Inflammopharmacology 17 (2009) 50–540925-4692/09/010050-5DOI 10.1007/s10787-008-8038-3© Birkhäuser Verlag, Basel, 2008

Inflammopharmacology

Abstract. The anti-pyretic activity of a standardized metha-nol/water (50/50) extract of Orthosiphon stamineus Benth. (SEOS) was investigated for its effect on normal body tem-perature and yeast-induced pyrexia in Sprague Dawley (SD) rats. The SEOS showed no effect on normal body tempera-ture. Doses of 500 and 1000 mg/kg body weight of SEOS significantly reduced the yeast-induced elevation in body temperature. This effect persisted up to 4 h following the ad-ministration of the extract. The anti-pyretic effect of SEOS was comparable with that of paracetamol (acetaminophen in U.S) (150 mg/kg p.o.), a standard anti-pyretic agent. HPLC study revealed that rosmarinic acid, sinensetin, eupatorin and tetramethoxyflavone were present in SEOS in the amounts of 7.58 %, 0.2 %, 0.34 % and 0.24 % respectively. The LD50 of the extract in rats was higher than 5000 mg/kg body weight. Therefore, the present study ascertained that SEOS possesses a significant anti-pyretic activity.

Key words: Orthosiphon stamineus – Antipyretic effect – Rosmarinic acid – Eupatorin – Sinensetin – 3’-hydroxy-5,6,7,4’-tetramethoxyflavone.

Introduction

Fever is a complex physiologic response triggered by infec-tions or aseptic stimuli. Elevation in body temperature occurs when the concentration of prostaglandin E2 (PGE2) increases within parts of the brain. Such an elevation contributes to a considerable alteration in the firing rate of neurons that con-

Short Communication

Evaluation of the anti-pyretic potential of Orthosiphon stamineus Benth standardized extractM. F. Yam1, 2*, L. F. Ang1, R. Basir2, I. M. Salman1, O. Z. Ameer1 and M. Z. Asmawi1

1 School of Pharmaceutical Sciences, University Sains Malaysia, 11800 Minden, Penang, Malaysia, e-mail: yammunfei@yahoo.com2 Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti, Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia

Received 10 July 2008; accepted 10 October 2008

Published Online First 26 December 2008

trol the thermoregulation process in the hypothalamus. It is now evident that most anti-pyretics exert their action by in-hibiting the enzymatic activity of cyclooxygenase and conse-quently reducing the levels of PGE2 within the hypothalamic region. Recently, other mechanisms of action for anti-pyretic drugs have been presumed. These mechanisms have shed the light on the ability of ani-pyretic agents to reduce pro-in-flammatory mediators, enhance anti-inflammatory signals at sites of injury or boost anti-pyretic messages within the brain (Simon, 1999; Flower and Vane; 2004; Aronoff and Neilson; 2001).

Orthosiphon stamineus Benth. (Lamiaceae) has tradi-tionally been used as a medicinal herb for many centuries in Southeast Asian countries like Indonesia and Malaysia. In Malaysia, this plant is used for management of fever. Moreo-ver, it is considered as a reputable remedy for catarrh of the bladder, and as treatment for various disorders such as ne-phritis, nephrolithiasis, hydronephrosis, vesical calculi, arte-riosclerosis, gout and rheumatism (Perry, 1980). In Malaysia, tea prepared from the O. stamineus leaves is consumed as a beverage to improve health and also for the treatment of kid-ney or bladder inflammation, gout and diabetes (Akowuah et al., 2005).

