Baicalein (5,6,7-trihydroxyflavone) is a flavonoid derivedfrom the root of Scutellaria baicalensis, a traditional Chinesemedicine used for hundreds of years. This flavonoid demon-strates a variety of biological activities, such as antibacterial,antiinflammatory, antioxidant, antitumor, antiproliferative,anticoagulant, and vascular-protective effects.1,2)

Baicalein is also an inhibitor of reverse transcriptase of thehuman immunodeficiency virus (HIV), interferes with theRNA replication of HIV, and induces apoptosis in infectedcells in vitro.3) Baicalein is the most potent inhibitor of thehuman cytomegalovirus (HCMV), displaying an IC50 valueof 0.4�0.04 mM in the colorimetric assay and 1.2�0.8 mM inthe titer reduction assay. It also significantly reduces the lev-els of early and late proteins of HCMV, as well as viral DNAsynthesis.4)

Influenza, one of the major pandemic diseases worldwide,represents a grave threat to human health. At present, onlytwo types of chemical drug are used clinically in the treat-ment of influenza. One type is the M2 proton channel block-ers (amantadine and rimantadine), and the other is neu-raminidase inhibitors (zanamivir and oseltamivir). However,resistance to these drugs has been reported.5—8) Furthermore,the high price and lack of a sufficient supply prevent os-eltamivir (Tamiflu) from being widely used in developingcountries. Traditional Chinese medicine, which is widespreadin nature with good efficacy, is beginning to play a more im-portant role in this area.

It has been reported that baicalein demonstrates inhibitoryeffects on viruses and that more than 90% of baicalein is rap-idly metabolized into baicalin, which is transported to themesenteric blood when perfused through the rat jejunum.9,10)

However, the mechanism of its antiviral effect is unknown.Here, we report for the first time that baicalein has an in-hibitory effect on the influenza virus A/FM1/1/47(H1N1) invivo and that this effect is mainly due to its metabolitebaicalin in serum.

MATERIALS AND METHODS

Reagents Baicalein was a gift from Prof. Qinglong Guo

of the Deptartment of Physiology, China PharmaceuticalUniversity. It was dissolved in 0.5% sodium carboxymethylcellulose (CMC) for in vivo experiments and in dimethyl sulf-oxide (DMSO) for in vitro tests. Ribavirin injection was ob-tained from the China Pharmaceutical University Pharma-ceutical Co., Ltd., Nanjing, China. Reference samples ofbaicalein and baicalin used in HPLC analysis were obtainedfrom the National Institution for the Control of Pharmaceuti-cal and Biological Products, China. Methanol, acetonitrile,and phosphoric acid (HPLC grade) were purchased fromSinopharm Chemical Reagent Co., Ltd., Shanghai, China.

Virus and Cells The influenza A/FM1/1/47(H1N1)virus used in this study was preserved in the Department ofMicrobiology, School of Life Science and Technology, ChinaPharmaceutical University. The virus was grown in the allan-toic cavity of 10-d-old embryonated hen eggs at 37 °C for2 d.11) The allantoic fluid was harvested and filtered with a0.22-mm cellulose acetate membrane. The filtered liquid wasstored in small aliquots at �70 °C until further use.

Madin–Darby canine kidney (MDCK) cells were grown inDulbecco’s modified Eagle’s medium (DMEM) supplementedwith 10% (v/v) fetal calf serum (FCS). The cells were incu-bated in a humidified atmosphere of 5% CO2 at 37 °C.

Animals Ninety-six female BALB/c mice (18—22 g)and 10 Sprague-Dowley (SD) rats were obtained from theLaboratory Animal Center, Nanjing Medical University,China, and treated according to the “Guide for the Care andUse of Laboratory Animals” prepared by the National Acad-emy of Sciences and published by the National Institutes ofHealth.

Antiviral Effects of Baicalein in Vivo After a 2-d accli-mation period, the mice were slightly anesthetized by the in-halation of diethyl ether and intranasally infected with 8�MLD50 of mouse-adapted influenza A/FM1/1/47(H1N1) virusin 100 m l of phosphate buffered saline (PBS). The mice weretreated with various doses of baicalein solution (240, 480,960 mg/kg/d) by oral gavage twice daily for 4 d beginning24 h after virus inoculation. Ribavirin (400 mg/kg/d) or ster-ile sodium chloride (100 m l) was given as a positive controlor placebo control, respectively. All doses were determined

238 Vol. 33, No. 2

Inhibitory Effects of Baicalein on the Influenza Virus in Vivo IsDetermined by Baicalin in the Serum

