Antiviral and Immunomodulatory Properties of Prunella vulgaris

Asian Journal of Traditional MedicinesAntiviral and Immunomodulatory Properties of Prunella vulgarisChuen-lung Cheng, Hongxi Xu *Hong Kong Jockey Club Institute of Chinese Medicine, Unit 703, 7/F, Bio-informatics Building,Hong Kong Science Park, Shatin, Hong Kong, ChinaPrunella vulgaris L. is a traditional Chinese medicine that has been used to treat a variety of ailmentsfor hundreds of years. In recent decades, P. vulgaris has been shown to have antiviral andimmunomodulatory properties. Potent antiviral effects of P. vulgaris polar fractions against HIV-1, HSV-1and HSV-2 have been reported. In addition, it has been shown that the extracts and chemical componentscan modulate various immune factors including histamine, TNF-alpha, IgG, IgG1, IgG2b, nitric oxide,LTB 4 , IFN-gamma, IL-2 in T cell activation and Src family protein kinase. This review will describe thenovel discoveries and potential therapeutic applications of P. vulgaris extracts and its chemical componentswith regard to their antiviral and immunomodulatory effects.Key wordsPrunella vulgaris; antiviral ; HIV; HSV; immunomodulationIntroductionPrunella vulgaris L., (Chinese name: Xia-Ku-Cao) alow sprawling perennial herb, is a traditional Chineseherbal n China, its water extract for therapeutic use isprepared from the dried fruit-spikes of medicine that hasbeen used for the treatment of headache, dizziness due tohypertension, tinnitus, conjunctivitis, dry cough,dermatitis and boils, high blood pressure and skinallergic diseases[1-3] . This plant, also known as“self-heal”, is commonly found in China, the BritishIsles and other parts of Europe where it has been used asan astringent for internal and external applications [4] . Itwas popular in traditional European medicine in the 17 thcentury for treating sore throat, reducing fever andaccelerating wound healing [5] . Currently, it is scheduledin the Chinese Pharmacopoeia and listed in the ChineseMedicine Ordinance of the Laws of Hong Kong as acommonly used Chinese herbal medicine. However, P.vulgaris is not currently classified as a medicinal plant inEurope [6] .In China,its water extract for therapeutic use isprepared from the dried fruit-spikes of P. vulgaris. Thisextract tastes bitter and is nontoxic. Traditionally, theaqueous extract of P. vulgaris is administered orally andthe typical dosage for an adult is 9 to 15 g per day.Nowadays, it is also popularly used in the form of herbaltea in China and Hong Kong [7] .As long as more viral mutants with single- ormultiple-drug resistance continue to be appeared,primarily due to prolonged antiviral treatment, it is vitalto search and develop more effective and less toxic newantiviral agents. Traditional Chinese medicinal herbs area rich potential source of new antiviral medicines.______*Corresponding author: Dr Hongxi Xu .Tel.: +852 3406 2873fax: +852 3551 7333 email: xuhongxi@hkjcicm.orgHundreds of aqueous extracts of Chinese medicinalherbs are demonstrated with antiviral effects [8-10] . P.vulgaris is one of the most promising CM herbs withantiviral properties and its immunomodulatory effectshave also attracted increasing attention in recent years.Studies have shown that P. vulgaris contains oleanolicacid, ursolic acid, vulgarsaponin A, vulgarsaponin B,beta-amyrin, alpha-spinasterol, stigmasterol,stigmast-7-en-3beta-ol, flavanoids, fenchone, tannins,prunellin, betulinic acid, 2 alpha, 3alpha-dihydroxyurs-12-en-28-oic acid, 2 alpha, 3alpha-dihydroxyursolic acid and caffeic acid [2, 11-14] .Also, a review paper has reported that 28 triterpenoidcompounds have been isolated from the Prunella genusincluding P. vulgaris L., P. vulgaris L. var. lilacinaNakai and P. grandiflora L. [15] . The extract of P.vulgaris can be divided into polar (aqueous) and organicfractions. In the aqueous fraction, a 10 kDa anionicpolysaccharide called prunellin has been isolated [16] anda polysaccharide with a molecular weight estimated as3,500 Da has been discovered with antiviral activity[10] . An organic fraction (25.7% w/w of rosmarinic acid)of P. vulgaris