Investigation of antihypertensive effect of Nigerian varieties of Solanum lycopersicon on rats

Hypertension remains a global challenge even in the 21st century with attendant increase in mortality rate. The quest for alternative management medications suffixed this investigation using three varieties. Thirty male hypertension induced wistar rats divided into 6 groups of 5 rats each were used. Group A served as the normal control group and was administered 0.9% normal saline as placebo. Group B, C and D were fed with Jos, UTC, Gboko varieties, respectively. Group E was treated with lisinopril at 2.5 mg/kg orally, while group F served as the hypertensive untreated group. Administration lasted for 28 days and all animals were allowed access to food and water ad libitium. Standard methods of noninvasive blood pressure assessment was used to access systolic blood pressure, diastolic blood pressure, heart rate, while the lipid panel was assessed using Cardiocheck test meter (lipidocare) in all the groups. ANOVA was used to analyze data and probability level of p<0.05 considered significant. Results show that rats given Jos and UTC tomatoes performed better as compared to those given Gboko tomatoes but the 3 groups did less well as compared to the Lisinopril group. Groups A and F, the Normotensive and hypertensive controls remained status quo. Groups B and C also did better in having an improvement in the lipid profile, as compared to all the other groups. All these indices put together suggest that the Jos and UTC varieties of tomatoes show a better antihypertensive effect as compared to the Gboko variety and could be used in the management of hypertension owing to the presence of high concentration of antioxidants in them. And if these results are applicable to man, the consumption of Jos and UTC varieties of tomatoes should be encouraged.


INTRODUCTION
Hypertension is defined as a persistent increase in blood pressure of >140 mmHg (systolic) and or ≥ 90 mmHg (diastolic) (Onwubere et al., 2012).Not less than 46.4% Nigerian over 15 years of age has hypertension'' (Ogah et al., 2012) and it is positively and independently associated with high morbidity and mortality rates in Africa (Ediale, 2011).Hypertension and overweight places an excessive financial burden on population and health systems consuming a scarce resources and thus places a lot of economic burden on the individual, loss of productivity and pre-mature death at younger age (Macmahon et al., 2005).Hypertension remains a global challenge even in the 21st century with attendant increase mortality rate.Considering the uncomfortable side effects of antihypertensive drugs and the fact that many hypertensive patients need more than two kinds of drugs per day, alternative and supplementary treatment for blood pressure control has been suggested such as life style modification, especially dietary intervention.Thus, the quest for alternative management medications suffixed our investigation using different varieties of Solanum lycopersicon (tomatoes) commonly consumed within Makurdi, Benue State, Nigeria.Tomatoes outside its juiciness and rich flavor are quietly gaining a place in the prevention and management of hypertension.This attribute is suggestive of the presence of lycopene, potassium, beta carotene and antioxidants in tomatoes (Xinli and Jiuhong, 2013).
Tomatoes play an active role in the management of hypertension, coronary heart disease, Ischemic stroke, type II diabetes and certain diseases.Worldwide, about 58% of diabetic mellitus and 21% of Ischemic heart disease are attributed to high blood pressure (Onwubere, 2012).Blood pressure (BP) is the pressure exerted by circulating blood upon the walls of blood vessels and is one of the principal vital signs.During each heartbeat, blood pressure varies between maximum (systolic) and minimum (diastolic) pressure.The mean BP, due to pumping by the heart and resistance to flow in the blood vessels decreases as the circulating blood moves away from the heart.Though that of healthy adult human is 120 (systolic) and 80 mmHg diastolic (written 120 / 80 mmHg, and called {in US and UK} one twenty over eighty), systolic and diastolic arterial BPs are not static but undergo natural variations from one heart beat to another and throughout the day in a circadian rhythm, they also change in response to stress, nutritional factors, drugs, diseases, exercise and momentarily from standing up.Persistently raised blood pressure exceeding about 120 (systolic) and 90 mmHg (diastolic) at rest is called hypertension.Cardiovascular diseases are associated with oxidative stress, inflammatory processes and vascular dysfunction.Lycopene, a carotenoid found in tomatoes is an antioxidant with protective effect on lipid peroxidation and antiatherosclerotic capacity (Ried and Fakler, 2011).A meta-analysis suggest that lycopenetaken in dosage ≥25 mg daily is effective in reducing LDL cholesterol by about 10% which is comparable to the effect of low dose of statins in patients with slightly elevated cholesterol levels.

