Novel Oxidation Oligomer of Chlorogenic Acid and (-)-Epigallocatechin and Its Quantitative Analysis during the Processing of Keemun Black Tea.

Not only do flavan-3-ols participate in the formation of chromogenic oxidation products such as theaflavins, but chlorogenic acid (3-caffeoylquinic acid, CQA) is also involved in the enzymatic oxidation during black tea processing. The critical oxidation product of CQA and (-)-epigallocatechin (EGC) were identified as an adduct containing benzobicyclo[3.2.2]nonenone structure, which was named as the dichlorogeniccatechin (DCGC) oligomer. It was composed of two molecules of CQA and one molecule of EGC. The effects of the initial reactant ratio and reaction time on the generation of DCGC were also analyzed. A high proportion of CQA promoted the production of DCGC, but a high proportion of EGC inhibited the DCGC formation. In addition, the content of DCGC in Keemun black tea during processing was determined. The content of DCGC highly increased after withering but decreased after drying. This study provides a new perspective for the investigation of other oxidation oligomers in black tea.

QSAR Research of Novel Tetrandrine Derivatives against Human Hepatocellular Carcinoma.

BACKGROUND: The new tetrandrine derivative is an anti-human liver cancer cell inhibitor which can be used to design and develop anti-human-liver-cancer drugs. OBJECTIVE: A quantitative structure-activity relationship (QSAR) model was established to predict the physical properties of new tetrandrine derivatives using their chemical structures. METHODS: The best descriptors were selected through CODESSA software to build a multiple linear regression model. Then, gene expression programming (GEP) was used to establish a nonlinear quantitative QSAR model with descriptors to predict the activity of a series of novel tetrandrine chemotherapy drugs. The best active compound 31 was subjected to molecular docking experiments through SYBYL software with a small fragment of the protein receptor (PDB ID:2J6M). RESULTS: Four descriptors were selected to build a multiple linear regression model with correlation coefficients R2, R2CV and S2 with the values of 0.8352, 0.7806 and 0.0119, respectively. The training and test sets with a correlation coefficient of 0.85 and 0.83 were obtained via an automatic problem-solving program (APS) using the four selected operators as parameters, with a mean error of 1.49 and 1.08. Compound 31 had a good docking ability with an overall score of 5.8892, a collision rate of -2.8004 and an extreme value of 0.9836. CONCLUSION: The computer-constructed drug molecular model reveals the factors affecting the activity of human hepatocellular carcinoma cells, which provides directions and guidance for the development of highly effective anti-humanhepatocellular- carcinoma drugs in the future.

Neuroprotective effects of Longxue Tongluo Capsule on ischemic stroke rats revealed by LC-MS/MS-based metabolomics approach.

Objective: The present study aimed to evaluate the therapeutic effect and explore the underlying mechanisms of Longxue Tongluo Capsule (LTC) on ischemic stroke rats. Methods: Twenty-six rats were randomly divided into four groups, including sham group, sham + LTC group, MCAO group, and MCAO + LTC group. Ischemic stroke rats were simulated by middle cerebral artery occlusion (MCAO), and LTC treatment group were orally administrated with 300 mg/kg of LTC once daily for seven consecutive days. LTC therapy was validated in terms of neurobehavioral abnormality evaluation, cerebral infarct area, and histological assessments. The plasma metabolome comparisons amongst different groups were conducted by UHPLC-Q Exactive MS in combination with subsequent multivariate statistical analysis, aiming to finding the molecules in respond to the surgery or LTC treatment. Results: Intragastric administration of LTC significantly decreased not only the neurobehavioral abnormality scores but also the cerebral infarct area of MCAO rats. The interstitial edema, atrophy, and pyknosis of glial and neuronal cells occurred in the infarcted area, core area, and marginal area of cerebral cortex were improved after LTC treatment. A total of 13 potential biomarkers were observed, and Youden index of 11 biomarkers such as LysoPC, SM, and PE were more than 0.7, which were involved in neuroprotective process. The correlation and pathway analysis showed that LTC was beneficial to ischemic stroke rats via regulating glycerophospholipid and sphingolipid metabolism, together with nicotinate and nicotinamide metabolism. Heatmap and ternary analysis indicated the synergistic effect of carbohydrates and lipids may be induced by flavonoid intake from LTC. Conclusion: The present study could provide evidence that metabolomics, as systematic approach, revealed its capacity to evaluate the holistic efficacy of TCM, and investigate the molecular mechanism underlying the clinical treatment of LTC on ischemic stroke.

