Introduction

Exposure of offshore drilling workers to chemical stressors/ hazards is inevitable. Gardner (2003) reported that although base oils have attracted the most interest, workers in off shore drilling operations are potentially exposed to a range of particulates   especially during handling of additives like barium sulphate. Stress/ hazardous exposure  is one of the basic factors in the philosophical investigation of the causes and origin of a number diseases such as atherosclerosis, coronary heart diseases,  diabetes, aging, liver disease  (Selye, 1950) experienced by oil workers. ASTDR (1999) reported neurological effect, carcinogenic effect, pulmonary effect as some health effects associated with inhalation exposure. McDougal et al (2000), also demonstrated petroleum  distillates like crude oil and diesel oil associated renal, hepatic, neurological, immunological and pulmonary toxicity. The biochemical feature that are brought into focus in relation to the aetiology and prevention of these stress-induced diseases are serum cholesterol and its factors like low density lipo-protein cholesterol (LDL-C), high density lipo protein cholesterol (HDL-C). Eliot (1974), in his work, showed a good correlation between cholesterol and lipo-protein levels. Increase stress level was also reported to cause an increase in the secretion of cortisol in blood. Cortisol reacts with insulin to cause high fat deposition in visceral tissues.

In animal studies, it was found that stress raises serum and tissue cholesterol level of rats on normal diet (Horst et al, 1960; 1963.), Berger et al (1980), also reported that serum cholesterol level increased after different psychological stress in rats. There are various reports that stress can change the level of certain hormones like insulin, cortisol and epinephrine (Daniel, 1989; River et al, 1986; Ghulam et al, 2009). All these hormones affect lipid profile of the body to a great extent. Cardiovascular system is more prone to be affected by stress either directly or indirectly (Koolhass et al, 1991). Presently, cardiovascular accidents are reported to be the major killer especially in this modern human life that is full of hassles. The use of natural products and secondary metabolites from plants for the treatment of numerous human diseases like cancer, coronary artery disease, diabetes mellitus and infectious diseases have increased over the decades ( Omoregie and Okugbo, 2013). These plants natural products and secondary metabolites include alkaloids, tannins, flavonoids phenolic compounds , vitamins and minerals which in their mode of action act as antioxidants.  Hibiscus sabdariffa, l is one of such plants. The leaf is reported to contain protein, fat, carbohydrate, fibre, ash, calcium, phosphorus, iron, thiamine, B- carotene, riboflavin, niacin and ascorbic acid ( Chane, 1949; Duke,1978; Duke,1979; Duke et al.,1984; Morton, 1975;Watt and Breyer, 1962; Perry,1980).

This study was therefore conducted to evaluate the effect of Hibiscus sabdariffa calyx extract on crude oil associated biochemical changes in the blood and organs of the rabbit (Oryctalagus cuniculus).

Materials and Method

Plant materials

The dried flowers (calyces) of H.sabdariffa, l. locally known as Zobo in Nigeria was bought from Hausa quarters in Igbudu market, Warri, Delta State, Nigeria and was authenticated in the Department of  Plant Biology and Biotechnology, Faculty of life science, University of Benin, Benin city, Nigeria.

Animals

A total of 28 rabbits weighing between 700g and 800g bred in Ajuwawa market, Benin city, were used for this study. The rabbits were housed in a wooden frame steel mesh cage under standard environmental conditions. The rabbits were given standard rabbit food (pelleted growers mash) and tap water ad libitum and left to acclimatize to the laboratory conditions for 7days before the commencement of the study.

Crude oil sampling

Sample of crude oil from a land rig of KCA-Deutag Oil Company located in   Rivers state, Nigeria, was used for this study.

Preparation of extract

100g of dried calyces of Hibiscus sabdariffa, L., was soaked in 600ml ice cold de-ionized water for 4hours and filtered, a solution of the red pigment obtained and the residue discarded. The filtrate was made up to 1000ml with ice cold de-ionised water.

Determination of solid residue of H.sabdariffa Extract

The solid residue of the filterate was determined by drying 1ml of the filtrate at 200°C in a preweighed watch glass.  The solid content was found to be 52.0mg/ml.  A 5% (v/V) solution of the extract was prepared by making 5ml of the filtrate up to 100ml with deionised water.

Preparation of crude oil fortified diet

Growers’ mash bought from PTI road was air dried at room temperature until completely dried. It was sieved using a 2mm mesh. The sieved growers’ mash was supplemented or fortified with 2% crude oil/growers’ mash w/W. the mixture was homogenized  sieved again and air dried at room temperature. The fortified diet was packed in a celophene and stored in a refrigerated until required for feeding animals. The CFD feeding protocol was once weekly for 2 weeks.