has been found to exhibit a range ofbiological activities [6] .Studies have shown that P. vulgaris possesses a varietyof pharmacological properties which include[6,anti-oxidative17-18] , free radical scavenging[17] ,anti-allergic and anti-inflammatory [3, 13] , cytotoxic [19] ,anti-microbial [6] , anti-mutagenic [20] , anti-HIV [16, 21-27] ,anti-HSV [8-10, 28] , anti-HBV [30] [30, 31]and anti-arthriticeffects. It has been reported that the crude aqueousextract of P. vulgaris has been used for clinical treatmentof herpetic keratitis [8] and a herbal-based dentifrice withP. vulgaris was effective in reducing symptoms ofgingivitis in a double-blind, placebo controlled clinicaltrial [32] . Since new scientific evidence continues to bepresented regarding the antiviral and immunomodulatory1

Asian Journal of Traditional Medicineseffects, this review will focus on these novel propertiesand future therapeutic potential.Antiviral activities of Prunella vulgaris againstHIV-1, HSV-1 and HSV-2A number of antiviral studies of P. vulgaris extractshave been carried out and active compounds against HIVand HSV have been identified (Table 1), especially in itspolar fractions. A 10 kDa anionic sulfatedpolysaccharide called prunellin was found to inhibit thereplication of HIV-1 [16] . In addition, the extract of P.vulgaris demonstrated a significant inhibitory effect onHIV-1 replication with relatively low cytotoxicityin-vitro and it was able to arrest the cell-celltransmission of HIV-1, prevent syncytium formation andinterfere with both HIV-1 and purified gp120 bindingto CD4 receptors [21] . Similar results have been obtainedin further studies involving the inhibitory mechanism ofHIV-1 infection in vitro by a purified extract of P.vulgaris [25] . The polyphenols and tannins of P. vulgarishave been suggested to be the major inhibitors of HIV-1gp41 six-helix bundle formation which is a critical stepin the membrane fusion between the HIV and its targetcells [17] . Also, it has been reported that an aqueousextract of P. vulgaris inhibited HIV-1 integrase activity[26] .Previously, anti-HSV activity of the aqueous extractof P. vulgaris has been described [8] . Out of one hundredand forty two kinds of traditional medicines, it has beenfound that the hot aqueous extract of P. vulgarisselectively inhibited HSV-1 [9] . Xu and co-authorsreported that a water soluble polysaccharide componentin P. vulgaris, which could be extracted with hot water,precipitated in ethanol and then purified by gelpermeation column chromatography, exhibited potentanti-HSV activity. The 50% plaque reduction dose of thepolysaccharide for HSV-1 and HSV-2 was 10 mg/ml andsimilar inhibitory effects were also exhibited againstacyclovir-resistant(TK-deficient or polymerase-defective)strains of HSV-1 and HSV-2 [10] . Furthermore, a studyusing the polysaccharide fraction of P. vulgaris showed areduction in the expression of HSV-1 and HSV-2antigens in their host Vero cells. Interestingly, thepolysaccharide fraction of P. vulgaris was also shown tohave an inhibitory effect on an acyclovir-resistant strainof HSV-1, ranging from 24.8 to 92.6% at 25-100 mg/ml,and it was suggested to have a different mode ofanti-HSV action from that of acyclovir [28] .Immunomodulatory effects of P. vulgarisThe immunomodulatory effects of P. vulgaris havereceived increasing attention in recent years. The keypromising immunomodulatory effects of differentextracts and chemical constituents isolated from P.vulgaris have been demonstrated in different models andthese are summarized below (Table 2).Table 1Antiviral properties of Prunella vulgaris and its active componentsAntiviral actions Active components ReferencesInhibition of HIV replication Prunellin [16]Prevention of viral attachment to CD4 receptors Active component with a molecular weight of 10 kDa [21]Suppression of the replication of HIV-1(IC100=16 microgram/ml)Hot water extract [22]Inhibition of HSV-1 plaque formation Hot water extract [9]Inhibition of HIV-1 reverse transcriptase activity Aqueous extract [23]Suppression of HSV and acyclovir-resistant strains of HSV-1and HSV-2 by