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Abstract

Toddalia asiatica (Linn) Lam (T. asiatica) as a traditional Miao medicine was investigated to find rational alternative medicinal parts for T. asiatica root bark and its antitumor chemical constituents by quantitative pharmacognostic microscopy, high performance liquid chromatography (HPLC) fingerprint and multivariate statistical analysis. A bivariate correlation analysis method based on microscopic characteristics and content of chemical constituents was established for the first time, there were some regular discoveries between powder microscopic characteristics and common chromatographic peaks of T. asiatica through quantitative pharmacognostic microscopy, cork cells, calcium oxalate square crystal, brown clump, starch granule and phloem fiber, as powder microscopic characteristics may be placed where the main chemical constitutes were enriched. Scores plot of principal component analysis (PCA) and dendrogram of hierarchical clustering analysis (HCA) showed that 18 T. asiatica samples were distinguished correctly, clustered clearly into two main groups as follows: S01∼S03 (root bark) and S07∼S09 (stem bark) in cluster 1, S04∼S06 and S10∼S18 in cluster 2. Nineteen common peaks were obtained in HPLC fingerprint of T. asiatica, loadings plot of PCA indicated seven compounds played important roles in different part of samples (P10 > P08 > P07 > P14 > P16 > P17 > P19), peaks 04, 06, 07, 08, 10 were identified as hesperidin, 4-methoxycinnamic acid, toddalolactone, isopimpinlline and pimpinellin. MTT assay was used to determine the inhibitory activity of different medicinal parts of T. asiatica on human breast cancer MCF-7 cells, all parts of T. asiatica had different inhibitory effects on MCF-7 cell lines, root and stem barks of T. asiatica showed the best inhibitory activity. The relationship between chemical constituents and the inhibitions on MCF-7 cell had been established, significant antitumor constituents of T. asiatica were identified by correlation analysis, the order of the antitumor effect of the main compounds was P07 (toddalolactone) > P16 > P06 (4-methoxycinnamic acid), P11 > P18 > P10 (pimpinellin) > P08 (isopimpinellin) > P01 > P19 > P14 > P04 (hesperidin) > P17, which were antitumor chemical constituents of T. asiatica root bark. T. asiatica stem bark was the most rational alternative medicinal part for T. asiatica root bark.

Abstract

Toddalia asiatica (Linn) Lam (T. asiatica) as a traditional Miao medicine was investigated to find rational alternative medicinal parts for T. asiatica root bark and its antitumor chemical constituents by quantitative pharmacognostic microscopy, high performance liquid chromatography (HPLC) fingerprint and multivariate statistical analysis. A bivariate correlation analysis method based on microscopic characteristics and content of chemical constituents was established for the first time, there were some regular discoveries between powder microscopic characteristics and common chromatographic peaks of T. asiatica through quantitative pharmacognostic microscopy, cork cells, calcium oxalate square crystal, brown clump, starch granule and phloem fiber, as powder microscopic characteristics may be placed where the main chemical constitutes were enriched. Scores plot of principal component analysis (PCA) and dendrogram of hierarchical clustering analysis (HCA) showed that 18 T. asiatica samples were distinguished correctly, clustered clearly into two main groups as follows: S01∼S03 (root bark) and S07∼S09 (stem bark) in cluster 1, S04∼S06 and S10∼S18 in cluster 2. Nineteen common peaks were obtained in HPLC fingerprint of T. asiatica, loadings plot of PCA indicated seven compounds played important roles in different part of samples (P10 > P08 > P07 > P14 > P16 > P17 > P19), peaks 04, 06, 07, 08, 10 were identified as hesperidin, 4-methoxycinnamic acid, toddalolactone, isopimpinlline and pimpinellin. MTT assay was used to determine the inhibitory activity of different medicinal parts of T. asiatica on human breast cancer MCF-7 cells, all parts of T. asiatica had different inhibitory effects on MCF-7 cell lines, root and stem barks of T. asiatica showed the best inhibitory activity. The relationship between chemical constituents and the inhibitions on MCF-7 cell had been established, significant antitumor constituents of T. asiatica were identified by correlation analysis, the order of the antitumor effect of the main compounds was P07 (toddalolactone) > P16 > P06 (4-methoxycinnamic acid), P11 > P18 > P10 (pimpinellin) > P08 (isopimpinellin) > P01 > P19 > P14 > P04 (hesperidin) > P17, which were antitumor chemical constituents of T. asiatica root bark. T. asiatica stem bark was the most rational alternative medicinal part for T. asiatica root bark.

Introduction

Toddalia asiatica (Linn) Lam (T. asiatica) as a traditional Miao medicine of Chinese Materia Medica (CMM), belonging to Toddalia Juss genus, Rutaceae family was first recorded in “Zhi Wu Ming Shi Tu Kao” of Qing Dynasty, it is widely distributed in Guizhou, Guangxi and other provinces in Southwest China, its root bark as a traditional medicinal part of T. asiatica, which has obvious pharmacological activities of hemostasis, anti-tumor, anti-inflammatory and analgesic, is widely used in the minority Miao for clinical treatment of traumatic injury, knife wound hemorrhage, tumor and rheumatic pain [1–5]. CMM as an important kind of natural medicines on the guidance of traditional Chinese medicine (TCM) theory has been used clinically for thousands of years in China, CMM have been followed up to now by their own exact pharmacological action and clinical effect. But, there is a main reason that TCM or CMM has always been questioned by western medical scholars due to complex chemical constituents and unclear pharmacological mechanisms of CMM [6], how to scientifically clarify the effective compounds of CMM has become an urgent problem to be solved with the continuous development of modern medicine.

