An effective and high-throughput sample preparation method involving demalonylation followed by an ultrahigh-performance liquid chromatography-charged aerosol detector for analyzing gypenoside XLIX and gypenoside A in Gynostemma longipes.

Gypenosides (Gps) are the major bioactive components in Gynostemma species. They include neutral Gps and acidic malonylgypenosides (MGps). MGps are abundant in Gynostemma species and can be transformed into corresponding Gps via extraction, concentration, and drying. If only the Gps were quantified and MGps were ignored, the quality of Gynostemma species would be underestimated. This study aimed to develop a sample preparation method involving demalonylation and ultrahigh-performance liquid chromatography-charged aerosol detector (UHPLC-CAD) analysis to determine the contents of gypenoside XLIX (Gp XLIX) and gypenoside A (Gp A). First, the optimized ultrasonic extraction method was established to extract G. longipes powder ultrasonically. Then, the extracted solution was put into a closed container (centrifuge tube) and heated in a water bath at 95 °C. Then, MGps were converted into corresponding Gps. The proposed preparation method was compared with the other three methods, including water bath reflux heating, alkali hydrolysis, and extraction of heated powder, and was shown to exhibit higher conversion and better convenience. Subsequently, an UHPLC-CAD method was established and validated. Gp XLIX and Gp A showed excellent linear correlations between 15.55 and 248.8 µg/mL and 24.10-385.5 µg/mL, respectively (R2 > 0.999). The limit of detection was 1.40 ng (Gp XLIX) and 2.41 ng (Gp A), and the limit of quantification was 7.77 ng and 14.46 ng, respectively. The relative standard deviation for precision, stability, and repeatability was 0.63-3.15%. The average recovery of Gp XLIX and Gp A was 98.97% and 98.23%, respectively. The established method was applied for determining Gp XLIX and Gp A contents in wild or cultivated G. longipes samples collected from the Qinba Mountains area. The contents of Gp XLIX and Gp A were 5.16-23.02 mg/g and 15.78-54.55 mg/g, respectively. Conclusively, the proposed sample preparation and analysis method could be used for the quality control and evaluation of G. longipes.

[Rapid prediction of flavonoid content in Epimedium sagittatum by infrared spectroscopy].

Epimedii Folium is a well-known Chinese herbal medicine with the effect of nourishing kidney and strengthening Yang. Its main active ingredients are flavonoids. In this study, 60 samples of Epimedium sagittatum were collected for the determination of total flavonoids(TF) including the total amount of epimedin A, epimedin B, epimedin C, and icariin(abbreviated as ABCI) specified in the Chinese Pharmacopoeia as well as rhamnosylicariside Ⅱ and icariside Ⅱ. The calibration parameters of "first derivativemultiva-riate scattering correction in 1 900-650 cm~(-1) band(4-point smoothing)" and "first derivativestandard normal variable correction in 4 000-650 cm~(-1) full band(4-point smoothing)" were confirmed respectively. The quantitative model was established via Fourier infrared spectroscopy plus attenuated total reflection(FTIR-ATR) accessory combined with partial least squares(PLS) method and then used to predict the flavonoid content of 11 validation sets. The average prediction accuracy for ABCI in calibration set and validation set was 98.985% and 96.087%, respectively. The average prediction accuracy for TF in calibration set and validation set was 98.998% and 94.771%, respectively. These results indicated that FTIR-ATR combined with PLS model could be used for rapid prediction of flavonoid content in E. sagittatum, with the prediction accuracy above 94.7%. The establishment of this method provides a new solution for the detection of a large number of E. sagittatum samples.

New prenylated flavonoid glycosides derived from Epimedium wushanense by ß-glucosidase hydrolysis and their testosterone production-promoting effects.

Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A-D (1-4) and two new prenyl flavonoid derivatives wushepimedoside E-F (5-6), and one know analog epimedkoreside B (7) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1-7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3ß-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.

Quality analysis of hawthorn leaves (the leaves of Crataegus pinnatifida Bge. var major N.E.Br) in different harvest time.

