| In order to find new types of natural analgesic constituents,84 medicinal plants from west Yunnan were selected on purpose for analgesic activity screening based on literature research,traditional Chinese medicine theory,previous drug-using experience and modern clinical practice.Bauhinia brachycarpa Benth,which has the best analgesic activity was selected for the further studies,including isolation,identification,and analgesic activity evaluation of its active ingredients,as well as the interaction of main active monomers.Moreover,the antimalarial activities of 113 plants from west Yunnan were studied in this paper.Objective: To find new types of natural analgesic active ingredients and explore their relevant mechanism.To lay the foundation for the exploitmention and utilization of rich plant resources in Yunnan.Methods: A total of 84 species from 65 families and 80 genus collected in west Yunnan were selected for analgesic activity screening.After screening,Bauhinia brachycarpa Benth,which with best analgesic activity was selected for the further research by the activity tracing experiment.The pharmacological activity of the total extract,fractions,and active ingredients were studied.First,the analgesic activity of 84 plants was evaluated by the model of acetic acid writhing in mice.The evaluating index of the inhibition rate of writhing times(It was regarded as effective when the crude extract was 1000 mg/kg,the inhibition rate of acetic acid writhing reached or exceeded 50%).The plant with the best analgesic activity was selected for the activity tracking studies.The active components were separated and purified by chromatography,and the structures of the isolatedcompounds were identified by modern spectroscopic analysis and physicochemical properties analysis.Analgesic effects of the crude extract,the solvent fractions of the crude extract,the active parts and the main active monomer of the plant were evaluated by acetic acid writhing experiment,hot plate experiment and hot water tail flick test in mice.Finally,the monomer components with better analgesic activity were selected to evaluate their single or combined effects.Anti-malarial activities screening were performed with ?-hematin formation inhibitory experiment.Results:1.Analgesic activity screening of west Yunnan plants: Among the 84 tested plants,the samples from 16 plants which numbered as 0030 AE,0054BE,0064 AE,0097BE,0099 AE,0120BE,0129 BE,0152BE,0185 BE,0185CE,0204 A,0218BE,0238 AE,0283AE,0318 AE,0336BE and 0365 AE,revealed different analgesic activity.Their inhibition rates of writhing were 52.19%,54.63%,52.31%,68.85%,55.31%,61.62%,52.09%,53.85%,62.18%,57.21%,52.74%,49.38%,55.31%,62.48%,55.14%,54.17% and 51.02%,respectively.Compare with them,the inhibition rate of writhing of aspirin was 44.14%.Among these samples,Bauhinia brachycarpa Benth(BBB),whose number is 0097 BE,showed the best analgesic activity.2.Analgesic activity tracking of BBB.(1)The time-effect relationship of the analgesic effect of BBB crude extract.The analgesic activities of 1000 mg/kg BBB crude extract at 30,60,90 and 120 minute were studied by hot plate experiment in mice.It was demonstrated that BBB could significantly prolong the duration of licking feet at 60 minutes after intragastric administration.(2)The dose-effect relationship of BBB crude extract.The analgesic activities of BBB crude extract in different dosing were studied by using acetic acid writhing experiment in mice.The results showed that BBB crude extract could significantly reduce the writhing times,and the writhing inhibition rates of in dose of 200,500 and1000 mg/kg(i.g.)were 52.50%,55.31%,and 70.00%,respectively,which showed astrong does-dependent relationship.(3)Analgesic activity evaluation of BBB in different solvent extraction fractions.After BBB crude extract was suspended in water,the resulting solution was partitioned with petroleum ether,ethyl acetate,and n-butanol to acquire petroleum ether fraction(BBB-PEF),ethyl acetate fraction(BBB-AF),n-butanol fraction(BBB-BF),and water parts(BBB-WF),respectively.The analgesic activity of each fraction was evaluated by acetic acid writhing experiment,hot plate experiment and hot water tail flick test in mice.As a result,the writhing inhibition rate of different solvent fractions were as follows: BBB-AF(60.80%)> BBB-WF(43.82%)>BBB-BF(41.49%)>BBB-PEF(39.31%).Thus,BBB-AF activity was best.However,the BBB solvent fractions could not extend the duration of mice licking feet or flicking tail in mice.