唐古特紅景天舒張肺小動脈的有效部位及機制的初步研究*

李更兄，蓋祥云，李占強，常 榮，祁玉娟，扎西東主，靳國恩，聶生斌，吳 萍**，蘆殿香***

(1.青海大學(xué)醫(yī)學(xué)院；2.青海民族大學(xué)藥學(xué)院；3.青海省人民醫(yī)院)

唐古特紅景天舒張肺小動脈的有效部位及機制的初步研究*

李更兄1，蓋祥云2，李占強1，常 榮3，祁玉娟3，扎西東主1，靳國恩1，聶生斌1，吳 萍1**，蘆殿香1***

(1.青海大學(xué)醫(yī)學(xué)院；2.青海民族大學(xué)藥學(xué)院；3.青海省人民醫(yī)院)

目的 研究唐古特紅景天舒張大鼠離體肺小動脈的有效部位及機制。方法 制備唐古特紅景天95%、70%醇提物及水提物，利用HPLC-DAD分析提取物的含量特征。測定不同提取物中總黃酮、總酚酸含量。采用大鼠肺小動脈血管環(huán)離體灌流技術(shù)，確定藥物舒血管作用的有效部位。利用去甲腎上腺素(NE)及氯化鉀(KCl)預(yù)收縮血管環(huán)，初步分析有效部位舒血管作用的機制。結(jié)果 藥物水提物中總黃酮及總酚酸的含量均為最高。水提物中具有末端吸收化合物的種類及含量較醇提物中的少，醇提物中僅化合物的含量差異較大。唐古特紅景天舒張肺小動脈的有效部位主要富集于95%醇提物。藥物有效部位可以濃度依賴性地舒張肺小動脈(0.76～7.6mg/mL)，其舒血管作用與抑制細胞膜受體門控性鈣通道的機制有關(guān)。結(jié)論 唐古特紅景天95%醇提物是其舒張肺小動脈的主要有效部位。有效部位可能通過抑制細胞膜受體門控性鈣通道，減少Ca2+流入血管平滑肌細胞而發(fā)揮舒張血管的作用。

唐古特紅景天 提取物 部位 血管舒張 機制

Introduction

Pulmonary arterial hypertension(PAH)is a group of clinical conditions presenting with abnormal elevation in the pulmonary circulation pressure and characterized by pulmonary arterial remodeling，leading to increased pulmonary vascular resistance and increased pulmonary arterial pressure[1].PAH is a debilitating disease and results ultimately in right ventricular failure and death. It is reported that PAH appears approximately in 12～50 per million in adult people[2].

Hypoxic pulmonary arterial hypertension(HPAH)is one of PAH occurred at high altitude and are associated with significant morbidity and mortality.Chronic exposure to high altitude induced pulmonary vasoconstriction，lead to medial hypertrophy and pulmonary arterial hypertension，which have been reported that HPAH appears about 5%～10% in people who lived at high altitude[3].Individuals living at high altitude，either temporarily or permanently，may present mild to moderate pulmonary hypertension，and those with greater susceptibility to hypoxia may have exaggerated pulmonary vasoconstriction，leading to HPAH[4].However，so far，no significant benefit from the use of targeted PAH therapies has been demonstrated in treatment of HPAH[5].

Rhodiola rosea is one of medicinal plants having stimulating and adaptogenic properties.RhodiolaalgidaVar.tangutica(Crassulaceae)has been used for a very long time in Qinghai and Tibetan folk medicine.In our previous research，we investigated the pharmacological role of 95% ethanol extract ofRhodiolaalgidavar.tanguticaon the reversal of HPAH and the related molecular mechanism.In our experiment，Sprague Dawley rats were kept in a hypobaric chamber which simulated the environment of a 4，500 m altitude except control animals.The rats were administrated 95% of ethanol extract ofRhodiolaalgidavar.tanguticafor 30 days.We found that the herb attenuated HPAH and reduced remodeling of pulmonary small artery in HPAH rats[6].However，to the best of our knowledge，there has not been a study investigating its active part ofRhodiolaalgidavar.tanguticaand effects on the vascular tone in pulmonary small arteries.

The aims of this report are as follows：(1)to find the active part inRhodiolaalgidavar.Tangutica.(2)to explore the effect of active part on inducing vasorelaxation in rat pulmonary arteriesinvitroand its mechanism.