INTRODUCTION: Harvest time plays an important role on the quality of medicinal plants. The leaves of Crataegus pinnatifida Bge. var major N.E.Br (hawthorn leaves) could be harvested in summer and autumn according to the Pharmacopoeia of the People's Republic of China (Pharmacopoeia). However, little is known about the difference of the chemical constituents in hawthorn leaves with the harvest seasonal variations. OBJECTIVE: The chemical constituents of hawthorn leaves in different months were comprehensively analysed to determine the best harvest time. METHODS: Initially, the chemical information of the hawthorn leaves were obtained by ultra-high-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Subsequently, principal component analysis (PCA) was applied to compare the chemical compositions of hawthorn leaves harvested in different months. Then, an absolute quantitation method was established using high-performance liquid chromatography-charged aerosol detector (HPLC-CAD) to determine the contents of five compounds and clarify the changes of these components with the harvest seasonal variations. Meanwhile, a semi-quantitative method by integrating HPLC-CAD with inverse gradient compensation was also established and verified. RESULTS: Fifty-eight compounds were identified through UHPLC-Q-TOF-MS. PCA revealed that the harvest season of hawthorn leaves had a significant effect on the chemical compositions. The contents of five components were relatively high in autumn. Other four main components without reference standards were further analysed through the semi-quantitative method, which also showed a high content in autumn. CONCLUSIONS: This work emphasised the effect of harvest time on the chemical constituents of hawthorn leaves and autumn is recommended to ensure the quality.

[Content correlation of eight phenolic acids and flavonoids in different medicinal parts of PA-type Perilla germplasms].

In this study, 10 PA-type Perilla germplasms were selected to detect the content of two phenolic acids, i.e., rosmarinic acid(RA) and caffeic acid(CA), and six flavonoids, including scutellarin-7-O-diglucuronoside(SDG), luteolin-7-O-diglucuronoside(LDG), apigenin-7-O-diglucuronoside(ADG), scutellarin-7-O-glucuroside(SG), luteolin-7-O-glucuroside(LG), and apigenin-7-O-glucuroside(AG) in leaves, stems, and fruits. The total content of phenolic acids and flavonoids in leaves was 3.991-12.028 mg·g~(-1) and 12.309-25.071 mg·g~(-1), respectively, which was much higher than that in stems(0.586-2.015 mg·g~(-1) and 0.879-1.413 mg·g~(-1), respectively) and fruits(0.004-2.222 mg·g~(-1) and 0.651-1.936 mg·g~(-1), respectively). RA was detected in five fruit samples, and RA content between leaves and fruits showed a significant negative correlation in the other five samples. For flavonoids, only LG and LDG could be detected in stems, and SG and SDG were not detected in fruits, while other flavonoids were not detected in some samples. The content of total flavonoids and LG in leaves and fruits was significantly positively correlated, and the content of LG in stems and fruits was significantly positively correlated. In 10 stem samples, seven met the standard that the content of RA in the stem should be not less than 0.1% specified in the Chinese Pharmacopoeia(2020 edition). Only one fruit sample reached the standard of RA content in the fruit not less than 0.25% specified in the Chinese Pharmacopoeia.

[Contents determination of eight phenolic compounds in Perilla frutescens leaves of different cultivation years and harvesting periods].