[Chemical constituents of diterpenoids from Boswellia carterii].

This paper explored the chemical constituents of Boswellia carterii by column chromatography on silica gel, Sephadex LH-20, ODS column chromatography, and semi-preparative HPLC. The structures of the compounds were identified by physicochemical properties and spectroscopic data such as infrared radiation(IR), ultra violet(UV), mass spectrometry(MS), and nuclear magnetic resonance(NMR). Seven diterpenoids were isolated and purified from n-hexane of B. carterii. The isolates were identified as(1S,3E,7E,11R,12R)-11-hydroxy-1-isopropyl-4,8,12-trimethyl-15-oxabicyclo[10.2.1]pentadeca-3,7-dien-5-one(1),(1R,3S,4R,7E,11E)-4,8,12,15,15-pentamethyl-14-oxabicyclo[11.2.1]hexadeca-7,11-dien-4-ol(2), incensole(3),(-)-(R)-nephthenol(4), euphraticanoid F(5), dilospirane B(6), and dictyotin C(7). Among them, compounds 1 and 2 were new and their absolute configurations were determined by comparison of the calculated and experimental electronic circular dichroisms(ECDs). Compounds 6 and 7 were obtained from B. carterii for the first time.

Platelet-inhibitory phenolic constituents from the fruits of Daemonorops draco.

Eight previously undescribed phenolic compounds, dracoropins A - H (1-8), along with two known analogues (9 and 10) were isolated from the fruits of Daemonorops draco. Four pairs of isomers (1a/1b, 2a/2b, 3a/3b, and 4a/4b) were resolved by using chiral-phase HPLC separation. Their structures, including the absolute configurations of the resolved isomers, were elucidated by analysis of spectroscopic data (1D and 2D NMR, IR, and HRESIMS), single-crystal X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compounds 1, 2, and 3 bear a rare 2-phenylbenzo[d]-1,3-dioxepine skeleton. All the isolates were evaluated for their inhibitory activity against ATP release in thrombin-activated platelets. Compounds 2b, 3a, and 6 could significantly inhibit ATP release in thrombin-activated platelets.

Isoquinoline alkaloids from Corydalis edulis Maxim. Exhibiting insulinotropic action.

Eleven undescribed isoquinoline analogues, namely edulisines A-K, along with sixteen known alkaloids, were isolated from the whole plants of Corydalis edulis. The structures of the isolated alkaloids were established on the basis of extensive spectroscopic data (1D and 2D NMR, UV, IR, and HRESIMS). Their absolute configurations were determined by single-crystal X-ray crystallographic analysis and ECD. Compounds (+)-1 and (-)-1 are a pair of undescribed isoquinoline alkaloids bearing a unique coupled pattern of coptisine and ferulic acid via Diels-Alder [4 + 2] cycloaddition, while compounds (+)-2 and (-)-2 feature benzo [1,2-d:3,4-d]bis [1,3]dioxole moiety. Compounds (+)-2, (-)-2, (-)-5, 10, 13, 15, 20, 22, and 23 significantly triggered the secretion of insulin in the HIT-T15 cells at a concentration of 40 µM.

Formation Mechanism of Di-N-ethyl-2-pyrrolidinone-Substituted Epigallocatechin Gallate during High-Temperature Roasting of Tea.

Four di-N-ethyl-2-pyrrolidinone-substituted epigallocatechin gallate (EGCG) and two di-N-ethyl-2-pyrrolidinone-substituted gallocatechin gallate (GCG) flavan-3-ols (di-EPSFs) were prepared by the thermal simulation reaction. The effects of reaction temperature and time, initial reactant ratios, and pH values on the content of di-EPSFs were studied. The formation of six di-EPSFs was most favored when the initial reactant ratio of EGCG and theanine was 1:2 and heated under 130 °C at pH 10 for 120 min. The contents of di-EPSF1, di-EPSF2, and di-EPSF5 in large-leaf yellow tea (LYT) increased with the increase of roasting degree. Through quantitative analysis, it was found that EGCG would interact with the Strecker degradation products of theanine to form EPSFs, which further combined with the Strecker degradation products of theanine to form di-EPSFs. This study further improved the understanding of the transformation pathways of EGCG and theanine during tea processing and contributed to exploring the flavor characteristics and health benefits of di-EPSFs.