Experimental design

Twenty eight (28) rabbits were used for this study. The rabbits were divided into 4 groups of seven rabbits each. The rabbits were weighed and left to acclimatize for seven days. They were housed in wooden framed cages with top and side metal mesh. They were given water and pelleted growers mash ad libitum.  At the end of the acclimatization period, they were weighed again just before the commencement of the study. Group I Rabbits: were given neither crude oil fortified diet nor given the extract (Minus CFD minus HSE). Group II Rabbits: were not given CFD but received the extract (100mg kg^-1 bd.wt). (Minus CFD Plus HSE). Group III Rabbits:  were given CFD (2% w/W) once weekly for 2 weeks, but not given the extract. (Minus HSE Plus CFD). Group IV Rabbits received the extract (100 mg kg^-1 bd wt) and also received the crude oil fortified diet  once weekly for 2 weeks (2% w/W) (Plus HSE Plus CFD).

Blood Sample Collection

Blood samples were collected from the pinna of each rabbit just before commencement of study and analyzed for glucose using the glucometer. At the end of the study duration (day 14), the external ear of each rabbit was pricked again for glucose assay using the glucometer. The rabbits were later sacrificed and blood samples collected into heprinized tubes for plasma cortisol, total cholesterol via cardiac puncture.  The blood samples were centrifuged thereafter at 3000 rpm for 5 minutes and the plasma was stored at -20^oC until required for the analyses.

Methods of analysis

Body and organ weights were obtained using a Mettler electronic balance as outlined by Emmanuel et al., (2013).  Total cholesterol and HDL-cholesterol were estimated by the cholesterol oxidase method (Richmond, 1973).  LDL- cholesterol was estimated using Freidewald’s formulae. Glucose was estimated based on the glucose oxidase method by means of  digital glucometer. Cortisol was analyzed by the Enzyme linked immuno-sorbent assay (ELISA) method outlined by Ramnik (2006).

Statistical Analysis

Results are expressed as mean ±SEM. Analysis of variance (Anova) was used to test for differences between treatments effects while Turkey multiple comparism test was used to test for significant differences between treatment means.  Values were considered significant at P<0.05.

Results

Gravimetry

The effects of crude oil fortified diet and  H.sabdariffa extract (HSE) on the body weight gain and organ body weight ratios of the experimental rabbits is presented in tables 3.1 and 3.2.

Administration of the HSE caused significant (P<0.05) reduction in body weight gain of rabbits relative to the control (group I) (Table 3.1.).  CFD also caused significant (P<0.05) reduction in body weight gain relative to the control (Table 3.1.). When animals were given the extract and subsequently CFD, there was a significant (P<0.05) reduction in body weight gain relative to the CFD only group (group III) (Table 3.1.)

Table 3.1: Effects of  CFD and aqueous extract of H.sabdariffa  on body weight gain of rabbits

^a value significantly different from group I (P<0.05)

^b value significantly different from group II (P<0.05)

^c value significantly different from group III (P<0.05)

The effects of crude oil fortified diet and aqueous extract of   H. sabdariffa,l on organ body weight ratio of  rabbits are presented in Table (3. 2).

Table 3. 2:  Effect  of  H. sabdariffa  aqueous extract on organ/body weight ratio of crude oil-exposed rabbits.

^avalue not significantly  different from  corresponding group I  value(P>0.05)

^bvalue significantly different from corresponding group I  value(P<0.05)

^cvalue significantly different from corresponding group II  value(P<0.05)

^dvalue not significantly different from corresponding group II value (P>0.05)

^evalue significantly different from corresponding group III value (P<0.05)

^fvalue not significantly different from corresponding group III value (P>0.05)

*see table 3.1 for interpretation of abbreviation and crude oil fortified diet  protocol

Administration of extract caused significant increase (P<0.05) in heart body weight ratio relative to group I (Table 3.2). CFD also caused a significant (P<0.05) decrease in kidney and heart / body weight ratios relative to their respective group II. In this study, liver body weight was not significantly (P>0.05) affected by any one of the treatments.

Cholesterol Status

Table 3.3: Effect of CFD and H. Sabdariffa calyx aqueous extract on plasma cholesterol status

^a value significantly (p<0.05) different from corresponding group I value (P<0.05)

^b value not significantly different from corresponding group I value (P>0.05)

^c value significantly different from corresponding group II value ( P<0.05)

^d value not significantly different from corresponding group II value (P>0.05)

^e value significantly different from corresponding group III value ( P<0.05)

 *see table 3.1 for interpretation of abbreviation and CFD protocol .

Administration of the extract caused a slight decrease in plasma total cholesterol status (Table 3.3) but it was not statistically significant (P>0.05). the extract also caused a significant increase in HDL-Cholesterol status (P<0.05), significant decrease in LDL-Cholesterol and Total Cholesterol/HDL ratio (P<0.05) relative to their corresponding group I. CFD caused a significant (P<0.05) increase in total cholesterol  levels, LDL-Cholesterol status, total cholesterol/HDL  ratio and a significant  (P<0.05) decrease in HDL-Cholesterol status relative to group II.

