Urinary parameters were evaluated in urine collected from treated animals 5 and 24 hours after the administration of the test compounds. In terms of the ischemia-reperfusion model, the triterpenoids conferred a protective effect comparable to that of class II and IV antiarrhythmic drugs.
Follow-up changes in blood pressure (mm Hg) and heart rate (beats/min) of Wi-Star rats after a single i.p. 106 Follow-up changes in blood pressure (mm Hg) and heart rate (beats/min) of wi-star rats treated with the phytochemical extractives for six consecutive years.
INTRODUCTION
This suggested that the active principles of the African wild olive had a different chemical structure and activity than that of the European olive plant. Thus, the cardiovascular effects of European olive leaf extracts have been attributed to two secoiridoids, oleuropein and
LITERATURE REVIEW
Secondary Hypertension
Some of the reasons for this are; Cushing's syndrome, coarctation of the aorta, kidney and urinary tract diseases such as nephritis, diabetes, amyloid kidney damage, chronic pyelonephritis, renal artery disease, obstructive sleep apnea, aldosteronism, endocrine disorders, excess catecholamines, excess erythropoietin and drugs ( Pickering, 1995 ; Onusko, 2003).
Essential Hypertension
DIAGNOSIS OF HYPERTENSION
Although new methods for assessing hypertension are being introduced, conventional blood pressure monitoring remains the standard method for diagnosing hypertension. As mentioned earlier, a blood pressure reading of more than 140/90 mm Hg is defined as hypertensive.
REGULATION OF BLOOD PRESSURE
MODELS OF HYPERTENSION
- Spontaneously hypertensive rats (SHR)
- Stroke-prone Spontaneously hypertensive rats (SHR-SP)
- Dahl/Rapp salt-sensitive rats
- Diabetic hypertensive rats
- Transgenic Rats
- Models for studying renovascular hypertension
- Models for Pulmonary hypertension
The development of hypertension and heart failure in these rats can be controlled by titrating the amount of salt in their diet. Thirdly, there is the two-kidney one clip model whereby one clip is placed on the renal artery of one of the kidneys.
MEASUREMENT OF BLOOD PRESSURE IN EXPERIMENTAL HYPERTENSION
Indirect blood pressure recording
The advantages of using the tail cuff method are primarily that no surgical intervention is required before starting to measure blood pressure. On the other hand, the main limitation of this method is that it does not allow continuous recording of blood pressure.
Direct blood pressure recording
Depending on the assessment, intervention can either take place through lifestyle changes or/in connection with drug treatment. Treatment can be with lifestyle changes alone for up to a year if there are no other risk factors.
Intervention through lifestyle modifications
Before starting treatment, it is necessary to assess blood pressure and the absolute risk of cardiovascular disease. On the other hand, drug treatment is indicated in patients with stage 2 hypertension or higher, or in patients with high cardiovascular risks, and in patients with diabetes.
Intervention through drug treatment
- Chemically induced arrhythmias
- Mechanically induced arrhythmias
- Electrically induced arrhythmias
- Class III anti-arrhythmic drugs
- Class IV anti-arrhythmic drugs
The severity of these arrhythmias is therefore highly dependent on the duration of the ischemia. There is therefore an increase in the development of cardiac stress due to the increased concentration of intracellular free calcium.
Selecting a Plant
When used as herbal tea or other homemade remedies, it is with the assumption that the plant is a medicinal one. The strategy widely used to select a plant is careful observation of the use of the plant resources in folk medicine, commonly referred to as ethnopharmacology or ethnobotany.
Preparation of the plant material and isolation of active compounds
The elucidation of the structure can be done using spectroscopic methods such as; ultraviolet/visible spectrum, infrared (IR) spectrum, mass spectrum and nuclear magnetic resonance (NMR) spectra (Houghton and Raman, 1998). It is also vital to prepare derivatives or analogues of the compounds for the investigation of structure-activity relationships.
BOTANY OF PLANTS USED IN THIS STUDY
Combretum molle (COMBRETACEAE)
Chemical constituents that have been isolated from the plant include malic acid, xyloside, arabinoside, and large amounts of triterpenoids (Rogers and Thevan, 1986; Lawton and Rogers, 1993). Although many studies have shown the hypoglycemic activity of this plant, one study reported that the plant does not exhibit this activity. Ethanol extracts of the plant showed significant inhibitory activity against castor oil-induced diarrhea and PGE2-induced enteropooling in rats.
