[Determination of Bioactive Components in Paeonia lactiflora Roots Cultivated in Various Areas by UHPLC].

Objective: To assess the quality of Paeonia lactiflora roots by the multi-bioactive chemical markers. Methods: Total of66 Paeonia lactiflora roots samples were collected from Sichuan,Zhejiang and Anhui. An UHPLC-DAD technique was employed to quantify the contents of paeoniflorin,hydroypaeoniflorin,abiflorin,pentagalloyglucose,benzoypaeoni-florin,paeonol,gallic and catechin in these samples. These chemical components in each sample were also calculated by Principal Component Analysis( PCA). Results: The eight bioactive components were good separated in 30 min on the UHPLC chromatogram. The correlation coefficients between peak areas and concentration for these bioactive components were not less than 0. 9990( n = 6). And their recoveries were in the range of95. 94% ~ 100. 92%( n = 6). The contents of paeoniflorin in Paeonia lactiflora roots samples collected from Sichuan,Zhejiang and Anhui were 40. 54 mg / g( n = 23),33. 09 mg / g( n = 22) and 39. 47 mg / g( n = 21),respectively. The values of PCA were 0. 4435( n = 23)for the samples from Sichuan,0. 0122( n = 22) for the samples from Zhejiang and- 4. 9850( n = 21) for the samples from Anhui. The content of paeoniflorin in biennial,triennial,four-year,five-year and six-year old Paeonia lactiflora roots were 24. 76( n = 2),37. 17( n= 16),37. 83( n = 23),39. 71( n = 16) and 37. 45 mg / g( n = 7),respectively. Conclusion: The developed method can accurately quantify the content of principal bioactive compounds in Paeonia lactiflora roots. The quality is various among Paeonia lactiflora roots cultivated in Sichuan, Zhejiang and Anhui on the basis of paeoniflorin content or the value of PCA,but the quality of Paeonia lactiflora roots cultivated in Sichuan is the best. Moreover, it is suggested Paeonia lactiflora roots should be harvested in third or fourth year based on the output and quality.

[Analysis of different parts and tissues of Panax Notoginseng by Fourier transform infrared spectroscopy].

The techniques of Fourier transform infrared (FTIR) spectroscopy were applied to analyze the different parts and tissues of Panax Notoginseng (Sanqi, SQ), i.e. rhizome, main root, rootlet, fibrous root, xylem, cambium, phloem and epidermis. Both the FTIR spectra and second derivative spectra of these various parts and tissues of SQ samples were found to be similar. Their dominant component is starch resulting from the characteristic peaks of starch observed at 3 400, 2 930, 1 645, 1 155, 1,080 and 1,020 cm(-1) on the spectra of all these SQ samples. However, the varieties of peaks were found on the spectra among these specific samples. The rhizome contains more saponins than others on the basis of the largest ratio of the peak intensity at 1,077 cm(-1) to that at 1,152 cm(-1). The peaks located at 1 317 and 780 cm(-1) on the FTIR spectra of the rhizome and its epidermis indicate that the two parts of SQ samples contain large amount of calcium oxalate, and its content in the latter is relative larger than that in former. The fibrous root contains much amount of nitrate owing to the obvious characteristic peaks at 1 384 and 831 cm(-1). For the difference among the various tissues of SQ samples, the peaks at 2,926, 2,854 and 1,740 cm(-1) on the FTIR spectra of epidermis is the strongest among the various tissues of main root indicating the largest amount of esters in epidermis. Protein was also found in the cambium of the main root based on the relative strong peaks of amide I and II band at 1,641 and 1,541 cm(-1), respectively. The results indicate that FTIR spectra with its second derivative spectra can show the characteristic of the various parts and tissues of SQ samples in both the holistic chemical constituents and specific chemical components, including organic macromolecule compounds and small inorganic molecule compounds. FTIR spectroscopy is a useful analytical method for the genuine and rapid identification and quality assessment of SQ samples.

[Chemical change of chuanxiong raw materials during storage].