A method was established for content determination of two kinds of phenolic acids, including rosmarinic acid)(RA) and caffeic acid(CA), and six kinds of flavonoids including scutellarein-7-O-diglucuronide(SDG), luteolin-7-O-diglucuronide(LDG), apigenin-7-O-diglucuronide(ADG), scutellarin-7-O-glucuronide(SG), luteolin-7-O-glucuronide(LG), and apigenin-7-O-glucuronide(AG) in Perilla frutescens leaves. The content of eight chemical components was measured based on ten P. frutescens germplasms of different chemotypes of volatile oil, different cultivated years, and different harvesting periods. The results showed that there was a great difference between the two kinds of constituents of different germplasms. The total content of the two phenolic acids was 2.24-34.44 mg·g~(-1), and the total content of the six flavonoids was 11.55-34.71 mg·g~(-1). Then according to content from most to least, the order of each component was RA(2.13-33.97 mg·g~(-1)), LDG(1.31-14.80 mg·g~(-1)), SG(1.97-8.45 mg·g~(-1)), ADG(2.68-7.60 mg·g~(-1)), SDG(1.16-5.87 mg·g~(-1)), LG(0.78-1.91 mg·g~(-1)), AG(0.56-1.00 mg·g~(-1)), and CA(0.11-0.68 mg·g~(-1)). The chemical contents of the 5 PA-type germplasms in 2017 were mostly higher than those in 2018 showing a large variation with the cultivation years. These contents of two kinds of phenolic acids of 9 germplasms fluctuated with the harvesting time. The content decreased before early flower spike(the 3~(rd) to 18~(th) in August) at first and began to increase in flowering and fruiting period(the 18~(th) in August to 2~(nd) in September). However, these contents had slowly decreasing trend after 2~(nd) in September till 17~(th) in the same month. Interestingly, the content raised again in the maturity of fruits. The variation tendency of contents in six kinds of flavonoids components was inconsistent in different germplasms with the variation of harvesting time. The content of flavonoids in part of germplasms was negatively correlated with the fluctuation of phenolic acids. There was no correlation between phenolic acids and chemical type of the volatile oil. This paper may provide a reference for the high-quality germplasm of P. frutescens cultivation.

[Qualitative analysis of Gynostemma longipes for medicinal usage].

The Qinling-Daba Mountains area is the main producing areas of Gynostemma longipes for medicinal usage, and samples of wild whole plants in Pingli, Shaanxi Province and Qingchuan, Sichuan Province were collected. The ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS~E) was used to profile the chemical compositions and analyze the similarities and differences of G. longipes samples in these areas. Based on the accurate molecular weight and fragment information obtained from Q-TOF-MS~E, the structures of the main components were identified by combining with the mass spectra, chromatographic behaviors of reference standards and related literatures. The results showed that the components of wild G. longipes from different places among Qinling-Daba Mountains area were similar. Forty-five chemical components were identified in the whole plant of G. longipes from Pingli, Shaanxi Province, including 43 triterpenoid saponins and 2 flavonoids which contain all main peaks in its fingerprint. The main components are dammarane-type triterpenoid saponins, such asgypenoside ⅩLⅨ, gypenoside A and its malonylated product of glycosyl.

New steroidal glycosides from the roots of Asparagus cochinchinensis.

Steroidal saponins were the main active constituents of the traditional medicinal herb Asparagus cochinchinensis. A phytochemical investigation of A. cochinchinensis roots led to the isolation of nine new steroidal glycosides (1-9) and seven known analogues (10-16). Their structures were established by spectroscopic analyses as well as necessary chemical evidence.

[Effects of light quality on growth and icariin flavonoid content of Epimedium pseudowushanense under different light intensity].

In this study, the growth index including plant height, compound leaf area, specific leaf area, leaf water content, number of branches, and leaf biomass per plant and the icariin flavonoids such as epimedin A, epimedin B, epimedin C and icariin of Epimedium pseudowushanense were determined on 30 d and 60 d under light intensity(18.2±2.5) µmol·m~(-2)·s~(-1)(L1) and(90.9 ±2.5) µmol·m~(-2)·s~(-1)(L2), and white light as control, red light, blue light and yellow light were used as three light quality treatments, to study the effect of light quality on the growth and flavonoids accumulation of E. pseudowushanense. The E. pseudowushanense was sui-table for growth under L1 light intensity, the blue light treatment significantly reduced the leaf area, but had little effect on the stem height, the red light treatment and the yellow light treatment had no obvious effect on the stem height and leaf area, but the yellow light treatment significantly increased the germination of new branches, and had a sustained promoting effect, and the biomass was significantly higher than the white light treatment at 60 d. The content of icariin flavonoids in red light, blue light and yellow light treatment was higher than that in white light treatment at 30 d and 60 d under L1 light intensity, while yellow light treatment promoted the synthesis of icariin flavonoids to the largest extent, which was 1.8 and 1.9 times of white light treatment(30 d and 60 d).Under L2 light intensity, the effect of strong light on promoting stem germination became the main factor, while the yellow light treatment showed no significant effect on promoting stem germination, and the red light treatment exhibited a significant effect on reducing leaf area. Icariin flavonoids under red light, blue light and yellow light treatment were all lower than that under white light treatment, that is, the effect of white light treatment on the synthesis of icariin flavonoids is better than red light, blue light and yellow light treatment. When the time of strong light treatment was longer, the degradation range of icariin flavonoids in other light treatment appeared, while red light treatment promotes the synthesis of icariin flavonoids. Therefore, the influence of light quality on E. pseudowushanense is quite different under different light intensity, no matter from growth index or flavonoid content index. The results support that the biomass and icariin flavonoid content can be increased by providing appropriate red and yellow light.

