Rapid and global detection and characterization of aconitum alkaloids in Yin Chen Si Ni Tang,a traditional Chinese medical formula,by ultra performance liquid chromatography–high resolution mass spectrometry and automated data analysis

An improved method employing Metabolynx XS with mass defect filter (MDF), a post-acquisition data processing software, was developed and applied for global detection of aconitum alkaloids in Yin Chen Si Ni Tang, a traditional Chinese medical formula (TCMF). The full-scan LC–MS/MS data sets with extra mass were acquired using ultra performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) with the MS^E mode in a single injection. To remove the interferences, Metabolynx XS was optimized to extract the ions of aconitum alkaloids located at the lower abundance. As a result, 62 ions were assigned rapidly to aconitum alkaloids and identified tentatively by comparing the accurate mass and fragments information with that of the authentic standards or by mass spectrometry analysis and retrieving the reference literatures. Compared with the previous studies on Fuzi-containing TCMF, the report detected more aconitum alkaloids, and the analysis process was accelerated by automated data processing. It is concluded that the screening capability of Metabolynx XS with MDF, together with the utilization of MS^E in structural elucidation, can facilitate a rapid and comprehensive searching and effective structural characterization of aconitum alkaloids in TCMF.     

Qualitative and quantitative analysis of steroidal saponins in crude extracts from Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis by high performance liquid chromatography coupled with mass spectrometry

High performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC–ESI-MSⁿ) and triple quadrupole mass spectrometric detection (HPLC–ESI-MS/MS), respectively, had been employed for the simultaneous identification and quantification of steroidal saponins in the rhizomes of Parispolyphylla var. yunnanensis and P. polyphylla var. chinensis, which are the qualified plants of “Chonglou” in Chinese. The HPLC experiments were performed by means of a reversed-phase C-18 column and a binary mobile phase system consisting of 0.1% aqueous formic acid and acetonitrile under gradient elution conditions. The characteristic fragmentation patterns of diosgenin- and pennogenin-type steroidal saponins were investigated using ESI-MSⁿ in negative ion mode. The MSⁿ data of the [M−H]⁻ ions provided structural information on the sugar sequence of the oligosaccharide chains and the aglycones of steroidal saponins. As a result, ten and seven saponins were determined in P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, respectively, including four unknown compounds. One unknown compound was tentatively identified as diosgenin-3-O-α-l-rhamnopyranosyl(1 → 4) [α-l-rhamnopyranosyl(1 → 2)]-β-d-glucopyranoside and the aglycones of the other three new compounds were reported from Chonglou for the first time. The developed HPLC–ESI-MS/MS method was validated and found to be satisfactorily linear, selective and robust. The limits of detection (LODs) and quantitation (LOQs) ranged, respectively, from 0.5 to 10 ng/mL and 2 to 34 ng/mL depending on six various compounds. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. Recoveries ranged from 92% to 104% for all compounds. The established quality evaluation method was successfully used for simultaneous quantification of six predominant steroidal saponins in the rhizomes of these two Paris species.     

Analysis of the bioactive constituents of ChanSu in rat plasma by high performance liquid chromatography with mass spectrometric detection

A plasma pharmacochemistry analysis of the bioactive constituents in rat plasma after oral administration of ChanSu was performed. High performance liquid chromatography coupled with the electrospray ionization mass spectrometry techniques of HPLC/ESI-IT-MS/MS and RRLC/ESI-Q-TOF-MS were used for the chemical profiling of samples of dosed plasma, control plasma and ChanSu extract. Comparative analysis of the resulting chemical profiles revealed 20 prototype compounds and 4 metabolites derived from ChanSu as potential bioactive components. By MS/MS analysis and accurate molecular weight assessments, the majority of these bioactive components (19 prototype compounds and 2 metabolites) were structurally identified. Moreover, seven were confirmed by comparing their retention behaviors using MS and MS/MS analysis. This study proposes a series of potential bioactive components of ChanSu, which we hope will lead to new drug discovery based on ChanSu and other traditional Chinese medicines.     

