PHYTOTHERAPY RESEARCH, VOL. 9,452-454 (1995)

SHORT CO MM U NlCA TI0 N Hypotensive Action of Syringin from Syringa vulgaris

Mansoor Ahmad* and Khalid Aftabl- Department of Pharrnacognosy, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan t HEJ Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan

Naturally occurring syringin was isolated from the bark of Syringa uulguris (Oleaceae), and its pharmacological action examined on anaesthetized Wistar rats. When the drug was introduced intravenously, it caused a dose dependent fall in systolic, diastolic and mean arterial blood pressure, whereas heart rate also decreased at a slightly higher dose. The hypotensive activity was not inhibited by antihistamine or antimussarinic agents. Syringin has no effect on the pressor effect induced by norepinephrine or carotid occlusion.

Keywords: syringin; hypotensive; Syringa vulgaris; NMR.

INTRODUCTION

Many therapeutic agents in current use have their prototype derived from natural origin. In this commu- nication we describe the antihypertensive activity of naturally occurring syringin from Syringa vulgaris. In European folk medicine the fruits and bark of S. uulgaris were used as a tonic and astringent, whereas fresh leaves were employed in the treatment of malaria (Hegi, 1926). The present research work was carried out with the objective of isolating the major active constituents of this plant which may provide an expla- nation for the folk uses.

MATERIALS AND METHODS

Plant material. The bark of Syringa uulgaris (an orna- mental plant) was collected near Zurich, Switzerland and was dried in a hot air oven. The plant material was identified by Professor Dr 0. Sticher, Department of Pharmacy, ETH-Zurich, Switzerland, and a voucher herbarium specimen was deposited in the Department of Pharmacy, ETH-Zurich.

Chromatography. For TLC, silica gel FP254 type 60 (Merck, Germany) and for column silica gel 60 particle size 0.063-0.2OO mm (70-230 mesh ASTM, Merck, Germany) was used. The spray reagent was vanillin-H,SO, (1 % vanillin in concentrated H2S04) and after development was heated at 120 "C in a hot air oven for 5min. UV spectra were recorded on Perkin-Elmer Spectrophotometer model 550 and IR spectra with KBr pellets on Perkin-Elmer grating IR- Spectrophotometer model 257. 'H-NMR: Varian spec- trograph model H-100 (100 MHz). I3C-NMR: Bruker Spectrospin (75.47). Mass spectrometer: Hitachi

Author to whom correspondence should be addressed.

Perkin-Elmer RMU 6M. For blood pressure a pressure transducer was used, model P23 ID, Gould Statham Instruments Inc. (Hato Rey, Puerto Rico), tachograph (Grass Instruments Co., Quincy, MA, USA), Grass polygraph model 7D (Grass Instruments Co., Quincy, MA, USA).

The following reference materials were obtained from the sources specified: acetylcholine chloride (Sigma), atropine sulphate (Sigma), chlorophenira- mine maleate (Sigma), norepinephrine (Sigma) and pentothal sodium (Abbott Laboratories, Pakistan).

Isolation and identification. Dried bark was chopped into small pieces and extracted with methanol at 40 "C under reflux. The extract was concentrated in uacuo, water was added and the insoluble material was removed by filtration through celite. The filtrate was extracted with petroleum ether and the soluble part was discarded. The aqueous phase was concentrated and lyophilized to give crude glycosides (30g). This crude extract was chromatographed on silica gel column using methylene chloride-methanol-water in the ratio of 80 : 20: 2 and 70: 30: 3 as eluent. The impure fractions were further chromatographed and pure compounds were isolated. One fraction of column chromatography gave a compound which was crystallized in pure metha- nol. Later this was identified as syringin (Chaudhuri and Sticher, 1981; Yoshizawa el a l . , 1990). The descrip- tion of syringin is: molecular formula CL7H2.,09, mole- cular weight 372, melting point 191.9 "C, [a]g -22.3 " (C = 0.80; MeOH), UV max. in MeOH: 220, 264 nm, IR (KBr): 3560 (free OH), 3380 (br. OH), 1650 ((+ C), 1510 and 1465 (aromatic ring) cm-'.

1 CHZOH I

CCC 0951-418X/95/060452-03 0 1995 by John Wiley & Sons, Ltd Accepted (revised) 4 June 1994

HYPOTENSIVE ACTION OF SYRINGIN

BP MHK loo

0

453

1.0 4.0 m w

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In viuo experiments. Wistar rats of either sex (200-250 g) were anaesthetized with sodium pentothal (40 mg/kg, intraperitoneal injection). The trachea was exposed and cannulated with a polyethylene cannula to facilitate spontaneous respiration. Drugs and syringin (dissolved in distilled water) were slowly injected via the cannula inserted into an external jugular vein. The maximum volume of injection was 0.2mL. The systemic blood pressure was recorded from the carotid artery via the arterial cannula connected to a pressure transducer (model P23 ID, Gould Statham Instruments Inc., Hato Rey, Puerto Rico) and the heart rate was recorded using a tachograph (Model 7P 44C, Grass Instrument Co., Quincy, MA) driven by the blood pressure waves. These were displayed on a Grass Polygraph model 7D (Grass Instruments Co., Quincy, MA). An equilibrium period of 30 min was allowed before administration of any drug (McLeod et al., 1970).

The mean arterial blood pressure values (MABP) were measured in millimeters of mercury (mmHg) and were calculated by the following formula (Steinmetz ef a f . , 1990):

Systolic BP - Diastolic BP 3 MABP = Diastolic BP +

Responses were taken as O/O change in control values obtained immediately before the addition of test sub- stances. Results are expressed as mean ? standard error of means (SEM). Student's t-test was used to test the significance in difference of means.