Materials and methods

NE(H12020621)was purchased from Jin Yao amino acid co.，LTD(Tianjin，China)，acetylcholine(ACh，≥99%)was purchased from Sigma Chemical Co(St Louis，MO，USA)，rutin，with a purity of more than 98%，was purchased from Melone Pharmaceutical Co.，Ltd(Dalian，China)，gallic acid，with a purity of more than 98%，was purchased from Melone Pharmaceutical Co.，Ltd(Dalian，China)，F(xiàn)oline-Phenol was purchased from Solarbio Science & Technology Co，Ltd (Beijing，China)，methanol(HPLC grade)was purchased from Yu Wang Industrial Co.，Ltd(Shandong，China).All other reagents from local sources were of analytical grade.

Rotary evaporator(RE-52，Ya Rong biochemical instrument factory，Shang Hai，China)，lyophilizer(Alpha 1-2，Christi company in German)，force transducer(JH-2，Space Medico-Engineering Institute，Beijing，China)，biological function experiment system(BL-420，Tai Meng Technology Co.，Ltd，Chengdu，China)，invitrotissue and organ perfusion system(HV-4，Tai Meng Technology Co.，Ltd，Chengdu，China)，ultraviolet spectrophotometer(DU800)，HPLC-DAD(1260，Agilent Co)，chromatographic column(ZORBAX SB-C18，4.6×250mm，5μm，Agilent Co).

Extract preparation ofRhodiolaalgidavar.tangutica

Rhodiolaalgidavar.tanguticawas provided by traditional Tibetan hospital in Zhiduo county，Qinghai province and authenticated by Prof.Dejun Zhang(College of Eco-Environmental Engineering in Qinghai University).

Herb dry roots ofRhodiolaalgidavar.tangutica(50.0g)were extracted three times，each time for one hour，with 95%，70% of ethanol and deionized water at room temperature.The extracting solution was collected，revolved evaporation and lyophilized.The dry powder was used to study their chemical constituents.

Analysis of HPLC

2.0 mg/mL solutions of three kinds of extracts were prepared and 10 μL of each of them were used for HPLC-DAD analysis.Chromatographic condition was as follows：mobile phase：0～5 min 15% methanol，5～36 min 15% to 70% methanol；detection wave length：210 nm；column temperature：35 ℃；flow rate：1 mL/min.

Detection of total flavonoids content

Total flavonoids content was determined by a colorimetric assay.The properly diluted sample(1.0mL)was mixed with deionized water(4.0mL)in a 10.0 mL volumetric flask，and NaNO2(0.3mL，5.0%，w/v)was added.After 5 min，AlCl3(0.3mL，10%，w/v)was added.Then，after 6 min，NaOH(2.0mL，1.0M)was added to the mixture，and followed by the addition of deionized water.The solution was mixed and shaken vigorously，and the absorbance at 510 nm was measured against a blank.A standard solution of rutin was used to prepare a calibration curve(regression equation：y=1.1199x-0.058，R2=0.990).The results were expressed as mg rutin/g of dry extract.

Detection of total phenolic acid content

The total phenolic acid content was determined according to the Folin-Ciocalteau method.Properly diluted sample(100μL)was mixed with Folin-Ciocalteau reagent(4.5mL)which was pre-diluted 10 times with deionized water.After standing for 5 min at room temperature，NaCO3(3.0mL，7.5%，w/v)was added，and this mixture solution was incubated at room temperature for 60 min.The absorbance was measured at 765 nm.A standard solution of gallic acid was used to prepare a calibration curve(regression equation：y=0.7665x+0.0115，R2=0.994).The total phenolic content was calculated and expressed as mg gallic acid/g of dry extract.

Experimental animals

All procedures and protocols were approved by the Animal Care and Use Committee of the Medical College of Qinghai University.Male SD rats，6～8 weeks old，250～300 g body weight，were purchased from the Animal Center of Xi′an Jiaotong University(Shanxi，China)and maintained on a standard laboratory diet and tap wateradlibitumat an ambient temperature of(22±2)℃ and a relative humidity of 45%～55% throughout the experiments.

Invitropulmonary artery perfusion

Rats weighing 250～300 g were anesthetized by intraperitoneal injection of urethane(1.5g/kg，i.p.)，and then their hearts and lungs were removed and immersed immediately in ice-cold KH solution containing(in mmol/L)：118 NaCl，4.7 KCl， 2.5 CaCl2，1.2 MgSO4·7H2O，1.2 KH2PO4，25 NaHCO3，and 11.1 glucose(pH 7.4).The intrapulmonary arteries(0.7～1.5mm in diameter)were dissected free of surrounding connective tissue and adventitia and then cut into rings of approximately 2～3 mm in length.The rings were suspended in organ chambers filled with 10 mL of KH solution at 37 ℃ and gassed with 95% O2+5% CO2，and isometric tension was measured using a force transducer(JH-2，Space Medico-Engineering Institute，Beijing，China).Contraction percentage=(Tension induced by herb/Tension induced by NE)×100%

Relaxation percentage=(Tension induced by vasodilator/Tension induced by NE) ×100%[7].

