American Journal of Biochemistry and Biotechnology 6 (3): 231-238, 2010 ISSN 1553-3468 2010 Science Publications Uterus-Relaxing Study of a Sudanese Herb (El-Hazha)
1Aimun Abdelgaffar Elhassan Ahmed, 1Robert Gaspar, 1Arpad Marki, 3Andrea Vasas, 2Mahmoud Mudawi Eltahir Mudawi, 3Judit Hohmann and 1George Falkay 1Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy, University of Szeged, H-6720 Szeged, Eotvos u. 6, Hungary 2Department of Therapeutics, Faculty of Pharmacy, Omdurman Islamic University, Sudan 3Department of Pharmacognosy, Faculty of Pharmacy, University of Szeged, H-6720 Szeged, Eotvos u. 6, Hungary Abstract: Problem statement: The aim of this study is to investigate the pharmacological effects of
the Methanolic-extract (AH2) of El-Hazha and its sub-fractions. Approach: These investigations were
carried out on in vitro isolated uterus preparations from Non-Pregnant (NP) and Late-Pregnant rats
(LP). In parallel displacement radio-ligand binding assay was performed for β-Adrenergic Receptors
(β-ADR). Results: Showed that the herb and its different fractions produced dose-dependent relaxant
effect (p<0.05, t-test, n = 6) on uterine contraction elicit by 25 mM KCl in both NP and LP uteri that
affected significantly by fractionation, however, the effect of the most active fraction (AH2-11) was
reversed by prior addition of propranolol (non-specific β-antagonist), but not affected by progesterone
pre treatment of the LP rats. In addition, AH2 only in high concentration displaced isotopes from β-
ADR, this affinity changed markedly by fractionation. Conclusion: We validate the fractionation
effect on its relaxant activity and found partial role for β-ADR on mediating this activity. Future study
was recommended to isolate and investigate its active components to enhance this activity or to
discover a new novel natural therapeutic agent(s).
Key words: Haplophylium tuberculatum, radio-ligand binding assay, β-Adrenergic Receptors (β-
ADR), Non-Pregnant (NP), Late-Pregnant rats (LP), Sudanese plant, charles-river laboratories, medicinal and aromatic plants, tocolytic agent, pharmacological effects, progesterone treatment, uterus-relaxing study INTRODUCTION
while the Medical prevention consists of antibiotic or progesterone administration (Tara and Thornton, 2004). Despite extensive efforts, the incidence of preterm One of the most well-known mechanism of action birth has not decreased recent several decades (Monga through which a tocolytic agent acts was to relax the and Creasy, 1995; Hannah, 2000). Preterm labor is still a uterus, so generally any agent has ability to relax the health challenge, because there are as yet no effective uterus can be considered as a tocolytic agent (Clouse et al., primary means of its prevention (Koucky et al., 2009). 2007) demonstrates that relaxation of rat myometrium is Tocolytic agents are drugs designed to inhibit the mediated by β2-adrenoceptors. also α1/β-adrenoceptor contractions of myometrial smooth muscle cells. The ratio determines not only the spontaneous motor activity aim of tocolysis is not only to stop uterine contractions of the rat uterus, but also the potency of the agents with and to prevent preterm delivery, but also to decrease the tocolytic effect (Zupko et al., 1998). However, the efficacy and safety of tocolytics are prenatal morbidity and mortality associated with not adequate, new agents are therefore required preterm birth (Tsatsaris et al., 2004). The main drugs including substances from natural sources. Many plants used as tocolytics are indomethacin and other have been recently investigated world-wide in the prostaglandins inhibitors (Vermillion and Landen, search for tocolytic or uterus-relaxing agent such as 2001), calcium channel blockers such as nifedipine Curcuma aeruginosa Roxb. Rhizome (Thaina, 2009), (Pryde et al., 2001; Giles and Bisits, 2007), β- Scutellaria baicalensis root (Shih et al., 2009) and adrenergic agonists and oxytocin receptors antagonist, Ficus capensis Thunb (Owolab et al., 2009). Corresponding Author: George Falkay, Department of Pharmacodynamics and Biopharmacy, Faculty of Pharmacy,
University of Szeged, H-6720 Szeged, Eotvos u. 6, Hungary Tel: +36 62 341974 Fax: +36 62 545567 Am. J. Biochem. & Biotech., 6 (3): 231-238, 2010 El-Hazha (Haplophyllum tuberculatum) (Forssk.) applications such as muscle relaxant and contracting A. Juss. (Rutaceae); is an herb indigenous to the northern part of Sudan, North Africa and other areas of This study was an attempt to proof and evaluate the Middle East (Boulus, 1983). Named locally in some of its traditional uses in gynaecological area as Sudan as “a plant of all disease”, it is to be found in uterus-relaxing agent and to see the influence of every Sudanese home as an emergency drug that used extensively by old Sudanese in the rural areas. The herb is utilized in Sudan as an antispasmodic, as an MATERIALS AND METHODS