Prior administration of the extract to rabbits before exposure to CFD caused a significant decrease in total cholesterol status (P<0.05), a significant (P<0.05) decrease in LDL-cholesterol and a significant (P<0.05) decrease in total cholesterol/HDL ratio relative to their respective CFD only group (group III).

Crude Oil Contamination and Associated Biochemical Changes

Blood Glucose

The effects of CFD and whole aqueous extract of H.sabdariffa, L on blood glucose of rabbits are represented in Table 3.4.

Table 3.4: Effects of CFD and whole aqueous extract of H.sabdariffa,l calyx on blood glucose concentration of rabbit

^avalue (s) not significantly different from corresponding group I value (P>0.05)

^bvalue(s) significantly different from corresponding group II  (P<0.05).

^cvalue(s) not significantly different from corresponding group II (P>0.05)

^dvalue(s) significantly different from corresponding group III  (P<0.05).

^e value(s) not significantly different from corresponding group III (P>0.05)

 *see table 3.1 for interpretation of abbreviation and CFD protocol

 values on the same row having  **superscript are statistically significant

The effects of CFD and aqueous extract of H.sabdariffa, L calyx on blood glucose concentration of rabbit are presented in Table 3.4.

CFD caused a significant (P<0.05) increase in blood glucose concentration of rabbits relative to group II, post treatment (Table 3.4.). Treatment of the rabbits with HSE prior to exposure to CFD caused significant (P<0.05) decrease in blood glucose concentration relative to group III value, post treatment (Table 3.4.)

Blood Cortisol Status

The effects of stress and aqueous extract of H.sabdariffa, l calyx on plasma cortisol status are  presented in (Table 3.5).

Table 3.5:   Effects of stress and aqueous extract of H.sabdariffa,L. calyx on plasma cortisol status of  rabbit

^avalue not significantly different from group I (P>0.05)

^bvalue significantly different from group I (P<0.05)

^cvalue significantly different from group II (P<0.05)

^dvalue not significantly different from group III (P>0.05)

 *see table 3.1 for interpretation of abbreviation and CFD protocol

HSE caused a slight increase in plasma cortisol status relative to group I but it was not statistically significant. CFD caused a significant (P<0.05) increase in cortisol status relative to group I and II.

Prior treatment of rabbits with HSE before exposure to CFD caused a significant (P<0.05) increase in plasma cortisol status relative to group I and II but group III remained unaffected.

Discussion and Conclusion

This study examined the effects of  H.sabdariffa,L calyx aqueous extract  (HSE) on crude fortified diet linked biochemical changes in rabbit (Oryctalagus cuniculus).

Alterations in body weight gain are usually seen as toxicity indices ( Horiguchi et al., 1996; Timbrell, 1991). The significant (P<0.05) reduction in body weight gain (Table 3.1) and organ body weight ratio (Table 3.2) of rabbits in the crude fortified diet groups (III and IV) relative to the body weight gain of  their respective  non-crude diet groups (I and II) observed in this study, reveals the toxic nature of crude oil . This result aligns with the works of Timbrell, 1991; Horiguchi et al., 1996; and the later works of (Asagba, et al., 2004; Asagba, et al., 2007, ) who demonstrated how Cd, a chemical stressor caused a significant reduction  in male reproductive organ /body weight  ratios in rats and HSE completely reversed  the effect of Cd.  In the present study, prior administration of the extract before exposure of rabbits to the crude oil fortified diet resulted in further reduction in body weight gain instead of a reversal as observed by (Asagba et al., 2004, Asagba et al., 2007. This could be due to the weight reducing property of the extract. This aligns with the work of Emmanuel et al., 2013). The result obtained in table 3.1 is not surprising, this is because on examination of Table 3.4, plasma cortisol (Table 3.4) concentration was also reduced in group (IV) rabbits, and this may also be responsible for the observed weight loss. This is because under stressful situation( whether physical, chemical, biological or psychological stress), cortisol floods the blood with glucose supplying immediate energy, this leads to cell starvation and the consequence may be over-eating in the crude oil fortified diet fed rabbits (group III). The unused glucose can later be stored as body fats which could lead to increase body weight gain. Examination of   Table 3.4 revealed a reduction in cortisol concentration of group IV rabbits, although, it was not statistically significant (P>0.05).  The reduced cortisol level of group IV rabbits (Table 3.4) could also explain the reduction in body weight gain observed by group IV rabbits (Table 3.1) in this study. An investigation of Table 3.2 showed that the extract caused a significant (P<0.05) increase in kidney and heart body weight ratio of extract treated CFD exposed rabbits (group IV) when compared to CFD only group, (group III). HSE is seen to be repairing the kidney and heart organs in this study.  This restoration potential of the extract on the organs in this study could possibly be due to some of the bioactive principles of HSE. Phyto chemical screening of HSE revealed the presence of flavonoids and phenolic compounds like anthocyanins, glycosides and hydroxycitric acid.  This agrees with the works of Ali (2003), Tseng (1996) and Obi et al (2005) who demonstrated the protective effect of the extract and its anthocyanin component on paracetamol, tertbutyl  hydroperoxide and carbon tetrachloride – induced cell and organs toxicity in rats respectively.