Gastroprotective 2-(2-phenylethyl)chromone-sesquiterpene hybrids from the resinous wood of Aquilaria sinensis (Lour.) Gilg.

Six previously unprecedented 2-(2-phenylethyl)chromone-sesquiterpene hybrids, aquisinenins A-F (1 - 6), were isolated from the resinous wood of Aquilaria sinensis by a LC-MS-guided fractionation procedure. Their structures were determined by extensive spectroscopic analysis (1D and 2D NMR, UV, IR, and HRMS) and experimental and computed ECD data. Compounds 1 - 6 were rare dimeric 2-(2-phenylethyl)chromone-sesquiterpene derivatives featuring 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone hybridized with different sesquiterpene (eudesmane/guaiane type) moieties via ester bond. Furthermore, all the isolated compounds were evaluated for their protective effects on taurocholic acid (TCA)-induced GES-1 cell injury. The most effective aquisinenin F (6) was used to elucidate the involved mechanism on protection against TCA-induced gastric mucosal damage. Our results indicated that 6 protected against gastric mucosal cell insult by downregulation of the ER stress triggered by TCA.

Anti-inflammatory flavonoid derivatives from the heartwood of Dalbergia odorifera T. Chen.

Two pairs of flavonoid enantiomers (1a/1b and 2a/2b) together with three known analogues (3-5) were isolated from the heartwood of Dalbergia odorifera T. Chen. Their structures were elucidated by extensive spectroscopic analysis (1 D and 2 D NMR, UV, IR, and HRMS) and experimental and calculated ECD data. Compound 2 features an unusual 2-methyl-3(2H)-furanone moiety forming the C-ring of flavonoid, and its putative biosynthetic pathway is also proposed. Compounds 3‒5 exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells with IC50 values of 14.7 ± 0.3 µM, 40.2 ± 1.1 µM, and 3.2 ± 0.1 µM, respectively.

A 5-Pathway Signature Predicts Prognosis Based on Immune-Derived lncRNAs in Patients with Breast Cancer.

Background: Currently, predictive models were not developed based on the signaling pathway signatures of immune-related lncRNAs in breast cancer (BRCA) patients. Methods: We selected unsupervised hierarchical clustering algorithm to classify patients with BRCA based on the significant immune-derived lncRNAs from the TCGA dataset. And different methods including ESTIMATE, ImmuneCellAI, and CIBERSORT were performed to evaluate the immune infiltration of tumor microenvironment. Using Lasso regression algorithm, we filtered the significant signaling pathways enriched by GSEA, GSVA, or PPI analysis to develop a prognostic model. And a nomogram integrated with clinical factors and significant pathways was constructed to predict the precise probability of overall survival (OS) of BRCA patients in the TCGA dataset (n = 1,098) and another two testing sets (n = 415). Results: BRCA patients were stratified into the PC (n = 571) and GC (n = 527) subgroup with significantly different prognosis with 550 immune-related lncRNAs in the TCGA dataset. Integrated analysis revealed different immune response, oncogenic signaling, and metabolic reprograming pathways between these two subgroups. And a 5-pathway signature could predict the prognosis of BRCA patients between these two subgroups independently in the TCGA dataset, which was confirmed in another two cohorts from the GEO dataset. In the TCGA dataset, 5-year OS rate was 78% (95% CI: 73-84) vs. 82% (95% CI: 77-87) for the PC and GC group (HR = 1.63 (95% CI: 1.17-2.28), p = 0.004). The predictive power was similar in another two testing sets (HR > 1.20, p < 0.01). Finally, a nomogram is developed for clinical application, which integrated this signature and age to accurately predict the survival probability in BRCA patients. Conclusion: This 5-pathway signature correlated with immune-derived lncRNAs was able to precisely predict the prognosis for patients with BRCA and provided a rich source characterizing immune-related lncRNAs and further informed strategies to target BRCA vulnerabilities.