Meanwhile, under the huge clinical demand for T. asiatica root bark and current drug market driven by actual economic interests, whole root slice and whole stem slice of T. asiatica are the main forms of T. asiatica instead of its traditional medicinal part – root bark. This chaotic use of CMM made it necessary for us to carry out research on rational medicinal parts of T. asiatica, and find out more suitable alternative parts of T. asiatica for T. asiatica root bark. In recent years, some researchers had tried to address these pratical challenges through studies on phytochemistry, LC fingerprinting and LC-MSMS analysis of T. asiatica [7–10], our group also had carried out a series of pharmacodynamic material basis studies on T. asiatica root bark in the previous research work, its hemostatic activity was systematically verified through animal model experiments, a lot of natural furocoumarins from T. asiatica root bark had been detected, isolated and finally identified [11–15]. However, the findings of these studies still did not pay attention to the rationality of medicinal parts of T. asiatica, as there were no comparative studies of all possible medicinal parts of T. asiatica, some of them such as root core of T. asiatica even had been used in drug market by default until now without any relevant drug quality standards and scientific research data support.

High performance liquid chromatography (HPLC) fingerprint technology as an important analytical method had got quick development in many areas such as TCMs, food and biological samples. It can be a relatively effective method to evaluate the quality of complex TCMs as a whole [16–18]. In order to further explore the differences of all medicinal parts of T. asiatica, the obtained data were statistically processed by multivariate statistical analysis including bivariate analysis, hierarchical clustering analysis (HCA) and principal component analysis (PCA). Multivariate statistical analysis was used to evaluate the intrinsic quality of T. asiatica and to identify the chemical constituents that are most responsible for quality control of different medicinal parts of T. asiatica [19, 20]. HPLC fingerprint profiling combined with quantitative pharmacognostic microscopy, pharmacological activity and multivariate statistical analysis was a novel strategy for assessing different medicinal parts of T. asiatica in this study. Our research group had conducted deep and systematic research on different parts of T. asiatica and their antitumor effects for the first time, so as to solve current chaos of T. asiatica, clarify on its antitumor material basis and to scientifically confirm the rational alternative medicinal part of T. asiatica.

Materials and methods

Plant materials

Eighteen batches of different medicinal parts of T. asiatica including root bark, root core, stem bark, stem core, near-leaf stem and leaf collected from Qixing mountain area, Duyun, Guizhou in the southwest of China were investigated (Table 1). T. asiatica samples were ground into powder of the homogenous 24 mesh before the experiment. All samples had been identified by Professor Zhiyou Guo, Qiannan Normal University for Nationalities, and the specimens were deposited in Herbarium of Chinese Materia Medica and Ethnomedicines, School of Pharmacy, Guizhou Medical University.

Table 1.

The origins of different medicinal parts of Toddalia asiatica

Sample no.Medicinal parts of T. asiaticaOriginsCollecting time
01Root barkDuyun, GuizhouJuly, 2018
02Root barkDuyun, GuizhouJuly, 2018
03Root barkDuyun, GuizhouJuly, 2018
04Root coreDuyun, GuizhouJuly, 2018
05Root coreDuyun, GuizhouJuly, 2018
06Root coreDuyun, GuizhouJuly, 2018
07Stem barkDuyun, GuizhouSept, 2018
08Stem barkDuyun, GuizhouSept, 2018
09Stem barkDuyun, GuizhouSept, 2018
10Stem coreDuyun, GuizhouSept, 2018
11Stem coreDuyun, GuizhouSept, 2018
12Stem coreDuyun, GuizhouSept, 2018
13Near-leaf stemDuyun, GuizhouAugust, 2018
14Near-leaf stemDuyun, GuizhouAugust, 2018
15Near-leaf stemDuyun, GuizhouAugust, 2018
16LeafDuyun, GuizhouAugust, 2018
17LeafDuyun, GuizhouAugust, 2018
18LeafDuyun, GuizhouAugust, 2018

Cells, chemicals and reagents

MCF-7 cells were purchased from China Center for Type Culture Collection (CCTCC) of Wuhan University, less than 10 generations of cell passage. Eleven authentic compounds were used in the present study, namely, hesperidin (P04), 4-methoxycinnamic acid (P06), toddalolactone (P07), isopimpinellin (P08), pimpinellin (P10), methyl trans-4-hydroxycinnamate (P20), avenalumic acid methyl ester (P21), ferulic acid methyl ester (P22), bergapten (P23), moellendorffiline (P24) and phellopterin (P25). These were isolated and prepared from T. asiatica, identified by our research group in our previous research, and their purities in HPLC are more than 98%. DMEM culture medium (Lot No 8118403, GIBCO), Fetal bovine serum (Lot No 1P1701, SeraPro S601-500), Trypsin digestive fluid (Lot No 1951208, GIBCO), Phosphate buffer saline (Lot No 1015M022, Solarbio), MTT (Lot No 829Z0513, Solarbio), DMSO (Lot No 1213C0222, Solarbio), 75% medical alcohol (Lot No 190414, Guizhou Kangtai Lijian), Methanol (HPLC pure, Shanghai Sinopharm Chemical), Glycerol (Shanghai Sinopharm Chemical), Chloral hydrate (Shanghai Macklin Biochemical), Pure water (Watsons), 96-well Cell culture plate (Lot No 180809-078, Guangzhou Jet Bio-Filtration) were purchased for the analysis.

Instruments

Agilent 1260 infinity HPLC-DAD system (Agilent), R-1001VN rotary evaporator (Zhengzhou Greatwall Scientific), SHB-Ⅲ water circulating multi-purpose vacuum pump (Zhengzhou Greatwall Scientific), SB-5200D ultrasonic cleaning machine (Ningbo Scientz), TS100 Inverted Microscope (Nikon), Epoch microplate reader (Biotek), Thermo Forma 3131 carbon dioxide cell incubator (Thermo), TDL-40B centrifuge (Changzhou Langyue), HH-501 thermostatic water bath (Changzhou Langyue) and HK-UP-20 Ultra-pure water preparation system (Hefei Hongke) were used for the analysis.