The extracts also showed a significant reduction in gastrointestinal motility in charcoal meal tests in rats. The results thus obtained demonstrate the effectiveness of plant materials as antidiarrheal agents (Mukherjee, et al., 1998).
MATERIALS AND METHODS
MATERIALS
PLANT IDENTIFICATION AND COLLECTION OF PLANT MATERIAL
BIOASSAY-DIRECTED ISOLATION OF ACTIVE PRINCIPLES OF PLANTS
- Extraction of plants
- Thin layer chromatography
- Isolation and purification of compounds
- STRUCTURE ELUCIDATION
Crude ethyl acetate extracts were initially chosen over other crude extracts because a preliminary screening showed a strong presence of triterpenoids in this fraction (personal communication from Prof. F. O. Shoda). As preliminary experiments showed that crude ethyl acetate extracts contained most of the active compounds, i.e. triterpenoids, only crude ethyl acetate extracts were subjected to a further purification process. The crude extracts were fractionated sequentially on silica gel 60 column chromatography (particle size mm) with gradient elution (100% hexane to 60% hexane/ethyl acetate).
The eluates that had similar TLC profiles to the authentic triterpenoids were combined and subjected to further chromatographic purifications.
TEST FOR TOXICITY
Urine electrolyte content was analyzed using the Beckman Synchron EL-ISE Electrolyte System (Germany). To evaluate the diuretic effect, the volume of urine excreted per 100 g of body weight was calculated for each group. The results were expressed as the "Lipschitz value", i.e., the TIU ratio, in which T is the response of the test compound and U is that of the urea treatment.
To characterize the duration of the diuretic effect, the "Lipschitz value" was calculated for both the 5-hour and 24-hour elimination periods.
BLOOD PRESSURE MEASUREMENTS
Indirect BP measurements
The blood pressure monitor works with a hardware lITC system to determine blood pressure and heart rate. The use of the photoelectric sensor allows measurement at the lowest possible ambient temperature reducing heat stress on the animal and as such gives results that are closer to values obtained by cannulation. After a single intraperitoneal application of the test compound, systolic and diastolic blood pressure and heart rate were monitored for 60 minutes at 10-minute intervals.
The same parameters were monitored for 6 consecutive days just before the animals received a daily single intraperitoneal (Lp.) injection of the test compound.
Direct BP measurements
BIOCHEMICAL DETERMINATIONS
CARDIOTONIC EXPERIMENTS
Cardiotonic measurements in guinea pig atria
The tissues were then washed 3 times and equilibrated for 15 min and washed 3 more times. Test compounds were added to the tissue bath in a cumulative (mg/ml) manner at 3 minute intervals to obtain a complete dose-response curve. The tissue was then washed three times and equilibrated for 15 minutes before being checked for its stability and compared to the effect of isoprenaline.
After thorough washing, the tissue was further equilibrated for 15 min and washed again before testing the second compound.
After baseline values were obtained, cumulative doses of each test compound were added to the tissue bath, and their inotropic and chronotropic effects were recorded for a total of 10 minutes. Involvement of the cholinergic mechanism was performed by introducing the extracts to the tissue bath before and after the addition of the atropine sulfate. For chemically induced arrhythmia, we used the CaCh-, BaCh-, and adrenaline-induced models of arrhythmia.
As positive controls, we used all four classes of reference antiarrhythmic drugs according to the Vaughan Williams (1991) classification.
Chemically induced models of arrhythmia
- BaCh -induced arrhythmia
- eaCh - induced arrhythmia
- Adrenaline-induced arrhythmia
CaCh-induced arrhythmia was used to evaluate the potential antiarrhythmic calcium antagonist effects of the test compounds. Arrhythmia was induced by administration of a 2.5% solution of CaCh (140 mg/kg) (Papp et al., 1966) on a background of the test compound administered 20 min previously. Changes in ECG were recorded after application of CaCh before and against the background of the test compounds.
Just as above, changes in ECG were recorded after the administration of CaCh before and on the background of the test compounds.
RESULTS
PLANT MATERIALS AND EXTRACTS
Diuretic effects of the test compounds
The results of the Lipschitz test analyzed usmg unne collected 5 hours after intraperitoneal injection indicate that all the test compounds except Psidium guajava (small fruit) and Psidium guajava (white fruit) had a diuretic effect comparable to hydrochlorothiazides (Figure 5a). On the other hand, the results from the urine collected 24 hours after intraperitoneal administration show that only oleanolic acid, ursolic acid, Syzygum cordatum, Eugenia jambolona, Plantago lanceolata, Tetradenia riparia and Psychotreia serpens exhibited a diuretic effect comparable to that of hydrochlorothiazide ( figure 5b).