Ge XU,a,# Jie DOU,a,# Lei ZHANG,a Qinglong GUO,*,b and Changlin ZHOU*,a

a School of Life Science & Technology, China Pharmaceutical University; and b Department of Physiology, ChinaPharmaceutical University; 24 Tong Jia Xiang, Nanjing, Jiangsu 210009, P. R. China.Received August 28, 2009; accepted October 13, 2009; published online November 10, 2009

Baicalein, an extract from Scutellaria baicalensis, was evaluated for its ability to inhibit the influenza virusin vivo. Oral administration of baicalein to BALB/c mice infected with the influenza A/FM1/1/47(H1N1) virusshowed significant effects in preventing death, increasing the mean time to death, inhibiting lung consolidation,and reducing the lung virus titer in a dose-dependent manner. These effects are believed to be due to baicalin,the metabolite of baicalein in the serum. At a concentration of baicalin 2 mmg/ml in overlay medium, it showed sig-nificant inhibition in the plaque assay, and the mean IC50 value of baicalin was calculated as 1.2 mmg/ml in the cytopathic effect assay. Our results showed that baicalein warrants further research as a potential antiinfluenzaviral agent.

Key words baicalein; baicalin; antiviral activity; serum pharmacology

Biol. Pharm. Bull. 33(2) 238—243 (2010)

© 2010 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: cl_zhou@cpu.edu.cn# These authors contributed equally to this work.

in advance in toxicity experiments. Normal controls weretreated with sodium chloride. Ten animals per group wereobserved for 14 d for clinical signs of infection or death.

An additional 6 mice per group were used to assess lunginfection parameters on day 5 of infection with the influenzavirus. Mice were weighed and killed, and then the lungs wereremoved and weighed. The lung index and lung index inhibi-tion were calculated as:

lung index�A/B�100%

lung index inhibition (%)�(C�D)/C�100%

where A is lung weight, B is body weight, C is mean lungindex of the placebo control group, and D is mean lung indexof the drug-treated group.

Lung consolidation scores ranging from 0 (normal appear-ance) to 4 (100% of the lung exhibiting a plum color) wereassigned. After being fixed in 10% formalin and embeddedin paraffin, four lungs per group were sectioned for histologicexamination. Pathologic changes were evaluated based on thefollowing aspects: hyperemia and bleeding of the lungs;leukopedesis; bronchiole epithelium cell necrosis; exudate oflung; alveolus interstitial pneumonia; and lung abscess.

The remnant lungs were homogenized in PBS to give a10% (w/v) suspension, and the homogenate was centrifugedat 2000�g for 10 min. The supernatant was serially dilutedand inoculated into 10-d-old embryonated hen eggs. Virustiters in the lungs were calculated using the method of Reedand Muench12) and egg infective dose (EID) expressed asmean log10 EID50/ml�S.D.

Preparation of Medicine Serum Ten SD rats were usedin this experiment. Six rats were orally administered abaicalein solution at a dose of 500 mg/kg once daily for 5 d.The other 4 rats were treated with saline as controls. On day5, after 2.5 h of treatment with baicalein, the rats were anes-thetized with an intraperitoneal injection of 10% (m/v) amo-barbital sodium at a dose of 500 mg/kg, and blood was col-lected under sterile conditions from the main ventral artery.After standing at 4 °C for 30 min, the serum was obtained bycentrifugation at 3000�g for 20 min and stored in smallaliquots at �20 °C.13)

HPLC was used for quantitative analysis of baicalein andits metabolite baicalin in the serum. The LC-2010 ShimadzuHPLC system was employed with a Class VP workstation fordata collection. Liquid chromatographic separation wasachieved using a Lichrospher C18 analytical column (6.0mm�150 mm, 5 mm) connected to a C18 guard column (Han-bon Science & Technology Co., Ltd., Jiangsu, China). Refer-ence substances of baicalein and baicalin were dissolved inmethanol to concentrations of 196 mg/ml and 19.8 mg/ml, re-spectively, for use as internal standards. To prepare for HPLCanalysis, 400 m l of methanol–acetonitrile (1 : 1, v/v) and100 m l of 0.4% phosphoric acid were added to a 200-m lserum sample. After vortex mixing for 3 min, the mixturewas centrifuged for 10 min at 12000�g. Six hundred micro-liters of the upper layer was obtained, evaporated under N2,and reconstituted with 200 m l of the mobile phase prior toHPLC analysis. Blank serum and blank serum with 20 m l of the internal standards were prepared in parallel as controls.14,15) The mobile phase consisted of methanol–0.2%phosphoric acid (48 : 52, v/v) at a constant flow rate of

1.0 ml/min. The column temperature and the detection wave-length were set at 30 °C and 275 nm, respectively. The prepa-rations were analyzed both qualitatively and quantitatively bycomparisons of retention times and analyzed peak areas withthose of the internal standards.