Reports have demonstrated that O. stamineus contains several active constituents such as terpenoids and polyphe-nols (Tezuka et al., 2000). Most of the therapeutic effects and health befits of O. stamineus have been ascribed mainly to its polyphenolic contents (Akowuah et al., 2005). The latter are the most dominant constituents of the plant’s leaves (Holl-man and Katan, 1999). Akowuah and colleagues have dem-onstrated the presence of rosmarinic acid (RA), sinensetin (SEN), eupatorin (EUP) and 3’-hydroxy-5,6,7,4’- tetrameth-oxyflavone (TMF) in the leaves of O. stamineus. Moreover, the RA contents of this plant were also reported by Tezuka et * Corresponding author

Vol. 17, 2009 Anti-pyretic effect of O. stamineus 51

al. (Tezuka et al., 2000). Lipophilic flavonoids isolated from O. stamineus have been found to possess a remarkable free radical-scavenging activity towards the diphenylpicrylhy-drazyl radical adding to their abilities to inhibit the enzyme 15-lipoxygenase from soybeans, a model for mammalian 15-lipoxygenase (Lyckander and Malterud, 1996). Researches have indicated that the flavones, SEN and TMF isolated from O. stamineus exhibit a potent diuretic activity in rats (Schut and Zwaving, 1996). The diterpenes, on the other hand, iso-lated from Orthosiphon sp. have been shown to exhibit anti-proliferative activities on rat thoracic aorta (Tezuka et al., 2000; Awale et al., 2001; Awale et al., 2002a; Awale et al., 2002b). Furthermore, studies on these diterpenes revealed their substantial capabilities to antagonize nitric oxide ac-tion (Awale et al., 2004) and inhibit the tumour promoter-induced inflammation of 12-O-tetradecanoylphorbol-13- ac-etate (TPA) in mouse ears (Masuda, 1992). Present studies have also shown that O. stamineus extract retains a notable hepatoprotective action against CCl4-induced hepatopathy (Yam et al., 2007), anti-inflammatory and non-narcotic anal-gesic activities (Yam et al., 2008) and remarkable hypoglyc-emic and anti-diabetic properties in normal and diabetic rats respectively (Sriplang et al., 2007).

The well-known traditional use of O. stamineus for fe-ver in Malaysia, the previous studies on this plant which re-ported the use of this herbal remedy to alleviate gout and rheumatism accompanying inflammatory processes (Perry, 1980), and the recent evidences that this plant possesses a significant anti-inflammatory and non-narcotic analgesic activities, conditions in which the prostaglandin pathway exert a considerable effect, have drawn attention toward for-mulating a hypothesis that O. stamineus could exert a clear anti-pyretic action if its effect is investigated and scientifi-cally proven using an animal model of fever and a standard experimental protocol. Accordingly, the aim of this study is to investigate and characterize the anti-pyretic effect of O. stamineus leaves extract in an attempt to support its tradi-tional use and further add to previous works on this plant in which prostaglandin pathway and inflammatory processes are thought to play a role.

Materials and methods

Materials and chemicals

Standard compounds of sinensetin, eupatorin, 3’-hydroxy-5,6,7,4’-te-tramethoxyflavone and rosmarinic acid were purchased from Indofine Chemical Co. (Hillsborough, NJ, USA). Solvents used for HPLC were tetrahydrofuran, methanol (HPLC grade) and water (HPLC grade) were obtained from Merck (Darmstadt, Germany). Yeast, methylcellulose and paracetamol (acetaminophen) were purchased from Sigma Chemical Co. (St. Louis, MO). Carboxymethylcellulose was purchase from Brit-ish Drug House (UK).

Experimental animals

Sprague Dawley (SD) rats of either sex weighing 180–200 g obtained from the Animal Care Facility of the School of Pharmaceutical Sci-ences, Universiti Sains Malaysia (USM) were used in this study. The animals were maintained at 28–30 ºC and allowed a free access to food (normal laboratory chow, Gold Coin) and tap water ad libitum. All the

experimental animals were fasted 14–16 h prior to the employment in any experimental protocol. The animals were acclimatized to laboratory conditions for seven days before commencement of experiments. The experiments were approved by The Animals Ethic Committee, USM.

Plants materials

Plants were grown from cuttings using standard agronomic practices at Kepala Batas, Penang, Malaysia. The leaves of O. stamineus were col-lected after they flowered. The plant was identified by a taxonomist and voucher specimens number 10106 and number 027 were deposited at the School of Biological Sciences and School of Pharmaceutical Sciences herbariums, USM respectively.