Plant collection and identification
The samples used for this research were fresh and ripe tomato fruits which include these three varieties (Lycopersicon hiresitium (LH) called Jos tomatoes, Lycopersicon perivianum (LP), UTC variety and Lycopersicon cheesmani (Gboko variety) and were purchased from railway market in Makurdi Nigeria.Identified and authenticated at the Herbarium Unit of the Department of Plant Science and Animal Breeding of the Federal University of Agriculture, Makurdi, Benue State, Nigeria and their samples were collected after identification and kept at their herbarium units with voucher number UAM/1773.

Phytochemical screening
In order to determine the presence and the concentrations of the alkaloids, glycosides, flavonoids, tannins, soluble carbohydrates, steroids, saponin, reducing sugar, standard methods of Harbone (1983) were used, while for vitamin C, beta-carotene and lycopene, method of Alexander and Griffiths (1993) were used.A preliminary study was performed with the blended ripe tomato fruits using the standard methods for various phytoconstituents as stated in Table 1.

Lipid profile assessment
The total cholesterol level (TC), high density lipoprotein (HDL-C) and triglycerides (TG) for all the experimental animals were determined using the hand held cardiocheck self-test meter (lipidocare), a device made by SD Biosensor Inc USA, and was used for the determination of the lipid profiles in the experimental animals using whole blood collected from the tail vein using Insulin syringe.The equipment has already being calibrated prior to its use after purchase.The memo clips was inserted and the device switched ON, the test strip for each sample was inserted after which two drops (0.2 ml) of blood samples were placed on the strips and within few seconds that the blood samples were dropped, a pink coloration was observed respectively and the automatic button was pressed which led to the instant display of the result (TC, TG, HDL-C, etc) and were recorded in milligram per deciliter (mg/dl).

Determination of body weights
The body weights of the experimental animals were determined prior to the commencement of the research and during the experiments on weekly basis (7 days interval) throughout the experimental periods for 28 days.Their weights were taken using the digital top loading weighing balances by Harvard Apparatus Ltd.A cylindrical transparent glass rat restrainer was weighed and the weight tarred to zero before introducing the rats individually and their weights were recorded in gram (g).

Determination of the systolic, diastolic and the heart rate
Non-invasive blood pressure meter (NIBP) (LE 5001) by PANLAB Equipment was used for the determination of the cardiovascular parameters (systolic, diastolic and pulse rates).The sensitive blood pressure meter was switched on and allowed to acclimatize for about 20 min; the selector switch located at the back of the equipment was switched to area marked for rats.The rats' tail was briefly immersed in water at temperature of 45°C with a thermostat and allowed for about 30 s for the dilatation of the tail veins to increase blood flow to the tail region.Before this, the rats were introduced into the transparent glass restrainer before the immersion of the tails into the hot water.The animals were covered with pieces of dark clothes for reduction of anxiety.The tail cuff/transducer were introduced into the base of the tail region and the selector switch turned on immediately the pulse waves indicated "ready", the readings were displayed on the screen of the apparatus.The foot control switch was matched to save the reading and then recorded.

Preparation of tomato sauce
The tomato fruits were washed thoroughly with clean water to reduce the microbial load and other contaminants that might adhere to the surface of the tomatoes.The tomato fruits were chopped into smaller sizes with a kitchen knife and were blended and homogenized using a homogenizer.After which it was mixed with the normal rats chow and dried using an oven in the ratio of 92:8 g and kept for the animals ad libitum.

