideas for improving the specificity of analytical methods. Bupleurum scorzonerifolium Willd and Bupleurum chinense DC are both included in the Chinese Pharmacopoeia, but National Institutes for Food and Drug Control can provide only the reference material of Bupleurum chinense DC. Hence, the lack of reference material of Bupleurum scorzonerifolium Willd is a problem for quality control of Bupleuri Radix.

Setting Reasonable Content Range of Index Components from Bupleuri Radix
Bupleuri Radix is the JUN of XCH formula. Thus far, qualitative identification using the reference material of Bupleuri Radix was adopted in Chinese Pharmacopoeia. However, considering drug safety and efficacy, the contents of saikosaponins should be controlled in specific ranges. Studies have indicated that saikosaponins are important active ingredients of Bupleuri Radix, which has antipyretic, anti-inflammatory and antitumor activities. Therefore, it is necessary to set up lower limits for their contents [93,94]. Moreover, some reference materials have reported that Bupleuri Radix has a certain degree of toxicity when taken in a large dose for a long period of time, and its toxic side effects are often caused by its saponins and volatile substances, which mainly affect the liver [95]. Therefore, from the perspective of drug safety, it is necessary to set up upper limits for saikosaponins. At present, the upper and lower limits of the saikosaponin B2 content are set up in the Japanese Pharmacopoeia, which is worth referencing. When setting up the lower limit, companies can consider collecting big data from clinical practice. Accordingly, the needs of drug quality control indicators can be taken into consideration, such as drug interactions and medications for special populations.

Strengthening the Standard of Limited Detected Items
In recent years, great progress in the control of heavy metals, pesticides, and biological toxins in Chinese medicines and extracts was achieved. The Chinese Pharmacopoeia has specially listed items General Principle for Inspection of Crude Drugs and Decoction Pieces and Guidelines for Establishment of Limit for Harmful Residue of Traditional Chinese Medicine, which have provided guidance for controlling heavy metals, pesticides and biological toxins for medicinal materials. The Chinese Pharmacopoeia stipulates that Jujubae Fructus needs to be tested for aflatoxin, Glycyrrhizae Radix needs to be tested for heavy metals, harmful elements and pesticide residues, and Codonopsis Radix needs to be tested for sulfur dioxide residues, all of which help to guarantee the safety of XCH preparations. However, the current guidelines for XCH preparations still require more relevant limiting items for heavy metals, pesticides and biological toxins, and other toxic ingredients. The Japanese Pharmacopoeia stipulates the limits of heavy metals and arsenic in XCH preparations. It takes the increase in heavy metals during the production process into account, which is more rigorous and improves the level of quality control.
Therefore, from the perspective of drug safety, XCH preparations require an upper limit for the amounts of certain active ingredients, heavy metals, pesticides, biotoxins, and other toxic components. Similar quality control problems exist for many other Chinese medicines. Therefore, the development direction of quality control presented in this work can also be referenced for that of other Chinese medicines.