Oven-dried (45 ºC) and powdered leaves (150 g) of O. stamineus were extracted cold extraction procedure in which the leaves were mac-erated in a mixture of (5 l) water/methanol (50:50) and allowed to stand at room temperature for 3 days. The extract was concentrated on a ro-tary evaporator and subsequently lyophylized to solid material using a freeze dryer. The yield obtained from the leaves was found to be 6 % of SEOS. The extract was kept in tightly stoppered bottles and stored in a dessicator.

HPLC analysis of SEOS

HPLC analysis was performed using Agilent Technologies series 1100 system/series equipped with an automatic injector, a column oven, and a diode array UV detector. A LiChrosorb RP-18 (250 mm x 4.6 i.d. mm, 5 μm particle size) (Merck Darmstadt, Germany) was used. The tem-perature was maintained at 25˚C, with injection volume of 20 μl and flow rate of 1 ml/min. All the markers were separated using reverse-phase LiChrosorb C-18 column with the methanol-water-tetrahydro-furan (45:50:5 v/v) mobile phase. The peaks were detected at 340 nm and identified using standard substances [RA, SEN, EUP and TMF] (Akowuah et al., 2005).

Toxicity study

Acute toxicity relating to the determination of LD50 value was performed with a range of doses of the SEOS according to the method described by Ghosh (1984).

Effect on normal body temperature

Rats of either sex were divided into five groups (n = 6). The body tem-perature of each rat was measured rectally at predetermined intervals be-fore and for 5 h after oral administration of either 1 % w/v carboxymeth-ylcellulose (control) or SEOS at doses of 125, 250, 500 and 1000 mg/kg body weight, orally (Murugesan et al., 2000).

Induction of yeast-induced pyrexia

The yeast-induced pyrexia model was used to investigate the anti-pyretic activity of the extract. The rats were divided into six groups (n = 6). The body temperature of each rat was recorded by measuring rectal tempera-ture at predetermined time intervals. Fever was induced according to the method described by Smith and Hambourger (1935). An electronic thermometer (Omron MC 101, USA) was inserted 3–4 cm deep into the rectum. After measuring the basal rectal temperature, animals were given a subcutaneous injection of 10 ml/kg of 15 % w/v yeast suspended in 0.5 % w/v methylcellulose solution and then the rats were returned to their housing cages. After 19 h of yeast injection, the rats were again restrained in individual cages for the recording of their rectal tempera-ture. The SEOS was administered orally at doses of 125, 250, 500 and 1000 mg/kg body weight, to four groups of rats respectively. The fifth group, on the other hand, received 10 ml/kg of 1 % w/v carboxymethyl-

52 M. F. Yam et al. Inflammopharmacology

cellulose as control. Finally, the sixth group was given 150 mg/kg body weight of paracetamol as control. Eventually, rectal temperature of all the rats was recorded at the 19th h, immediately before extract, vehicle or paracetamol administration, and again at 1-hour intervals up to the 23rd h, after yeast injection.

Statistical analysis

Data are expressed as mean ± S.E.M. Statistical significance was ana-lyzed using Two Way ANOVA followed by LSD multiple comparison test, SPSS version 10.0.

Results and discussion

HPLC showed that all the markers (Figure 1a) were present in SEOS (Figure 1b). The percentage of RA, SEN, EUP and TMF in SEOS were 7.58 %, 0.2 %, 0.34 % and 0.24 % re-spectively (Table 1). These percentages were employed to calculate the quantity of each of these constituents in terms of the amount delivered to the rat per dose (Table 3). Effects of the SEOS on normal body temperature in rats were pre-sented in Figure 2. It was found that the SEOS did not influ-ence the basal body temperature.

