Research On Synthesis Of Catecholic Tetrahydroisoquinolines From Portulaca Oleracea And Their Anti-asthmatic And Anti-inflammatory Effects

Portulaca oleracea L.is an annual succulent herb of Portulacaceae.The aerial part of P.oleracea is used as an edible vegetable and also a traditional Chinese medicine.It has many kinds of functions such as heat-clearing,detoxifying,dispelling heat from blood to stop bleeding.Modern pharmacological studies have shown that P.oleracea exhibits a variety of activities,including antibacterial,anti-inflammatory,analgesic,sedative,anti-asthmatic,skeletal muscle relaxation,hypoglycemic,hypolipidemic,anti-oxidatant,anti-aging,and neuroprotective effects.Bencao Jingshu states that P.oleracea "can dissipate the heat in lungs",and the formulations based on P.oleracea as the principle drug combined with other drugs possessing heat-phlegm removing efficacy can treat lung abscess(lung carbuncle),pneumonia,acute and chronic bronchitis,purulent bronchitis,pediatric pertussis,pulmonary tuberculosis cough,chronic cough and other diseases of pulmonary system which belong to phlegm-heat-obstructing-lung induced cough and asthma.Traditional Iranian medicine records that P.oleracea can treat asthma and other respiratory diseases.Moreover,clinical trials and animal experiments have proved the anti-asthmatic effect of P.oleracea.These findings suggest that P.oleracea has the basis of anti-asthma clinical application and the potential for the discovery of novel anti-asthma drugs.At present,the research on the anti-asthmatic effect of P.oleracea is only limited to its water extract,and its anti-asthmatic ingredients as ?2-AR agonist remain unclear and need to be intensively discovered.Section 1:Synthesis and purification of catecholic tetrahydroisoquinolines(THIQs)from P.oleracea and their derivativesIn the previous study,a series of novel water-soluble catecholamine derivatives including THIQs that exhibited different degree ?2-AR agonist and anti-inflammatory activities were discovered from P.oleracea,which were hypothesized to be the anti-asthmatic ingredients in this plant.However,the low contents of the isolation of these compounds from natural resources limited their pharmacological study.To provide large amounts of compounds for further pharmacological research,the chemical synthesis of catecholic THIQs is necessary.Starting from dopamine hydrochloride and aldehydes as the substrates,three catecholic THIQs(1,2,12)present in P.oleracea,together with their analogues were biomimetic synthesized by phosphate-mediated Pictet-Spengler reaction.Among them,14 was a new alkaloid,except 1,3,4,5,8,12,13,seven other alkaloids were synthesized through phosphate-mediated Pictet-Spengler reaction for the first time.Owing to the limitation of the existing purification protocols,separation of water-soluble catecholic THIQs products from the water-soluble reaction system(containing phosphate buffer and vitamin C)and unreacted dopamine substrate became a great challenge.AB-8 macroporous resin column chromatography was firstly applied in this study,and after elution with H2O as the first step,water soluble constituents such as phosphate,and vitamin C can be quickly removed,and after elution with EtOH as the second step,the target catecholic THIQs can be completely separated from dopamine,thereby yielding purified product.AB-8 macroporous resin column chromatography provided a simple,green,economical,and general method for the high efficacy separation and purification of water-soluble catecholic THIQs from phosphate-mediated Pictet-Spengler reaction system.Section 2.Research on anti-asthmatic effects of catecholic THIQs from P.olerarceaIn this study,the effects of 100 ?M of 14 synthesized catecholic THIQs and seven compounds isolated from P.oleracea on ?2-AR and their selectivity on ?1-and?1B-AR were systematically investigated,as assayed by calcium determination in CHO-K1/G?15 cell lines that stably express ?1-,?2-,or ?1B-AR,with isoproterenol and epinephrine as the positive control.Meanwhile,their structure-activity relationships were discussed.It was found for the first time that 12 possessed the potent ?2-AR agonist activity,however,it also displayed better agonist effect on?1-AR subtype;2 and OE were ?2-AR selective agonists and they possessed the weak?1B-,?1-AR agonist activities.It was further demonstrated that all of the ?2-AR agonists 12,2 and OE dose-dependently inhibited histamine-induced isolated guinea-pig tracheal contraction,with EC 50 values of spasmolysis being 0.8,2.8 and 7.0 ?M,respectively.Moreover,their bronchodilatory effects at the lower concentration can be competitively antagonized by ?