Antiviral Effects of Medicine Serum in Vitro Antiviralactivities of medicine serum were measured in the plaque in-hibition assay. MDCK cells (1�105 cells/ml) were culturedin 24-well plastic plates for 24 h and inoculated with 100plaque forming units (PFU) per well of influenza A/FM1/1/47(H1N1) virus. After 60 min for virus absorption, the so-lution was removed and replaced with 1 ml of overlaymedium (DMEM medium containing 1% carboxymethyl cel-lulose, without serum) containing different concentrations ofmedicine serum. The final concentrations of baicalin in theoverlay medium were 0.5 mg/ml, 1 mg/ml, and 2 mg/ml. Afterincubation at 37 °C for 3 d, the solution was removed and thecells were fixed with 10% formaldehyde solution for 60 minat room temperature. Then the cells were stained with 0.5%(w/v) crystal violet solution for 15 min, and the number ofplaques was counted for each well.

Cytopathic Effect Assay To examine whether baicalincan inhibit the cytopathic effect (CPE) of cells infected by in-fluenza A/FM1/1/47(H1N1) virus, a 96-well tissue cultureplate was seeded with 100 m l of 1�105 cells/ml in DMEMwith 10% FBS. After cells were incubated for 18—24 h at37 °C, the solution was removed and the cells were infectedwith 100 PFU per well of influenza A/FM1/1/47(H1N1)virus at 37 °C for 1 h. The viral inocula were removed andreplaced with growth medium (DMEM medium withoutserum) containing different concentrations of medicine serum.The final concentrations of baicalin in the growth mediumwere 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, 2 mg/ml, and 3 mg/ml.Each treatment was performed in quadruplicate. After incu-bation for 3 d at 37 °C, the cells were fixed with 100 m l of10% formaldehyde for 1 h at room temperature. After re-moval of the formaldehyde, the cells were stained with 0.5%(w/v) crystal violet solution for 15 min at room temperature.The plate was washed and dried, and the intensity of crystalviolet staining for each well was measured at 570 nm. TheIC50 value of baicalin was calculated from the absorbancevalues.16)

Statistical Analysis Data from the in vivo studies arepresented as mean�S.D. Comparisons were made betweendrug-treated and placebo groups. Differences in the meanday to death, lung consolidation scores, and lung virus titerswere analyzed using the two-tailed Mann–Whitney U-test.The probability of survival was estimated with the Kap-lan–Meier method, and survival estimates were comparedusing the log-rank test.17)

RESULTS

Antiviral Effects of Baicalein in Vivo Influenza viralinfection leads to high mortality, lung virus titers, and pneu-monia.18) Therefore the efficacy of treatment was evaluatedon the basis of survival rate, mean day to death, lung virustiters, and lung parameters including lung consolidation andthe inhibition of the lung index. The results showed thatbaicalein displayed a protective effect on mice infected withinfluenza A/FM1/1/47(H1N1) virus (Table 1). Oral gavage

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with all doses of baicalein (240, 480, 960 mg/kg/d) resultedin protection from death and statistically significant pro-longed survival time of mice. A survival rate of 50% was ob-tained even at the lowest dose of 240 mg/kg/d, while all theplacebo control mice died. Compared with the other twodoses, treatment with baicalein at the dose of 960 mg/kg/dhad the most effect on life protection with a survival rate of70% and the mean day to death of 12.4�2.6 d. Furthermore,in the 960 mg/kg/d group, mortality occurred on day 8 afterinfection, which was 3 d later than in the placebo group, 2 dlater than in the 240 mg/kg/d group, and 1 d later than in the240 mg/kg/d group (Fig. 1).

Lung parameter data showed that treatment with baicaleinprovided a dose-dependent protective effect from viral pneu-monia (Table 1). Inhibition of the lung index was detected at26%, 30%, and 35% with the doses of 240, 480, and 960mg/kg/d, respectively. Stronger inhibition of the lung index(35%) and less lung consolidation (1.2�0.75) were observedin the mice treated with baicalein 960 mg/kg/d than in thosetreated with ribavirin (32%; 1.3�0.52). Meanwhile, virusreplication was reduced in all treatment groups. The lowestvirus titer detected was 3.8�0.8 in 960 mg/kg/d group. Theresults of the histologic examination were consistent with thelung parameter data (Fig. 2). Protection from bronchitis and

interstitial pneumonia was found in all groups treated withbaicalein, and the degrees of protection were different de-pending on the baicalein dose.