The subcutaneous injection of yeast suspension markedly elevated the rectal temperature after 19 h of administration. Treatment with SEOS at doses of 500 and 1000 mg/kg body weight significantly decreased the rectal temperature of the rats (Figure 3). The anti-pyretic effect of SEOS started as ear-ly as 20 h (1 hour after SEOS administration) and the effect persisted for 4 h after its administration. Yeast-induced fever is a pathogenic type of fever, the etiology of which includes

Table 1. Percentage of reference compounds in SEOS. SEOS = Stand-ardized leaf extract of O. stamineus Benth.

Compound Percentage present in SEOS

Rosmarinic acid 7.58

Sinensetin 0.2

Eupatorin 0.3

3’-hydroxy-5,6,7,4’-tetramethoxyflavone 0.24

Fig. 1. (a): HPLC chromatogram of standard markers. Peaks: RA (ros-marinic acid), TMF (3’-hydroxy-5,6,7,4’-tetramethoxyflavone), SEN (sinensetin) and EUP (eupatorin). (b): HPLC chromatogram of SEOS. Peaks: RA (rosmarinic acid), TMF (3’-hydroxy-5,6,7,4’-tetramethoxy-flavone), SEN (sinensetin), EUP (eupatorin).

Fig. 2. Effect of SEOS on normal body temperature in rats. Each value represents mean ± S.E.M. (n = 6). Control (1% carboxymethylcellulose). SEOS: Standardized leaf extract of O. stamineus Benth.

Fig. 3. Effect of SEOS on yeast-induced pyrexia in rats. Each value rep-resents mean ± S.E.M. (n = 6). Control (1% carboxymethylcellulose). *P < 0.05, **P < 0.01, ***P < 0.001 significant as compared to control values at corresponding hour. SEOS = Standardized leaf extract of O. stamineus Benth.

production of prostaglandins, known mediators that set the thermo-regulatory center at a lower temperature (Howard, 1993). So inhibition of prostaglandin synthesis could be the possible mechanism of anti-pyretic action of the SEOS, an effect that is comparable to that of paracetamol (Graham and Scott, 2005). Morimoto et al. (1998) suggested that there are several mediators or multi-processes underlying the patho-genesis of fever. Inhibition of any of these mediators may bring about antipyresis; however, further studies need to be carried out to determine its exact mechanism of action.

Previous work has demonstrated that RA has potential anti-pyretic action (Kurokawa et al., 1998), a key constituent

Vol. 17, 2009 Anti-pyretic effect of O. stamineus 53

of SEOS which was displayed in considerably large amount as compared to the other polyphenols based on the HPLC profile and therefore, to which the anti-pyretic action of SEOS can be attributed.

The fact that neither toxicity nor lethality was observed at any dose of the extract explains the wide safety margin of SEOS within the doses range (Table 2). This observation also hints that the LD50 of the extract is much higher than the highest dose employed (Abdullah et al., in press). The present study reveals that the leaves extract of O. stamineus Benth. show a significant anti-pyretic effect in yeast-induced eleva-tion of body temperature. The results of this investigation support the traditional use of the plant in reducing fever.

Acknowledgements. This study was supported by an Intensifying Re-search Priority Areas (IRPA) grant from the Ministry of Science, Tech-nology, and Environment.

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Table 2. Toxicity study of SEOS in rat. SEOS= Standardized leaf extract of O. stamineus Benth.

Treatment Dose (mg/kg body weight) No. of animals No. of survivors No. of deaths LD50 value

Control 10 ml/kg of 1% w/v carboxymethylcellulose

10 10 0 -

SEOS 313 10 10 0 -

625 10 10 0 -

1250 10 10 0 -

2500 10 10 0 -

5000 10 10 0 > 5 g/kg

SOES dose (mg/kg) Amount delivered per dose (mg/kg)

RA SEN EUP TMF

125 9.5 0.25 0.43 0.3

250 19 0.5 0.86 0.6

500 38 1 1.72 1.2

1000 78 2 3.44 2.4

Table 3. Amount of compounds in SEOS delivered per dose.

54 M. F. Yam et al. Inflammopharmacology

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