antiflatulant, to relieve toothache and to treat allergic rhinitis (Mohamed AH et al., 1996), malaria, Plant material:
Crude plant collection and identification: The aerial
difficulties, renal disorders and others. parts of Haplophylum tuberculatum (El-Hazha) were This plant is also well known among herbalists and collected freshly during the 1rst week of November widely used traditionally in other counties such as 2008 from their natural habitats in the North part of Saudi Arabia (Mohammed et al., 1991) and Oman Sudan (Abu-hamad, Nahr El-Neel State). The voucher specimens (No. M23/08) were identified by Dr. Wai’l E. Abdalla and Yahia S. Mohamed of Herbarium of Its essential oils were investigated for antimicrobial Medicinal and Aromatic Plants Research Institute activity by (Al-Burtamani et al., 2005) and were found (MAPRI), Khartoum, Sudan, where the specimens were to cause partial inhibition of the growth of Escherichia coli, Salmonella choleraesuis and Bacillus subtilis to Extraction, fractionation and preparation of the
plant material for pharmacological tests: The
(Mohamed AH et al., 1996), who reported that its aqueous extract significantly decreased the contractility tuberculatum (AH) (2 kg) were extracted with and the heart rate, but did not affect the flow rate of the Methanol for 1h. The MeOH extract (AH2) was isolated perfused rabbit heart. This action was not concentrated and completely dried under vacuum to blocked by atropine, but the muscarinic antagonist yields 315 g of the crude extract which reconstituted in blocked the fall in blood pressure seen when the extract distilled water to get the desired concentration and was administered to anaesthetized cats. The extract also tested pharmacologically (Ganguly et al., 2007), then stimulated rabbit aortic strip, rat vas deferens and rat fractionated and tested. The most active fraction in each anococcygeus muscles. These adrenergic effects were step was selected based on comparing the in vitro pharmacological results, then fractionated and tested. Its hepatoprotective activity was investigated on The most active fractions are (AH2-11) 88.31 g, (AH2- the liver damage induced by paracetamol in mice by 11-4) 4.31 g and (AH2-11-4-6) 161mg (Fig. 1). (Ali et al., 2001) and proved to be relatively ineffective protecting only 16% of the animals against the lethal Pharmacological studies:
effect of paracetamol (1 g kg ) in comparable to that of Ethical considerations for housing and handling the
the standard hepatoprotective agent silymarin. animals: The animals were treated in accordance with
When its cytotoxic activity was checked against 11 tumor cell lines, where strong cytotoxic activity was (86/609/ECC) and the Hungarian Act for the Protection of Animals in Research ( §). All observed (Varamini et al., 1992). experiments involving animal subjects were carried out On the other hands and from phytochemical's point with the approval of the Hungarian Ethical Committee of view, some compounds of this Sudanese plant were for Animal Research (registration number: IV/1758- isolated by (Khalid and Waterman, 1981). Both its uses to relax the uterus and to treat Laboratories, Hungary) were kept at 22 ± 3°C, the asthma and inspiration difficulties catalyzed us to relative humidity was 30-70% and the light/dark cycle carry out this study to evaluate their effects in order to was 12/12 h. They were maintained on a standard find a new therapeutic agent(s) to aid in solving of two major medical challenges (preterm labor inhibition Hungary) with tap water available ad libitum. The animals were sacrificed by CO2 inhalation. Literature survey revealed that the aqueous extract of this plant obviously possess contracting activity, Mating of the animals: Mature female (180-200 g) and