Saluretic effects of the test compounds
T/u "Lipschitz value" ratio where T is the response of the compound tested and U is the urea response. T/u "Lipschitz value" ratio where T is the response of the compound tested and U is the urea response.
Natriuretic effects of the test compounds
The results of the effects of phytochemicals on blood pressure and heart rate after a single intraperitoneal administration (100 mg! kg body weight) are presented in Table 8 and Figures 24a–26b. All phytochemicals showed a trend to lower blood pressure 10 minutes after a single intraperitoneal application, except those from Clerodendrum capense, Prunus a/ricana, Psidium guajava (hybrid), Terminalia sericea and Plantago lanceolata. All phytochemicals except those from Psidium guajava (Pink fruit), Psidium guajava (hybrid), and Terminalia sericea occasionally reduced blood pressure.
Clerodendrum capense lowered blood pressure every other day, while Syzygum cordatum lowered blood pressure only on the second day.
EFFECTS OF PHYTOCHEMICALS ON BODY WEIGHT
Some phytochemicals caused a significant reduction in animal body weight when the test compounds were administered for six days (Table 10). The most pronounced reduction was observed in rats injected with Syzygum cordatum. In these animals, the body weight decreased from the second to the fifth day, but from the sixth day the weight started to increase again.
Psidium guajava (hybrid) reduced body weight from day 4 onwards, while Plantago lanceolata caused a significant decrease in weight from day 5.
ACUTE EFFECTS OF PHYTOCHEMICALS ON BLOOD GLUCOSE LEVELS
CARDIOTONIC ACTIVITY OF TEST COMPOUNDS
- Cardiotonic effects of the phytochemicals and pure triterpenoids in guinea pig atria
Dose-response relationship for the effect of the phytochemicals on the force of contraction measured in electrically driven left guinea pig atria (mean SEM; n = 3). The effects of oleanolic acid, ursolic acid, and uvaol were reversible, as washing the tissue reversed inotropic effect to their baseline values. The inotropic and dromotropic effects of the triterpenoids in anesthetized rats are shown in Table 13.
All the triterpenoids had a positive inotropic effect on the rats as shown by the increase in the amplitude of the QRS complex.
ANTIARRHYTHMIC ACTION OF TEST COMPOUNDS
- On BaCh-induced arrhythmia
- On Ischemia-reperfusion arrhythmia
Tetradenia riparia showed an antiarrhythmic effect comparable to that of propranolol on this model. Ursolic acid and oleanolic acid showed antiarrhythmic effects comparable to those of amiodarone. Ursolic acid and methyl maslinate exhibited antiarrhythmic activity on ischemic reperfusion-induced arrhythmia comparable to that of verapamil (Table 17).
On the other hand, uvaol and oleanolic acid exhibited antiarrhythmic effects similar to those of propranolol.
Baseline 3 minute 5 minute 10 minute 20 minute 30 minute 40 minute 60 minute
- DISCUSSION
- INTRODUCTION
- Isolation of crude and pure compounds
- Hypotensive effects of the phytochemicals and the triterpenoids
- Cardiotonic effects of the phytochemicals and the triterpenoids
- Antidysrhyhmic effects of compounds
- Interdependent link between structure of compounds and the observed cardiovascular effects
- FUTURE WORK AND RECOMMENDATIONS
- CONCLUSION
- REFERRENCES
- APPENDICES
It is very likely that some of the triterpenoids remained in the crude extracts of methanol, hexane and dichloromethane. The study found that the isolated triterpenoids and most phytochemicals had a hypotensive effect in Wistar rats. This inhibition demonstrates that the positive inotropic effects of the triterpenoids were mediated via ~-adrenergic receptors.
The last possibility is that the triterpenoids may have increased the sensitivity of the contractile apparatus to Ca2+. There is therefore an interdependent relationship between the chemical structures of the triterpenoids isolated from Africa. The study therefore showed that there is a connection between the chemical structure of the triterpenoids and its cardiovascular activity.
Weights of crude extracts from different plants
Name of plant Code Weight of extract Jgl
PICTURES OF EXPERIMENTAL SET UP