Statistical analysis

Results

The result of HPLC-DAD characteristic of different extracts fromRhodiolaalgidavar.tangutica(figure 1).

Figure 1 HPLC characteristic of 95%，70% of ethanol extract and aqueous extract ofRhodiolaalgidavar.tangutica

The result of content determination of total flavonoids and total phenolic acid in different extracts fromRhodiolaalgidavar.tangutica(table 1).

Table 1 Content determination of total flavonoids and total phenolic acid in 95%，

The vasorelaxant effects of different extracts on isolated pulmonary artery pre-contracted by NE(table 2).

Group(extract)nConcentrationofrawmedicinalherb(μL)(mg/mL)502.31506.930013.850023.0695%ethanol(152mg/mL)623.17±2.5728.56±3.8264.01±7.9179.24±1.9670%ethanol(200mg/mL)420.52±3.8424.89±4.4463.10±4.1268.17±1.04▲aqueous(232mg/mL)30.00*0.00*0.00*15.86±2.59*F73.79063.528128.563689.526P<0.001<0.001<0.001<0.001

*：compared with effect of 95%，70% ethanol extractP<0.05；▲：compared with effect of 95% ethanol extractP<0.05.

The result of vasorelaxant effects comparison of 95% ethanol extract on isolated pulmonary artery pre-contracted by NE and KCl(table 3).

GroupnConcentrationofextract(mg/mL)0.762.284.567.6NE623.17±2.5728.56±3.8264.01±7.9179.24±1.96KCl30.000.000.0015.26±1.94t15.06512.52213.54733.489P<0.001<0.001<0.001<0.001

Discussion

The result of HPLC-DAD showed that different extracts fromRhodiolaalgidavar.tanguticahad different content of end absorption compound.Among them， the types and content of end absorption compound from aqueous extract were less than that of ethanol extract.Only the compound content was different between ethanol extracts of 95% and 70%.

Phenolic compounds such as flavonoids and phenolic acids are considered to be major contributors to the capacities of plants. According to reports，the major chemical composition inRhodialaeincluded flavonoid，phenolic acid，phenylethanoid glycosides and coumarins compound.Table 1 showed the results of total phenolic acid content in three different extract，expressed in terms of mg gallic acid/g.The results revealed that the total phenolic acid of aqueous extract was much higher than that of other extract. Table 1 also showed the total flavonoid content of three different extracts.The results of total flavonoid were expressed in mg rutin/g dry extract by comparison with standard rutin treated in the same conditions.The results revealed total flavonoid of aqueous extract was also much higher than that of other extracts.In ethanol extracts of 95% and 70%，the content of total flavonoids and total phenolic acid were high，respectively.

To the best of our knowledge，this is the first study evaluating the effect of extract fromRhodiolaalgidavar.tanguticaon vascular tone of rat pulmonary small artery.We found that three kinds of extracts induced vasorelaxation effect in different levels.Among them，aqueous extract showed lowest vasorelaxation effect and only 15% of vasorelaxation effect at high pharmacological concentrations.Ethanol extracts of 95% and 70% also showed vasorelaxation effect in a concentration dependent manner.In the concentration range of 2.3～13.8 mg/mL(equal raw medicinal herb concentration)，there was no significant vasorelaxation difference between these two ethanol extract.However，at the concentration of 23.06 mg/mL(equal raw medicinal herb concentration)，95% of ethanol extract showed maximum vesorelaxation effect(compared with effect of 70% ethanol extract，P<0.05).In our previous animal experiment，we found that 95% of ethanol extract attenuated hypoxic pulmonary hypertension and remodeling of pulmonary small artery in HPAH rats.In this test，the result ofinvitropulmonary artery perfusion was same as that of animal experimentinvivo，we selected 95% of ethanol extract to be studied in the following experiment.

To maintain angiotasis，it is important to keep balance between contraction of vascular smooth muscle and endothelium dependent relaxation.Calcium，entering from extra-cellular and releasing from intra-cellular，is important in maintaining contraction of blood vessel smooth muscle.The receptor gated calcium channel and voltage dependent calcium channel control inflow of calcium into intra-cellular.In this experiment，we compared the effects of 95% ethanol extract on KCl(60mM)and NE(1μmol·L-1)pre-contraction endothelium-intact pulmonary artery ring.95% ethanol extract induced vasorelaxation of rat pulmonary artery in a concentration- dependent manner(0.76～7.6mg/mL).However，on 60 mM KCl-precontracted strips，this extract induced vasorelaxation was lower than that of obtained on 1 μmol·L-1NE-contracted samples.It could be deduced that such effect is produced via inhibition of extracellular Ca2+entry through receptor gated calcium channel.And the vasorelaxation effect of 95% extract based less on inhibiting voltage dependent calcium channel.