male (240-260 g) rats were mated in a special mating Am. J. Biochem. & Biotech., 6 (3): 231-238, 2010 cage. A metal door, which was movable by a small Animals were sacrificed by rapid cervical dislocation, electric engine, separated the rooms for the male and both side of the skull were cut from back to forward. female animals. A timer controlled the function of the The intact brain was expose and removed carefully engine. Since rats are usually active at night, the using forceps. The brain were freed from other tissues separating door was opened before dawn. Within 4-5 h and homogenized in ice-cold homogenizing buffer (20 after the possibility of mating, vaginal smears were mM NaHCO3) in a ratio of 1:5. The homogenate was taken from the female rats and a sperm search was centrifuged at 15500 rpm speed for 40 min, the re- performed under a microscope at a magnification of suspended pellets were centrifuged under the same 1200 times. If the search proved positive, or if smear conditions. Finally the pellet were re-suspended in taking was impossible because of an existing vaginal binding buffer (50 mM tris + 0.5 mM EDTA with pH = sperm plug, the female rats were separated and were 7.5) and divided into small stock aliquots 2.6 mL each regarded as first day- pregnant animals. and frozen at -70°C which diluted and used in radio- Isolated organ bath studies: Uteri were removed from
non-pregnant (180-200 g), 22-day-pregnant (270-350 g) Displacement assay: The affinities of the tested extract
rats. Muscle rings 5 mm long were sliced from the and its fractions for β-adrenergic receptors were uterine horns and mounted in an organ bath (8 parallels) containing 10 ml de Jongh solution (in mM: 137 NaCl, measured on above membrane preparation using [3H] Dihydroalprenolol (DHA) (β-adrenergic antagonist) as glucose, pH 7.4). The organ bath was maintained at 37 o radioligand (~1.5 nM).Radioligand were purchased from Amersham International plc (UK). Under standard it. After mounting, the rings were equilibrated for about 1 assay conditions, the final incubation system volume h before experiments were undertaken; with a solution was 300 µl consisted of diluted membrane preparation change every 15 min. The initial tension was set to about (protein content approximately 0.5-1 mg ml ), radio- 1.5 g, which was relaxed to about 0.5 g at the end of ligand and incubation buffer or with the tested extract equilibration. The tension of the myometrial rings was or its fractions (its concentration ranging from 10 -104 measured and recorded with a gauge transducer and an S.P.E.L. Advanced ISOSYS Data Acquisition System µg mL ), following the incubation period, the (Experimetria Ltd., Hungary), respectively. Contractions membranes were collected on a Whatman GF/C filter, were elicited with KCl (25 mM) and cumulative dose- using a Brandel M24 Cell Harvester. Filters were response curves were constructed in each experiment by collected in liquid scintillation vials and the addition of plant extract or its fraction(s) in different radioactivity was measured with LKB Wallac liquid concentrations (range from 10 -100 µg ml ) alone or in scintillation counter. The experiment were performed at the presence of propranolol (β-blocker) (10 mM). 25°C for 45 min, the nonspecific binding were Recording was performed for 5 minutes. Dose-response determined using 10 M Alprenolol. Displacement curves were fitted from areas under curves (AUCs) experiments were analyzed individually with the using Prism 4.0 computer program. Emax and EC50 values were calculated (E computer program Prism 4.0 to determine the inhibition constants (Ki) of the investigated agents. of extract or its fraction (s) alone which elicits half of Statistical analysis: The statistics was done by using
the maximum relaxant effect of extract or its fraction Prism 4.0 (GraphPad Software, USA) computer program. For the statistical evaluations, data were Progesterone treatment of pregnant rats: The
collected from at least 6 animals and analyzed by progesterone treatment of the pregnant rats was started performing two-tailed unpaired t-test to compare the on day 15 of pregnancy. Progesterone was dissolved in significance mean differences for various results. The corn oil and injected subcutaneously every day up to differences were considered to be significant at levels day 21 at a concentration of 0.5 mg 0.1 ml kg . On day 22, the uteri were collected and the organ bath studies were performed as described above. The experimental data on the non-treated and the Phytochemical extraction and fractionation: The
Methanolic-maceration of the plant produced a yield's percentage of (5.5%), while the steps of fractionations Radio-ligand binding assay:
were highlighted in a summary diagram (Fig.1) Membrane preparation: The selected tissue of
emphasizing on that mentioned as most active abundant receptors of interest was rat brain membrane. pharmacologically and used throughout the study. Am. J. Biochem. & Biotech., 6 (3): 231-238, 2010
Fig. 1: Diagram illustrated the main steps of Bioactivity-guided fractionation of the Methanolic-extract of El-Hazha
emphasized on the most active fractions resulted and used throughout the study.