OBJECTIVE: To evaluate the chemical changes in Chuanxiong raw material (CX), the rhizome of Ligusticum chuanxiong Hort., during CX storage and further assess its quality variety. METHODS: Four CX samples were sealed and stored at ambient temperature in room for two years. These samples were quantified on the amounts of characteristic chemical compounds by HPLC-DAD-(APCI) MS techniques. RESULTS: Eight characteristic peaks in HPLC fingerprint were found to be good separation and assigned as vanillin (1), ferulic acid (2), senkyunolide I (3), senkyunolide H (4), coniferyl ferulate (5), senkyunolide A (6), Z-ligustilide (7) and levistolide A(8), respectively based on their on-line APCI-MS data and UV spectra. After CX being stored, compounds 1 - 4, and 8 were decreased by 44.4%, 52.1%, 37.6%, 52.8% and 47.5% (n = 4), respectively, whilst compounds 5 - 7 were increased by 59.1%, 40.1% and 47.5% (n = 4), respectively. CONCLUSION: Multiple chemical compounds are found to be changed during CX storage, which results in the variety of quality and therapeutic effect because most of the tested compounds have been demonstrated to be bioactive by pharmacological study and clinical trials. It is suggested that CX should be stored under dark, cool and dry condition.

Bioactivity-guided fractionation of the volatile oil of Angelica sinensis radix designed to preserve the synergistic effects of the mixture followed by identification of the active principles.

In natural product research, it is a common experience that fractionation of biologically-active crude extracts can lead to the loss of their original activity. This is attributed to synergistic effects, where two or more components are required to be present together for full activity of the sample. Our previous study showed that a volatile oil of Angelica sinensis radix (VOAS) inhibited endothelial cell proliferation in culture. Here we have used a bioactivity-guided fractionation method to preserve any synergistic effects of VOAS combining countercurrent chromatography (CCC), the MTS cell viability assay and gas chromatography (GC). Using a two-phase CCC solvent system (heptane-ethyl acetate-methanol-water at a volume ratio of 27:23:27:23%), forty-five fractions were isolated, nine of which exhibited anti-endothelial properties. GC analysis showed two bioactive alkylphthalides, Z-ligustilide and n-butylidenephthalide (BP) were the major compounds detected in the bioactive fractions, and were absent in non-bioactive fractions. Our results indicate that Z-ligustilide and BP are the main constituents responsible for the anti-endothelial properties of VOAS. This rapid and reliable approach in preserving sample activity while isolating and identifying its active compounds suggests that this protocol can be a powerful tool for drug discovery from natural products.

Effect of electro-acupuncture intervention on cognition attention bias in heroin addiction abstinence-a dot-probe-based event-related potential study.

OBJECTIVE: To study the changes of cognitive attention-related brain function in the heroin addicts before and after electro-acupuncture (EA) intervention for exploring the concerned neuro-mechanism of addictive relapse and the central action role of EA intervention. METHODS: Adopting event-related potential (ERP) technique, the ERP at 64 electrode spots in 10 heroin addicts (test group) were recorded before and after EA intervention with dot-probe experimental form during implementing cognitive task on positive emotional clue (PEC), negative emotional clues (NEC), and heroin-related clue (HRC). The P200 amplitude components on the selected observation points (Fz, Cz, and Pz) were analyzed and compared with those obtained from 10 healthy subjects as the control. RESULTS: Before EA, the ERP of attention on HRC in the test group was higher than that on PEC and NEC (P<0.05) and significantly higher than that in the control group (P<0.05); after EA, the P200 amplitude of attention on HRC at Cz and Pz was significantly lowered (P<0.05) and that on PEC at Fz was significantly elevated (P<0.05). After EA, the P200 amplitude at Pz was ranked as NEC > PEC > HRC, but in the control group, it showed PEC > HRC at all three observation points and PEC > NEC at Pz. CONCLUSION: Heroin addicts show attention bias to HRC, which could be significantly reduced by EA intervention, illustrating that EA could effectively inhibit the attention bias to heroin and so might have potential for lowering the relapse rate.

Improved chromatographic fingerprints for facile differentiation of two Ganoderma spp.