[Planting density of Perilla frutescens PA-type].

Two medicinal PA type Perilla germplasms were planted at five planting densities(D1,2 500 plants/Mu;D2,5 500 plants/Mu;D3,8 500 plants/Mu;D4,11 500 plants/Mu;D5,14 500 plants/Mu;1 Mu≈667 m~2). A total of 17 traits, including leaf shape, plant type, yield, volatile oil content and composition, were recorded and studied. With the planting density increased, the leaves appeared narrow, plants small, the deciduous leaves increased, and the leaf yield per plant was low, but the leaf yield per Mu increases significantly with the planting density, and was basically stable after reaching D4. The extraction rate of volatile oil from leaves at planting density D2-D5 was about 0.1% higher than that of D1, and there was no significant difference in the relative content of perillaldehyde, among 5 density. In order to achieve high leaf yield, it is recommended to plant at a density of D4, 11 500 plants/Mu(plant spacing is 15 cm, and row spacing is 40 cm); while comprehensive leaf yield and leaf morphology are recommended to be planted at a density of D2, 5 500 plants/Mu(plant spacing is 30 cm, and row spacing is 40 cm). At the same time, dense planting resistance of two germplasms were different. Number of secondary branches, first section with leaves and plant types were most important feature for the evaluation of the density tolerance of PA-type Perilla. This study provided a reference for the suitable density of PA-type Perilla, and laid a foundation for further study of the tolerance characteristics of different Perilla.

Steroidal saponins from Trillium tschonoskii rhizomes and their cytotoxicity against HepG2 cells.

A phytochemical study on the rhizomes of Trillium tschonoskii led to the isolation of fourteen new steroidal saponins, trillitschosides S1-S14 (1-14), along with ten known analogues (15-24). Their structures were established mainly by spectroscopic analyses as well as necessary chemical evidence. All isolated compounds were screened for the cytotoxicity against HepG2 cells, and the results demonstrated that only the known compounds 21-24 exhibited the remarkable cytotoxic activity against HepG2 cells which is much better than the positive control of 5-FU.

[Change of icariin content in five Epimedium species by heating process].

The change of icariin( ICA) content in thirty-three samples of five Epimedium species listed in the Chinese Pharmacopoeia( 2015 edition),including E. brevicornu,E. sagittatum,E. pubescens,E. koreanum,and E. wushanense has been investigated in this study. The results indicated that the optimized process procedure was baking at 150 ℃ for 30 min,and 3'''-carbonyl-2″-ß-L-quinovosyl icariin( CQICA) could not be translated into ICA and ICA could be converted under this heating process condition. ICA increased remarkably after the heating process by 1-3 times in E. brevicornu,E. wushanense and E. koreanum,and increased lightly in E. brevicornum and E. pubescens,while ICA slightly increased or decreased in E. sagittatum and E. wushanense.

[Investigation of aflatoxins,mycobiota,and toxigenic fungi during post-harvest handling of Ziziphi Spinosae Semen].

