Anwar H. Gilani ( Departments of Pharmacology, The Aga Khan University Medical College, Karachi. )
K.H. Janbaz ( Departments of Pharmacology, The Aga Khan University Medical College, Karachi. )
M. Zaman ( Departments of Pharmacology, The Aga Khan University Medical College, Karachi. )
A. Lateef ( Departments of Pharmacology, The Aga Khan University Medical College, Karachi. )
A. Suria ( Departments of Pharmacology, The Aga Khan University Medical College, Karachi. )
H.R Ahmed ( Departments of Physiology, The Aga Khan University Medical College, Karachi. )
Crude extract of Rubia cordifolia (PC) was tested In isolated tissue preparations for its possible calcium channel antagonislic activity. PC suppressed the spontaneous contractions of guinea- pig atria, rabbit jejunum and rat uterus in a concentration dependent manner (0.1-3 mg/ml). In rabbit aorta, It inhibited norepinephrine (10 uM) and KCl (80 mM) induced contractions. Replacement of physiological salt solution with calcium free solution abolished the spontaneous movements of rabbit jejunum. However, addition of calcium (25 pg/mi) In the tissue bath restored the spontaneous movements. When the tissues were pretreated with plant extract (1 mg/mi) or verapamil (0.5 ug/ml) addition of calcium failed to restore spontaneous contractions. These results Indicate that the plant extract exhibits spasmolytic activity similar to that of verapamil suggestive of presence of calcium channel blocker like constituent(s) in this plant (JPMA 44:82, 1994).
Rubia cordifolia Linn. (Family: Rubiacea; Synonmy: R Purpuria DC) locally known as “Manjit” is a perennial climbing herb growing wild in northern hilly areas of Pakistan1. The plant has been employed to dye cotton, silk and wooly garments since ancient time2,3. Dried roots are considered to be useful in alleviating dropsy, paralysis, jaundice, amenorrhoea and visceral obstructions4. Plant is also known to exhibit antineoplastic5, antibacterial6 and antiviral7 properties Moreover, it exerts uricolytic and nephrolytic8 activities and thus promotes disintegration as well as elimination of urinary stone9. Furthennore, itis used as an ingredient of a native recipe "Qurse-rewand" prescribed by traditional healers to cure chronic diseases and trauma of liver10. In an attempt to validate this traditional use of the plant, we tested crude extract of this plant against acetaminophen and CCI4-induced liver damage in animal model and found it hepatoprotective11 with diuretic activity (unpublished data). On the other hand, Ca++ + channel blocking drugs have recently been reported to exhibit hepatoprotective, as well as antilithiatic and diuretic properties12,13 in addition to their well established uses in cardiovascular disorders. It has recently been reported that Ca++ channel blockers are present in plants14,15. The aim of this study was therefore, to see if this plant also contains Ca++ channel blocker like activity which could explain in part some of the folldoric uses of the plant.
Materials and Methods
Preparation of crude extract
Rubia cordifolia roots were purchased from local herbal store and authenticated with the help of a botanist at The University of 2Xarachi. The plant material was powdered and macerated in 80% aqueous-methanol (BDH Ltd., Poole, England) for one week with occasional shaking. The extract was filtered and concentrated to dark reddish brown residue under reduced pressure on a rotary evaporator, with approximate 11% yield.
Drugs and animals
The following reference materials were obtained from the sources specified; norepinephrine hydrochloride, potassium chloride (Sigma Chem. Co., St. Louis, MO, USA) and calcium chloride (E. Merck, Darmstadt, P.R Germany). All drugs were dissolved in distilled water and dilutions were made fresh in normal saline (0.9% NaCl) on the day of experiment. Animals used in this study were housed in the Animal House of the Aga Khan University at 23-25°C and were given a standard diet and tap water ad libitum.
These experiments were carried out by the method previously used in our laboratory16,17.
Gunea-pigs of either sex (400-600g) were killed by cervical dislocation. Paired atria were removed carefully and mounted in a 20 ml tissue bath filled with Krebs-Henseleit solution maintained at 32°C and aerated with 5% carbon dioxide in oxygen. The composition of physiological salt solution was (mM): NaCl, 118.2; KCl, 4.7; MgSO4, 1.2; KH2 PO4 1.2;D-glucose, 11.7; NaHCO3, 25.0 and CaCl2, 2.5 (PH, 7.4). The spontaneous atrial contractions were recorded via an isotonic transducer (T3) coupled with Bioscience (PR200) chart recorder. Each preparation was allowed to equilibrate under ig resting tension for atleast 30 min before administration of any drug.
White rabbits (local breed) of either sex weighing 2-3 kg were killed by a blow to the back of the head. The descending thoracic aorta was quickly removed and cut into rings of 2-3mm width which were opened by cutting perpendicular to the axis of symmetry of the cylindrical vessel to make strips. Each strip preparation was mounted in a 20 ml tissue bath containing Krebs Henseleit solution, maintained at 37°C and continuously bubbled with a mixture of 95% oxygen and 5% carbon dioxide. A resting tension of2g was applied to each tissue and an equilibrium period of one hour was allowed before drug-induced changes in isometric tension of the strips were measured on Gould 3000 polygraph.