Also in this study, a decrease in total cholesterol and LDL-cholesterol concentration occurred in the extract treated CFD exposed rabbit group, (group IV).  These are all component of total lipid. It is reasonable therefore to suggest that the lipid reducing effect of the extract observed in this study could be as a result of the reduction of other components of plasma total lipid besides cholesterol. This result aligns with the the work of (Ajay et al 2007;   Faraji et al., 1999; Onyenekwe et al., 1997 ; Odigwe et al., 2003; Herrera-arellamo et al., 2004; who demonstrated that HSE extract exhibits some anti hypertensive and cardio protective effects in rats.

The hormone responsible for stimulating glucose uptake also regulates blood lipid concentration as well. The decrease in plasma glucose concentration of extract treated -crude fortified diet exposed rabbit group (group IV) relative to CFD only group agrees with  the finding. The  findings from this present study is in agreement with the works of El Saadany et al (1991),   Chau et al (2004), Carajal- Zarrabel  et al (2005) , Hirunpanich et al ( 2006).

Reduction in total cholesterol status and an increase in HDL-Cholesterol is usually an indication of low risk of coronary artery diseases. In this study (Table 3.3), there was a significant (P<0.05) decrease in total cholesterol  LDL-Cholesterol status  and Cholesterol/HDL ratio. There was also a significant (P<0.05) increase in HDL-cholesterol in extract only treated rabbit group (group II).  The decrease in plasma cholesterol observed in the extract treated CFD group (group IV) could be as a result of increase HDL-C which may have caused the increase removal of cholesterol from tissues for metabolism by the liver. This report aligns with the work of  Prince et a,l 1999 .The decreases in plasma total cholesterol status and LDL-C of rabbits treated with the extract before giving CFD observed in this study could also be due to  the inhibitory effect of HSE on LDL-C oxidation . This could also be as a result of the glycoside delphinidine-3- glucoside, content of HSE. This confirms the works of Chau et al (2004); Obi et al, 2013, where the authors demonstrated the inhibitory effects of HSE extract on low density lipoprotein oxidation; the hypocholesterolemic; the antioxidants effects of the extract.

Stress has been reported to cause glycogen immobilization Inoguchi and his co authors in a previous work demonstrated the direct relationship between high palmitate level and increase level of reactive oxygen species in patients. In the present study, crude oil fortified diet increased post treatment whole blood glucose concentration while prior administration of the extract caused a reduction in whole blood glucose concentration. This glucose reducing effect of the extract could be due to either hormonal stimulation of blood glucose or its ability to reduce plasma total lipid.   Prior administration of extract (HSE) before exposure to the crude oil fortified diet (CFD) caused a decrease in blood glucose concentration relative to the extract only group (group II) although, the value was not statistically significant (P>0.05).  This finding agrees with the previous works of   Irondell et al, 1977, Kim et al., 1966 and Merson et al.,1978. Who demonstrated how stress leads to the immobilization of glycogen and enhance production of pyruvate.

Cortisol is a glucocorticoid hormone synthesized from cholesterol. Cortisol aids the synthesis of triacylglycerol to sustain energy during stress. Cortisol acts by binding to cortisol binding globulin (CBG). Stress increases cortisol level in blood and increase cortisol level enhances the synthesis of cortisol binding globulin (CBG) allowing for more cortisol- CBG interaction and drastically reducing the presence of free   cortisol  thereby preventing the negative effect of prolong cortisol in circulation during prolong stress. But in this study, the chemical stressor (crude oil fortified diet) elevated cortisol level but test animals who received HSE before exposure to the CFD showed an insignificant (P>0.05) reduction in cortisol concentration. This seemingly cortisol increasing potency of the extract (HSE) may be due to the presence of  mavidin and  delphinidine anthocyanins reported from the previous work done by (Ologundudu et al 2009), who demonstrated  the structural  resembles of the structure of collapsed cortisol  and delphinidine. This may have been responsible for the observed   increased cortisol concentration observed in this study. The mechanism could be that since HSE structurally resembles collapsed cortisol, there is competition for cortisol binding site on CBG and hence the apparently increased pool of cortisol observed in this study.

In conclusion, this study advocates the regimented and regular  intake of H.sabdariffa aqueous extract amongst offshore workers for organ protection. However, further studies are needed to determine the exact remedial concentration.

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