Ziziphi Spinosae Semen is one of the Chinese herbal medicine being susceptible to aflatoxins contamination. To investigate the sources of aflatoxins contamination and toxigenic fungi species on Ziziphi Spinosae Semen,32 samples were collected from multiple steps during the post-harvest processing in this study. Aflatoxins in these samples were determined by immunoaffinity column and HPLC coupled with post-column photochemical derivatization. The dilution-plate method was applied to the fungi isolation. The isolated fungi strains were identified by morphological characterization and molecular approaches. The results showed that aflatoxins were detected in 28 samples from every step during the processing of Ziziphi Spinosae Semen. Three samples were detected with aflatoxin B_1 and 2 samples with both aflatoxin B_1 and total aflatoxin exceeding the limit of Chinese Pharmacopoeia. Especially the samples from the washing step,with the highest detected amounts of AFB_1 and AFs were reached 94. 79,121. 43 µg·kg~(-1),respectively. All 32 samples were contaminated by fungi. The fungal counts on the newly harvested samples were 2. 20 × 10~2 CFU·g~(-1). Moreover,it increased as tphreocessing progresses,and achieved 1. 16×10~6 CFU·g~(-1) after washing. A total of 321 isolates were identified to 17 genera. Aspergillus flavus was the main source of aflatoxins during the processing and storage of Ziziphi Spinosae Semen. One isolate of A. flavus was confirmed producing AFB_1 and AFB_2. The fungal count was significantly increased by composting,and Aspergillus was the predominant genus after shell breaking. The contamination level of aflatoxins was increased by composting and washing.

[Study on morphological classification and chemical-type of Perilla frutescens cultivated germplasm].

Fifty cultivated Perilla seeds were collected all over the country and planted in Beijing experiment field for morphology and chemical-type researches. Twenty morphological characteristics were selected and observed, and the essential oil from leaves was extracted by steam distillation and analyzed by GC-MS to confirm chemical-types. There were significant diversities in plant height, leaf color and morphology, and fruit color and weight. Clustering analysis was carried out based on these morphological characteristics. Six types were divided with their chemical-type designated. Type Ⅰ: Six germplasms, attributed to P. frutescens var. crispa, with dwarf plants, thin creased purple leaf, named Crispa, their chemical types were diversified, including EK, PAPK, PA and PK. Type Ⅱ: Six germplasms, attributed to P. frutescens var. crispa, plants were taller than type I and with thin and creased green leaf, named Big Crispa, all PK type. Type Ⅲ: Seventeen germplasms, attributed to P. frutescens var. frutescens with leaf color upside green and underside purple, tall plant and wide distribution all over the China, named Ordinary Frutescens, all PK. Type Ⅳ: Four germplasms, attributed to P. frutescens var. acuta with tall plant and small seed, named Acuta, all PK. Type Ⅴ: Seven germplasms, attributed to P. frutescens var. frutescens with green leaves, tall plants and long clusters, named Long-spike Frutescens, all PK. Type Ⅵ: Ten germplasms, attributed to P. frutescens var. frutescens with big, thick and creased leaf, named Thick-leaf Frutescens, including PK, PP, PL and PA. The morphological classification of this paper would lay the foundation for the taxonomic naming and following evaluation of the Perilla germplasm resources.This study also showed that there was no correspondence but a certain correlation between volatile oil chemical-types and subspecies classification and morphological characteristics of Perilla.

[Comparative analysis on chemical constituents in Bupleurum chinense,B. marginatum,B. marginatum var. stenophyllum and B. smithii var. parvifolium].

UPLC-Q-TOF-MS was used to analyze the chemical differences in Bupleurum. chinense,B. marginatum,B. marginatum var. stenophyllum and B. smithii var. parvifolium. Chromatographic separation was carried out on an Acquity HSS T3 C_(18) column( 2. 1 mm ×100 mm,1. 8 µm,Waters) with the mobile phase composed of 0. 1% formic acid in water-acetonitrile in the gradient elution. A hybrid quadrupole time-of-flight tandem mass spectrometry( Q-TOF-MS~E) was used for mass spectrometric analysis. Finally,25 peaks were identified based on their exact mass data and fragmentation characteristics. B.chinense,B.marginatum,B. marginatum var. stenophyllum and B. smithii var. parvifolium were obviously clustered into 3 types through processing by principal component analysis( PCA). There was almost no difference between B. chinense and B. marginatum. However,the compounds existed in B. chinense were different from those in B. marginatum var. stenophyllum,and B. smithii var. parvifolium.