competing for cell receptors (50% plaquereduction=10 microgram/ml); and inhibitory effects at earlyand late stages of virus replicationInhibition of HIV-1 integrase activity(EC50=45 microgram/ml)Suppression of HIV-1 entry by disrupting the gp41 six-helixbundle formation (at 50 microgram/ml, 86.2% inhibition)Suppression of the expression of HSV-1 and HSV-2 antigensand also the acyclovir-resistant strain of HSV-1 (EC50 of theHSV-1 and HSV-2 antigens were 20.6 and 20.1microgram/ml, respectively)A polysaccharide with a molecular weight of 3,500Da[10]Tannin or polyphenols [26]Tannin or polyphenols [27]A polysaccharide fraction [28]2

Asian Journal of Traditional MedicinesTable 2 Immunomodulatory properties of Prunella vulgaris and its active componentsImmunomodulatory actions Active components ReferencesInhibition of nitric oxide production Ursolic acid, 2 alpha-hydroxyursolic acid [13]Inhibition of histamine and TNF-alpha release Aqueous extract [3]Inhibition of interleukin-2 gene expression Methanol extract, rosmarinic acid [33]Organic fraction containing 25.7% andInhibition of 5-lipoxygenase activity0.32% rosmarinic acid and caffeic acidrespectively[6]Inhibition of Src family protein kinase, IL-2 and INF-gamma Rosmarinic acid [37]Reduction of total IgG, IgG1 and IgG2b levels Ethanol extract of Spica Prunellae [34]Stimulation of superoxide and nitric oxide Polysaccharide fraction PV2 [35]Stimulation of proteins levels of iNOS and mRNA TNF-alpha Polysaccharide fraction PV2IV [36]The effect of an aqueous extract of P. vulgaris (PVAE)has been tested on immediate-type allergic reactions. Inrats, when pretreatment with PVAE at concentrationsranging from 0.005 to 1 g/kg was applied, the serumhistamine levels induced by compound 48/80 werereduced. Also, PVAE (0.001 to 1 g/kg) inhibited thepassive cutaneous anaphylaxis activated byanti-dinitrophenyl(DNP) IgE antibody in adose-dependent manner and inhibited the histaminerelease induced by compound 48/80 or anti-DNP IgEfrom rat peritoneal mast cells (RPMC). Moreover,significant suppression of anti-DNP IgE-mediated tumornecrosis factor-alpha production from RPMC has beenreported [3] .It has also been reported that the methanol extract of P.vulgaris with rosmarinic acid inhibited interleukin-2(IL-2) gene expression by 50 % in Jurkat cellsstimulated with anti-CD3 and anti-CD4 antibodies. Inaddition, it has been shown to have an inhibitory effecton the intracellular [Ca 2+ ] increase in Jurkat cells after Tcell activation. It has been suggested that rosmarinic acidhas the potential to specifically inhibit lymphocytecell-specific kinase (Lck) Src -homology 2 (SH2)domain binding to its cognate ligand, including ZAP-70,Cbl, HS-1, and PLCgamma1, and the Lck-dependentCa 2+ signaling pathway of its downstream effector and tomodulate IL-2 gene expression after T cell activation [33] .Recently, the ethanol extract of Spica Prunellae(EESP) was shown to significantly inhibit concanavalinA-, lipopolysaccharide- and ovalbumin-inducedsplenocyte proliferation in immunized mice. It wasfound that the total IgG, IgG1 and IgG2b levels in theimmunized mice were significantly reduced by EESP.Moreover, the suppression of the antibody responses toovalbumin in the immunized mice was dose-dependentlyenhanced in parallel with the increase in EESP. Theresults suggest that EESP is able to reduce cellular andhumoral responses in mice [34] .In an activity-guided fractionation of the P. vulgarisextract, a compound, 2 alpha, 3 alpha-dihydroxyursolicacid, was isolated and shown to significantly inhibit therelease of beta-hexosaminidase from cultured RBL-2H3cells. Other isolated compounds, including ursolic acidand 2 alpha-hydroxyursolic acid, were found to inhibitthe production of nitric oxide in cultured murinemacrophages, RAW 264.7 cells [13] .An organic fraction of P. vulgaris, containing 25.7% and 0.32% rosmarinic acid and caffeic acid, respectively, markedly inhibited 5-lipoxygenase, a key enzyme in leukotriene B4 biosynthesis, in bovine pol-ymorphonuclear leukocytes in vitro [6] .A polysaccharide fraction isolated from P. vulgaris,named PV2, markedly stimulated the production ofsuperoxide and nitrite, representing nitric oxide, frommurine macrophage RAW264.7 and brain macrophageBV2 cells. The amount of nitrite and superoxideproduced after PV2 stimulation was as high as that seenafter stimulation using bacterial endotoxinlipopolysaccharide (LPS) [35] . Furthermore, the PV2IVfraction also stimulated the production of protein levelsof iNOS and mRNA TNF-alpha [36] .In a large-scale screening of plant extracts, rosmarinic3