Induction of hypertension
Hypertension was induced in the rats after their initial baseline physical and cardiovascular parameters were assessed.For a rough estimation, a typical young male adult rats weighing between 300 and 350 g consumes around 20 g of normal rats chow per day, that is, about 48 g/kg body weights per day and this is equivalent to g NaCl per 325 g of body weights per day or about 5 g of NaCl per kilogram weight for day.Based on this, 8 g of NaCl (Uncle Palm Iodized salt with Batch No; FT 256) was weighed using a digital weighing balance by Ohaus USA and transferred to a 500 ml beaker and made up to 100 ml of clean water using a delivery pipette to prepare sodium chloride solution and was mixed with approximately 92 g of normal rats chow and 1% NaCl in drinking water and was kept for them ad libitum (Sofola et al., 2002).They were allowed to feed on the diet for 28 days followed by blood pressure measurements on weekly basis till there was sustained increased in blood pressure to a hypertensive state before the treatment with the different varieties of L. esculentum commenced and lasted for another 28 days.The values were also reported and tabulated in the results.

Experimental animals
A total of thirty hypertensive male Albino rats of 3 to 4 months of age weighing between 100 and 200 g were used for the experiment.They were procured from the Animal House Unit, Department of Pharmacology, and College of Health Sciences of the Benue State University, Makurdi and kept in well aerated laboratory cages in a room with 12 h and dark cycle in clean disinfected cages in the animal house with free access to feed (standard pelletized growers feed from UAC-vital feed Jos, Plateau State) and clean drinking water ad libitium.The animals were treated according to the international guidelines for the care and maintenance of laboratory animals and allowed to acclimatize to the environment for one week (7 days) before commencement of the experiment.The animals were handled according to the protocols approved by the Research Ethics Committee of the Benue State University, Makurdi with protocol number, NHREC/BSUM/2016/00234b.

Experimental design
The thirty male hypertensive wistar rats after acclimatization were divided into six (6) groups each and their basal parameters measured and recorded before the commencement of the experiment.
Group A (n=5) Normotensive (control group); Group B (n=5) hypertensive and treated with Jos variety; Group C (n=5) hypertensive and treated with UTC variety; Group D (n=5) hypertensive and treated with Gboko variety; Group E (n=5) hypertensive and treated with standard angiotensin converting enzyme inhibitor (lisinopril) 2.5 mg/kg body weight P.O; Group F (n=5) hypertensive untreated but received water and feed ad libitium.