Sample preparation

About 6.0 g of root bark, root core, stem bark, stem core, near-leaf stem or leaf of T. asiatica was soaked separately in methanol for 24 h at a solid–liquid ratio of 1:50 (g:mL), extracted three times by reflux method for 2 h each time in a 65 °C-constant temperature water bath [9, 11, 21]. Six extracts from different parts of T. asiatica were finally prepared. Each accurately-weighed extract of T. asiatica was dissolved in methanol and fixed in their respective 25-mL volumetric flasks. All sample solutions were filtered through 0.22-μm lipophilic microporous filters before LC analysis.

The standard reference solutions were prepared by adding an accurately-weighed amount of above-mentioned eleven natural compounds to a 10-mL volumetric flask, respectively, dissolved with methanol to make 100.0 μg/mL of the final concentrations as the stock solutions prepared for analysis.

Microscopical character analysis of pharmacognostical powder

The root bark, root core, stem bark, stem core, near-leaf stem and leaf of T. asiatica were pulverized by a pulverizer, sieved by a 24-mesh sieve to obtain the medicinal material powders to be tested (Fig. 1). The right amount of the powder from different parts of T. asiatica was taken out separately, placed on a glass slide of 7.5 cm × 2.5 cm and dripped with 2∼3 drops of chloral hydrate-glycerol test solution for further 2–3 s of heat treatment under the flame of an alcoholic lamp, then each sample on the glass slide was covered with a cover glass without generating bubbles. All these glass slides with sample powders were observed under an inverted microscope to do microscopical character analysis of pharmacognostical powder.

Fig. 1.
Fig. 1.

The appearance of differental parts of Toddalia asiatica and their powders.

1: Root bark of T. asiatica and its powder.

2: Root core of T. asiatica and its powder.

3: Stem bark of T. asiatica and its powder.

4: Stem core of T. asiatica and its powder.

5: Near-leaf stem of T. asiatica and its flake.

6: Leaf of T. asiatica and its powder

Citation: Acta Chromatographica Acta Chromatographica 2020; 10.1556/1326.2020.00762

Chromatographic condition

Chromatographic separation was performed on a Diamonsil C18 column (250 mm × 4.6 mm, 5 μm) in a HPLC-DAD system (Agilent), the mobile phase was methanol (A)–water (B) with gradient elution of 0–10 min A:B (5:95) → (25:75), 10∼50 min A:B (25:75) → (50:50), 50∼80 min A:B (50:50) → (80:20), 80∼95 min A:B (80:20) → (90:10) and 95–100 min A:B (90:10) → (90:10) [9, 10]. The flow rate was 1.0 mL/min, detection wavelength was 230 nm, column temperature was maintained at 25 °C and the injection volume was 20.0 μL by automatic sampling system at 4 °C.

Evaluation of anti-tumor activities of different parts of T. asiatica

MCF-7 cells in logarithmic growth stage were selected and inoculated into 96-well plates at a cell density of 1 × 104/well with 100.0 μL DMEM culture medium, six duplicates in each group 12.5∼200.0 μg/mL extracts of six medicinal parts of T. asiatica (root bark, root core, stem bark, stem core, near-leaf stem and leaf) in 0.1%-DMSO DMEM culture medium were added respectively and incubated at 37 °C, 5% CO2 for 24 h in the dark, while 0.1%-DMSO DMEM culture medium without drug treatment was set up as Blank control group. Then, 15 μL of 5% MTT solution was added to each well in the dark. These cells were continuously incubated at 37 °C, 5% CO2 for 4 h, the supernatant was discarded, 150 μL DMSO was added to each well and shaken for 10 min. Subsequently, the optical density (OD) of each well at 490 nm was determined by a microplate reader (Bio-Rad Laboratories, Inc.), the inhibitory rate (%) was finally calculated by the following formula:

Inhibitoryrate(%)=ODBlankODSampleODBlank×100%

Correlation analysis

Quantitative pharmacognostic microscopy and multivariate statistical analysis of antitumor chemical constituents of T. asiatica need to be analyzed by means of correlation analysis method, which contained bivariate analysis, HCA and PCA. Bivariate analysis and HCA were performed by SPSS 22.0 (SPSS Inc), PCA was carried out by SIMCA 14.1 Software (Umetric, Sweden).

Results and discussion

Pharmacognostical powder analysis

Microscopic characteristics of powders of different medicinal parts of T. asiatica are: 1) powder of the root bark was brown, the powder microscopic characteristics were the most abundant, there were much more brown cork cells, calcium oxalate square crystals, brown clumps and reticulate vessels. Xylem fibers, non-glandular hairs, oil cells, starch granules and phloem fibers could also be characterized in the root bark; 2) powder of the root core was yellowish green, its microscopic characteristics contained yellowish brown cork cells and reticulate vessels, no calcium oxalate square crystals and stone cells were found here; 3) T. asiatica stem bark was brown, more cork cells, calcium oxalate square crystals, brown clumps and starch granules were its main microscopic powder characteristics, which were similar to T. asiatica root bark; 4) T. asiatica stem core was brown yellow, there were many wood fibers containing calcium oxalate square crystals in the powder, cork cells and reticulate vessels were also common herein; 5) the near-leaf stem was brown, it had wood fibers, cork cells, non-glandular hairs and reticulate vessels; 6) the leaf was light green in color, brown oil cells, non-glandular hairs, vessels, parenchyma cells and wood fibers were its main powder microscopic characteristics (Fig. 2).

Fig. 2.
Fig. 2.

Microscopic characteristics of fine powders of different parts of Toddalia asiatica (10 × 40).

1: Stone cells.

2: Calcium oxalate square crystals.

3: Reticulate vessels.

4: Wood fibers.

5: Fiber containing crystals.

6: Catheters.

7: Cork cells.

8: Parenchyma cells.