(4)Analgesic activity evaluation of different parts of BBB-AF.Different parts of BBB-AF were administrated in dose of 200 mg/kg(i.g.).As a result,AF-A2,AF-B,AF-C,AF-E,and AF-G could differently inhibit the writhing times in varying degrees on the acetic acid writhing experiment in mice,with writhing inhibition rates of58.33%,55.20%,48.55%,64.98%,and 59.11%,respectively.In the mice hot plate experiment and hot water tail flick experiment in mice,only AF-F and AF-H parts could extend the duration of licking feet in varying degrees,and other parts could not extend the duration about licking feet and flicking tail.(5)Analgesic activity evaluation of the major constituents in BBB-AF.Four compounds,?-sitosterol,daucosterol,betulinic acid,and isoquercetin were able to inhibit the writhing times in varying degrees on the acetic acid writhing experiment in mice,with writhing inhibition rates of 46.39%,16.39%,33.61%,and 48.09%,respectively.Isoquercetin and ?-sitosterol showed better analgesic activity.By the acetic acid writhing experiment in mice,the dose-effect relationship of isoquercetin and ?-sitosterol were further studied.As a result,isoquercetin in the dose of 50,80,130,220,and 336 mg/kg could dose-dependently reduce the writhing times,with 50%effective dose(ED50)value at 80.43 mg/kg(95% confidence interval: 65.49 to 98.77mg/kg).?-Sitosterol also in dose of 100,130,150,and 336 mg/kg coulddose-dependently reduce the writhing times,with 50% effective dose(ED50)value at65.15 mg/kg(95% confidence interval: 56.17 to 75.56 mg/kg).(6)Analgesic activity evaluation after combination of quercetin and ?-sitosterol.With the acetic acid writhing experiment in mice,when isoquercetin or ?-sitosterol was used alone,ED50 value was 68.25 mg/kg(95% confidence interval: 38.83 to116.90 mg/kg)and 59.63 mg/kg(95% confidence interval: 31.54 to 112.70 mg/kg),respectively.However,when the two constituents were used together,the combination index at ED50,ED75,and ED90 were 0.48,0.58,and 0.70,respectively,indicating that the combination of the two constituents had a strong synergistic analgesic effect.(7)Phytochemical constituents of BBB: By means of various chromatographic separation and purification techniques,as well as spectroscopic structure analysis and identification methods(including NMR,IR and MS),a total of 13 compounds were isolated from the analgesic activity of the BBB ethyl acetate fraction,and identified as?-sitosterol(1),lupeol(2),3,4,5-trimethoxyphenol O-?-D-glucopyranoside(3),betulinic acid(4),luteolin(5),quercetin(6),quercetin-3-O-glucoside(7),daucosterol(8),epicatechin(9),texifolin(10),aplgenin(11),eriodictyol(12),and kaempferol(13).3.In the work to study antimalarial activities,a total of 198 samples from 113 plants were screened,among which 25 samples from 24 plants displayed varied?-hematin formation inhibitory activity,with IC50 values less than 1388.9 ?g/mL.Conclusion:1.There were 16 plants in 84 representative plants collected from Western Yunnan with varying degrees of analgesic activity.2.The analgesic activity of BBB crude extract showed a strong time-effect and dose-effect relationships.3.After BBB crude extract was suspended in water and partitioned with different organic solvents,the resulting BBB-AF revealed the best analgesic activity.And the AF-A2,AF-B,AF-C,AF-E,and AF-G parts derived from BBB-AF were the parts with strong analgesic activity.4.Both quercetin and ?-sitosterol showed a strong dose-dependent analgesicactivity.When the two compounds were used in combination,the synergistic analgesic effect was observed.5.A total of 13 chemical constituents were isolated and identified from the analgesic active parts.The type of structure involved steroids,terpenoids,and flavonoids.And flavonoids were the major constituents.6.There were 24 test samples with different degrees of ?-hematin formation inhibitory activity,of which the IC50 values of the five test samples could be calculated.This study had pointed out the direction for further research and has certain academic significance in antimalarial drug development. |
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