[Effects of plant growth regulators and nitrogen on germination of Epimedium pseudowushanense after its stems and leaves reaped].

In order to explore appropriate measures to promote germination after the harvest of Epimedium pseudowushanense, 6-BA, urea, ammonium bicarbonate and GA3 were chosen to spray on the root and rhizomes, and then the biological indicators such as branches, leaf length, leaf width, plant height and so on, were measured in different periods, and the contents of epimedin A, epimedin B, epimedin C and icariin in the dry leaves were detected by HPLC. Results showed that 6-BA 90 mg·L⁻¹(B1), 6-BA 60 mg·L⁻¹(B2),6-BA 30 mg·L⁻¹+urea 300 mg·L⁻¹ (C1), 6-BA 60 mg·L⁻¹+urea 300 mg·L⁻¹(C2),6-BA 60 mg·L⁻¹+ ammonium bicarbonate 300 mg·L⁻¹(C4) significantly increased bud germination in the early period, and the plants quickly set up new system of photosynthesis, the branches in a month of which were higher than the control group respectively by 165.9%, 115.76%, 103.86%, 104.50%, 81.67%.However the branches developed the next year and the dry weight of leaves per plant in group B1 and B2 were much lower than that in control group. The groups that use 6-BA and nitrogen at the same time reaped a good yield of leaves even though the treatment had no significant influence on the branches developed the next year. The dry weight of leaves of C1, C2, C4 treatments were 36.80%, 32.84%, 45.97% more than the control group respectively. Therefore, C1, C2 and C4 treatments are the more appropriate to promote recovery after harvest. Furthermore, different groups, except 10 mg·L⁻¹ 6-BA treatment significantly reduced the content of epimedin C, other groups didn't have any significant effect on the contents of such flavonoids.

[Genetic relationship and population genetic diversity of Epimedium pubescens and its related species based on ISSR and non-glandular hair characteristic].

In this study, 128 individuals form 14 Epimedium pubescens populations and 1 E. stellulatum population were analyzed by ISSR marker. The data were calculated by POPGENE software and clustered by UPGMA method. Optical microscope was used to observe the main types of the non-glandular hairs and their characteristics in each population. It is found that the following conclusions: Non-glandular hairs can be divided into five morphological categories, long straight pubescent, curly pubescent, appressed curly pubescent, pseudo short appressed hairs and long appressed. Eight primers were screening and a total of 94 bands were detected in ISSR, among which 90 were polymorphic bands. Based on the results of ISSR cluster analysis, 15 populations were divided into 3 clades. E. stellulatum populations should be incorporated into the E. pubescens or as avariety under E. pubescens not be independent and as it has no separate phylogenetic branch for a cluster. The genetic relationship among the populations of E. pubescens was closely related with its geographical distribution and non-glandular hair features. But there were also some inconsistency, which provided a good hint for the further study on the interspecific relationship and natural speciation manner of Epimedium species. Population diversity analysis showed Nm=0.354 4, Nei's=0.585 2. It was showed that E. pubescens has high genetic diversity among populations, for which the main reason was probably the high inbreeding rate and the small range of seed dispersal.

[Morphological characteristics of Chinese Perilla fruits].