9: Non-glandular hairs

Citation: Acta Chromatographica Acta Chromatographica 2020; 10.1556/1326.2020.00762

In our opinion, the bioactive chemical constitutes of T. asiatica were definitely from this TCM, which showed us its own powder microscopic characteristics in the field of view of a microscope, and this prompted us to try to build correlation between powder microscopic characteristics of T. asiatica and 19 common chromatographic peaks through quantitative pharmacognostic microscopy. Correlation analysis method helped us to calculate their correlation coefficents, and the final data was listed in Table 2. Cork cells, calcium oxalate square crystal, brown clump, starch granule and phloem fiber obtained relatively high frequency of occurrence with common peaks (01∼19) from these results, these microscopic characteristics may be placed where the main chemical constitutes were concentrated and enriched, it can guide us to more purposefully extract and purify the target natural compounds. For example, phloem fiber had rich compounds of P06 (0.805, P < 0.01), P11 (0.539, P < 0.05) and P18 (0.787, P < 0.01).

Table 2.

Correlation coefficents between nineteen common chromatographic peaks and powder microscopic characteristics of different parts of Toddalia asiatica

Cork cellsXylem fiberNon-glandular hairCalcium oxalate square crystalVesselsOil cellsParenchyma cellsBrown clumpStone cellsStarch granulePhloem fiber
P010.685**−0.741**−0.628*0.552*−0.823**−0.715**−0.4000.772**−0.2180.4550.879**
P02−0.294**0.882**0.906**−0.3200.659**0.898**0.834**−0.3250.725**0.211−0.514
P03−0.2660.595*0.815**0.2380.2750.688**0.783**−0.0450.724**0.468−0.538
P040.748**−0.661**−0.4000.951**−0.727**−0.547*−0.3360.504−0.0860.4240.312
P05−0.0030.1830.4150.579*−0.1140.2930.4550.3020.710**0.772**0.180
P06−0.115−0.161−0.2740.073−0.065−0.175−0.2780.0950.1580.2360.805**
P070.563*−0.691**−0.592*0.683**−0.661**−0.636*−0.5080.501−0.1120.3930.799**
P080.425−0.487−0.3410.726**−0.577*−0.411−0.2430.546*0.1900.631*0.809**
P09−0.814**0.981**0.971**−0.4170.766**0.987**0.882**−0.3970.783**0.192−0.520*
P100.435−0.507−0.3670.718**−0.586*−0.433−0.2680.547*0.1630.613*0.821**
P11−0.2960.1400.0870.0760.1250.1530.0540.0060.4440.3600.539*
P120.486−0.287−0.0120.731**−0.536*−0.1850.1190.559*0.2280.621*0.229
P13−0.796**0.840**0.821**−0.2060.654**0.864**0.724**−0.2970.918**0.4130.073
P140.634*−0.563*−0.3130.882**−0.757**−0.463−0.1460.735**0.1580.735**0.689**
P150.751**−0.445−0.0850.862**−0.759**−0.3290.1180.752**0.0830.648**0.132
P160.244−0.402−0.3490.530*−0.414−0.354−0.2930.3920.1840.5090.862**
P170.611*−0.473−0.1930.874**−0.707**−0.364−0.0310.703**0.2010.732**0.516*
P18−0.120−0.144−0.2360.145−0.057−0.144−0.2590.0790.2180.2800.787**
P190.0110.0650.1740.246−0.1600.1050.2890.3660.4160.563*0.375

Notes: * P < 0.05 statistically significant correlation, ** P < 0.01 statistically very significant correlation. Bold values are chromatographic peaks.

Chromatographic fingerprint analysis

The chromatographic fingerprints of root bark, root core, stem bark, stem core, near-leaf stem and leaf of T. asiatica had been established, a total of 19 common chromatographic peaks (01∼19) in different medicinal parts of T. asiatica were confirmed, in Fig. 3, showing large peak areas and good separation from adjacent peaks. The total peak areas of 19 common peaks were more than 90% of the total peak areas. By comparing with the chromatographic peaks of our eleven reference substances, the five common chromatographic peaks 04, 06, 07, 08 and 10 of T. asiatica were determined to be hesperidin, 4-methoxycinnamic acid, toddalolactone, isopimpinlline and pimpinellin, respectively.

Fig. 3.
Fig. 3.

HPLC-UV chromatograms of standard references (A) and different medicinal parts of Toddalia asiatica (B)

Citation: Acta Chromatographica Acta Chromatographica 2020; 10.1556/1326.2020.00762

The LC fingerprints were matched automatically by use of the Similarity Evaluation System for Chromatographic Fingerprint of TCM (Version 2004), similarities of LC fingerprints of samples no. 1∼18 of T. asiatica were calculated by the cosine value method of vectorial angle, and the results were detailed below: 0.9507–0.9765 of samples no. 1–3, 0.8306∼0.8570 of samples no. 4∼6, 0.9824∼0.9881 of samples no. 7∼9, 0.8207∼0.8940 of samples no. 10∼12, 0.5571∼0.5667 of samples no. 13∼15 and 0.1328∼0.1366 of samples no. 16–18. The degree of chromatographic fingerprint similarity between T. asiatica root bark and T. asiatica stem bark was much higher than that of other parts. HCA was used in our study to find relatively homogeneous clusters of all six different medicinal parts of T. asiatica based on the peak areas of the 19 common peaks as the measured characteristics, and the results were shown in Fig. 4D, it was clear that the six parts of T. asiatica were able to be classified into two broad categories. Samples no. 1∼3 of T. asiatica root bark and samples no. 7∼9 of T. asiatica stem bark were in the first category, and the other samples of T. asiatica were in the second category, which was further classified into two subclusters: samples no. 4∼6 of T. asiatica root core, samples no. 10∼12 of T. asiatica stem core and samples no. 13∼15 of T. asiatica near-leaf stem as one subcluster, and samples no. 16∼18 of T. asiatica leaf as the other subcluster.