O ' 0.5 1.0 2.0 4.0 mg/kg

Dose Figure 2. Dose-dependent effect of syringin on blood pressure and heart rate.

al., 1989), atropine (an antimuscarinic agent) and chloropheniramine (an antihistamine agent) on the hypotensive effect of syringin were investigated. The syringin was given 5 min after the administration of atropine (2 mg/kg) or chloropheniramine (10 mg/kg) which were demonstrated to the block effect of acetyl- choline (1 pg/kg) and histamine (1 pglkg), respectively, did not effect the hypotensive response of syringin (Fig. 3). Each experiment was repeated three times (n = 3).

DISCUSSION

RESULTS Natural product chemistry research work has already been published on S . oulgaris but few reports are available on the pharmacological aspects. Prior to our investigation Balinet (1971) reported the analgesic and antiphlogistic properties of the crude extract, obtained from the leaves of S . oulgaris. The natural products reported from this plant are iridoid, secoiridoid, flavo- noids, phenylpropanoids and lignans (Sticher et al. , 1982; Ahmad, 1985; Ahmad and Salama, 1987; Ahmad et a f . , 1987; Boros and Stermitz, 1990, 1991). The aim of our study was to explore the pharmacological behav- iour of natural products, and in this connection we have already reported the hypotensive action of two pure compounds, rutin and kaempferol-3-O-rutinoside, iso- lated from the fresh flowers of Syringa uulgaris (Ahmad et al., 1993).

The present findings demonstrate that syringin pos- sessed a marked hypotensive action in anaesthetized Wistar rats and this also confirms the hypotensive action of crude extract of leaves of S . vulgaris in anaesthetized cats (Balinet, 1971). This compound, in

10 mylrs

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carotid occlusion

Bulmin 2.70 I

Figure3. Syringin shows no effect on the pressor effects induced by carotid occlusion in anaesthetized rats.

454 M. AHMAD AND K. A R A B

Table 1. Effect of syringin on mean arterial blood pressure in anaesthetized rats

Dose (mglkgl Number of Change in MBP body weight experiments (rnm Hg)+SEM

0.5 3 12.30 k 2.33 1 .o 4 20.10+1.91 2.0 5 29.80 + 1.66 4.0 4 43.71 f 2.24

intravenous doses ranging from 0.5-4.0 mg/kg, low- ered the blood pressure in a dose dependent manner with an apparent change in heart rate (Fig. 2). The hypotensive effect appeared immediately after the injection of substances (Fig. l ) , reaching a maximum within 1 min and was followed at 3 min by full recovery. Table 1 illustrates the percentage effect of the four doses assayed.

It is also interesting to note that the hypotensive effect of syringin was not affected by chlorophenira- mine (antihistamine) or atropine (antimuscarinic). Therefore, it would appear that the syringin exhibited hypotension by an action not mediated through hista- minic and muscarinic receptors. In addition a mecha- nism such as a-adrenoceptor blockade, or an interfer- ence with sympathetic transmission can be excluded (Ghosh, 1984), since the syringin did not block the pressor effect of norepinephrine or the reflex pressor response to bilateral occlusion of the common carotid arteries (Fig. 3).

Considering the structure-activity relations it has been observed in the experiments that the efficacy/ action of drugs always increases upon the decrease in number of hydroxyl group in the aromatic ring. For example, comparing the hypotensive effect, rutin (two OH in aromatic ring) showed no activity at any dose and kaempferol-3-0-rutinoside (one OH in aromatic

ring) showed a better activity (41.60% f 3.62% at 10mg/kg dose) than rutin (Ahmad et al . , 1993; Yildizoglu-Ari et al . , 1991; Funayama and Hikino, 1981). Syringin is a caffeoyl derivative and has two methoxy groups at C-3’ and C-5’ and a glucose moiety at C-4’ position in the aromatic ring. So in comparison to the aromatic ring of rutin and kaempferol-3-0- rutinoside, it has no hydroxyl group, and its hypoten- sive activity is relatively much higher (43.71% f 2.24% at 4 mg/kg dose) than both.

Evidence in support of this theory can be obtained from the report of Izzo et al. (1994). In this report is is stated that quercetin, rutin (two O H in aromatic ring) showed an inhibitory effect of 73.6% and 76.3% at 50 mg/kg dose, i.p., respectively while kaempferol and naringin (one OH in aromatic ring) showed 47.3% and 2.6% at 100 mg/kg dose, i.p., respectively. The hyd- roxyl groups of the aromatic ring are active in stomach ulcer and not in blood pressure.

From the above findings it can be postulated that the hypotensive action of those drugs, not mediated through histaminic and muscarinic receptors and hav- ing an aromatic ring in the structure, is dependent on the occurrence of an O H group in the aromatic ring. Further pharmacological research work on the chemi- cal constituents of S. vulgaris is in progress.

Acknowledgement

One of the authors (M.A.) is grateful to the Swiss Federal Scholarship Committee for a scholarship and financial support in the isolation and structure elucidation of the chemical constituents. He is also thankful to Professor Dr 0. Sticher, Department of Pharmacy, ETH-Zurich, Switzerland, for h is help and valuable suggestions. We are very grateful to Professor Dr Attaur Rehman and Dr A. Gilani for permission to carry out pharmacological work at their laboratory, HEJ Research Institute of Chemistry, University of Karachi, Karachi, Pakistan.

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