Cytotoxic sesquiterpenoid dimers from the resin of Commiphora myrrha Engl.

Seven undescribed sesquiterpenoid dimers, commiphomyrones A - G, together with three known analogs, were isolated from the resin of Commiphora myrrha Engl.. The structures of the undescribed compounds were elucidated based on a comprehensive analysis of spectroscopic data (NMR, UV, IR, and MS), and the absolute configurations were defined by comparing the experimental and calculated ECD spectra as well as by performing X-ray crystallographic analysis. All the isolated dimeric sesquiterpenoids feature a 7-oxabicyclo [2.2.1] hept-2-ene moiety formed by the [4 + 2] cycloaddition of two sesquiterpenoids. Commiphomyrones C and G and commiphoratone D showed cytotoxic activity against the HGC-27 cell line with IC50 values of 22.76, 25.01, and 27.51 µM, respectively.

[Sesquiterpenoids from Aquilariae Lignum Resinatum].

Eight sesquiterpenoids were isolated from petroleum ether extract of Aquilariae Lignum Resinatum by various column chromatography techniques including silica gel, ODS, and semi-preparative HPLC. Their structures were identified on the basis of physicochemical properties, UV, IR, MS, and NMR spectroscopic data as(4S,5S,7R,10S)-5,7-dihydroxy-11-en-eudesmane(1),(7R,10S)-eudesma-4-en-11,15-diol(2),(2R,4S,5R,7R)-2-hydroxyeremophila-9,11-dien-8-one(3), 7α-H-9(10)-ene-11,12-epoxy-8-oxoeremophilane(4),(+)-9ß,10ß-epoxyeremophila-11(13)-en(5), 4(14)-eudesmene-8α,11-diol(6), 12,15-dioxo-selina-4,11-dien(7), and 2ß,8 aα-dihydroxy-11-en-eremophilane(8). Compounds 1 and 2 are new compounds, and their absolute configurations were determined by calculating ECD. Compounds 1, 4, and 6-8 could significantly improve taurocholic acid(TCA)-induced gastric mucosal GES-1 cell injury at a concentration of 20 µmol·L~(-1), and the cell protection rates were 23.51%±2.79%, 16.10%±1.25%, 24.45%±4.89%, 17.48%±2.93%, and 21.44%±2.39%, respectively.

Phenolic Glucosides from the Leaves of Vitex negundo var. cannabifolia.

Four new phenolic glucosides, cannabifolins G-J (1-4), together with four known ones (5-8), were isolated from the leaves of Vitex negundo var. cannabifolia. Their structures were established by comprehensive analysis of 1D and 2D NMR data and comparison of their spectroscopic and physical data with the literature values. Compound 7 exhibited weak inhibition of nitric oxide production stimulated by lipopolysaccharide in BV-2 microglial cells with IC50 value of 132.8 µM.

2-(2-phenylethyl)chromone-enriched extract of the resinous heartwood of Chinese agarwood (Aquilaria sinensis) protects against taurocholic acid-induced gastric epithelial cells apoptosis through Perk/eIF2α/CHOP pathway.

BACKGROUND: Injury of gastric epithelial cells is one of the most important pathological features of bile reflux gastritis. Chinese agarwood (the resinous heartwood of Aquilaria sinensis) has been used to treat stomach problems for thousands of years in China. However, the pathological mechanism of epithelial cells death induced by bile acids and the therapeutic target of Chinese agarwood for improving bile reflux gastritis have not yet been fully clarified. PURPOSE: This study aimed to investigate the pro-apoptotic effect of taurocholic acid (TCA) by regulating the ER stress pathway. Moreover, the role of Chinese agarwood 2-(2-phenylethyl)chromone-enriched extract (CPE) to inhibit gastric epithelial cell death induced by TCA was also been demonstrated. METHODS: We adopted human gastric epithelial GES-1 cells to explore the mechanism of TCA-induced cell death in vitro. Then the cell viability, apoptosis rate, and protein expressions were evaluated to explore the protective effects of CPE on GES-1 cells by TCA injury. The therapeutic effect of CPE on bile reflux gastritis was further confirmed by the bile reflux mice in vivo. RESULTS: Our results demonstrated that TCA activated GES-1 cell apoptosis by increased cleavage of caspase-7 and PARP. Further experiments showed that TCA up-regulated endoplasmic reticulum (ER) stress, subsequently triggered the apoptosis of the epithelial cells. Our research explored that CPE is the main effective fraction in Chinese agarwood by preventing the TCA-induced gastric epithelial cell injury. CPE effectively suppressed GES-1 cell apoptosis activated by TCA through inhibiting Perk/eIF2α/CHOP pathway. The anti-apoptotic effect of CPE on gastric mucosa had also been confirmed in vivo. Moreover, the main effective components in CPE corresponding to the protection of epithelial cells were also been identified. CONCLUSION: Our finding suggested that CPE recovered the TCA-induced epithelial cell apoptosis by mediating the activation of ER stress, which explored potential medicine to treat bile reflux gastritis.