Fig. 4.
Fig. 4.

Scores plot of PCA (A, B), loadings plot of PCA (C), dendrogram and heatmap of HCA (D) of different parts of Toddalia asiatica. Red color represents to higher concentration, while blue color corresponds to lower concentration.

S1, S2, S3: Root bark of T. asiatica.

S4, S5, S6: Root core of T. asiatica.

S7, S8, S9: Stem bark of T. asiatica.

S10, S11, S12: Stem core of T. asiatica.

S13, S14, S15: Near-leaf stem of T. asiatica.

S16, S17, S18: Leaf of T. asiatica.

P: Liquid chromatography peak

Citation: Acta Chromatographica Acta Chromatographica 2020; 10.1556/1326.2020.00762

PCA involves a mathematical procedure that transforms a number of possibly correlated variables into a number of uncorrelated variables called principal components. It is the unsupervised multivariate data analysis approach, and appropriate when a function of many attributes is involved in differences between samples. PCA was able to provide the accurate component that played the most important role in the discrimination of the different medicinal parts of T. asiatica, PCA on 19 common peaks of LC fingerprints of T. asiatica samples was obtained to find the possible chemical markers for the discrimination of different medicinal parts of T. asiatica. The 3D matrix was composed of 18 observations and 19 variables, which indicated samples and the various markers measured by HPLC, respectively. Based on eigenvalues higher than 1, the first (λ 1 = 10.3), the second (λ 2 = 3.32) and the third (λ 3 = 2.84) uncorrelated principal components accounted for 57.3%, 18.5% and 15.8% contribution rate of variance, respectively, and cumulative contribution rate of these three principal components had reached 91.5% (Fig. 4B). Eighteen batches of samples were divided into four groups in 3D score plot: Group 1 – S01∼S03; Group 2 – S07∼S09; Group 3 – S04∼S06, S10∼12 and S13∼15; and Group 4 – S16∼S18. Group 1 and Group 2 would be merged into one group in 2D score plot (Fig. 4A). The three principal components were assigned to evaluate the similarities and differences of the samples. The loading plot (Fig. 4C) indicated that seven compounds played important roles in the samples, P10 > P08 > P07 > P14 > P16 > P17 > P19. In particular, P10, P8 and P7 showed more influence on the discrimination of different medicinal parts of T. asiatica than other peaks, which might be the chemical markers for quality control of T. asiatica. Peaks 10, 08 and 07 were also confirmed to natural compounds pimpinellin, isopimpinlline and toddalolactone.

Antitumor activities of different medicinal parts of T. asiatica

Antitumor activities of different medicinal parts (root bark, root core, stem bark, stem core, near-leaf stem and leaf) of T. asiatica were considered to deeply evaluate their differences through classical MTT experimental method. Anti-tumor mean inhibitory rate (%) of 100.0 μg/mL different medicinal parts of T. asiatica on human breast cancer MCF-7 cells showed clear pharmacodynamic action law: root bark (58.23%), stem bark (48.03%) > root core (27.01%), stem core (33.01%) > near-leaf stem (7.69%), leaf (6.64%), and the inhibition rate showed a significant upward trend with the increase of test sample concentration of treated MCF-7 cells. T. asiatica root bark and root core can significantly inhibit the proliferation of MCF-7 breast cancer cells (P < 0.01) than other parts (Fig. 5). Through bivariate analysis, correlation between 19 characteristic peaks and antitumor inhibition rate of different medicinal parts of T. asiatica had been established, and reflected in the form of correlation coefficients of peaks 01∼19 (*P < 0.05, **P < 0.01): 0.739**(P01), −0.719**(P02), −0.673**(P03), 0.555*(P04), 0.166(P05), 0.783**(P06), 0.854**(P07), 0.751**(P08), −0.537**(P09), 0.769**(P10), 0.783**(P11), 0.337(P12), −0.096(P13), 0.600*(P14), 0.054(P15), 0.813**(P16), 0.550*(P17), 0.775**(P18), 0.717**(P19). The above results of statistical analysissuggested that some compounds in T. asiatica showed strong antitumor activities, their orders were P07 (toddalolactone) > P16 > P06 (4-methoxycinnamic acid), P11 > P18 > P10 (pimpinellin) > P08 (isopimpinellin) > P01 > P19 > P14 > P04 (hesperidin) > P17 in turn.

Fig. 5.
Fig. 5.

The inhibitory effect of extracts from different parts of the Toddalia asiatica on MCF-7 cells

Citation: Acta Chromatographica Acta Chromatographica 2020; 10.1556/1326.2020.00762

Conclusion

A novel strategy integrating quantitative pharmacognostic microscopy, pharmacological activity and multivariate statistical analysis was successfully set up herein for assessing different medicinal parts of T. asiatica, an HPLC-UV method was established to evaluate the quality of different medicinal parts of T. asiatica, antitumor chemical constituents of T. asiatica and rational alternative parts for the root bark by quantitative pharmacognostic microscopy and multivariate statistical analysis. The powder microscopic characteristics of different medicinal parts of T. asiatica had gone through systematic analysis and comparison. Cork cells, calcium oxalate square crystal, brown clump, reticulate vessels, oil cells, stone cells and xylem fibers were the main powder microscopic characteristics of T. asiatica. The main natural compounds of T. asiatica were concentrated in cork cells, calcium oxalate square crystals, brown clumps, starch granules and phloem fibers. The results of multivariate statistical analysis illustrated that different medicinal parts of T. asiatica could be classified, and pimpinellin, isopimpinlline and toddalolactone were highlighted as potential chemical markers for discrimination of different medicinal parts of T. asiatica and quality control of T. asiatica. This study fills the blank space for the research work of rational medicinal parts of T. asiatica, and is helpful in establishing a scientific and rational method for explaining T. asiatica stem bark as a rational alternative medicinal part for T. asiatica root bark and solving current chaos of T. asiatica in the drug market.