To investigate the active part and mechanism of 95% ethanol extract ofRhodiolaalgidavar．tanguticaon inducing rat pulmonary artery vasorelaxation，different eluting parts by macroporous adsorptive resins were prepared and further test was going on.

Acknowledgments

This study was supported by grants from Natural Science Foundation of Science and Technology Department in Qinghai Province，China (2014-ZJ-915)；Natural Science Foundation for Young Scientists of Science and Technology Department in Qinghai province，China(2015-ZJ-928Q)；Natural Science Foundation of China(81160243)；Fund for Development of Applied Basic Key Laboratory of High Altitude medicine Research of Qinghai Province(2014-Z-Y-07).

REFERENCES

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[3]Ge R L，Hackett P．Life on the Qinghai-tibetan plateau[M].Beijing：Peking University Medical Press，2007，7：100.

[4]Cahill E，Rowan S C，Sands M，et al．The pathophysiological basis of chronic hypoxic pulmonary hypertension in the mouse vasoconstrictor and structural mechanisms contribute equally[J].Experimental Physiology，2012，97( 6)：796.

[5]Simonneau G，Gatzoulis MA，Adatia I，et al.Updated clinical classification of pulmonary hypertension[J].J Am Coll Cardiol，2013，62(D)：34-41.

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[7]XY Gai，YH Wei，W Zhang et al.Echinacoside induces rat pulmonary artery vasorelaxation by opening the NO-cGMP-PKG-BKCa channels and reducing intracellular Ca2+levels[J]. Acta Pharmacologica Sinica，2015，36(5)：587-596.

Preliminary study of active component and mechanism ofRhodiolaalgidavar．tanguticaon inducing rat pulmonary artery vasorelaxation*

Li Gengxiong1，Gai Xiangyun2，Li Zhanqiang1，Chang Rong3，Qi Yujuan3，
Zhaxi dongzhu1，Jin Guoen1，Nie Shengbin1，Wu Ping1**，Lu Dianxiang1***

(1.Medical college of Qinghai University；2.Pharmaceutical college of Qinghai nationalities University；
3.People’s hospital of Qinghai province)

Objective To investigate the effective part and mechanism ofRhodiolaalgidavar．tanguticaon pre-contracted pulmonary artery vascular ring.Methods Ethanol extracts of 95%，70% and aqueous extract ofRhodiolaalgidavar．tanguticawere prepared.The content characteristic of extracts were analyzed by HPLC-DAD.The total flavonoids and total phenolic acid were determined.Vascular ring perfusion methodinvitrowas used to detect effective part of extract.The effect of effective part on noradrenaline(NE)and potassium chloride(KCl)pre-contracted pulmonary artery vascular rings was compared to test related mechanism.Results The result of HPLC-DAD showed that the type and content of end absorption compound from aqueous extract was less than that of ethanol extract.Whereas，only compound content was different between ethanol extract of 95% and 70%.The contents of total flavonoids and total phenolic acid from aqueous extract were in highest levels compared to ethanol extracts.The result of vascular ring perfusioninvitroshowed that 95% of ethanol extract was the effective part which relaxed NE pre-contracted rat pulmonary arteries in a concentration-dependent manner(0.76～7.6mg/mL).Conclusion The active part ofRhodiolaalgidavar．tanguticawas mainly accumulated in 95% ethanol extract.The vasorelaxation effect of active part was based on inhibiting receptor-operated calcium channel which could reduce intracellular Ca2+levels.

Rhodiolaalgidavar.tanguticaExtract Part Vasorelaxation Mechanism

*：青海省科技廳自然科學(xué)基金面上項目(2014-ZJ-915)；青海省科技廳自然科學(xué)基金青年項目(2015-ZJ-928Q)；國家自然科學(xué)基金(81160243)；青海省高原醫(yī)學(xué)應(yīng)用基礎(chǔ)重點實驗室項目(2014-Z-Y-07) 李更兄(1990～)，女，漢族，青海籍，在讀碩士研究生.**：通訊作者，教授，wupingyixue@163.com；***：通訊作者，教授，ludianxiang@qhu.edu.cn

R96

A

10.13452/j.cnki.jqmc.2016.01.007

2015-11-12