Young female rats (1 70-200 g) were killeci by a blow on the head. The middle 2 cm of the uterine horns were cut longitudinally and each strip was mounted in a 10 ml tissue bath containing Tyrode’s solution maintained at 37°C and gassed with a mixture of 5% of CO2 in oxygen. The composition of the Tyrode\\\'s solution was (mM):NaCl, 136.9; KCI, 2.7; MgCl2, 1.1; NaH2PO4, 0.4; NaHCO3,11.9; D-glucose,5.6; CaCI2,1.8(pH 7.4). The tissue was allowed to equilibrate under 0.5g basal tension for 20 min before isotonic contractions were recorded as described for atria.
White rabbits (2-3 kg) of either sex starved for 24 hours were killed by cervical dislocation and exsanguina tion. Segments of jejunum about 2 cm length were mounted in 20 ml tissue bath containing Krebs-Henseleit solution, maintained at 37°C and bubbled with a gas mixture of 95% 02 and 5% CO2. A preload of 1.Og was applied and spontaneous contractions were recorded isotonically via T-3 isotonic transducer on Bioscience MD4 recorder. The tissue were allowed to equilibrate for one hour before addition of any drug. The concentrations mentioned in the text or in the figures represent the final bath concentrations.
Plant extract at the concentration of 0.1-3 mg/nil, caused a concentration-dependent inhibition of spontaneous contractions of guinea-pig paired atria, rabbit jejunum and rat uterus as shown in Figure 1.
In jejunum and uterus plant extract at the concentration of 1 mg/nil, completely abolished spontaneous movements whereas in atrial preparation, 3 times greater concentration produced only 50% inhibitory response indicating that the plant extract is approximately 10 times more potent in smooth muscle preparations. This inhibitory effect on all free preparations was reversible as each tissue regained its spontaneous activity after washing the tissue a couple of times with the fresh bathing fluid. In rabbit aorta, both norepinephrine (10MM) and K+ (80 mM) caused maximal reproducible contractions and the tissue attained its resting state 20-30 minutes after wash out. Then tissue were pretreated with plant extract at different concentration (0.1- 1mg/kg) 30 minutes before redetermination of norepinephrlne or K+ responses.
As shown in Figure 2, plant extract inhibited norepinephrine or C-induced contractions in a concentration- dependent manner. The inhibitory effect of plant extract was similar against both spasmogens and was reversible after frequent washings. Plant extract was also tested in calcium free bathing fluid for its inhibitory effect on calcium supplementation in rabbit jejunum and the results were compared with that of verapamil (Figure 3).
When bathing fluid was replaced with the calcium free krebs solutions the spontaneous movements were abolished within one minute. Addition of calcium to the tissue bath restored spontaneous activity of the tissue in a concentration-dependent manner (15-25 ug/ml). However, addition of calcium failed to restore spontaneous movements of tissues which were treated with either plant extract (1 mg/mi) or verapamil (0.5 ,ug/ml) indicating blockade against calcium effect.
The plant extract suppressed the spontaneous movements of guinea- pig atria, rabbit jejunum and rat uterus thus showing its spasmolytic activity with approximately 10 times more potent on smooth muscle preparations than in cardiae tissue. The rabbit aorta preparation was used for the study of mechanism of spasmolytic action. Pretreatment of tissue with the plant extract inhibited aorta contractions induced by norepinephrine (10 uM) or K+ (8OumM). It has been shown that high K+ - concentrations cause marked contractions in blood vessels by depolarizing smooth muscle cells and increasing the influx of calcium through L-type voltage operated channels (VOC) 18. In contrast, contractions induced by norepinephrine and by other neurotransmitters is due partly to calcium release by intracellular stores and partly to influx of extraceilular calcium via receptor operated channels (ROC)18-20. In this study, plant extract inhibited contractions induced by 80 mM Kt This vasorelaxant effect is not due to opening of K+ channels because cromokalin and other C channel openers do not inhibit contractions induced by K+ concentrations greater than about 30 mM21,22. The fact that the inhibitory effect of plant extract against K+ induced and norepinephrine-induced contractions was observed at similar concentrations, suggests that plant extract acts intracellularly and/or on the cell membrane blocking calcium influx through voltage dependent and receptor operated channels in aortic tissue. The calcium blocking activity of plant extract was further confirmed when tested on spontaneous movements of rabbit jejunum suspended in calcium free solution. The spontaneous movements of the intestine is regulated by periodic depolarization and repolarization. At the height of depolarization, action potential appears as a rapid influx of Ca ++ + via VDCs23. As the plant extract inhibited the spontaneous movements of rabbit jejunum, it may be due to interference either with the depolarization process or with the Ca++ influx through VDCs. The spontaneous movements of rabbit jejunum were abolished when exposed to Ca++ + free solution, however, these movements were resumed on addition of Ca++ to the bathing fluid. Whereas, the same dose of calcium failed to restore such phasic movements when the tissue was pretreated with plant extract or verapamil rendering the added Ca++ unavailable to contractile elements in cytoplasm. Thus, hindrance to Ca++ influx via VDCs may be assigned to the chemical constituent(s) of Rubia cordifolia in a manner similar to verapamil, a standard Ca++ channel blocker24. The results of this study indicate that the crude extract of Rubia cordifolia produces direct depressant effects possibly through Ca++ channel blocking action. Since calcium channel blocking drugs are known to exhibit antilithiastic effect12,13 the presence of calcium channel blocker constituent(s) in Rubia cordifolia may be responsible for the folkloric use of this plant for disintegration of urinary stones9, though direct antilithiastic effects remain to be elucided.
This study was supported by funds provided by The National Scientific Research Development Board and The Aga Khan University.
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