Identification of the absorbed components and metabolites in rat plasma after oral administration of Rhizoma Chuanxiong decoction by HPLC-ESI-MS/MS

An HPLC-ESI-MS/MS method was established to identify the absorbed components and metabolites in rat plasma after oral administration of Rhizoma Chuanxiong decoction (RCD), a well-known traditional Chinese medicine. By comparing the extracted ion chromatograms (EICs) obtained from dosed rat plasma, blank rat plasma and RCD, a total of 25 compounds were detected in dosed rat plasma. Among them, 13 compounds were absorbed into rat plasma in prototype and identified as ferulic acid, senkyunolide J, senkyunolide I, senkyunolide D or 4,7-dihydroxy-3-butylphthalide, senkyunolide F, senkyunolide M, senkyunolide Q, senkyunolide A, E-butylidenephthalide, E-ligustilide, neocnidilide, Z-ligustilide, levistolide A, according to the retention times, UV, MS, MS/MS spectra. In addition, 12 conjugated metabolites including 6 senkyunolide I-related metabolites, 4 senkyunolide J-related metabolites and 2 butylidenephthalide-related metabolites were also detected and identified by comparing their MS, MS/MS spectra with that of corresponding original components. Conjugated with glutathione, cysteine, glucuronic acid and sulphuric acid were the main metabolic reactions of phthalides. Finally the in vivo metabolic pathways of chemical constituents of Chuanxiong in rat plasma were proposed in this study.     

Characterization of steroidal saponins in crude extracts from Dioscorea zingiberensis C. H. Wright by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Steroidal saponins are the major bioactive constituents of Dioscorea zingiberensis C. H. Wright (D. zingiberensis). In this work, ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS/MS) was applied to the separation and characterization of steroidal saponins in crude extracts from D. zingiberensis. The results showed that fragment ions from glycosidic and cross-ring cleavages gave a wealth of structural information related to aglycone skeletons, sugar types and the sequence of sugar units. According to the summarized fragmentation patterns, identification of steroidal saponins from D. zingiberensis could be fulfilled, even when reference standards were unavailable. As a result, a total of thirty-one saponins with five aglycone skeletons, including fourteen new trace saponins, were identified or tentatively elucidated in crude extracts from D. zingiberensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data.     

Alkaloid profiling of the traditional Chinese medicine Rhizoma corydalis using high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry

Since alkaloids are the major active constituents of Rhizoma corydalis (RC), a convenient and accurate analytical method is needed for their identification and characterization. Here we report a method to profile the alkaloids in RC based on liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (LC–Q-TOF-MS/MS). A total of 16 alkaloids belonging to four different classes were identified by comparison with authentic standards. The fragmentation pathway of each class of alkaloid was clarified and their differences were elucidated. Furthermore, based on an analysis of fragmentation pathways and alkaloid profiling, a rapid and accurate method for the identification of unknown alkaloids in RC is proposed. The method could also be useful for the quality control of RC.KEY WORDS: LC–Q-TOF-MS/MS, Alkaloid, Fragmentation pathway, Rhizoma corydalis     

Characterization of polymethoxylated flavones in Fructus aurantii by off-line two-dimensional liquid chromatography/electrospray ionization-ion trap mass spectrometry

Off-line two-dimensional reversed-phase liquid chromatography (2D-RPLC) coupled to electrospray ionization-ion trap mass spectrometry (ESI-ITMS) was operated in positive mode (PI) to characterize polymethoxylated flavonoids (PMFs) in botanical sample. The fragments of [M+H−n×15]⁺ produced by loss of one or more methyl group from the protonated molecules, as well as [M+H−14]⁺, [M+H−29]⁺, [M+H−33]⁺, [M+H−43]⁺, [M+H−46]⁺ and [M+H−61]⁺ fragments formed the multiple MS (MSⁿ) “fingerprint” of PMFs. 42 target compounds were tentatively identified from the extract of Fructus aurantii (F. aurantii) based on this “fingerprint”. Experimental outcomes indicated that the application of 2D separation method can reduce the matrix suppression of analytes caused by the coelution with interferential components and the column overloading of interferential components. 42 versus 23 target compounds were detected through 2D versus 1D method, which confirm the superiority of 2D coupled to MS in elimination of matrix effects.     