Method of data analysis
The data are presented as mean±standard deviation and were analyzed using one-way analysis of variance (one-way ANOVA) then multiple comparison with post hoc Tukey test (least standard deviation) to compare their means using computer software (SPSS version 21.0) and Excel for windows.P < 0.05 was considered to be statistically significant.
Table 1 shows the results of the qualitative and quantitative analysis of the phytochemical present in the three varieties of Solanum lycopersicon.The table shows the different concentration of the phytochemical present in Nigerian tomatoes.Jos variety of tomatoes has the highest concentration of antioxidants (flavonoids, lycopene, beta-carotenes and vitamin C) as compared to UTC and Gboko varieties, respectively.
Table 2 shows the result of the mean±SD of the SBP of the baseline values and the final treatment values.There was a decrease in their SBP (E>B>C>D), that is, A (-1.0±1.2 mmHg), B (-32.0±1.5 mmHg), C (-21.8±1.3 mmHg), representing 0.8, 19.6, 14.5 and 20.1%, respectively when compared with that of lisinopril (33.4±0.1 mmHg), representing 20.1% with the exception of group D (18.4±4.9 mmHg) and F (16.4±12.2mmHg) that increased instead of decrease with 11.3 and 16.2%, respectively.They were compared statistically and found that there was a significant difference (P<0.05) between Groups B, C, and F when compared with A. Also, Groups B, C and E were significantly difference (P<0.05) when compared with Group D. Groups B, C, D and E were also significantly different (P<0.05) when compared with Group F. The group treated with the Jos variety was found to be more potent in SBP reduction than the UTC and Gboko varieties, respectively.
Table 4 shows the changes in their TCL.Groups B, C and E decreased significantly by B (-13.1±0.2),C (-6.8±2.3) and E (-8.8±0.5)representing 21.3, 11.7 and 15.7%, respectively, while D (9.4±0.1) and F (92.2±4.5)represents 18.0 and 150.4% increase in the TCL.They were compared statistically and found that there was a significant difference (P<0.05)among all the groups compared with Group F.
Table 5 shows the results of the high density lipoprotein (HDL-C).All the groups showed a significant increase in their HDL-C levels after treatment period with the exception of group F (HPT-U) that decreased and there was a significant difference (P<0.05)among Groups A, B, C, D and E as compared to Group F .
Table 6 represents the result of the triglycerides (TG-C) levels.After treatment, tomatoes treated groups showed a significant decrease in their TG-C levels when compared with the control Groups A and F that increased significantly with more significant increase seen in HPT-U groups.Groups B, C and D were  significantly difference (P<0.05) when compared with HPT-U also, all the groups showed a significant difference with the control group A.
Table 7 similarly show the HR decrease by 1.4±16.27beats/min, 20.2±0.3, 11.0±2.0,30.6±8.9 representing 0.4, 5.9, 3.2 and 8.8%, respectively for Groups A, B, C and D. While Group E (15.0±1.3 mmHg) and F (19.1±1.6mmHg)represent 4.4 and 5.6% increase in the HR.They were also compared statistically and observed that there were significant differences between groups B, C and D when compared with Groups F and B, C significantly different (P<0.05) when compared with Group E. Also, Group D was significantly difference (P<0.05) when compared with group A.
In Table 8, body weights decreased in Groups B, C and D with 8.0±3.1 g, 3.8±0.1 and 4.6±0.8g representing 5.2, 2.6 and 3.5%, respectively.Groups A, E and F increased in their body weights with 9.76±2.2and 3.7±3.7 and 20.7±1.7 g representing 6.5, 2.6 and 13.2%, respectively and there was no statistical significant difference (P>0.05)among all the groups at the end of the experimental period.At the end of the treatments, the blood pressures returned to normal in Groups B (132/85), C (129/72) and E (119/79 mmHg), whereas this was not achieved in group D (145/85) and F (195/118).No death/sickness was reported during this study which shows that tomatoes fruits do not have any adverse effect on the body.

