Advances in Studies on Total Flavone of Abelmoschus manihot(L.)Medic.

Lei ZHOU

Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, The Department of Endocrinology, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing 210029, China

Abstract Flos Abelmoschus manihot has a long history of application.Recent studies show that total flavone of A. manihot(L.)Medic(TFA)has a wide range of pharmacological activity.This article is a review mainly on chemical composition, method of determination and pharmacologic action of TFA, and it raises research and development direction.

Key words Total flavone of Abelmoschus manihot(L.)Medic(TFA), Chemical composition, Method of determination, Pharmacological activity

1 Introduction

Dry corolla ofAbelmoschlmanihot(L.)Medic is often used as traditional Chinese medicine[1], and it was firstly recorded in theJiayouMateriaMedicawritten by Zhang Yuxi in Northern Song Dynasty.A.manihot(L.)Medic has wide distribution and rich resources, and is mainly produced in Jiangsu, Anhui, Jiangxi, Hebei, Henan, Gansu, Shandong, Sichuan and Yunnan-Guizhou region.In theCompendiumofMateriaMedica, it was recorded that its flower smell is sweet, cold, slippery and non-toxic.It is mainly used for urination, gonorrhea and hastening parturition, and can cure all kinds of evil sores and abscesses that have not been cured for a long time.It can heal immediately after the last application.The flower ofA.manihot(L.)Medic is an important medicine for the sore family, could eliminate gangrene, and spread fire injury by soaking oil[2].Modern pharmacology has proved that flowers ofA.manihot(L.)Medic mainly contains flavonoids, reducing sugars, acids, long-chain hydrocarbons and other compounds.Flavonoids are its main components, among which the content of hypericin, isoquercetin and quercetin-3′-glucoside is the highest[3-5].At present, the researches on chemical compositions of flowers ofA.manihot(L.)Medic are mostly concentrated in total flavone ofA.manihot(L.)Medic(TFA)and its monomer.In recent years, with the continuous updating of separation and extraction methods, the research on TFA has been deepened day by day.Great progress has been made in the research on the chemical components, content determination methods and pharmacological effects of TFA, and the potential clinical application value of TFA has attracted more and more attention.Therefore, the above research results are briefly summarized in this paper, which could provide a reference for further study of the medicine.

2 Chemical compositions

Wang Xianrongetal.[6]separated five kinds of flavonoids from alcohol extract of flowers ofA.manihot(L.)Medic in 1981.Via spectral analysis, preparation of derivatives, acid hydrolysis, and determination of physicochemical constants, they were respectively identified as quercetin-3-robinobioside, quercetin-3′-glucoside, hyperin, quercetin, and myricetin.It was the earliest research on flowers ofA.manihot(L.)Medic in China.After that, Wang Xianrongetal.used chromatography and fractional crystallization to separate the components of flowers ofA.manihot(L.)Medic, and identified the structure of the compound according to physicochemical properties and spectroscopy.Results showed that two new flavonol monoglycosides were found, and they were identified as gossypetin-3′-O-β-glucoside and isoquercetin[7].

Lai Xianyinetal.[8]carried out petroleum ether extraction from ethanol extract of flowers ofA.manihot(L.)Medic.Through silica gel column chromatography, SephadexLH-20 column chromatography, preparation of TLC and recrystallization, new flavonoids were separated and obtained: cannabiscitrin and myricetin-3-O-β-D-glucopyranoside.

Taking flowers ofA.manihot(L.)Medic as the research object, Chi Yumeietal.[9]used HPLC-ESI/Q-TOF MS/MS to firstly carry out systematic analysis on TFA.By analyzing UV spectrum and secondary mass spectrometry, it was speculated that flowers ofA.manihot(L.)Medic isolated by HPLC contained 19 flavonols.Among them, seven compounds(hyperoside, isoquercetin, myricetin, quercetin-3′-O-glucopyranoside, quercetin, gossypetin, and hibiscoside)isolated by HPLC were all consistent with previous literature reports.Li Chunmeietal.[10-13]isolated and identified 21 flavonoid monomers from the macroporous adsorption resin eluate of 95% ethanol extract of flowers ofA.manihot(L.)Medic.According to their physical and chemical properties and spectroscopy method, especially NMR method, their structure was identified.It contained 10 flavonoids with quercetin as mother nucleus, and they were quercetin(1), hyperoside(2), quercetin-3-O-β-D-glucopyranoside(3), quercetin-3-O-β-D-6″-acetylglucopyranoside(4), quercetin-3-O-robinoside(5), quercetin-3-O-rutinoside(6), quercetin-3-O-β-D-xylopyranosyl(1→2)-β-D-galactopyranoside(7), quercetin-3′-O-β-D-glucopyranoside(8), quercetin-7-O-β-D-glucopyranoside(9), and quercetin 3-O-[β-D-xylopyranosyl(1→2)-α-L-rhamnopyranosyl(1→6)]-β-D-galactopyranoside(10); eight flavonoids with myricetin as mother nucleus, and they were respectively myricetin(11), myricetin-3-O-β-D-glucopyranoside(12), cannabiscitrin(13), myricetin-3-O-β-D-galactopyranoside(14), myricetin-3-O-rutinose(15), myricetin-3-O-robinoside(16), myricetin-3-O-β-D-xylopyranosy-(1→2)-β-D-glucopyranoside(17), and myricetin-3-O-β-D-xylopyranosyl-(1→2)-β-D-galactopyranoside(18); additionally, there were gossypetin8-O-β-D-glucuronide(19), gossypetin-3-O-β-glucopyranoside-8-O-β-glucuronopyranoside(20), and gossypetin 3′-O-β-D-glucopyranoside(21).Adding gossypetin and hibiscoside, so far, 23 flavonoids have been found in flowers ofA.manihot(L.)Medic.