Characterization of flavonoids in the extract of Sophora flavescens Ait. by high-performance liquid chromatography coupled with diode-array detector and electrospray ionization mass spectrometry

A method coupling high-performance liquid chromatography (HPLC) with diode-array detector (DAD) and electrospray ionization mass spectrometry (ESI) was established for the separation and characterization of flavonoids in Sophora flavescens Ait. Based on the chromatographic separation of most flavonoids present in S. flavescens Ait., a total of 24 flavonoids were identified. Fourteen compounds were unambiguously identified comparing experimental data for retention time (t(R)), UV and MS spectra with those of the authentic compounds: 3',7-dihydroxy-4'-methoxy-isoflavone (13), trifolirhizin (14), kurarinol (18), formononetin (19), 7,4'-dihydroxy-5-methoxy-8-(gamma,gamma-dimethylallyl)-flavanone (22), maackiain (21), isoxanthohumol (23), kuraridine (26), kuraridinol (27), sophoraflavanone G (30), xanthohumol (31), isokurarinone (33), kurarinone (35) and kushenol D (38), and additional 10 compounds were tentatively identified as kushenol O (10), trifolirhizin-6'-malonate (15), sophoraisoflavanone A (20), norkurarinol/kosamol Q (24), kushenol I/N (25), kushenol C (28), 2'-methoxykurarinone (29), kosamol R (32), kushecarpin A (34) and kushenol A (37) by comparing experimental data for UV and MS spectra with those of literature. Furthermore, fragmentation pathways in positive ions mode of 24 flavonoid compounds of types of flavanone, flavanonol, flavonol, chalcone, isoflavone, isoflavanone and ptercocarpane were summarized. Some common features, such as CH(3)., H(2)O, CO, CO(2), C(3)O(2) and C(2)H(2)O losses, together with Retro-Diels-Alder fragmentations were observed in the prenylated flavonoids in S. flavescens Ait. The loss of the lanandulyl chain was their characteristic fragmentation, which might help deducing the structure of unknown flavonoid compounds. The present study provided an approach to rapidly characterize bioactive constituents in S. flavescens Ait.     

Rapid identification of acetophenones in two Cynanchum species using liquid chromatography–electrospray ionization tandem mass spectrometry

Acetophenones in Cynanchum species, especially cynandione A and its derivatives, whose utilization and toxicity in herbal drugs and folk medicines has caused great interest in the chemical investigation, have extensive biological activities. In this paper, a facile method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MSⁿ) was developed for the analysis of cynandione A derivatives in the roots of the Cynanchum wilfordii and C. auriculatum. ESI-MS/MS and ESI-MSⁿ analysis of cynandiones A and B in negative ion mode were firstly performed employing two mass spectrometers each equipped with an ion-trap and a quadrupole time-of-flight (Q-TOF) mass analyzer. The results drawn from both instruments were similar to each other. Characteristic fragmentation pathways were proposed by comparing the spectra of two standards acquired in the experiments. The fragment ions at m/z 283 and 268 were obtained, and then were used as diagnostic ions to screen and identify cynandione A derivatives from the roots of above two species, together with an HPLC-MSⁿ method. Total of 28 cynandione A derivatives comprising 4 reported and 24 novel components were identified or tentatively identified. Furthermore, breakdown curves were constructed to distinguish two types of isomers among these compounds. To our knowledge, this is the first report on characterization of acetophenones by HPLC-ESI-MSⁿ, which allows a rapid and complete analysis of cynandione A derivatives in roots of Cynanchum species.     