Conflicts of interest

The authors declare that there is no conflict of interest.

Acknowledgments

This work was financially supported by National Natural Science Foundation of China (81360681), Guizhou Provincial Natural Science Foundation (2020-1Y404), Open Undergraduate Experimental Project of Guizhou Medical University (2018-8), Teaching Engineering Project of Guizhou Medical University (WK2018-8), and Reform Project of Undergraduate Teaching Content and Course System of Guizhou Medical University (2019-60).

Abbrevations
CMM

Chinese materia medica

HCA

hierarchical clustering analysis

HPLC

high performance liquid chromatography

MTT

methyl thiazolyl tetrazolium

PCA

principal component analysis

TCM

traditional Chinese medicine

References

  • 1.

    Iwasaki, H. ; Okabe, T. ; Takara, K. ; Toda, T. ; Shimatani, M. ; Oku, H. Tumor-selective cytotoxicity of benzo[c]phenanthridine derivatives from Toddalia asiatica Lam . Cancer Chemoth. Pharm. 2010, 65(4), 719726.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Li, X ; Qiu, Z. D. ; Jin, Q. H. ; Chen, G. L. ; Guo, M. Q. Cell cycle arrest and apoptosis in HT-29 cells induced by dichloromethane fraction from Toddalia asiatica (L.) Lam. Front. Pharmacol. 2018, 9, 629.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Zhu, Y. Z. ; Pan, Y. Y. ; Zhang, G. B. ; Wu, Y. C. ; Zhong, W. C. ; Chu, C. X. ; Qian, Y. ; Zhu, G. F. Chelerythrine inhibits human hepatocellular carcinoma metastasis in vitro. Biol. Pharm. Bull. 2018, 41(1), 3646.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Lu, Y. ; Zhu, Y. Z. ; Cuo, C. X. ; Han, C. ; Zhu, G. F. ; Pan, Y. Y. Analgesic effect of Toddalia Asiatica's extract and its peripheral analgesic mechanism. Shanghai J. Tradit. Chin. Med. 2015, 49(7), 8286.

    • Search Google Scholar
    • Export Citation
  • 5.

    Zhou, W. ; Sun, W. B. ; Zeng, Q. F. ; Li, L. ; Hao, X. Y. ; Liang, Y. Pharmaceutical research progress on Toddalia asiatica . China J. Tradit. Chin. Med. Pharm. 2018, 33(8), 35153522.

    • Search Google Scholar
    • Export Citation
  • 6.

    Zhang, X. Y. ; Shang, H. C. Current situation and problems of clinical research on traditional chinese medicine in china in recent 10 years. J. Tradit. Chin. Med. 2018, 59(21), 18081811.

    • Search Google Scholar
    • Export Citation
  • 7.

    Chen, X. X. ; Simayi, R. ; Long, S. J. Studies on chemical constituents of Toddalia asiatica stems. Northwest Pharm. J. 2013, 28(4), 337339.

    • Search Google Scholar
    • Export Citation
  • 8.

    Cao, C. ; Du, P. ; Zhu, X. ; Yan, H. ; Song, X. ; Zhu, H. ; Geng, Y. ; Wang, D. Rapid screening and purification of potential alkaloid neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. roots via ultrafiltration liquid chromatography combined with stepwise flow rate counter-current chromatography. J. Sep. Sci. 2019, 42(16), 26212627.

    • Search Google Scholar
    • Export Citation
  • 9.

    Wei, M. C. ; Liu, K. ; Yi, Q. ; Zhang, L. Y. ; Zhou, H. H. ; Dou, S. S. ; Zhang, J. Analysis on fingerprint for root barks and stem barks of Toddalia asiatica . Chin. Tradit. Herbal Drugs 2016, 47(14), 25402544.

    • Search Google Scholar
    • Export Citation
  • 10.

    Zhu, M. J. ; Wei, P. Q. ; Peng, Q. ; Qin, S. Y. ; Zhou, Y. ; Zhang, R. ; Zhu, C. C. ; Zhang, L. Simultaneous qualitative and quantitative evaluation of Toddalia asiatica root by using HPLC-DAD and UPLC-QTOF-MS/MS. Phytochem. Anal. 2019, 30(2), 164181.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Zhou, W. ; Zeng, Q. F. ; Luo, C. R. ; Wan, L. ; Zhang, X. Y. Analysis of chemical constituents of polar extract of toddalia asiatica root bark by LC-Q-TOF. China J. Tradit. Chin. Med. Pharm. 2019, 34(12), 59145919.

    • Search Google Scholar
    • Export Citation
  • 12.

    Zhou, W. ; Luo, C. R. ; Liu, G. ; Dong, W. Q. ; Zhang, X. Y. , Shen, X. C. GC-MS analysis of the root bark oil of toddalia asiatica . J. Guizhou Med. Univ. 2019, 44, 147152.

    • Search Google Scholar
    • Export Citation
  • 13.

    Sun, W. B. ; Yang, Z. ; Liang, Y. ; Li, L. ; Hao, X. Y. ; Zuo, G. Y. ; Zhou, W. Chemical constituents from n-butanol part in Toddalia asiatica Root Bark. Chin. Pharm. J. 2018, 53, 10521056.

    • Search Google Scholar
    • Export Citation
  • 14.

    Zhang, X. Y. ; Sun, W. B. ; Yang, Z. ; Liang, Y. ; Zhou, W. ; Tang, L. Hemostatic chemical constituents from natural medicine Toddalia asiatica root bark by LC-ESI Q-TOF MSE . Chem. Cent. J. 2017, 11, 55.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Zhao, M. X. ; Zhang X.Y. ; Liu S. H. ; He, M. Q. ; Liang, Y. ; Hao, X. Y. ; Zhou, W. Pharmacognostic identification and hemostatic activity of Toddalia asiatica root bark. Chin. J. Exp. Tradit. Med. Formulae 2016, 22, 3236.