The morphological traits of 55 Chinese Perilla fruit samples (size, 100 grains weight, color, hardness, surface ridge height) are described and the statistically analyzed. It can be divided into 6 categories by cluster analysis, namely: Ⅰ, big grain (diameter 1.5 mm above and 100 grains weight above 0.16 g), low ridge, hard; Ⅱ, big grain, low ridge, soft; Ⅲ, big grain, high ridge, soft, fruit; Ⅳ, big grain. high ridge, gray brown or dark brown; Ⅴ, small grain (diameter 1.5 mm below and 100 grain weight 0.16 g below), low ridge, hard, dark brown; Ⅵ, small grain, low ridge, hard, yellow brown. The 38 fruit samples were planted, among which 31 ones were P. frutescens var. frutescens, 4 ones P. frutescens var. crispa and 3 ones P. frutescens var. acuta. By chemotype classification, they were 29 PK type, 3 PA type, 2 PL type, 2 PP type, 1 EK type and 1 PAPK type. According the description of herb Perillae Fructus in China Pharmacopoeia, the plant originates from P. frutescens var. frutescens. In contrast, not all fruits of P. frutescens var. frutescens have accord features. The fruits with white pericarp are mainly from P. frutescens var. frutescens with purple leaves. The materials with small grain, low ridge, hard, yellow brown or dark brown, are likely to be PA type and mainly P. frutescens var. crispa.

[Determination of three saponins in rhizoma and fibrous root of Trillium tschonoskii and Trillium kamtschaticum].

To compare the differences of main components between in rhizoma and fibrous root of Trillium tschonoskii and T. kamtschaticum, a simple, accurate and reliable high performance liquid chromatography coupled with the charged aerosol detector (HPLC-CAD) method was developed and then successfully applied for simultaneous quantitative analysis of three compounds, including polyphyllin Ⅶ (T1),pennogenin 3-O-α-L-rhamnopyranosyl-(1→2) [α-L-rhamnopyranosyl-(1→4)]-ß-D-glucopyranoside (T2),polyphyllin Ⅵ (T3), in 16 batches of rhizome and 14 batches of fibrous root. The analytes were well separated from other constituents on TSK gel ODS (4.6 mm×250 mm, 5 µm) with acetonitrile-water (43∶57) at a flow rate of 1.0 mL•min⁻¹. The injection volume was 20 µL. The nitrogen inlet pressure for the CAD system was 35 psi and the nebulizer chamber temperature was 35 ℃.The method was validated for linearity (r>0.999 0), intra and inter-day precision (0.29%-3.0%), repeatability (0.45%-1.4%), stability (1.9%-2.6%), recovery (100.1%-100.2%, 1.2%-1.8%), limits of detection (0.002 g•L⁻¹), and limits of quantification (0.005 g•L⁻¹).The obtained datasets were processed by principal component analysis (PCA) and it showed that there was almost no difference in rhizoma of T. tschonoskii and T. kamtschaticum from different areas of China. However, the 3 major compounds existed in rhizoma were different from those in fibrous root of T. tschonoskii and T. kamtschaticum.

[Research on effects of chemotype and components of Perilla frutescens leaf volatile oil I: different phenological periods].

This experiment researched on three kinds of Perilla frutescens including the widespread PK, PA and rare PL chemotype. The Perilla samples were the mature leaves collected in nutrition, flowering and frutescence three different phenological periods, and at 7 am, 12 pm and 6 pm three day time. The volatile oil was extracted by steam distillationand analyzed by GC-MS, as a result, the three chemotype samples'volatile oil yield was between 0.08% and 0.96%; volatile oil yield of different growth period was as follow: nutrition>flowering>fructescence, and the volatile oil yield of nutrition period: PA type>PK type>PL type. Each chemotype was not affected by the growth and development, indicating that the chemotype is determined by genetic factors. Characteristic and main components of PA and PK type are relatively stable, and the characteristic components of PL type are significantly decreased with the growth. There are still a large number of upstream metabolism components, and the chemical type may have their primitiveness and changeability. The relative content of perillaldehyde, characteristic components of PA type, is basically decreased from morning to night, in all the period. The relative content of perillaketone, characteristic components of PK type, in nutrition and flowering period, when samples were collected at 12 noon is relatively higher than that at 7 am and 6 pm, and contrary to samples collected in frutescence period. The relative content of perillene, characteristic components of PL type, in nutrition and frutescence period are highest at 12 noon, while in flowering period is highest at 6 pm. According to the volatile oil yield and relative content of maincomponents, the best harvest time of PA type is in the morning of the nutrition period; the best harvest time of PK type is in the morning of all the period; and the best harvest time of PL type is at dusk of the nutrition period.