Characterization of alkaloids in Sophora flavescens Ait. by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Sophora flavescens Ait., a well-known Chinese herbal medicine, is widely used in clinical practice for the treatment of viral hepatitis, cancer, gastrointestinal hemorrhage, and skin diseases. This paper is the first report on a method based on the combined use of high-performance liquid chromatography, photodiode array detection, and electrospray ionization tandem mass spectrometry for the comprehensive and systematic separation and characterization of bioactive alkaloids in Sophora flavescens Ait. A total of 22 constituents were identified on the basis of the extracted ion chromatograms for different [M+H](+) ions of the alkaloids present in S. flavescens Ait. Among these, 5 constituents were unambiguously identified by comparing the experimental data on their retention times and MS(n) spectra with those of the authentic compounds, and 17 other constituents were tentatively identified on the basis of their MS(n) fragmentation behaviors and/or molecular weight information from literatures. Furthermore, some characteristic fragmentation pathways of the alkaloids in S. flavescens Ait. were detected and examined. This information may be useful for characterizing the bioactive alkaloids present in S. flavescens Ait. and for possible applications in formulations.     

Profiling and Characterization of Sialylated N-glycans by 2D-HPLC (HIAX/PGC) with Online Orbitrap MS/MS and Offline MSn

Glycosylation is a critical parameter used to evaluate protein quality and consistency. N-linked glycan profiling is fundamental to the support of biotherapeutic protein manufacturing from early stage process development through drug product commercialization. Sialylated glycans impact the serum half-life of receptor–Fc fusion proteins (RFPs), making their quality and consistency a concern during the production of fusion proteins. Here, we describe an analytical approach providing both quantitative profiling and in-depth mass spectrometry (MS)-based structural characterization of sialylated RFP N-glycans. Aiming to efficiently link routine comparability studies with detailed structural characterization, an integrated workflow was implemented employing fluorescence detection, online positive and negative ion tandem mass spectrometry (MS/MS), and offline static nanospray ionization–sequential mass spectrometry (NSI–MSⁿ). For routine use, high-performance liquid chromatography profiling employs established fluorescence detection of 2-aminobenzoic acid derivatives (2AA) and hydrophilic interaction anion-exchange chromatography (HIAX) charge class separation. Further characterization of HIAX peak fractions is achieved by online (−) ion orbitrap MS/MS, offering the advantages of high mass accuracy and data-dependent MS/MS. As required, additional characterization uses porous graphitized carbon in the second chromatographic dimension to provide orthogonal (+) ion MS/MS spectra and buffer-free liquid chromatography peak eluants that are optimum for offline (+)/(−) NSI–MSⁿ investigations to characterize low-abundance species and specific moieties including O-acetylation and sulfation.     

Liquid chromatography/mass spectrometry-based structural analysis of new platycoside metabolites transformed by human intestinal bacteria

Platycosides, the main active constituents of Platycodi Radix, have been thoroughly studied for the characterization of their potent biological activities. However, metabolism of platycosides has not yet been characterized. A HPLC electrospray ionization-tandem mass spectrometry (LC/ESI-MSⁿ) approach was applied to new complex platycoside metabolites transformed by human intestinal bacteria to identify their structures and determine metabolic pathway. The molecular weights of metabolites were identified by LC/ESI-MS analysis in both positive and negative modes. Structures for the platycoside metabolites were proposed by the molecular weights and the expected enzymatic activity of intestinal microbes on platycoside. In the second step, successive LC–MSⁿ analysis was used to demonstrate the proposed structures. Under ESI tandem mass conditions, the sequential fragmentation patterns of [M+Na]⁺ ions exclusively showed signals, consistent with the cleavage of glycoside bonds, rearrangement and some cross-ring cleavage, thus allowing the rapid identification of platycoside metabolites. The metabolites identified in the time-dependent metabolism experiments enable us to propose several microbial pathways for platycosides. Even though the metabolites of some platycosides may have unknown structures and low levels, the analytical tools presented in this study made it possible to obtain a rapid and complete characterization of new metabolites and their metabolism pathway in human intestinal bacteria.     