    • Search Google Scholar
    • Export Citation
  • 16.

    Zhou, W. ; Xie, M. F. ; Zhang, X. Y. ; Liu, T. T. ; Yu, Y. J. ; Duan, G. L. Improved liquid chromatography fingerprint of fat-soluble Radix isatidis extract using multi-wavelength combination technique. J. Sep. Sci. 2011, 34, 11231132.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Zhu, L. L. ; Fang, L. X. ; Li, Z. J. ; Xie, X. M. ; Zhang, L. A HPLC fingerprint study on Chaenomelis Fructus. BMC Chem. 2019, 13, 7.

  • 18.

    Zhong, Y. C. , Wang, H. Y. ; Wei, Q. H. ; Cao, R. ; Zhang, H. L. ; He, Y. Z. ; Wang, L. Z. Combining DNA barcoding and HPLC fingerprints to trace species of an important traditional Chinese medicine fritillariae bulbus. Molecules 2019, 24, 3269.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Duan, S. N. ; Qi, W. ; Zhang, S. W. ; Huang, K. K. ; Yuan, D. Simultaneous quantification combined with multivariate statistical analysis of multiple chemical markers of Wu Ji Bai Feng Pill by UHPLC-MS/MS. J. Food Drug Anal. 2019, 27, 12751283.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Corleto, K. A. ; Singh, J. ; Jayaprakasha, G. K. ; Patil, B. S. A sensitive HPLC-FLD method combined with multivariate analysis for the determination of amino acids in L-citrulline rich vegetables. J. Food Drug Anal. 2019, 27, 717728.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Zhou, W. ; Zhang, X. Y. ; Lv, Y. P. ; Liu, X. D. ; Xu, C. ; Duan, G. L. RSM-Optimized IRAE sample pretreatment and HPLC simultaneous determination of tryptanthrin, indigo, and indirubin from Chinese herbal medicine Radix Isatidis . Acta Chromatographica 2013, 25(2), 297315.

    • Crossref
    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1.

    Iwasaki, H. ; Okabe, T. ; Takara, K. ; Toda, T. ; Shimatani, M. ; Oku, H. Tumor-selective cytotoxicity of benzo[c]phenanthridine derivatives from Toddalia asiatica Lam . Cancer Chemoth. Pharm. 2010, 65(4), 719726.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Li, X ; Qiu, Z. D. ; Jin, Q. H. ; Chen, G. L. ; Guo, M. Q. Cell cycle arrest and apoptosis in HT-29 cells induced by dichloromethane fraction from Toddalia asiatica (L.) Lam. Front. Pharmacol. 2018, 9, 629.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3.

    Zhu, Y. Z. ; Pan, Y. Y. ; Zhang, G. B. ; Wu, Y. C. ; Zhong, W. C. ; Chu, C. X. ; Qian, Y. ; Zhu, G. F. Chelerythrine inhibits human hepatocellular carcinoma metastasis in vitro. Biol. Pharm. Bull. 2018, 41(1), 3646.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 4.

    Lu, Y. ; Zhu, Y. Z. ; Cuo, C. X. ; Han, C. ; Zhu, G. F. ; Pan, Y. Y. Analgesic effect of Toddalia Asiatica's extract and its peripheral analgesic mechanism. Shanghai J. Tradit. Chin. Med. 2015, 49(7), 8286.

    • Search Google Scholar
    • Export Citation
  • 5.

    Zhou, W. ; Sun, W. B. ; Zeng, Q. F. ; Li, L. ; Hao, X. Y. ; Liang, Y. Pharmaceutical research progress on Toddalia asiatica . China J. Tradit. Chin. Med. Pharm. 2018, 33(8), 35153522.

    • Search Google Scholar
    • Export Citation
  • 6.

    Zhang, X. Y. ; Shang, H. C. Current situation and problems of clinical research on traditional chinese medicine in china in recent 10 years. J. Tradit. Chin. Med. 2018, 59(21), 18081811.

    • Search Google Scholar
    • Export Citation
  • 7.

    Chen, X. X. ; Simayi, R. ; Long, S. J. Studies on chemical constituents of Toddalia asiatica stems. Northwest Pharm. J. 2013, 28(4), 337339.

    • Search Google Scholar
    • Export Citation
  • 8.

    Cao, C. ; Du, P. ; Zhu, X. ; Yan, H. ; Song, X. ; Zhu, H. ; Geng, Y. ; Wang, D. Rapid screening and purification of potential alkaloid neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. roots via ultrafiltration liquid chromatography combined with stepwise flow rate counter-current chromatography. J. Sep. Sci. 2019, 42(16), 26212627.

    • Search Google Scholar
    • Export Citation
  • 9.

    Wei, M. C. ; Liu, K. ; Yi, Q. ; Zhang, L. Y. ; Zhou, H. H. ; Dou, S. S. ; Zhang, J. Analysis on fingerprint for root barks and stem barks of Toddalia asiatica . Chin. Tradit. Herbal Drugs 2016, 47(14), 25402544.

    • Search Google Scholar
    • Export Citation
  • 10.

    Zhu, M. J. ; Wei, P. Q. ; Peng, Q. ; Qin, S. Y. ; Zhou, Y. ; Zhang, R. ; Zhu, C. C. ; Zhang, L. Simultaneous qualitative and quantitative evaluation of Toddalia asiatica root by using HPLC-DAD and UPLC-QTOF-MS/MS. Phytochem. Anal. 2019, 30(2), 164181.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Zhou, W. ; Zeng, Q. F. ; Luo, C. R. ; Wan, L. ; Zhang, X. Y. Analysis of chemical constituents of polar extract of toddalia asiatica root bark by LC-Q-TOF. China J. Tradit. Chin. Med. Pharm. 2019, 34(12), 59145919.

    • Search Google Scholar
    • Export Citation
  • 12.