3 Comparison and influence factors of content determination methods

There are many methods for content determination of TFA.Zhou Zhenghuaetal.[15]used four kinds of methods to determine the content of TFA, and respectively compared the accuracy of various determination methods.Method A: rutin was taken as control, and it was determined by colorimetry at 510 nm after NaNO2-Al(NO3)3-NaOH coloration; method B: hyperoside was taken as control, and it was determined by colorimetry at 510 nm after NaNO2-Al(NO3)3-NaOH coloration; method C: hyperoside was taken as control, and it was determined by ultraviolet spectrophotometry at 258 nm; method D: hyperoside was taken as control, and it was determined at 398 nm after adding HAC-NaAC buffer and AlCl3solution for coloration.Taking NaNO2-Al(NO3)3-NaOH for coloration, quercetin-3′-glucopyranoside, gossypetin-3′-glucopyranoside and myricetin did not develop color.When determining TFA at 510 nm, the content of these three components was not included in the content of total flavonoids, while the content of quercetin-3′-glucopyranoside in flowers ofA.manihot(L.)Medic was higher.So, the content of total flavonoids determined by methods A and B was low, and the result determined by method C was higher.It was because that flowers ofA.manihot(L.)Medic contained a large amount of tannins, and they had strong UV absorption at 258 nm, which would affect the determination of TFA content.Finally, the conclusion was that the result determined by method D was relatively accurate, so method D was worth popularizing.

Different harvest time will affect the content of TFA[16], and the flowering period ofA.manihot(L.)Medic can be divided into bud stage, opening stage and withering stage.The content of total flavonoids varies greatly during different flowering periods, and its content is the highest in opening stage.In withering stage, its content is the lowest, and the content of total flavonoids is 1/5 of that in opening stage.However, the content of total flavonoids has little change when harvested at any time in the same flowering period.

Different drying methods also affect the content of TFA.If the natural drying method is adopted after harvest, that is, the open flowers will be used after drying in the sun, the content is higher in sunny days and lower in cloudy days.If the oven-drying method is adopted, that is, the flowers shall be dried in the drying room or oven with hot air circulation, the temperature must be strictly controlled.If temperature is too high, the drying speed of medicinal materials is fast, which is easy to scorch and change color, affecting the appearance; if temperature is too low, there is long drying time and low content of total flavonoids.So, the flowers ofA.manihot(L.)Medic should be harvested on the day of opening, and dried immediately after picking.It should be dried at 60-80 ℃ in the drying room or oven with hot air circulation.In this way, the content of extracted total flavonoids is high, otherwise the medicinal material is very easy to deteriorate and reduce the content of total flavonoids.

4 Pharmacological actions

4.1 Effect on myocardial ischemia injuryBy establishing whole animal models of myocardial ischemia, ischemia-reperfusion injury and myocardial infarction, Li Qinglinetal.[17-18]found that TFA(100 and 200 mg/kg, oral administration)inhibited T wave increase of ECG in rats injected with pituitrin.For the rats with reversible ligation of left anterior descending coronary artery, ischemia for 30 min and then reperfusion for 3.5 h, TFA(100 and 200 mg/(kg·d)×3, oral administration)could decrease the generation of MDA in myocardium, increase SOD activity in ischemia-reperfusion tissue, inhibit the generation of CPK in serum, raise bcl-2, and effectively inhibit cardiomyocyte apoptosis.For acute myocardial infarction rats caused by complete coronary artery ligation, TFA(100 mg/(kg·d)×3, oral administration)could significantly inhibit the rise of CPK and LDH in serum, and decline infarct size in rats with acute myocardial infarction.It indicated that TFA has a certain protective effect on myocardial injury, and its action mechanism may be related to its anti free radical formation and the induced expression of related anti apoptotic proteins.