Development of an in-line HPLC fingerprint ion-trap mass spectrometric method for identification and quality control of Radix Scrophulariae

Chromatographic fingerprinting has been widely accepted as a crucial method for qualitative and quantitative analyses of bioactives within traditional Chinese medicine. A fingerprint provides detailed information, specific for any given herb, thus facilitating the quality control measures of a given traditional Chinese medicine. In this article, quality assessment of Radix Scrophulariae was achieved by using high performance liquid chromatography combining diode-array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS). Eight batches of sample obtained from different origins in China were used to establish the fingerprint and quantitative analyses. By comparing the retention times, UV and MS spectral data with reference standards, four characteristic peaks in the chromatograms were confirmed as corresponding to acetoside, angoroside C, cinnamic acid, and harpagoside. In addition, other two characteristic peaks were tentatively identified, following the literature interpretation of HPLC-ESI-MS and LC-MS/MS (affording structural information) to be sibirioside A and scrophuloside B₄, respectively. The results indicated that the newly developed HPLC-DAD-MS fingerprint method would be suitable for quality control of Radix Scrophulariae.     

Identification and determination of the major constituents in Traditional Chinese Medicinal formula Danggui-Shaoyao-San by HPLC–DAD–ESI-MS/MS

Danggui-Shaoyao-San (DSS), a famous traditional Chinese medicine formula consisting of six herbal medicines (Paeonia lactiflora, Angelica sinensis, Ligusticum chuanxiong, Poria cocos, Atractylodis macrocephalae and Rhizoma Alismatis), has been used as a classical gynecological remedy in China for centuries. However, its active substances have remained unknown. In this paper, an HPLC/DAD/ESI-MS/MS method was developed for the qualitative and quantitative analysis of the major constituents in DSS. The ESI-MS/MS fragmentation behavior of the reference compounds was proposed for aiding the structural identification of components in DSS extract. Forty-one compounds including monoterpene glycosides, phenolic acids, phathalides, sesquiterpenoids and triterpenes were identified or tentatively characterized by comparing their retention times, UV and MS spectra with those of authentic compounds or literature data, and 14 of them (gallic acid, albiflorin, paeoniflorin, ferulic acid, benzoic acid, senkyunolide I, coniferyl ferulate, senkyunolide A, 3-butylphthalide, Z-ligustilide, Z-butylidenephthalide, atractylcnolide II, atractylcnolide I and levistolide A) were determined by HPLC–DAD using a C₁₈ column and gradient elution of acetonitrile/water–formic acid (100:0.1, v/v). The linearity, precision, accuracy, LOD and LOQ were validated for the quantification method, which proved sensitive, accurate and reproducible. The study might provide a basis for the quality control of DSS extracts and preparations.     

LC-MS/MS as a tool for identification of bioactive compounds in marine sponge Spongosorites halichondriodes (Dendy 1905)

The sensitivity, specificity and selectivity of liquid chromatography/mass spectrometry tandem mass spectrometry (LC-MS/MS) make it an essential tool for the characterization and identification of low molecular compounds such as fatty acids, sterols, cholastane derivatives, nucleosides etc. In the current work, the marine sponge Spongosorites halichondriodes (order Halichondrida, Family Halichondriidae); a particularly rich source of cytotoxic compounds is studied for the initial characterization of bioactive compounds. The composition of ethyl acetate and butanol extracts were subjected to LC-MS and LC-MS/MS. Many novel sterol derivatives compounds which were not reported in any marine sponge mainly belonged to the group of C₂₅-C₂₈ saturated and unsaturated esters like 3β, 4β, 7α, 12α-tetrahydroxy-5β-cholan-24-oic acid methyl ester, 7 α, 12 β-dihydroxy-5 β-cholan24-oic acid methyl ester, novel isocoumarin citrinolactone A, a triterpenoid glycyrrhetinic acid as well as other unknown compounds in this species such as nucleoside inosine was identified. Other compound investigated was 3β, 6β, 7α-trihydroxy-5β-cholan-24-oic acid methyl ester. All the sterol ester derivatives are reported here for the first time in marine sponge belonging to family Halichondriidae. However, the literature report supports the occurrence of 3β-hydroxy sterols which is considered as a biomarker for this family.     