    Zhou, W. ; Luo, C. R. ; Liu, G. ; Dong, W. Q. ; Zhang, X. Y. , Shen, X. C. GC-MS analysis of the root bark oil of toddalia asiatica . J. Guizhou Med. Univ. 2019, 44, 147152.

    • Search Google Scholar
    • Export Citation
  • 13.

    Sun, W. B. ; Yang, Z. ; Liang, Y. ; Li, L. ; Hao, X. Y. ; Zuo, G. Y. ; Zhou, W. Chemical constituents from n-butanol part in Toddalia asiatica Root Bark. Chin. Pharm. J. 2018, 53, 10521056.

    • Search Google Scholar
    • Export Citation
  • 14.

    Zhang, X. Y. ; Sun, W. B. ; Yang, Z. ; Liang, Y. ; Zhou, W. ; Tang, L. Hemostatic chemical constituents from natural medicine Toddalia asiatica root bark by LC-ESI Q-TOF MSE . Chem. Cent. J. 2017, 11, 55.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Zhao, M. X. ; Zhang X.Y. ; Liu S. H. ; He, M. Q. ; Liang, Y. ; Hao, X. Y. ; Zhou, W. Pharmacognostic identification and hemostatic activity of Toddalia asiatica root bark. Chin. J. Exp. Tradit. Med. Formulae 2016, 22, 3236.

    • Search Google Scholar
    • Export Citation
  • 16.

    Zhou, W. ; Xie, M. F. ; Zhang, X. Y. ; Liu, T. T. ; Yu, Y. J. ; Duan, G. L. Improved liquid chromatography fingerprint of fat-soluble Radix isatidis extract using multi-wavelength combination technique. J. Sep. Sci. 2011, 34, 11231132.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Zhu, L. L. ; Fang, L. X. ; Li, Z. J. ; Xie, X. M. ; Zhang, L. A HPLC fingerprint study on Chaenomelis Fructus. BMC Chem. 2019, 13, 7.

  • 18.

    Zhong, Y. C. , Wang, H. Y. ; Wei, Q. H. ; Cao, R. ; Zhang, H. L. ; He, Y. Z. ; Wang, L. Z. Combining DNA barcoding and HPLC fingerprints to trace species of an important traditional Chinese medicine fritillariae bulbus. Molecules 2019, 24, 3269.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 19.

    Duan, S. N. ; Qi, W. ; Zhang, S. W. ; Huang, K. K. ; Yuan, D. Simultaneous quantification combined with multivariate statistical analysis of multiple chemical markers of Wu Ji Bai Feng Pill by UHPLC-MS/MS. J. Food Drug Anal. 2019, 27, 12751283.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 20.

    Corleto, K. A. ; Singh, J. ; Jayaprakasha, G. K. ; Patil, B. S. A sensitive HPLC-FLD method combined with multivariate analysis for the determination of amino acids in L-citrulline rich vegetables. J. Food Drug Anal. 2019, 27, 717728.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 21.

    Zhou, W. ; Zhang, X. Y. ; Lv, Y. P. ; Liu, X. D. ; Xu, C. ; Duan, G. L. RSM-Optimized IRAE sample pretreatment and HPLC simultaneous determination of tryptanthrin, indigo, and indirubin from Chinese herbal medicine Radix Isatidis . Acta Chromatographica 2013, 25(2), 297315.

    • Crossref
    • Search Google Scholar
    • Export Citation

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Senior editors

Editor(s)-in-Chief: Kowalska, Teresa

Editor(s)-in-Chief: Sajewicz, Mieczyslaw

Editors(s) Danica Agbaba (University of Belgrade, Belgrade, Serbia);
Ivana Stanimirova-Daszykowska (University of Silesia, Katowice, Poland),
Monika Waksmundzka-Hajnos (Medical University of Lublin, Lublin, Poland)

Editorial Board

R. Bhushan (The Indian Institute of Technology, Roorkee, India)
J. Bojarski (Jagiellonian University, Kraków, Poland)
B. Chankvetadze (State University of Tbilisi, Tbilisi, Georgia)
M. Daszykowski (University of Silesia, Katowice, Poland)
T.H. Dzido (Medical University of Lublin, Lublin, Poland)
A. Felinger (University of Pécs, Pécs, Hungary)
K. Glowniak (Medical University of Lublin, Lublin, Poland)
B. Glód (Siedlce University of Natural Sciences and Humanities, Siedlce, Poland)
A. Grochowalski† (Cracow University of Technology, Kraków, Poland)
K. Kaczmarski (Rzeszow University of Technology, Rzeszów, Poland)
H. Kalász (Semmelweis University, Budapest, Hungary)
R. Kaliszan† (Medical University of Gdańsk, Gdańsk, Poland)
I. Klebovich (Semmelweis University, Budapest, Hungary)
A. Koch (Private Pharmacy, Hamburg, Germany)
Ł. Komsta (Medical University of Lublin, Lublin, Poland)
P. Kus (Univerity of Silesia, Katowice, Poland)
D. Mangelings (Free University of Brussels, Brussels, Belgium)
E. Mincsovics (Corvinus University of Budapest, Budapest, Hungary)
G. Morlock (Giessen University, Giessen, Germany)
J. Namiesnik† (Gdańsk University of Technology, Gdańsk, Poland)
J. Sherma (Lafayette College, Easton, PA, USA)
R. Skibiński (Medical University of Lublin, Lublin, Poland)
B. Spangenberg (Offenburg University of Applied Sciences, Germany)
T. Tuzimski (Medical University of Lublin, Lublin, Poland)
Y. Vander Heyden (Free University of Brussels, Brussels, Belgium)
A. Voelkel (Poznań University of Technology, Poznań, Poland)
B. Walczak (University of Silesia, Katowice, Poland)
W. Wasiak (Adam Mickiewicz University, Poznań, Poland)

KOWALSKA, TERESA
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl