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Sci..Int.(Lahore),24(2),133-138,2012 ISSN 1013-5316; CODEN: SINTE 8 133A SURVEY OF THE CHEMICAL CONSTITUENTS ANDBIOLOGICAL ACTIVITIES OF ORTHOSIPHON STAMINEUS*Yen Chin Koay and Faheem AmirSchool of Chemical Sciences, Universiti Sains Malaysia, Penang 11800, Malaysia(*yckoay.86@gmail.com)ABSTRACT: For centuries, Orthosiphon stamineus Benth, a native plant to Southeast Asia, has beenextensively used as a source of medicinal agents due to its wide range of medicinal properties. It isusually consumed as a herbal tea and it was believed to be useful in the treatment of diabetes, fevers,high blood pressure and bone or muscular pain, hypertension, kidney stone as well as promotinghealth and well being. The plant has also been reported to possess various novel secondarymetabolites such as diterpenes, flavones and triterpene. This review summarizes the phytochemicalinvestigations and pharmacological properties of O. stamineus.Keyword: Orthosiphon stamineus, diterpenes, flavones, antibacterial, anti-inflammatory1. INTRODUCTIONOrthosiphon stamineus Benth (Lamiaceae) [syn: O. aristatus(B1) Miq., O. grandiflorus Bold., O. spicatus (Thumb)] orlocally known as ‘‘cat whisker”, is a medicinal herb whichhave ethnomedicinal applications in South East Asia,particularly Malaysia and Indonesia. Historically, this herbwere used as a traditional folk medicine for the treatment ofdiabetes, edema, epilepsy, eruptive fever, gallstones,hepatitis, hypertension, renal stones, as well as rheumatism[1-3]. An infusion of the leaves has been reported to possessdiuretic, anti-fungal, anti-inflammatory, anti-hypertensiveand antibacterial properties [4-7]. The leaves are also foundto possess significant antioxidant activity and the lipophilicflavonoids from the plant were found to prevent theoxidative inactivation of 15-lipoxygenase [8-9].2. Isolation and chemical investigationThe earliest phytochemical investigation on O. stamineusstarted in 1989, when Guerin et al. isolated an oil, namely,methylripariochromene A from the plant [10].Phytochemical investigation of the dry leaves of O.stamineus has led to the isolation of two novel highlyoxygenated pimarane diterpenes, orthosiphols A ( 1), B ( 2)(Figure 1) [11]. Takeda et al. reported the presence of twonew diterpenes, orthosiphols D (3) and E (4), together withseven known compounds, namely, rosmarinic acid,sinensetin, scutellarein tetramethyl ether, salvigenin,orthosiphols A and B [12]. Four novel highly oxygenatedisopimarane-type diterpenes, namely, siphonols A-D ( 5-8)and a novel biogenetically interesting norisopimarane-typediterpene named siphonol E ( 9) were isolated from O.stamineus of Indonesia [2].Olah et al. revealed the presence of caffeic acid, cichoricacid rosmarinic acid, sinesetine and patorine from twodifferent O. stamineus tinctures [6]. Phytochemicalinvestigations of the aerial parts of Taiwanese O. stamineusby Nguyen et al. reported the isolation of two newstaminane-type diterpenes, staminol C ( 10) and D ( 11)(Figure 2), three new isopimarane-type diterpenes identifiedas orthosiphonone C ( 12) and D ( 13), and 14-deoxo-14-OacetylorthosipholY ( 14), along with the presence of 16known diterpenes, orthosiphols A, B, D, K, M, N, O, X, andY, nororthosiphonolide A, neoorthosiphol B, orthosiphononeA, secoorthosiphol B and C, 3-O-deacetylorthosiphol I, and2-O-deacetylorthosiphol J [13].Akowuah et al. and Loon et al. reported the presence ofthree methoxylated flavones, namely, sinensetin, eupatorinand 3’-hydroxy-5,6,7,4’-tetramethoxyflavone from themethanolic leaves extracts of O. stamineus of Malaysia.Akowuah et al. also reported the presence of rosmarinic acidfrom the leaves [9,14].Hossain & Ismail isolated a new lupene-type triterpene,namely 16β-hydroxybetulinic acid (15) (Figure 3) from theleaves of O. stamineus [15]. Hossain et al. isolated apolyhydroxyflavone, namely, 5,7,3',4'-tetrahydroxy-thoxyflavone from the leaves of O. stamineus [16].

134ISSN 1013-5316; CODEN: SINTE 8Sci..Int.(Lahore),24(2),133-138,2012Figure 2. Compounds 10-14Figure 1. Compound 1-9Figure 3. Compound 15The examination of the hydrodistilled essential leaves andstems oils of O. stamineus revealed the presence of β-caryophyllene, α-humulene, β-elemene, 1-octen-3-ol, β-bourbonene, β-pinene, caryophyllene oxide, camphene andlimonene as major components which were analysed by GC-MS [17]. Mohamed et al. isolated orthosiphol A from theextract of O. stamineus leaves using the reverse phase highperformance liquid chromatography (RP -HPLC) with UVdetection [3].3. Phytochemical investigations with bio‐activityThe presence of five new isopimarane-type diterpenes[orthosiphols F-J (16-20)] (Figure 4) and two new diterpenes[staminols A (21) and B (22)], as well as three new highlyoxygenatedstaminane-type diterpenes [staminolactones A(23) and B (24) and norstaminol A (25)] were reported fromthe methanol extract of of O. stamineus [18-20]. 16 known

Sci..Int.(Lahore),24(2),133-138,2012 ISSN 1013-5316; CODEN: SINTE 8 135compounds identified as 7, 3’, 4’-tri-O-methylluteolin,eupatorin, sinensetin, 5-hydroxy-6, 7, 3’, 4’-tetramethoxyflavone, salvigenin, ladanein,tetramethylscutellarein, 6-hydroxy-5, 7, 4’-trimethoxyflavone,vomifoliol, aurantiamide acetate, rosmarinic acid,caffeic acid, oleanolic acid, ursolic acid, betulinic acid andβ-sitosterol were also isolated from the methanol extract ofO. stamineus [19-20]. Tezuka et al. reported that all thecompounds isolated, except for orthosiphol I, 7, 3’, 4’-tri-Omethylluteolin,eupatorin, ladanein and 6-hydroxy-5, 7, 4’-trimethoxy-flavone exhibited a promising cytotoxic effect onhighly liver metastatic murine colon 26-L5 carcinoma cells[20].Figure 4. Compounds 16-25Awale et al. also isolated six new highly oxygenatedisopimarane-type diterpenes, namely, orthosiphols U-Z (26-31) ( Figure 5) together with 15 previously reportedditerpenes. All the isolated diterpenes indicated significantdose-dependent inhibitory effects on the nitric oxide (NO)production in lipopolysaccharide (LPS) -activatedmacrophage-like J774.1 cells [21].Hossain & Ismail isolated two new prenylated flavonoidderivatives, namely, 5,7,3',5'-tetramethoxy-8-C-prenylflavone(32) (Figure 6) and 5,7,3',5'-tetramethoxy-6-Cprenylflavone(33), along with four known flavonoids5,6,7,3',4'-pentamethoxyflavone,5-hydroxy-6,7,3',4'-tetramethoxyflavone, ladanein and 6-hydroxy-5,7,4'-trimethoxyflavone isolated from the leaves extracts of O.stamineus. The two new prenylated flavones, together withthe four known flavonoids displayed a promising cytotoxiceffect on liver metasttatic murine colon 26-L5 carcinomacells with an ED 50 value between 10-90 μg/mL [22].

136ISSN 1013-5316; CODEN: SINTE 8Sci..Int.(Lahore),24(2),133-138,2012Figure 5. Compounds 26-314. Biological activities4.1 Adenosine A1 receptor binding assayYuliana et al. isolated seven methoxy flavonoids from amethanol-water extract of O. stamineus leaves through thebioassay-guided fractionation using adenosine A1 receptorbinding assay, whereby the adenosine A1 receptor bindingwere associated with the diuretic action in the treatment forrenal lithiasis. The findings of this study thus further validatethe traditional use of O. stamineus for kidney stonestreatment [23].4.2 Antibacterial activityThe studies conducted by Ho et al. showed that thepromising antibacterial of O. stamineus extracts againstselected food-borne bacteria in vitro were attributable to thepresence of rosmarinic acid in the plant [24].Figure 6. Compounds 32 and 334.3 Anti-hyperglycemic effectInvestigations were carried out to evaluate the effect ofleaves extract of O. stamineus on streptozotocin (STZ) -induced diabetic rat and the chloroform fraction 2 (Cf2) wasfound to significantly reduced the blood glucose level onglucose-loaded fasting rats of 150 mg/kg body weight [3].4.4 Antihypertensive activityThe oral administration of O. staminensis together with thecombination of policosanol, red yeast rice extract, berberine,folic acid and coenzyme Q 10 have indicated an effectivecontrol of high blood pressure in patients with metabolicsyndrome, thus may be served as a antihypertensive agent[25].4.5 Anti-inflammatory activityMasuda et al. reported the isolation of orthosiphol A and Bshowed strong inhibitory activity against the inflammationinduced by a tumor promoter, TPA (12 -Otetradecanoylphorbol-13-acetate)on the ears of genetargetedmice [11].4.6 Antioxidant activityFree-radical scavenging activity and superoxide inhibitionactivity of the methanol extract of O. stamineus wereexamined using the DPPH radical method and nonenzymePMS/NADH-NBT systems and the methanol extract wasfound to exhibit comparable antioxidant activity than purequercetin and higher antioxidative potency than the syntheticantioxidant butylated hydroxylanisole (BHA) [26-27].Yam et al. and Ho et al. reported that the O. stamineusextracts possessed high high antioxidant activity of O.stamineus extracts determined by the DPPH free radicalscavenging assay, whereby Ho et al. [24] suggested the

Sci..Int.(Lahore),24(2),133-138,2012 ISSN 1013-5316; CODEN: SINTE 8 137presence of high concentration of rosmarinic acid in the O.stamineus extracts were associated with the high antioxidantactivity of O. stamineus extracts [24,28].4.7 Anti-proliferative activitySahib et al. reported the methanolic extract of O. stamineuswhen combined with Tamoxifen (TMX), an antagonist ofthe estrogen receptor (ER+) in breast tissue, further enhancethe anti-proliferative activity of TMX against MCF-7hormone responsive breast cancer cells in vitro as comparedto the activity of singly treated TMX [29].4.8 Anti-pyretic activityAnti-pyretic effect of the standardized methanol/water(50/50) extract of O. stamineus were investigated at doses of500 and 1000 mg/kg body weight using yeast-inducedpyrexia in Sprague Dawley (SD) rats. Administration of theextract produced significant reduction in the bodytemperature of the yeast-induced pyrexia rats. Thus theresults in the study suggested the extract of O. stamineuspossessed potent any-pyretic properties [30].4.9 Antitumor and antiangiogenic activityAbdul Majid et al. reported that phytochemical andpharmacology components comprising standardized O.stamineus leaf extract was found to prevent or treat diseasesassociated with angiogenesis and maglignant tumors. Thecomponents with the presence of standardized O. stamineusleaf extract may also comprised components that possessanti-angiogenic, anti-cancer, anti-inflammatory and antiagingproperties [31].Ahamed et al. also investigated antitumor effect of theethanol extract of O. stamineus leaves athymic mice bearingcolorectal tumor and antiangiogenic activity wasinvestigated by measuring the tube formation of humanumbilical vein endothelial cells (HUVECs). Administrationof the extract at 100 mg/kg and 200 mg/kg, significantlysuppressed the tumor growth by 47.62 ± 6.4% and 83.39 ±4.1%, respectively. The expression of vascular endothelialgrowth factor (VEGF) in vitro along with in vivo weresignificantly inhibited at 211 ± 0.26 pg/mL cell lysate and90.9 ± 2 pg/g tissue homogenate when compared to thecontrol (378 ± 5 and 135.5 ± 4 pg, resp.) at 200 mg/kg. Theresults further validate the use of O. stamineus for treating avariety of angiogenesis-dependent diseases [32].4.10 Cardioprotective activityThe cardioprotective studies methanol extract of O.stamineus leaves based on the biochemical parameters inisoproterenol induced myocardial infarction in ratssuggested methanol extract of O. stamineus leaves protectedmyocardium from isoproterenol-induced myocardialfunctional injury via normalization levels of diagnosticmarker enzymes. This study indicated O. stamineus has thepotential to be used as natural cardioprotective agent [33].4.11 Diuretic and hypouricemic activitiesThe study of diuretic and hypouricemic activity of differentO. stamineus methanol extracts were conducted by Arafat etal. using Sprague-Dawley rats model. The infusion of asingle dose (2 g/kg) of methanol and methanol : water (1:1)extracts possesses important diuretic action, where the effectis quantitatively similar to the control, hydrochlorothiazide.Repeated administration of 0.5 g/kg of methanol : water(1:1) extracts showed an increase in diuresis from the thirdday of treatment. Oral administration of 0.5, 1.0 and 2.0 g/kgof methanol : water (1:1) extracts significantly reduced theserum urate level in hyperuricemic rats at hour 6, wherebythe decrease in the uric acid level was also observed for thestandard, allopurinol at hour 6. These results suggested thepotency of O. stamineus methanol : water extracts towardsdiuretic property and hypouricemic activity [34].4.12 Inhibiion of CYP enzymesPan et al. examined the effects of four O. stamineus extractsand three isolated constituents sinensetin, eupatorin androsmarinic acid on on major human cDNA-expressedcytochrome P 450 (CYP) enzymes. Three substrate -probebased HPLC (high -performance liquid chromatography)assays were established to serve as activity markers forCYP2C9, CYP2D6 and CYP3A4. The extracts and isolatedconstituents showed differential modulatory effects ondifferent CYPs and none of isolated constituents showed anysignificant inhibition on CYP2C9, eupatorin stronglyinhibited CYP2D6 activity with a K i value of 10.2 μM,while CYP3A4 was moderately inhibited by the petroleumether and dichloromethane extracts with K i values of 44.9and 93.7 μg/mL, respectively. The activity was found to bedue to the presence of eupatorin in the extracts. CYP3A4was also strongly inhibited by eupatorin alone, with a K ivalue of 9.3 μM. Thus it was suggested that coadministrationof products based on O. stamineus extractswith high eupatorin content, with conventional drugs mayhave the potential to cause drug-herb interactions involvinginhibition of major CYP enzymes [35].4.13 In vitro antimycobacterial activityHussain et al. investigated the antimycobacterial activity ofthe aqueous extract as well as the fractions of O. stamineusand evaluated the potential interaction of the plant withisoniazid (INH). The aqueous extracts, hexane fraction,chloroform fraction, ethyl acetate fraction indicatedminimum inhibitory concentrations of 25.00, 3.12, 6.25 and0.39 μg/mL, respectively. The results showed there was nostatistically significant interaction being observed for thecombinations of the extracts along with the fractions withINH in various proportions, thus no significant interaction ofbetween the aqueous extract and INH was observed [36].4.14 Nephroprotective activityKannappan et al. have evaluated nephroprotective activity ofO. stamineus benth extracts using rat model. Gentamycin, anextensively used aminoglycoside antibiotic, has beenreported to produce nephrotoxicity even at normaltherapeutic dose level. Oral administration of the drug at adose of 80 mg/kg weight for 9 days showed markedglomerular, peritubular and blood vessel congestion asindicated by the histopathological sections. Administratedmethanolic extract of O. stamineus at 100 and 200 mg/kgbody weight doses in rats and found a remarkable decreasein serum creatinine, blood urea, urinary protein levels andreduced the extent of renal damage [37].4.15 5-α reductase activityVogelgesang et al. indicated the presence of O. stamineusleaf extract as a main ingredient in cosmetic products canreduced the skin imperfection associated to excessive sebumsecretion. O. stamineus leaf extract was also found topossess the ability to reduce 5-α reductase, particular type 1isoenzyme in normal human epidermal keratinocytes in vitroand when applied to ex vivo skin reduces 5-α reductase

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