Intervention effects and mechanism of Toddalia asiatica on cardiovascular damage in rats with collagen-induced arthritis / 中国药房

OBJECTIVE To investigate the intervention effects and potential mechanism of Miao medicine Toddalia asiatica on cardiovascular damage in rats with collagen-induced arthritis （CIA） based on vitamin D （VD） and neutrophil extracellular traps （NETs）. METHODS SD rats were randomly divided into the normal group （9 rats） and the modeling group （61 rats）. CIA model was prepared by multi-point injection of type Ⅱ bovine collagen+Fisher’s incomplete adjuvant； the model rats were randomly divided into the model group， methotrexate group （positive control， 1.5 mg/kg， twice a week）， vitamin D group [pathway validation， 1 000 IU/（kg·d）， once a day]， T. asiatica low-dose， medium-dose and high-dose groups [0.54， 1.08， 2.16 g/（kg·d）， calculated by crude drug， once a day]， with 9 rats in each group； they were given relevant medicine intragastrically for 4 consecutive weeks. Arthritis index scoring was performed after modeling and before administration， and plantar thickness was measured before and after the last administration； the histopathological changes of ankle joint， heart and abdominal aorta were observed in rats； the serum contents of myeloperoxidase （MPO）， interleukin-6 （IL-6） and 25-hydroxyvitamin D3 [25（OH）D3] were detected； the expressions of peptidylarginine deiminase 4 （PAD4）， NETs markers [citrullinated histone H3（CitH3）， MPO]， VD-related indicators [vitamin D receptor （VDR）， 25-hydroxyvitamin D-1α-hydroxylase （CYP27B1）] and IL-6 were determined in cardiac tissue. RESULTS Compared with the normal group， the plantar thickness of the arthritis index increased significantly in the model group （P＜0.01）. The obvious inflammatory cell infiltration and fibrous tissue hyperplasia were found in the ankle joint， the obvious myocardial fiber （No.2019YFC171250101） vacuoles and thickening of some surrounding blood vessel walls were found in the heart tissue， and the endothelial detachment was found in the abdominal aorta. The contents of MPO and IL-6 in serum increased significantly（P＜0.01），while the level of 25（OH）D3 decreased significantly （P＜0.01）； the protein expressions of PAD4， CitH3， MPO and IL-6 in myocardial tissue up-regulated significantly （P＜0.01）， while protein expression of VDR and CYP27B1 changed to acertain extent without significance （P＞0.05）. Compared with the model group， the pathological changes of ankle joints and cardiac tissue in rats were significantly improved in administration groups， and the above indicators were generally reversed （P＜0.05 or P＜0.01）. CONCLUSIONS T. asiatica can improve rheumatoid arthritis symptoms and cardiovascular damage by inhibiting the formation of NETs and inflammatory response， the mechanism of which may be associated with the regulation of VD expression.

Six new coumarins from the roots of Toddalia asiatica and their anti-inflammatory activities / 中国天然药物

We reported the discovery of six novel coumarins, toddasirins A-F (1-6), each endowed with modified isoprenyl or geranyl side chains, derived from the roots of Toddalia asiatica. Comprehensive structural elucidation was achieved through multispectroscopic analyses, single-crystal X-ray diffraction experiments, and advanced quantum mechanical electronic circular dichroism (ECD) calculations. Furthermore, the anti-inflammatory activity of these compounds was assessed. Notably, compounds 1-3 and 6 demonstrated notable inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells, with 50% inhibitory concentration (IC50) values of 3.22, 4.78, 8.90, and 4.31 μmol·L-1, respectively.

Alcohol extract of root and root bark of Toddalia asiatica alleviates CIA in rats through anti-inflammatory and proapoptotic effects / 中国中药杂志

This study aims to investigate the therapeutic effect of alcohol extract of root and root bark of Toddalia asiatica(TAAE) on collagen-induced arthritis(CIA) in rats through phosphatidylinoinosidine-3 kinase/protein kinase B(PI3K/Akt) signaling pathway. To be specific, CIA was induced in rats, and then the rats were treated(oral, daily) with TAAE and Tripterygium Glycoside Tablets(TGT), respectively. The swelling degree of the hind leg joints was scored weekly. After 35 days of administration, the histopathological changes were observed based on hematoxylin and eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the levels of cytokines [tumor necrosis factor-α(TNF-α), interleukin(IL)-6)]. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining was performed to detect the apoptosis of synoviocytes in rats. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma 2(Bcl-2)-associated X(Bax), Bcl-2, and caspase-3 and pathway-related proteins phosphoinositide 3-kinase(PI3K), phosphorylated(p)-PI3K, protein kinase B(Akt), and p-Akt. RT-qPCR was conducted to examine the mRNA levels of Bax, Bcl-2, caspase-3, TNF-α, IL-6, and IL-1β and pathway-related proteins PI3K, p-PI3K, Akt, and p-Akt. TAAE can alleviate the joint swelling in CIA rats, reduce serum levels of inflammatory cytokines, improve synovial histopathological changes, promote apoptosis of synoviocytes, and inhibit synovial inflammation. In addition, RT-qPCR and Western blot results showed that TAAE up-regulated the level of Bax, down-regulated the level of Bcl-2, and activated caspase-3 to promote apoptosis in synoviocytes. TAAE effectively down-regulated the protein levels of p-PI3K and p-Akt. In this study, TAAE shows therapeutic effect on CIA in rats and reduces the inflammation. The mechanism is that it suppresses PI3K/Akt signaling pathway and promotes synoviocyte apoptosis. Overall, this study provides a new clue for the research on the anti-inflammatory mechanism of TAAE and lays a theoretical basis for the better clinical application of TAAE in the treatment of inflammatory and autoimmune diseases.

Effect of Toddalia asiatica Alcohol Extract on Apoptosis of Non-small Cell Lung Cancer A549 Cells Based on Autophagy Pathway / 中国实验方剂学杂志

ObjectiveTo study the effects of Toddalia asiatica alcohol extract on autophagy and apoptosis of non-small cell lung cancer A549 cells， and to explore its possible mechanism. MethodA549 cells were cultured in vitro. Cell counting kit-8 （CCK-8） was used to detect the proliferation of A549 cells， and cell survival rate was calculated to screen the drug concentration. The apoptosis in each dose group and that after the use of 3-methyladenine （3-MA）， an autophagy inhibitor， were detected by flow cytometry combined with Annexin V-FITC/PI double staining. Western blot was used to detect the expression levels of apoptosis-related proteins such as B cell lymphocytoma-2（Bcl-2）， Bcl-2-associated X protein（Bax）， microtubule-associated protein 1 light chain 3 （LC3）， cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）， activated poly （Adenosine diphosphate） ribonucleotide polymerase （cleaved PARP1）， PARP1， activated death activator （t-Bid）， Bid， and ubiquitin-binding protein p62 in each group and those after the use of 3-MA. ResultCompared with the conditions in the control group， the cell survival rate in 0.25 g·L-1 group （P<0.05）， and 0.5， 1， 2， 4 g·L-1 groups （P<0.01） was decreased after 24 h intervention. Additionally， the cell survival rate was reduced in a concentration-dependent manner at 48 h and it was less than 10% at 4 g·L-1 （P<0.01）. Compared with the conditions in the control group， the total apoptosis rate in 0.5 g·L-1 group was increased （P<0.05）， and the apoptosis rate in 1 and 2 g·L-1 groups was also increased （P<0.01）. Compared with the 2 g·L-1 group and 3-MA group， the 3-MA combined with T. asiatica alcohol extract had significantly decreased apoptosis rate （P<0.01）. Compared with the conditions in the control group， elevated expression of pro-apoptotic proteins cleaved PARP1， Bax and t-Bid in 1 and 2 g·L-1 groups （P<0.05， P<0.01）， and reduced expression of Bid in the 2 g·L-1 group （P<0.01） were found. Compared with the conditions in the control group， the expression of anti-apoptotic protein Bcl-2 （P<0.05， P<0.01） and the level of p62 （P<0.01） were down-regulated in 0.5， 1， 2 g·L-1 groups， while the level of LC3 Ⅱ protein was up-regulated （P<0.01）， with certain concentration dependence. ConclusionT. asiatica alcohol extract could significantly inhibit the proliferation of A549 cells， which might be related to promoting autophagy and inducing apoptosis.

The effect of processing methods on the content of toddaline in Toddalia asiatica Lam / 国际药学研究杂志

Objective:To explore the effect of processing methods on the content of toddaline in Toddalia asiati- ca Lam., so as to provide reference for the development and clinical application of T. asiatica in the future. Methods: The root of T. asiatica was processed by the processing methods of water-washing, water-boiling, water-steaming, winesteaming and stir-frying with wine. The content of toddaline in the processed products of T. asiatica was determined by high performance liquid chromatography(HPLC). Further, the orthogonal test design methodology was used for the stirfrying with wine process to investigate the effect of frying fire power, time and solid-liquid ratio(the ratio of raw material to yellow rice wine)on the toddaline content in T. asiatica. Results: The processing of the T. asiatica roots by the pro- cessing methods of water-washing, water-boiling, water-steaming and stir-frying with wine could all reduce the toddaline content in T. asiatica roots and the reducing effect was especially significant in the stir-frying with wine process. In con- trast, the steaming with wine(the wine-steaming)could increase the toddaline content in T. asiatica. The results of the orthogonal test for the stir-frying with wine process showed that the processing time most greatly influenced the toddaline content, the influence on the content came next from the solid-liquid ratio, and the influence from the frying fire power was lowest. The toddaline content in T. asiatica roots was increased by processing with Wen Huo (the weak frying fire power), with processing time 10 min and solid-liquid ratio 1:1. In contrast, the toddaline content was most significantly reduced when the roots were processed with Zhong Huo(the medium frying fire power), with processing time 30 min and solid-liquid ratio 1:1. Conclusion: The effect of different processing methods on the content of toddaline in T. asiatica was differed in the tested five processing methods. For the stir-frying with wine process, the change of processing parame- ters also caused the different effect on the toddaline content in in T. asiatica.

Chemical Constituents from n-Butanol Part in Toddalia asiatica Root Bark / 中国药学杂志

OBJECTIVE: To study the chemical constituents from n-butanol part in Toddalia asiatica root bark. METHODS: A variety of chromatographic techniques were applied to extract, isolate and purify natural products from extract of Toddalia asiatica root bark, their structures were identified on the basis of spectral data. RESULTS: Thirteen compounds were isolated from n-butanol part in Toddalia asiatica root bark, there were seven coumarin compounds, including six furocoumarin compounds, identified as pimpinellin (1), bergapten (2), alloimperatorin (3), isopimpinellin (4), moellendorffiline (5), phellopterin (6), toddalolactone (7); four organic acid ester compounds identified as mono (2-ethylhexyl) terephthalate (8), ferulic acid methyl ester (9), 4-methoxycinnamic acid (10), methyl trans-4-hydroxycinnamate (11); one alkaloid identified as nitidine (12) and one flavonoid compound which was hesperidin (13). CONCLUSION: The compound 8 is isolated from the Rutaceae family for the first time, compound 3, 5, 9, 10, 11 are isolated from this plant for the first time.

Anti-inflammatory and analgesic activities of ethanol extract of Toddalia asiatica / 中成药

AIM To study the anti-inflammatory and analgesic activities of ethanol extract of Toddalia asiatica Lamn.and the mechanism.METHODS Both inflammatory rat model induced by carrageenan and pain model induced by formalin were applied to investigating the analgesic effect of extract of Toddalia asiatica Lam.ELISA kit was used to detect the contents of β-EP,5-HT and PGE2 in serum of carrageenan-treated rats,contents of TNF-αand IL-1β in skin tissue of inflammatory rats,and content of LTB4 in serum of formalin-treated rats;immunohistochemical method was used to observe the SP and FOS protein expressions in rat spinal cord.RESULTS The ethanol extract of Toddalia asiatica Lam.could significantly reduce the rate of toe swelling.In the formalin test,the ethanol extract of Toddalia asiatica Lam.reduced not only the total licking time,but also the content of PGE2,especially in the high dose group.And lowered serum 5-HT contents were observed in all the three dose groups,but a much better performance was found in both the high and low dose groups,and the high dose group's capability in increasing serum β-EP content was also noticed.TNF-α and IL-1β contents in skin tissue were reduced in various dose groups.Middle and high dose groups inhibited FOS protein expression.And the content of LTB4 in serum was obviously decreased in the high dose group.CONCLUSION The anti-inflammatory and analgesic activities of ethanol extract of Toddalia asiatica Lam.may associate with its power in increasing β-EP in serum,decreasing PGE2,5-HT,LTB4 contents,reducing TNF-α,IL-1β contents in skin tissue,and lowering SP and FOS protein expressions in spinal cord.

Effects of Toddalia asiatica extract on inflammatory cytokines in rats with myocardial ischemia and hyperlipidemia / 中国比较医学杂志

Objective To explore the effects of Toddalia asiatica extract(TAE)on inflammatory cytokines in rats with myocardial ischemia and hyperlipidemia. Methods The early changes of hyperlipidemia were caused by feeding high fat diet for 4 weeks. Fifty male SD rats were randomly divided into 5 groups:the control group,model group,simvastatin group(3.5 mg/kg),high-dose TAE group(280 mg/kg)and low-dose TAE group(70 mg/kg). At the end of the 4-weeks treatment,isoprenaline hydrochloride(Iso)was subcutaneous injected once a day for consecutive three days. The electrocardiogram(EKG),the heart,liver and fat indexes,the serum levels of TC,TG,HDL,LDL,TNF-α,INF-γ,IL-6 and IL-10, and the pathological changes of myocardium were examined. Results TAE significantly improved the Iso-induced EKG changes in rats,reduced the heart,liver and fat indexes,and decreased the levels of TC,TG,LDL,TNF-α,INF-γ and IL-6 and increased the contents of HDL and IL-10(P< 0.05 for all),and improved the pathological damages of myocardium. Conclusions The Miao nationality medicine Toddalia asiatica extract may play an important protective role in cardiovascular diseases by regulating the balance of anti-inflammatory and proinflammatory cytokines.

Protective effects of Toddalia asiatica extract on oxidative damages in mice with myocardial ischemia and hyperlipidemia / 中国比较医学杂志

Objective To explore the effects of Toddalia asiatica extract( TAE) on oxidative damages in mice with myocardial ischemia and hyperlipidemia. Methods Fifty SPF male KM mice were randomly divided into 5 groups:control group,model group,simvastatin group (5 mg/kg),high-dose TAE group (400 mg/kg) and low-dose TAE group (100 mg/kg) . Hyperlipidemia was induced by feeding milk for 4 weeks. At the same time,the corresponding drug was given by oral administration with 20 mL/kg for 28 d. At the end of four-week treatment, isoprenaline hydrochloride ( Iso ) was subcutaneously injected every 24 hours for three times. The body weight, electrocardiogram, heart and liver indexes, myocardial water content, and the serum levels of TC, TG, HDL, LDL, SOD, MDA and GPx were measured. Results TAE significantly improved the electrocardiogram changes induced by Iso and milk in the mice, weight lose, inhibited the heart and liver indexes, reduced the myocardial water content, decreased the levels of TC, TG, LDL and MDA, and increased the contents of HDL, SOD and GPx. Conclusions TAE may play an important role in cardiovascular protection by regulating oxygen free radical metabolism and improving oxidative damages.

Analysis on fingerprint for root barks and stem barks of Toddalia asiatica / 中草药

Objective: To promote the comprehensive utilization in the root barks and stem barks of Toddalia asiatica and to establish an HPLC method for the determination of the fingerprints for the root barks and stem barks of T. asiatica. Meanwhile, the similarity was compared by similarity evaluation software based on the fingerprints. Methods: HPLC analysis was performed on C18 (250 mm × 4.6 mm, 5 μm) with acetonitrile and 0.1% aqueous solution of phosphoric acid as mobile phase in gradient mode at the wavelength of 269 nm, and the flow rate was 1 mL/min. The similarity was calculated by comparison on the fingerprints derived from 10 bathes of T. asiatica root barks and 10 bathes of T. asiatica stem barks, all the samples were collected from the different areas in different seasons. Results: The mutualmode of HPLC fingerprints was set up and the similar degrees to the crude drugs of different sources were compared. The content ratio of active ingredients is different from samples in different areas and seasons. Conclusion: The fingerprint similarity is greater than 0.9 between T. asiatica root barks and T. asiatica stem barks, so the compositions are highly similar.

Chemical constituents in Toddalia asiatica Lam.: Research advances / 国际药学研究杂志

Radix Toddaliae asiaticae, belonging to Rutaceae family and Toddalia genus, is a folk traditional medicine in China. Chemical constituents isolated from this plant include coumarins, alkaloids, triterpenes, flavonoids, phenolic acids, lignanoids, steroids and fatty acid, etc. Quantitative analyses focus on total lactones, total flavonoids, total alkaloids and isolated compounds of coumarins and alkaloids. In this paper, chemical constituents and quantitative analyses are reviewed, which can provide references for further research.

Pharamacological action in Toddalia asiatica Lam.: Research advances / 国际药学研究杂志

Radix Toddaliae asiaticae which belongs to Toddalia genus of Rutaceae family is a folk medicine in China. Modern pharmacological studies have shown its anti-inflammatory, analgesic, antioxidative, antibacterial, antifungal, cardiovascular prot tive, antitumor activities and so on. The review systematically summarizes pharmacological effects, which can provide references reasonable utilization.

Effects of Alcohol Extract of Toddalia asiatica on the Inflammation-associated Cytokines of Model Rats with Adjuvant Arthritis / 中国药房

OBJECTIVE:To study the effect of alcohol extract of Toddalia asiatica on the inflammation-associated cytokines of model rats with adjuvant arthritis(AA). METHODS:70 SD rats were randomly divided into a normal control group,a model con-trol group,a positive control chemical medicine group(Leflunomide tablets,0.012 g/kg),a positive control TCM group(Tripter-ygium glycosides tablets,0.012 g/kg)and the groups of low,medium and high-dose [1,4,6 g(crude drug)/kg] alcohol extract of T. asiatica,with 10 rats in each group. The rats in all groups except for the normal control group were given complete Freund’s complete adjuvant id for the establishment of AA models. At the same time,the rats in the drug administration groups were given corresponding drugs ig,while those in the normal control group and the model control group were given isometric normal saline ig,twice a day,for 28 consecutive days. The degree of toe swelling,arthritis index and the levels of interleukin(IL)-1β,IL-6, IL-10,tumor necrosis factor α(TNF-α)and prostaglandin E2(PGE2)in serum and synovial membranes of all groups of rats were determined. RESULTS:Compared to the normal control group,the model control group demonstrated higher degree of primary and secondary toe swelling,arthritis index and levels of IL-1β,IL-6,TNF-αand PGE2 in serum and joint synovial membrane,and low-er level of IL-10 therein(P<0.01). Compared to the model control group,all the above-mentioned indexes of the rats in drug ad-ministration groups significantly improved(P<0.05 or P<0.01). CONCLUSIONS:The alcohol extract of T. asiatica. has a preven-tive and therapeutic effect on the model rats with AA by regulating the expression of anti-inflammatory and proinflammatory cyto-kines in serum and synovial membrane.

Pharamacological action in Toddalia asiatica Lam.:research advances / 国际药学研究杂志

Radix Toddaliae asiaticae which belongs to Toddalia genus of Rutaceae family is a folk medicine in China. Modern pharmacological studies have shown its anti-inflammatory,analgesic,antioxidative,antibacterial,antifungal,cardiovascular protec?tive,antitumor activities and so on. The review systematically summarizes pharmacological effects,which can provide references for reasonable utilization.