Determination of Baicalin in Serum The concentrations

240 Vol. 33, No. 2

Fig. 1. Effects of Baicalein on Mouse Survival

BALB/c mice were infected with 8�MLD50 of influenza virus (H1N1) and orallytreated with baicalein (240, 480, 960 mg/kg/d) twice daily for 4 d, beginning 24 h after in-fection. Ten mice per group were observed for 14 d for clinical signs of infection or death.The Kaplan–Meier method was used to estimate the probability of survival, which is expressed as a survival distribution function. A value of 1 corresponds to 100% survival.

Table 1. Protective Effects of Baicalein in Mice Infected with A/FM1/1/47/(H1N1)

Mean lung parameters

TreatmentDose Survivors/total

MDDa)�S.D.Score�S.D.

Lung Lung index Virus (mg/kg/d) (%)index�S.D. inhibition % titerb)�S.D.

Baicalein 960 7/10 (70)*** 12.4�2.6** 1.2�0.75** 0.71�0.07*** 35 3.8�0.8*480 6/10 (60)*** 11.5�3.3** 1.8�0.41** 0.76�0.06*** 30 4.9�0.5240 5/10 (50)*** 10.6�3.6* 2.8�0.41* 0.81�0.04*** 26 5.5�0.2

Ribavirin 400 6/10 (60)*** 10.7�4.3* 1.3�0.52** 0.74�0.09*** 32 4.4�0.4*Placebo — 0/10 (0) 6.8�1.1 3.5�0.55 1.09�0.06 — 6.1�0.5Normal — 10/10 (100) 14.0�0.0 0.0�0.0 0.71�0.09 — 0.0�0.0

a) Mean day to death of mice dying prior to day 14; b) log10 EID50/ml. ∗ p�0.05, ∗∗ p�0.01, ∗∗∗ p�0.001, compared with values in placebo controls.

Fig. 2. Pathologic Changes in Lungs in Mice Infected with A/FM1/1/47/(H1N1) (200�)

The mice were killed 3 d after infection. The lungs were removed and rinsed with sterile PBS. After fixing in 10% formalin and embedding with paraffin, the lungs were sec-tioned for histologic examinations. Pathologic changes were evaluated based on hyperemia and bleeding of lungs, leukopedesis, bronchiole epithelium cell necrosis, exudate oflung, alveolus interstitial pneumonia, and lung abscess.

of baicalein and its metabolite in the serum of rats treated withbaicalein (500 mg/kg) were analyzed using HPLC 2.5 h aftertreatment on day 5 (Fig. 3). The retention times of baicaleinand baicalin were 34.2 min and 10.6 min, respectively.Baicalin at the concentration of 17.0 mg/ml was detected inthe serum of treated rats, while baicalein was detected at theconcentration of 0.8 mg/ml, suggesting that the majority ofbaicalein had been metabolized into baicalin, and the amountof baicalein remaining was extremely small.

Antiviral Effects of Medicine Serum in Vitro MDCKcells were infected with influenza A/FM1/1/47(H1N1) virusand incubated in overlay medium with medicine serum con-taining various concentrations of baicalin for 72 h. It wasshown that baicalin inhibited influenza virus replication in adose-dependent manner (Fig. 4A). At the concentrations of1 mg/ml and 2 mg/ml, baicalin showed significant inhibitoryeffect. At 0.5 mg/ml, baicalin showed a slight inhibitory ef-fect. Therefore the concentration of baicalin that effectivelyreduced the plaque of influenza A/FM1/1/47(H1N1) viruswas between 0.5 mg/ml and 1 mg/ml. The level of protectionof MDCK cell monolayers provided by baicalin from viralCPE was measured using influenza A/FM1/1/47(H1N1) virus.The ratio of 100 PFU per well of virus was required to pro-duce 90% CPE at 3 d post infection. After staining of the cellmonolayers with crystal violet, four sets of absorbance val-ues were averaged to generate a dose-response curve (Fig.4B). Absorbance values were proportional to the overallhealth of the cell monolayers, as demonstrated by the reduc-tion in protection afforded by baicalin as the concentrationdecreased. Protection of the monolayers from the CPE in-duced by influenza A/FM1/1/47(H1N1) virus was observedat baicalin concentrations from 1 to 3 mg/ml. The IC50 valueof baicalin was calculated as 1.2 mg/ml from the inhibitioncurve.