Table1: The basic effects of the Methanolic-extract on non-pregnant
Effect of the different AH2 fractions after the 2 nd
and late-pregnant rat uterus in vitro results, general pilot and 3rd fractionation on rat uteri: AH2 and its
screening for the direct plant relaxant activity fractions showed different activity level on both uteri type (data not shown), while the most active fraction *: p<0.05; Emax: The maximal relaxing effect of the Methanolic- Effect of the most active fractions of AH2 on rat
extract against (KCl-induced contraction); EC50: The concentration uteri: In non-pregnant (Fig. 2A and Table 2), the basic
of the Methanolic-extract producing 50% of their maximal relaxing effect of Methanolic-extract against (KCl-induced contraction) in AH2 relaxant activity was increased significantly after fractionation while it decreased by further fractionation. In late-pregnant unlike non-pregnant results, (Fig. In vitro pharmacological studies:
2B and Table 2), the extract activity was not Rat uterus results:
significantly affected by different fractionations steps. Effect of the Methanolic-extract on rat uteri:
Basically, the extract showed relaxant activity in NP considered as the most active one and selected for and LP rat uterus without significant difference in the advanced β- adrenergic study using propranolol Emax (p = 0.094), but with significant difference in as standard blocker (Fig. 3 and Table 3) and Am. J. Biochem. & Biotech., 6 (3): 231-238, 2010 Table 2: Relaxant effect of the Methanolic-extract AH2 and its most active fractions on non-pregnant and late-pregnant rat uterus in vitro results ---------------------------------------------------------------- ------------------------------------------------------ *p<0.05, AH2 was used as control for its different fractions, Emax: the maximal relaxing effect of the Methanolic-extract AH2 or its sub fractions (AH2-11, AH2-11-4 and AH2-11-4-6) against KCl-induced contraction; EC50: the concentration of the Methanolic-extract AH2 or its sub fractions (AH2-11, AH2-11-4 and AH2-11-4-6) producing 50% of their maximal relaxing effect of Methanolic-extract against KCl-induced contraction in the system
Table 3: Propranolol antagonistic effect on the relaxant effect of the most active fraction AH2-11 on isolated non-pregnant and late-pregnant rat
------------------------------------------------------------- ----------------------------------------------------- *: p<0.05, Emax: the maximal relaxing effect of the most active fraction AH2-11 against KCl-induced contraction; EC50: The concentration of the most active fraction AH2-11 producing 50% of their maximal relaxing effect against KCl-induced contraction in the system Fig. 2: Dose-response curves of the relaxing effect of Fig. 3: Dose-response curves of the most active the Methanolic-extract (AH2) (continuous-line) fraction AH2-11 alone (continuous-lines) and and its most active fractions (dotted-lines) on with propranolol 10 6 (dotted-line) on non-- non-pregnant (a) and late-pregnant (b) rat pregnant (a) and late-pregnant (b) on isolated uterus in vitro against KCl-induced control rat uterus in vitro against KCl-induced control contractions. Values presented are means of 6-8 contractions. Values presented are means of 6-8 observations, while vertical bars denote Am. J. Biochem. & Biotech., 6 (3): 231-238, 2010 Effect of AH2-11 on late-pregnant progesterone-
treated rat uterus results:
The progesterone treatment
for late-pregnant rat uterus did not alter significantly
neither the Emax% nor the EC50 of the curve of the most
active fraction AH2-11 (Fig. 4).
Radio-ligand binding assay: The affinity of the
Methanolic-extract and its most active sub fractions
from different fractionation steps for β-adrenergic
receptors were tested on rat brain membrane
preparation, using Dihydroalprenolol [3H] DHA (2 nM)
as an isotopes radioligand. All of the ligands displace
the radioligand from the target receptor.