Simultaneous determination of four effective components in Toddalia asiafica(L.)Lam by RP-HPLC / 中国药学杂志

OBJECTIVE: To develop a RP-HPLC method for simultaneous determination of hesperidin, nitidine chloride, ehelerythrine, and toddalolactone in Toddalia asiatica(L)Lam. Methods: The RP-HPLC system consisted of a Diamonsil C18 column (4.6 mm×250 mm, 5 μm) with the mobile phase of acetonitrile-water solution containing 75% glacial acetic acid. UV detector was used, and the detection wave length was 290 nm. Gradient elution was adopted at a flow rate of 1.0 mL · min-1 and the column temperature was 30°C. For different constituents, external standard method was used with the peak area at 290 nm as the quantitative index. RESULTS: The liner ranges of hesperidin, nitidine chloride, ehelerythrine and toddalolactone were 0.4874-2.4372 μg(r= 0.9997), 0.0303-0.1515 μg(r=0.9992), 0.0623-0.3117 μg(r=0.9993), and 0.0249-0.1246 μg(r=0.9999), respectively. The average recoveries(n=6) were 100.76%(RSD 2.75%), 97.98%(RSD 1.25%), 100.07%(RSD 3.24%), and 100.10% (RSD 3.83%), respectively. CONCLUSION: The method is accurate, simple, rapid, and reproducible for the determination of hesperidin, nitidine chloride, ehelerythrine, and toddalolactone in Toddalia asiatica(L)Lam. The determination results can be used as a reference for the rational medication, quality control, and further study of Toddalia asiatica(L.)Lam.

Chemical Constituents in Methanol Parts of Toddalia Asiatica(Linn) Lam / 中国药师

Objective:To study the chemical constituents in methanol parts of Toddalia asiatica ( Linn) Lam. Methods: The compounds were isolated by silica gel, Sephadex LH-20 column chromatograph and TLC. The structures were elucidated by physico-chemical properties and spectral analysis. Results:Nine compounds were obtained and elucidated as dictamnine(1), hesperetin(2), 4-hydroxy-N-methylproline(3), 8-hydroxy-dihydrochelerythrine(4), hesperetin7-O-β-D-glucopyranoside(5), daucosterol(6), hes-peridin(7), diosmin(8) and neohesperidin(9). Conclusion:Compound 5, 6, 8 and 9 are isolated from the plant for the first time.

Effect of Toddalia Asiatica on Plasma Recalcification Time in Rabbits / 中国药师

Objective:To preliminarily study on the procoagulant activity of Toddalia asiatica Lam. Methods:The plasma recal-cification time affected by the water decoction extract, ultrasonic extract and organic solvent extract from T. asiatica were determined to observe the effects of different extracts from T. asiatica on plasma recalcification time of rabbits. Results:The ultrasonic extract of T. asiatica could reduce the plasma recalcification time (P<0. 05), the ethyl acetate and methanol extract could significantly reduce the plasma recalcification time (P <0. 01), while the water decoction extract prolonged the plasma recalcification time (0.01

Analysis of chemical composition and evaluation of antigenotoxic, cytotoxic and antioxidant activities of essential oil of Toddalia asiatica (L.) Lam

Objective: To analyse the chemical composition of essential oil isolated from the leaves ofToddalia asiatica (L.) Lam. and to test its bioactive properties. Methods: Gas chromatography-mass spectrometry (GC/MS) analysis and antigenotoxicity, cytotoxicity and antioxidant studies of isolated oil. Results: GC-MS analysis of oil revealed the presence of 42 compounds. The major compound in the oil was β-phellandrene (21.35%). DNA protecting activity of oil was considerably significant against H2O2 induced genotoxicity in human lymphocytes. Concentration of oil needed to protect 50% of DNA was calculated as 100 μg/mL. Cytotoxicity of oil against breast (MCF-7) and colorectal (HT-29) cancer cells were observed with the IC50 value of (7.80±0.03) μg/mL and (100.00±0.16) μg/mL respectively. Considerable DPPH free radical, hydroxyl radical scavenging, iron chelation and inhibition of lipid peroxidation activities of oil were also studied. Conclusions: The results of the present study clearly indicate oil could be a promising candidate for food and drug preparation.