Asian Journal of Traditional Medicinesacid in extracts of P. vulgaris consistently inhibited theinteraction between Lck SH2 and a peptide containingits consensus binding sequence at pYEEI. It was shownthat the inhibitory effect of rosmarinic acid was specificfor the SH2 domains of Src family protein tyrosinekinase. It was also found that rosmarinic acid inhibitedTCR-induced-Ca 2+ mobilization and IL-2 promoteractivation. Thus, this suggested the inhibition involvedthe membrane proximal site of TCR signaling andshowed that rosmarinic acid inhibited the expression ofcytokines, such as IL-2 and IFN-gamma [37] .Overall perspectivesThe above results provide ample evidence of theantiviral and immunomodulatory properties of P.vulgaris. Most importantly, its active components inaqueous extracts have been studied in detail and potentantiviral activity againt HIV, HSV and HSV mutantshave been demonstrated. Furthermore, severalcompounds like prunellin, tannins, polyphenols and itspolysaccharides exhibit strong inhibitory effects on HIVand HSV. The mechanisms of action may involvesuppression of the early or late stages of viral replication,integrase activity, blocking of surface glycoproteins andviral antigens. Interestingly, the immunomodulatoryeffects of P. vulgaris may act synergistically with itsanti-viral effects. The polysaccharide fractions of P.vulgaris could not only markedly stimulate theproduction of superoxide and nitrite, representing nitricoxide, from murine macrophage RAW264.7 and brainmacrophage BV2 cells, but also the protein levels ofinducible nitric oxide synthase (iNOS) and the mRNA ortumor necrosis factor alpha [35, 36] . The actions of P.vulgaris extracts on immune factors like histamine, IL-2,TNF-alpha, IgG, IgG1, IgG2b, nitric oxide, Src familyprotein kinase and T cell activation may be associatedwith specific cell targets during inflammatory andallergic actions. P. vulgaris exhibits significant dualproperties in the areas mentioned. These results confirmthat P. vulgaris is able to act as a new and effectiveantiviral agent with low toxicity. However, morein-depth studies on the chemical components of P.vulgaris need to be carried out to advance the search foreffective antiviral agents. Also, it is essential to carry outresearch on the co-administration of the active fractionswith currently used antiviral agents, a cocktail treatment,to examine their effect on HSV and HIV infections.Clinically, the extract of P. vulgaris has been developedand used in oral hygiene products for the control ofgingivitis and in eye drops for the treatment of keratitisdue to the herpes infection. Considering the differentpathways of transmission of viral infections, commonlythrough the mucosae (e.g. labial and genital herpes),novel topical agents with good antiviral andimmunomodulatory effects need to be developed toprevent sexually transmitted infections including HSVand HIV.To conclude, the combined properties of P. vulgarissuggest that it has great potential as a treatment for viralinfections as well as immunological disorders.References[1] World Health Organization. WHO Regional Publications,Western Pacific Series No.2, Medicinal Plants in China – Aselection of 150 commonly used species. 1989, P. 231[2] Namba T. The Encyclopedia of Wakan-Yaku (TraditionalSino-Japanese Medicines), vol. II. Hoikusha Publishing Co. Osaka,Japan. 1994. P.120-1[3] Shin TY, Kim YK, Kim HM. Inhibition of Immediate-typeallergic reactions by Prunella vulgaris in a murine model.Immunopharmacol Immunotoxicol 2001: 23(3): 423-35[4] Grieve M. A Modern Herbal. 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