[Quality standard of Lobeliae Chinensis Herba].

In light of related methods in Chinese Pharmacopoeia(2020 edition), this study established the quality standard for Lobeliae Chinensis Herba. The TLC identification method was established with silica gel GF_(254) thin layer plate, diosmin standard, linarin standard, and the reference material of Lobeliae Chinensis Herba. The loss on drying, total ash, acid-insoluble ash, and ethanol-soluble extracts of 18 batches of Lobeliae Chinensis Herba samples were determined according to the general principles in Chinese Pharmacopoeia. Then, HPLC was adopted in the establishment of characteristic chromatogram and content determination. The results showed that the established method can achieve good separation for diosmin, linarin, and lobetyolin. Based on the results of detection for 18 batches of Lobeliae Chinensis Herba samples, the draft quality standard was established, which was expected to provide reference for the revision of this medicinal herb in Chinese Pharmacopoeia.

Trewioidesine A, an unsaturated fatty acid from rhizomes of Alchornea trewioides, shows synergy with NGF in inducing differentiation of pheochromocytoma PC12 cells.

A new unsaturated fatty acid trewioidesine A (1), together with seven known compounds (2 - 8) were isolated from the rhizomes of Alchornea trewioides (Benth.) Muell. Arg. Their structures were established on the basis of extensive spectroscopic data interpretation (1 D and 2 D NMR, and HRESIMS). The absolute configuration of 1 was determined by electronic circular dichroism (ECD) calculations, confirming as trewioidesine A. The functionality of isolated compounds was tested in cultured PC12 cells, a cell line from rat pheochromocytoma. Trewioidesine A was the one showing robust activity in inducing neuronal differentiation: the induction was synergized when co-applied with nerve growth factor (NGF). In addition, a neurofilament 200 (NF200) promoter-luciferase (pNF200-Luc) reporter was used to evaluate the differentiating ability in the transfected PC12 cells for the isolated compounds. Trewioidesine A exhibited a strong NF200 promoter activation, and application of trewioidesine A with low dose of NGF significantly induced the promoter activity over 50%.

Full Collision Energy Ramp-MS2 Spectrum in Structural Analysis Relying on MS/MS.

Albeit frequently being overlooked, MS2 spectrum variation against collision energy (CE) implies auxiliary structural clues for m/z values. Online energy-resolved MS (ER-MS) provides the opportunity to acquire the trajectory of ion intensity against CE for any fragment ion of interest, thus exactly offering the desired momentum to empower the conventional MS2 spectrum at a certain CE forward to a full-CE ramp MS2 spectrum (FCER-MS2). Efforts were made here to construct an FCER-MS2 spectrum and to evaluate its potential toward structural analysis. Flavonoids were employed as a proof of concept. MS2 spectra of 76 compounds were recorded by LC-Q-Exactive-MS, and online ER-MS was subsequently programmed using LC-Qtrap-MS to build a breakdown graph for each obvious fragment ion. After defining the greatest value amongst all regressive apices as 100%, the normalized breakdown graphs comprised an FCER-MS2 spectrum for each compound. The FCER-MS2 spectrum contained the MS2 spectrum at any CE as well as optimal CE (OCE) and maximal relative ion intensity (RIImax) of each fragment ion. Except the pronounced isomeric discrimination potential, either OCE or RIImax reflected certain structural properties, such as aglycone, glycosidic bond, and hydroxy, methoxy, and glycosyl substituents. These rules were subsequently applied for flavonoid-focused characterization of a famous herbal medicine, namely Scutellariae Radix, and high-level structural annotation was accomplished for 75 flavonoids. Above all, the FCER-MS2 spectrum includes m/z, OCEs, and RIImax features, thus facilitating confidence-advanced structural analysis.