-AR antagonist propranolol hydrochloride(Pro).When these three compounds mixed together with the molar ratio of 1:1:1,they can synergistically exert antispasmodic effect at the lower concentration(0.01 ?M-1 ?M),stronger than that of a single compound,however,competitive inhibition among three compounds may occur at the higher concentration(>1 ?M).These results suggested that the anti-asthmatic activity of P.oleracea may be attributed to the synergistic effect of catecholic isoquinoline alkaloids.Using histamine-induced acute asthma model of guinea-pigs,it was further found that 12 dose-dependently prolonged the asthmatic latency in guinea pigs at 10,20,and 40 mg/Kg(p<0.05,p<0.01,p<0.01),and 12 also dose-dependently decreased the tumble number of guinea pigs.2 dose-dependently enhanced the asthmatic latency in guinea pigs only at the higher concentration of 40 and 80 mg/Kg(p<0.01),and OE dose-dependently prolonged the asthmatic latency in guinea pigs only at the higher concentration of 80 and 160 mg/Kg(p<0.05,p<0.01).2 and OE had little or no effect on the tumble number of guinea pigs.These results demonstrated that catecholic THIQs from P.oleracea possessed the in vivo anti-spasmodic effects with the sequence of 12>2>OE,similar with the bronchodilator effects on the histamine-induced contraction of isolated guinea pig tracheal smooth muscle.Using ovalbumin(OVA)-induced chronic asthma mice model,the effect of the most potent ?2-AR agonist 12 on airway inflammation was further evaluated.The results showed a dose-dependent decreasing tendency of 12 on the number of eosinophils in the blood,however,this difference didn't reach to the significant level as compared with the OVA model group(p>0.05).12 at only 80 mg/Kg alleviated airway inflammation,as indicated by significant reduction in the levels of IL-1?,IL-5 and IL-13 in BALF(p<0.05 or p<0.01),with the decrease being 34.64%,23.40%and 37.63%,respectively.However,it had no significant influence on the level of TNF-? in BALF(p>0.05).No anti-inflammatory effect was observed on 12 at low and medium dosage of 5 and 20 mg/Kg(p>0.05).Section 3.Research on anti-inflammatory effects of catecholic THIQs 12 and 2 from P.oleracea on LPS-induced sepsis mice modelUsing LPS-induced sepsis mice model,the anti-inflammatory effects of catecholic THIQs 12 and 2 were further evaluated,through measuring inflammatory cytokines NO,IL-6,TNF-? levels in plasma and IL-6,TNF-? levels in BALF.The results indicated that,single intraperitoneal injection of LPS(5 mg/Kg)caused hypersecretion in the eyelids,spirit droop and movement reduction,in comparison with the control group.In two individual experiments,LPS significantly increased plasma NO(p<0.05,p<0.0001),TNF-?(p<0.0001)and IL-6(p<0.0001)levels,as well as TNF-?(p<0.01)and IL-6(p<0.01,p<0.05)levels in BALF.No significant difference was observed in the excessive release of plasma NO for each group of 12 as compared with sepsis mice model(p>0.05).12 at only high dosage of 80 mg/Kg significantly decreased TNF-?(p<0.01,p<0.05)and IL-6(p<0.01,p<0.05)levels in plasma and BALF of sepsis mice,with the decrease of 65.16%,56.54%,73.53%and 54.75%,respectively,however,low and medium dosages of 12 had no anti-inflammatory effects(p>0.05).Low and medium dosages of 2 had no significant effect on the TNF-? and IL-6 levels in the plasma and BALF of sepsis mice(p>0.05),and only high dosage of 2(80 mg/Kg)significantly reduced the levels of TNF-?(p<0.01,p<0.05)and IL-6(p<0.05,p<0.05)in the plasma and BALF,with the decrease of 48.57%,54.62%,46.65%and 62.09%,respectively.The above results indicated that 12 and 2 at 80 mg/Kg had certain anti-inflammatory effects on LPS-induced sepsis mice model.