Fan Lietal.[19-21]used Langendorff isolated heart perfusion to establish ischemia hypoxia reperfusion model of isolated rat heart, and found that TFA could significantly decline the generation of MDA, enhance the activity of SOD, protect myocardial cell membrane, decrease the leakage of CPK and LDH, and increase NO content.Meanwhile, it also observed that the pretreatment of TFA also had a protective effect on myocardial ischemia-reperfusion injury in rabbits.The experimental results displayed that TFA can reduce the incidence of arrhythmia in different degrees, inhibit the leakage of LDH in myocardial tissue, up regulate the expression intensity of bcl-2 mRNA, down regulate the expression level of P53 mRNA, reduce the severity of myocardial ischemia-reperfusion pathological injury in rabbits, and down regulate the expression of intercellular adhesion molecule ICAM-1 mRNA in ischemic myocardium.Therefore, the results of overall andinvitroanimal experiments confirmed that TFA had a protective effect on myocardial ischemia.

4.2 Effect on cerebral ischemia injuryThe research by Gao Shanetal.[22]showed that TFA could obviously inhibit occurrence of brain edema, reduce the changes of brain histopathology, improve NO content in brain tissue, and decline cerebral infarction weight on the experimental rat model of cerebral ischemia injury.Gao Shanetal.[23]also studied the impact of TFA on brain cell apoptosis of middle cerebral artery occlusion(MCAO)rats.The experimental results showed that TFA treatment could significantly improve ultrastructural changes of brain cells under transmission electron microscope.The observation of TUNEI method found that TFA can reduce the number of apoptotic brain cells in a dose-dependent manner, indicating that TFA can play a protective role against cerebral ischemia injury by anti brain cell apoptosis.Wen Jiyue[24]found that TFA pretreatment had the protective effect of ischemic preconditioning on cerebral ischemia-reperfusion injury in rats, could increase the activity of NOS and the content of NO in serum, and decrease cerebral infarction area.So, TFA has a certain protective effect on cerebral ischemia injury.

4.3 Effect on nephritis and renal fibrosisBy reproducing the rat model of damp heat syndrome of chronic nephritis, Hu Cuiyunetal.[25]found that TFA could improve erythrocyte immune adhesion in rats, promote transport and clearance of immune complexes, inhibit mesangial cell proliferation, reduce renal immune injury mediated by circulating immune complex, improve renal function, and had certain therapeutic effect on chronic nephritis.Feng Yuanetal.[26]studied protective effect and mechanism of TFA on renal fibrosis in rats with unilateral ureteral obstruction.The experimental results showed that TFA can significantly reduce the renal pathological changes after unilateral ureteral obstruction(UUO)operation in rats, decrease the expression of connective tissue growth factor(CTGF)and osteopontin(OPN)in renal tissue, up regulate expression of co inhibitor SnoN protein of Smad7 and Smad nuclear transcription in renal tissue, and delay renal tubular injury and the progress of renal fibrosis.

4.4 Hypoglycemic effectIt has been found that TFA had a certain hypoglycemic effect in the experimental model ofinvitrocells.Chen Gang[27]established glucose consuming cell model of 3T3-L1 adipocytes at cellular level, and observed that TFA can promote glucose absorption in varying degrees(glucose consumption ratio>50%)in the range of 1-100 mg/L, and there was certain dose dependence.

4.5 Promoting angiogenesisApplying chicken embryo chorioallantoic membrane model, Pan Wu[28-29]observed that a certain concentration of TFA can significantly promote the angiogenesis of chicken embryo chorioallantoic membrane.Taking human umbilical vein endothelial cells(HUVEC)culturedinvitroas the model, it was verified that a certain concentration of TFA can significantly promote the proliferation, migration and tubular structure formation of vascular endothelial cells.It is speculated that TFA can promote the formation of neovascularization in the repair of ischemic injury, thereby favorable for formation of microvessels and collateral circulation in ischemic injured tissues, and accelerating tissue repair.

4.6 Antiviral, antibacterial and repairing oral mucosaThe research by Jiang Qinetal.[30]showed that TFA could inhibit herpes simplex virus(HSV1 and HSV2).Invitroantibacterial test results by Zhang Hongyanetal.[31]displayed that TFA can inhibitStaphylococcusepidermidis,StaphylococcusaureusandCandidaalbicans.For guinea pig model of infectious oral mucosal ulcer caused by the above pathogens, TFA can significantly shorten the healing time of oral mucosal ulcer in guinea pigs.The above results show that TFA has a certain effect on infectious oral ulcer.

In conclusion, the total flavonoids extracted from the flowers ofA.manihot(L.)Medic have complex chemical components, wide pharmacological effects and broad clinical application prospects.It is the drug with great development value, the achievement of the modernization of traditional Chinese medicine and the crystallization of condensing the wisdom of the ancients and modern people.In this paper, the amount of flavonoids extracted from the flowers ofA.manihot(L.)Medic from the earliest to now is firstly summarized, and the accuracy and influencing factors of various methods for the determination of TFA are summarized.Moreover, the pharmacological effects found in various experiments are summarized in detail, with certain reference significance for guiding clinical work.However, the current research on TFA is not thorough and comprehensive, and many aspects, especially the pharmacological effects, still need to be further studied.We expect it to give better play to the advantages of traditional Chinese medicine and better benefit mankind.