This paper addresses a comprehensive and comparative study of six phytochemical extraction methods for triterpenes from the fruiting body of Ganoderma spp. Quantitative analysis of extracts was performed by HPLC with photodiode array detection. In general, pressurized liquid extraction and microwave-assisted extraction under optimized conditions produce better yields, and the former also significantly reduces the total time of extraction and manipulation of a sample, as well as the amount of solvent used in comparison with conventional soxhlet, reflux, ultrasonic, and methanol-CO(2) supercritical fluid extractions. Based on the improved extraction protocol, the fingerprinting profiles for two species of Lingzhi were established using the consistent chromatographic features of 12 authentic samples. Eleven common peaks of ganoderic/ganoderenic acids were identified using LC-ESI-MS-MS. These specific triterpene groups were adopted as chemical markers for Lingzhi. Using chemometric analysis, the developed fingerprinting was successfully applied to differentiate between the two species under the Ganoderma genus and is applicable as a method for quality evaluation of this valuable medicinal fungus and its related proprietary products.

Identification and determination of the major constituents in traditional Chinese medicinal plant Polygonum multiflorum thunb by HPLC coupled with PAD and ESI/MS.

An HPLC-PAD-MS method has been developed for the qualitative and quantitative analysis of the major chemical constituents in Polygonum multiflorum Thunb. Chromatographic separation was conducted on an Alltima C18 column using water:acetonitrile:acetic acid as the mobile phase. Altogether nine compounds of various classes, including stilbene glucosides, anthraquinone glucosides and anthraquinone derivates, were identified by online ESI/MS. Their identities were ascertained by comparison with data derived from the literature and/or standard compounds. Five components were quantified by HPLC-PAD and the method was fully validated. All the linear regressions were acquired with R2 > 0.99 and the quantification limits (with a signal-to-noise ratio of 3) ranged from 0.63 and 1.57 ng. Repeatability was evaluated by intra- and inter-day assays and RSD value was within 2.47%. Recovery studies for the quantified compounds were found to be within the range 96.32-102.53% with RSD less than 2.35%. The overall procedure is rapid and reproducible and is considered suitable for qualitative and quantitative analysis of a large number of samples.

Comparative analysis of Ligusticum chuanxiong and related umbelliferous medicinal plants by high performance liquid chromatography-electrospray ionization mass spectrometry.

A highly precise and accurate method, based on high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS), was developed for the qualitative and quantitative comparison of the main constituents in the rhizome of Ligusticum chuanxiong (LC) and three related umbelliferous medicinal plants. A comprehensive validation of the developed method was conducted, and the method was highly sensitive, reproducible and accurate. The unique properties of the present method were validated by analyzing 20 related herbal samples including 5 LC samples, 5 Cnidium officinale samples (CO), 5 Angelica sinensis samples (AS) and 5 Angelica acutiloba samples (AA). Twelve compounds including phenolic constituents, alkylphthalides and phthalide dimers were identified by online ESI-MS and by comparison with literature data and standard compounds, and six of them were quantified by HPLC-DAD simultaneously. The results demonstrated that identical compound types were identified as the main constituents of LC, CO, AS and AA herbs. The results also support the alternative application of these medicinal plants in Chinese and Japanese folk medicines. In the present study, it was found that the variation in the abundance of senkyunolide A was significant in these related herbs; it is therefore feasible to choose senkyunolide A as a characteristic compound for quality evaluation and chemical authentication of these herbs.

Identification and comparative determination of senkyunolide A in traditional Chinese medicinal plants Ligusticum chuanxiong and Angelica sinensis by HPLC coupled with DAD and ESI-MS.