RESULTS AND DISCUSSION
This study investigated the antihypertensive effects of three varieties of ripe tomato fruits (Solanum lycopersicon) procured from Makurdi, North-central Nigeria.In this study, the experimental animals were induced to hypertension by salt loading method (Sofola et al., 2002).The five different cardiovascular parameters and non-cardiovascular parameters which include systolic blood pressure, diastolic blood pressure, total cholesterol, high density lipoprotein and triglycerides, heart rates and body weights were assessed.In this research work, the researcher discovered that the three varieties of the Solanum lycopersicon were found to have antihypertensive effects through their lowering effects on the parameters assessed but the most antihypertensive effect was observed in the Jos variety of tomatoes accompanied by the UTC and Gboko varieties, respectively, the standard antihypertensive drug (lisinopril) performed better than the three varieties of Solanum lycopersicon.The significant increase in body weights of Groups A and F may be an indication of an increase in fluid volume.
The decreased body weights in Groups B, C and D on the last day of treatment could be as a result of the antioxidant properties of some of the phytochemical present in ripe tomato fruits like the flavonoid, lycopene, etc. Dietary flavonoid protect against cardiovascular diseases.Emerging and largely consistent evidence suggests that flavonoids can improve human endothelial functions and may reduce blood pressure (Hodgson and Croft, 2006) through its vasorelaxative effect on isolated arteries from rats as there is evidence those flavonoids metabolism is an important factor influencing the biological activity and effect of dietary flavonoids.Lycopene, flavonoids, beta-carotenes, etc, are known as powerful antioxidants and free radical quenchers which have received attention for its pivotal role in inhibiting oxidative stress which is found to inactivate nitric oxide,  The reduction in most of the cardiovascular parameters like systolic blood pressure etc could also be a protective effect of the saponin present in the ripe tomato fruits (Liu et al., 2012).The presence of saponin resulted in the lowering of total cholesterol and reduction in inflammation (Peter et al., 1997).Saponin if regularly included in the diets may help the body itself from cancer and other cardiovascular diseases as saponin and saponin like compounds have shown evidence that they can buttress the body's ability to fight cancer and cardiovascular diseases.The lipid profile obtained in this study showed a significant decrease in the triglyceride level and an increase in the high density lipoprotein cholesterol of the tomatoes treated groups when compared with the hypertensive untreated group.Lipidemia observed in the hypertensive untreated group may be as a result of an increase in visceral adipose mass (Brown and Dunmore, 2013) or may be due to a low activity of cholesterol biosynthesis enzymes (Tanko et al., 2016).Though anabolic steroids may increase blood pressure due to the sodium retention property, high dose of steroids use inhibits the enzyme 11-beta hydroxylase which leads to excessive production of deoxycortisterone, a mineralocorticoids in the adrenal glands and in due course a water and sodium retention.This mechanism of action of anabolic steroids towards increasing blood pressure was inhibited probably because of the high concentration of the antioxidants present in the Jos, UTC and Gboko varieties, respectively.Also, the high concentration of glycosides found in Jos variety could be a contributive factor that enhanced the the best blood pressure lowering effect observed in the research due to the fact that glycosides acts as a calcium channel blocking agent in the treatment of hypertension and other cardiovascular disorders and it is a potent dilator of peripheral arteries and in isolated tissue preparation exerts potent negative chronotropic, inotropic and dromotropic effect (Peter et al., 1980).Moreover, the limitations such as interrupted power supply, tail cuff bursting, etc were overcome successfully.

CONCLUSION AND RECOMMENDATION
This study showed that SL can effectively reduce blood pressure in hypertensive rats and it authenticates the various animal antihypertensive studies in SL.This also provides evidence that daily consumption of SE especially the Jos variety has no side effect.Therefore, these findings in addition to its cheapness and availability attenuate its desirability as an alternative blood pressure reducing agent in both mild to moderate hypertensive subjects.Though, its blood pressure reducing property is not commensurable to a standard antihypertensive drug (lisinopril).It further recommends that consumption of more of the Jos variety be encouraged and patronized in the early management of hypertension.Also, further investigation should be carried out on the mild, moderate and severe hypertensive rats with the duration and dosages properly put into consideration.The researcher also suggests that the effect of these plants be assessed in urine microscopy, protein and creatinine ratio, etc to find out the effect of this plant in renal function.Also, method of preparation/consumption (cooked, fresh and thermoxidized) should be considered in order to achieve a better therapeutic effect.

Table 2 .
The mean± standard deviation of the systolic blood pressure (mmHg).
Values are expressed as mean ±SD.*P<0.05 compared with A; µ P<0.05 compared with B; £ P<0.05 compared with C; € P<0.05 compared with D; β P<0.05 compared with E; ¥ P<0.05 compared with F.

Table 3 .
The mean± standard deviation of the diastolic blood pressure (DBP) (mmHg).Values are expressed as mean ±SD.*P<0.05 compared with A; β P<0.05 compared with E; ¥ P<0.05 compared with F.

Table 4 .
The mean± standard deviation of the total cholesterol level (TCL) (mg/dl).

Table 5 .
The mean± standard deviation of the high density lipoprotein cholesterol (HDL-C) (mg/dl).
¥P<0.05 compared with F

Table 6 .
The mean± standard deviation of the triglyceride level (TG) (mg/dl).

Table 7 .
The mean± standard deviation of the heart rates (HR) (beats/minute).

Table 8 .
The mean± standard deviation of the body weights (gram).Values are expressed as mean ±SD.*P<0.05 compared with A; µ P<0.05 compared with B; £ P<0.05 compared with C; € P<0.05 compared with D; β P<0.05 compared with E; ¥ P<0.05 compared with F.