DISCUSSION

In this paper, the inhibitory activity of baicalein against influenza A/FM1/1/47(H1N1) virus in vivo was examinedand its mechanism investigated. The oral administration ofbaicalein showed good effect against influenza A/FM1/1/47(H1N1) virus infection in mice, such as increasing survivalrate, prolonging survival time, inhibiting lung consolidation,and reducing lung virus titers. Baicalin, with a glucoseresidue, was difficult to absorb. After oral administration, itwas converted into baicalein by b-glucuronidase of intestinalbacteria and absorbed. When transported to the liver,baicalein was transformed into metabolites by UDP-glu-

curonosyltransferases (UGT) in liver microsomes and ex-erted activity,19—21) including 7-O-glucuronide-6-methoxyl-5-hydroxyflavone, 6-O-glucuronide-5,7-dihydroxyflavone, and7-O-glucuronide-5,6-dihydroxyflavone (baicalin) (Fig. 5).Among them, baicalin was the main metabolite. This mecha-nism was similar to the activation of sennosides and saiko-saponin. Sennosides present in Sennae folium are metabo-lized to rhein and rhein anthrone to produce a laxativeaction.22—24) Saikosaponin extracted from the root of Bupleu-rum falcatum must be metabolized in the intestinal tract toexert antiinflammatory and antitumor activity.25—27)

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Fig. 3. Chromatograms of Serum

Blank serum (A), serum with 20 m l of internal standards (B), and serum sample (C). 1, baicalin; 2, baicalain.

Fig. 4. Antiviral Effects of Medicine Serum in Vitro

(A) Plaque assay. Confluent monolayers of MDCK cells were infected with 100 PFUper well of influenza A/FM1/1/47(H1N1) virus. Infected cells were incubated withserum containing baicalin for 72 h, and then the number of plaques was counted. (B)Baicalin dose-response curves. MDCK cells were infected with 100 PFU per well of in-fluenza A/FM1/1/47(H1N1) virus. The CPE was evaluated by plotting the absorbanceat 570 nm of crystal violet-stained cell monolayers versus the concentration of baicalin.The results represent the mean�standard deviations of four independent measurements.

According to our results, the serum of rats orally adminis-tered baicalein for 2.5 h exhibited an obvious inhibitory ef-fect on the duplication of influenza A/FM1/1/47(H1N1) virusin MDCK cells. Since the amount of baicalein detected in theserum was too small to exert that effect and baicalin is thepredominant metabolite of baicalein, it was reasonably con-cluded that baicalin plays an important role in antiinfluenzaactivity. From the data obtained, the IC50 value of baicalinagainst the influenza virus in vitro was 1.2 mg/ml in the CPEassay, and baicalin also showed significant effects against thevirus at the concentration of 2 mg/ml based on the plaque in-hibition assay. According to previous studies,28—30) the con-centration of ribavirin which was effective in inhibiting theinfluenza virus in vitro was from 2.5 to 10 mg/ml. These sug-gest that there are similar antiviral effects between serumcontaining baicalin and ribavirin in vitro. The data also showedthat baicalin at the plasma concentration of 17 mg/ml is suffi-cient to inhibit the duplication of influenza virus in vivo.

It was reported that the plasma concentration of baicalinreached a maximum 2.5 h after baicalein was absorbed withoral administration.31) However, with the same administrationof baicalin, about 10 h was required to reach the maximalplasma concentration, which is lower than with the oral administration of baicalein. Therefore we concluded thatbaicalein is more effective than baicalin in inhibiting thereplication of influenza virus with oral administration, and itis more likely to be a potential antiinfluenza viral agent forclinical use.

The mechanism of the antiinfluenza viral activity of baicalinhas not been revealed. Neuraminidase, a specific glycopro-tein on the surface of the influenza virus, is essential for viralreplication due to its role in facilitating the release of prog-eny virions from infected cells.32) It is a valid drug target forthe development of antiinfluenza therapeutics. In our initialneuraminidase inhibition assay, baicalin to some extent in-hibited neuraminidase activity (data not shown). Our subse-quent investigations will explore this effect. Furthermore, itwas reported that baicalin significantly inhibits hepatitis B virus (HBV) and herpes simplex virus-1 (HSV)-1 invitro33,34) and shows some effect against HIV.35) It is reason-

able to assume that baicalin possesses a common, nonspe-cific antiviral mechanism based on its inhibitory effects on alarge number of viruses. We will also search for the mecha-nism through its modulation of the immune system in our fu-ture investigations.

Acknowledgments We would like to thank Prof.Wenyuan Liu of the Department of Pharmaceutical Analysis,China Pharmaceutical University, for excellent analytical as-sistance. This study was sponsored by the Qing Lan Project(2008) from Jiangsu province, and the “111 Project” fromthe Ministry of Education of China and State Administrationof Foreign Expert Affairs of China (No. 111-2-07).

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