The AH2 showed the displacement of the isotopes Fig. 4: Dose-response curves of the most active only in very high concentration (104 µg mL ) with un- fraction AH2-11, untreated (continuous-lines) estimated Ki ,while its other sub fractions showed better displacement affinities and the Ki-values of AH2- isolated late-pregnant rat uterus (Day22) in 11-4 and AH2-11-4-6 were (1.2 ±1.0) ×10 , (1.1 ±1.1) vitro against KCl-induced control contractions. 10 and 3.8 ± 3.8 µg mL respectively (Fig. 5). observations, while vertical bars denote DISCUSSION
Plants still a rich drug source (Wahab et al., 2008), El-Hazha, is named locally in Sudan as “a plant of all disease”. Due to its extensive use traditionally this plant subjected to different studies in different directions, but the novelty of this study arises from many factors which can be summarized as; deep pharmacological study with detailed assay guided fractionation, while other studies either general or used only the crude extract without fractionation, It involve the use of a pharmacological methods like RLB-assay and It done on both non-pregnant and late-pregnant (D22) rat uterus. Traditionally El-Hazha is used as an aqueous infusion. In our investigation we made a Methanolic- Fig. 5: The displacement curves of AH2 and its most extract (AH2) from the plant, because the polarity of active fractions on β-adrenergic receptors using the two solvents are quite similar, the extract contains Dihydroalprenolol [3H] DHA as a radioligand probably the same components. Previously, the aqueous extract of the plant was investigated by (Mohamed AH preparation. Values presented are means of 6-8 et al., 1996) and gives potent contracting activity. The observations, while vertical bars denote chloroformic fraction contains non-polar compounds In general pilot screening, AH2 exerts relaxant Propranolol effect on the relaxant activity of the
effect in both uteri with insignificant difference in most active fraction on rat uteri: Propranolol in
concentration of 10 6 M had no significant effect on max%, but the EC50 was significantly differ, while the RLB assay revealed that the extract exerts binding neither the curve nor its parameters of the most active fraction AH2-11 on non-pregnant rat uterus (Fig. 3A affinity to β-ADR only in relatively very high On late-pregnant rat uterus (Fig. 3B and Table 3), propranolol in concentration of 10 6M exerts significant Fractionation gives different sub-fractions with different efficacies on both isolated uteri, but we deal only with the most active ones. Fractions that produce active fraction AH2-11 under investigation. relaxation ≤35% on NP and ≤25% on LP after first Am. J. Biochem. & Biotech., 6 (3): 231-238, 2010 fractionation and that produce relaxation activity ≤25% adrenergic activity was weaker than the non-pregnant, on NP and 20% on LP after the 2nd fractionation unfortunately the progesterone treatment did not potentiate the β-receptors sensitivity to this fraction, mentioned and considered as active. Moreover, the these findings can be explained by that, the β-ADR only fraction that showed best pharmacological activity was partially participated in this relaxation besides other possible mechanism(s) or may be hide by the presence of The fractionation effect on the biological extract other compounds in this semi-purified fraction. activity was achieved by relating AH2 to its most active TLC chromatogram showed the complexity of the fractions. In NP uterus, the extract activity was active fractions. Even the sub-sub-sub fraction contains increased significantly (p = 0.026) by fractionation then large number of structurally related compounds exists markedly decreases, but still at a level (2X) greater. in a very small amount, thus their isolation is a very In LP uterus the activity did not affected difficult process. These compounds may contribute the significantly by fractionation, but the original extract various traditional uses and the local name (plant of all relaxant activity still exist. In spite that on this stage diseases) of the plant. Finally, the separation may affect of pregnancy the sensitivity of the uterus to β- markedly its biological activity due to the well-known adrenergic activity was weaker than the non pregnant These findings were supported with and confirmed CONCLUSION
by radio-ligand binding assay experiments results. In which at the beginning fractionation improved AH2 Finally we can concluded that, by these findings affinity to β-ADR, but after the 3rd fractionation and demonstration we confirm and proof its mentioned process the affinity was deteriorated to a level similar to traditional use, even it seems contradictory from the first point of view, The fractionation significantly affect We continue the study using the most active sub fraction AH2-11, because we thought that this relaxant There is a partial role for β-ADR on mediating this effect may be attributed to synergistic effects of the relaxation activity or may be its complete, but inhibited extract compounds that can be explain by the huge by the existence of other contracting substances that number of compounds that appears in the TLC plate The suggested purification may lead to a discovery The selected Fraction AH2-11 as most active one of a new novel natural therapeutic agent(s) useful to aid when used to perform further experiments to verify the solving two major medical problems pre term labour role of β-adrenergic receptor in mediating the above mentioned relaxant activity using propranolol as a blocker, both rat uteri revealed that; in NP, propranolol REFERENCES
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