Using the HPLC/DAD/ESI/MS method, the qualitative and quantitative analysis of senkyunolide A (SA) in the rhizomes of Ligusticum chuanxiong (Rhizoma chuanxiong; CX) and roots of Angelica sinensis (DG) was established. As a result, it was found that SA is a characteristic standard compound for the quality evaluation and chemical differentiation between CX and DG. Methanol was chosen in the preparation of standard solutions and extraction of samples based on the stability data. The identity of SA in CX and DG was unambiguously determined based on the quasimolecular ions in ESI-MS. A comprehensive validation of the method, including sensitivity, linearity, reproducibility and recovery, was conducted using the optimized chromatographic conditions. The linear calibration curve was acquired with R2>0.999 and limit of detection (S/N=3) was estimated to be 12.5 mug/g. The reproducibility was evaluated by repeated sample injection and replicated analysis of samples with the relative standard deviation (RSD) value found within 0.68%. The recovery rates of SA varied within the range of 96.91-101.50% with RSD less than 2.38%. In the present work, the contents of SA were quantified within 3.94-9.14 mg/g and 0.108-0.588 mg/g for 12 batches each of CX and DG. The results demonstrated that SA is a useful standard compound for the quality evaluation and chemical differentiation between CX and DG. The analytical procedure is precise and reproducible and thus suitable for the analysis of a large number of samples.

Systematic evaluation of organochlorine pesticide residues in Chinese materia medica.

A systematic evaluation on the levels of organochlorine pesticide residues (OCP) was conducted on four selected, authentic Chinese materia medica, namely: Radix Angelicae Sinensis, Radix Notoginseng, Radix Salviae Miltiorrhizae and Radix Ginseng. Altogether ten representative batches of samples were analysed for each herb. Six batches were collected in the major cultivation areas of the Mainland whilst the remaining four batches were procured in the Hong Kong herbal market. All except Radix Angelicae Sinensis have been identified as containing quintozene and hexachlorocyclohexane in various levels. Hexachlorobenzene and lindane were also reported in samples of Radix Ginseng. The banned pesticide, DDT and its derivatives, was also observed in one of the Radix Notoginseng samples. The investigation will be continued for a target list of common used herbs in Hong Kong. All the results will be gathered and analysed for setting up regulatory permissible limits of OCP residues in Chinese materia medica used in Hong Kong.

Development of high-performance liquid chromatographic fingerprints for distinguishing Chinese Angelica from related umbelliferae herbs.

A high-performance liquid chromatographic (HPLC) fingerprint of Chinese Angelica (CA) was developed basing on the consistent chromatograms of 40 CA samples (Angelica sinensis (Oliv.) Diels). The unique properties of this HPLC fingerprints were validated by analyzing 13 related herbs including 4 Japanese Angelicae Root samples (JA, A. acutiloba Kitagawa and A. acutiloba Kitagawa var. sugiyame Hikino), 6 Szechwan Lovage Rhizome samples (SL, Ligusticum chuanxiong Hort.) and 3 Cnidium Rhizome samples (CR, Cnidium officinale Makino). Both correlation coefficients of similarity in chromatograms and relative peak areas of characteristic compounds were calculated for quantitative expression of the HPLC fingerprints. The amount of senkyunolide A in CA was less than 30-fold of that in SL and CR samples, which was used as a chemical marker to distinguish them. JA was easily distinguished from CA, SL and CR based on either chromatographic patterns or the amount of coniferyl ferulate. No obvious difference between SL and CR chromatograms except the relative amount of some compounds, suggesting that SL and CR might have very close relationship in terms of chemotaxonomy. Ferulic acid and Z-ligustilide were unequivocally determined whilst senkyunolide I, senkyunolide H, coniferyl ferulate, senkyunolide A, butylphthalide, E-ligustilide, E-butylidenephthalide, Z-butylidenephthalide and levistolide A were tentatively identified in chromatograms based on their atmospheric pressure chemical ionization (APCI) MS data and the comparison of their UV spectra with those published in literatures.

Assay of free ferulic acid and total ferulic acid for quality assessment of Angelica sinensis.