Simultaneous determination of five lignan constituents of Wuzhi capsule in rat plasma by LC–MS/MS: Application to pharmacokinetic study

A rapid sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of multiple bioactive lignan constituents of Wuzhi capsule in rat plasma. The extraction, separation, and analytical conditions were optimized. Five constituents of the Wuzhi capsule (schisandrin, schisandrol B, schisantherin A, schisanhenol, and deoxyshisandrin) were determined by the LC–MS/MS method. Liquid–liquid extraction with methyl tert-butyl ether was carried out using bifendate as the internal standard. The five bioactive constituents were separated on a Zorbax SB-C18 reserved-phase column (100 mm × 2.1 mm i.d., 3.5 μm) by isocratic elution using a mobile phase consisting of acetonitrile, methanol, and 0.1% aqueous formic acid (72:18:10, v/v/v) at a flow rate of 0.3 mL/min. The total run time was only 3.5 min. All analytes showed good linearity over a wide concentration range (r² > 0.99) and their lower limit of quantification was 0.5 ng/mL. The average extraction recovery of the five analytes from rat plasma was more than 85%, and the intra-day and inter-day accuracy and precision of the assay were less than 15%. Our method was successfully used for pharmacokinetic study of the five components in the Wuzhi capsule.     

Identification and determination of the major constituents in Deng's herbal tea granules by rapid resolution liquid chromatography coupled with mass spectrometry

Deng's herbal tea (DHT), a famous traditional Chinese herbal tea consisting of six traditional Chinese medicines (Honeysuckle, Chrysanthemum, Rhizoma imperatae, Folium mori, dandelion and liquorice), is widely used in China for its health benefits. In this paper, a rapid resolution liquid chromatography coupled with mass spectrometry (RRLC-MS) method was developed for the identification and determination of the major constituents in DHT granules. A good RRLC separation was achieved using an Agilent Poroshell 120 SB-C₁₈ column and gradient elution (0.5% formic acid in water/acetonitrile) within 30 min. Twenty-eight compounds were identified or tentatively characterized based on their exact molecular weights and fragmentation patterns. Fifteen major bioactive constituents of those 28 compounds were chosen as the benchmark substances. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple-reaction monitoring mode, and a full quantitative analysis of the 15 major constituents was performed by our developed RRLC-MS/MS method in only 10 min. Of the 16 DHT granule samples tested, the quality of the results was stable, which confirms that the developed method was efficient and robust for the quality control of DHT granules.     

Identification and characterization of flavonoids from semen zizyphi spinosae by high-performance liquid chromatography/linear ion trap FTICR hybrid mass spectrometry

Semen zizyphi spinosae (SZS) has been used to treat insomnia and anxiety for thousands of years. In this paper, a novel high-performance liquid chromatography coupled with the photodiode array detector/linear ion trap-MS ⁿ (HPLC-PDA/LTQ-MS ⁿ ) method was established to separate and identify flavonoids from the extract of SZS. Separation was performed on an HYPERSIL C₁₈ column by gradient elution using CH₃CN/H₂O–CH₃COOH as the mobile phase at a flow rate of 0.8 ml/min. UV spectral data, accurate molecular weights, and multi-stage MS/MS fragmentation information were obtained. Electrospray ionization/MS/MS fragmentation patterns were proposed. Nineteen flavonoid glycosides were identified or tentatively characterized based on their retention time, UV spectral data, accurate molecular weights, and mass fragmentation behavior. The method was useful for separation and identification of the flavonoid components from SZS and could be applied to other complex samples, especially for minor constituents.     

A high expression EGFR/cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Rhizoma Polygoni Cuspidati

The epidermal growth factor receptors (EGFRs) in some tumor cells are significant targets for drug discovery. In this work, we have developed an EGFR cell membrane chromatography and online high performance liquid chromatography/mass spectrometry system for screening active component from Rhizoma Polygoni Cuspidati. As a result, resveratrol from Rhizoma Polygoni Cuspidati was found to be the active component acting on EGFR like gefitinib. There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro. The EGFR/CMC-online-HPLC/MS system demonstrated fast and effective characteristics for screening leading compounds from traditional Chinese medicine.