Longxuetongluo Capsule protects against cerebral ischemia/reperfusion injury through endoplasmic reticulum stress and MAPK-mediated mechanisms.

Introduction: Longxuetongluo Capsule (LTC) is wildly applied to treat ischemic stroke in clinical practice in China. However, the pharmacological mechanism of LTC on ischemic stroke is still unstated. Objective: Our research was designed to study the protective effect of LTC against cerebral ischemia-reperfusion (I/R) injury and reveal the underlying mechanism both in vivo and in vitro. Methods: PC12 cells treated with glucose deprivation/reperfusion (OGD/R) were used to simulate in vitro ischemia/reperfusion (I/R) injury. The cell viability, apoptosis rate, and protein expressions of PC12 cells were evaluated. In vivo validation of the protective effect of LTC was carried out by middle cerebral artery occlusion (MCAO)/reperfusion treatment, and the underlying mechanism of its anti-apoptosis ability was further revealed by immunohistochemistry staining and Western blotting. Results: In the current study, we observed that LTC effectively inhibited oxygen-glucose deprivation/reperfusion (OGD/R) induced apoptosis of PC12 cells through suppressing the cleavage of poly ADP-ribose polymerase (PARP), caspase-3, and caspase-9. Further investigation revealed that OGD/R insult remarkably triggered the endoplasmic reticulum stress responses (ER stress) to induce PC12 cell apoptosis. LTC treatment alleviated OGD/R induced ER stress by inhibiting the activation of protein kinase RNA (PKR)-like ER kinase (PERK)/eukaryotic translation initiation factor 2 (eIF2α) and inositol requiring enzyme 1 (IRE1)/tumor necrosis factor receptor-associated factor 2 (TRAF2) pathways. Additionally, LTC also restrained the OGD/R-induced PC12 cell apoptosis by reversing the activated mitogen-activated protein kinase (MAPK) through IRE1/TRAF2 pathway. Animal studies demonstrated LTC significantly restricted the infarct region induced by middle cerebral artery occlusion (MCAO)/reperfusion, the activation of ER stress and apoptosis of neuronal cells had also been suppressed by LTC in the penumbra region. Conclusion: LTC protects the cerebral neuronal cell against ischemia/reperfusion injury through ER stress and MAPK-mediated mechanisms.

Trimeric chalchonoids from the total phenolic extract of Chinese dragon's blood (the red resin of Dracaena cochinchinensis).

Four new chalchonoid trimers, named cochinchinenins N-Q (1-4), along with a pair of known enantiomers (5-6), were isolated from the total phenolic extract of Chinese dragon's blood (the red resin of Dracaena cochinchinensis). The planar structures of 1-4 were elucidated by extensive spectroscopic analysis including HRESIMS and 1D/2D NMR. The absolute configurations of new compounds were established by ECD data. Compound 1 exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated BV-2 microglial cells with IC50 value of 11.5 ± 1.7 µM.

[Research on in vivo metabolites of Longxue Tongluo Capsules by liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectrometry].

Longxue Tongluo Capsules(LTC) has good efficacy against blood stasis syndrome during the recovery period of ischemic stroke. Its main active ingredient is the phenolic extract of Chinese dragon's blood. In our previous study, the primary mass fragmentation pathways of phenolic derivatives from LTC were clarified. Herein, the metabolites in rat plasma were characterized following the oral administration of loureirin A and loureirin C using liquid chromatography coupled with hybrid ion trap/time-of-flight mass spectro-metry(LC-IT-TOF-MS), with 18 and 55 metabolites identified, respectively. On this basis, with the help of the obtained accurate molecular weight, characteristic fragment ions, reference comparison, combined with LTC database and natural products database self-created in our group, 18 prototypes and 106 metabolites were tentatively identified in rat plasma after oral gavage of LTC at a dose of 500 mg·kg~(-1). Glucuronidation, sulfonation, and methylation were major biotransformation pathways of LTC. This study preliminarily clarified the LTC constituents absorbed into blood and laid the foundation for clarifying the effective substances of LTC.