Activity of Chinese Danggui (DG), the processed root of Angelica sinensis (Oliv.) Diels, is linked to the ferulic acid content but the stability of ferulic acid during extraction for medicinal use is not known. The stabilities of ferulic acid and coniferyl ferulate were evaluated in the extracts of DG using a variety of extraction solvents. These included various combinations and proportions of methanol, water, formic acid, 1 M aqueous hydrochloric acid and 2% sodium hydrogen carbonate (NaHCO3) in water. Coniferyl ferulate was found liable to hydrolyze into ferulic acid in neutral, strongly acidic and basic solvents, where heat and water could facilitate this hydrolysis. However, the hydrolysis was relatively resisted in weakly organic acid. Based on the stability evaluation, two new terms, namely: free ferulic acid and total ferulic acid, were suggested and defined. Free ferulic acid refers to the natural content of ferulic acid in herbs. Total ferulic acid means the sum of free ferulic acid plus the amount of related hydrolyzed components. Meanwhile, the high-performance liquid chromatographic (HPLC) method was developed to assay free ferulic acid and total ferulic acid in DG using methanol-formic acid (95:5) and methanol-2% NaHCO3 in water (95:5) as extraction solvents, respectively. Ten DG samples were investigated on their contents of free and total ferulic acid. The results indicated that the amount variety of free ferulic acid was larger than that of their counterparts, and the ratio of total ferulic acid to free ferulic acid was 4.07 +/- 2.73 (mean +/- SD, n = 10). The chemical assay of DG using total ferulic acid content would be a better choice to assess the herbal quality and was recommended.

Quantification of ligustilides in the roots of Angelica sinensis and related umbelliferous medicinal plants by high-performance liquid chromatography and liquid chromatography-mass spectrometry.

A reversed phase high-performance liquid chromatographic method was developed for quantifying E-ligustilide (1) and Z-ligustilide (3) in the roots of Angelica sinensis (Oliv.) Diels with confirmation using UV, atmospheric pressure chemical ionisation (APCI) MS and APCI-MS-MS techniques. Based on the UV spectra of compounds 1, E-butylidenephthalide (2), 3 and Z-butylidenephthalide (4), the absorption at 350 nm was chosen as measuring wavelength in which baseline separation of compounds 1 and 3 could be obtained but avoided the interference of compounds 2 and 4. The identity of compounds 1 and 3 in samples were unambiguously determined by the respective quasi-molecular ions ([M+H]+) in APCI-MS. According to the stability data, acetonitrile was chosen for the preparation of standard solutions in order to minimize the isomerization of compound 3. Compounds 1 and 3 were qualitatively and quantitatively analyzed in seven samples of the roots of Angelica sinensis (Oliv.) Diels, Angelica acutiloba Kitagawa, Angelica acutiloba Kitagawa var. sugiyamae Hikino and the rhizome of Ligusticum chuanxiong Hort. Analysis of an extract from a sample root of Angelica gigas Nakai using LC-MS for the first time could not detect the presence of ligustilide in this herb. The overall analytical procedure is rapid and reproducible which is considered suitable for quantitative analysis of large number of samples.

[Study on the identification of dangguitou and dangguiwei by two-dimensional infrared correlation spectroscopy].

Fourier transform infrared spectroscopy (FTIR) combined with two-dimensional correlation spectroscopy was used to identify the different parts of Danggui in this paper. Guitou and Guiwei are two parts of the same officinal plant, and similar to each other in normal. FTIR spectra revealed the comparabilities of these two parts in herbal characters, organizing structure and alkaloid components. However, variance in amino acids and naphtha contents, which is aroused by the different parts of the angelica, could be identified easily and clearly in 2D FTIR spectra. Thus the fast and accurate property of 2D FTIR spectroscopy makes it a powerful and new approach to evaluating the medicinal herbs impersonally.

[Primary research of pharmacological effects of PEC on mice].

OBJECTIVE: To study pharmacological effects of PEC (the oral liquid which consists of Panax quinquefolium, Epimedium brevicornum, Schisandra chinensis Bail and Cervus eplaphus) on mice. METHOD: Experiments were carried out through swimming test, step-through, spontaneous activity and sleeping time. RESULT: When 5-10 mL x kg(-1) of PEC was given orally for 7 days, it could prolong swimming duration of mice in water tank, and increase the tolerant ability against oxygen-deficiency. PEC could also improve cognitive-deficiency induced by taking off sleep with force in mice after given orally for 7 days. The PEC could increase the spontaneous activity in mice, antagonize the inhabitation induced by Valium, and shorten the sleeping time caused by sodium pentobarbital. CONCLUSION: PEC has strong potential neuro-pharmacological activities such as anti-fatigue, improving cognitive-deficiency in mice.