Chemotherapeutic Properties of Boerhaavia Diffusa and Black Caraway Oil on DMBA-Induced Carcinogenesis and Hypercholesterolemia in Animals

Supervision By
Dr. R.P. Thapliyal

Ph.D. Thesis Presentation By Subodh Kumar Chauhan

Department of Zoology & Biotechnology

Faculty of Life Science

H.N.B. Garhwal Central University, Srinagar (Garhwal)

Boerhaavia diffusa is a medicinal plant widely used in the Ayurvedic medicine. Ayurveda is an ancient traditional medical system of health care of the Veda civilization, flourishing in India many thousand years ago. The term AYURVEDA means Knowledge or Science of Life (From Ayus meaning life and Veda meaning acquaintance, science) in Sanskrit. In fact, Ayurveda focuses on the physical, spiritual and mental aspects of an individual. The plant was named in honor of Hermann Boerhaave, a famous Dutch physician of the 18th century (Chopra, 1969). B. diffusa (Spreading Hogweed in English) belonging to the family of the Nyctaginaceae, is mainly diffused perennial herbaceous creeping weed of India (known also under its traditional name as Punarnava). B. diffusa is up to 1 m long or more, having spreading branches. The stem is prostrate, woody or succulent, cylindrical, often purplish, hairy, and thickened at its nodes. The last two decades witnessed an enormous research rush to reveal the pharmacological actions of an annual spicy delicate and beautiful herb known by the Latin name Nigella sativa Linnaeus (Black Caraway Oil) variety hispidula (brachyloba) that belongs to the botanical family Ranunculaceae. The plant is an erect profusely branched herb that can attain heights of 40 and up to 70cm.

Cancer is the second most common cause of death after cardiovascular diseases (CVD) in most developed and in many developing countries, including India. In this country, every year around seven million new cases of cancer are being detected. The global burden of cancer doubled during the last 30 years of the last century. In 2008, it is estimated that there were over 12 million new cases of cancer diagnosed, 7 million deaths from cancer and 25 million persons alive with cancer within five years of diagnosis. The continued growth and ageing of the worlds population has immediate consequences on the cancer burden. By 2030, it is estimated that there will be over 26 million incident cases of cancer annually.(IARC-W.H.O. Report 2008). Lung, stomach, liver, colon and breast cancer cause the most cancer deaths each year. About 30% of cancer deaths can be prevented. The change may be started by external agents and inherited genetic factors. About 72% of all cancer deaths in 2007 occurred in low- and middle-income countries. Deaths from cancer worldwide are projected to continue rising, with an estimated 12 million deaths in 2030, (WHO-2010).

The 20th century saw unparalleled increases in life expectancy and a major shift in the causes of illness and death throughout the world. A century ago, cardiovascular disease (CVD) accounted for fewer than 10% of all deaths; today, it accounts for approximately 30% worldwide (Gaziano, T.A., 2005; Yusuf S. et al., 2001). The increase in CVD, through a proliferation of risk factors that are heavily influenced by lifestyle choices, is the new challenge for many developing countries. Cardiovascular diseases accounted for 16.7 million or 29.2 % of the total global deaths in 2002, according to the World health report 2003, around 80 % of deaths due to CVD took place in low- and middle-income countries. By 2010, The contribution of developing countries to the global burden of CVD, in terms of disabilityadjusted life-years lost, was 2.8 times higher than that in developed countries (Reddy and Yusuf, 1998). By 2020, about 42 % of the total deaths in India are projected to be due to cardiovascular causes (Murray and Lopez, 1996). During the period 2000-2030, about 35 % of all deaths due to CVD in India are projected to occur in the age group of 35-64 years (Leeder et al., 2004).

Several lines of research have established that both CVD and most prominent ras growth protein-induced cancers are coupled and controlled by a common mechanism involving the up regulation of HMG-CoA reductase, the rate controlling enzyme in the cholesterol biosynthetic pathway. In tumour cells, the feed-back mechanism at the level of HMG-CoA reductase is flopped, resulting in a several fold increase in its activity and cholesterol formation, which also results in hypercholesterolemia and subsequently CVD. Increased HMG-CoA reductase activity in tumour cells is associated with unrestricted supply of farnesyl pyrophosphates, for rapidly proliferating tumours.

We investigated the effect of aqueous ethanolic extract of B. diffusa and Black Caraway Oil

on DMBA-Induced carcinogenesis and hypercholesterolemia in animals which was not

studied earlier.

The first pharmacological studies have demonstrated that the root of punarnava exhibits a wide range of properties: anti-inflammatory (Bhalla et al., 1968, 1971), diuretic (Gaitonde et al., 1974), laxative (Chopra et al. 1956), anti-urethritis (Nadkarni, 1976), anticonvulsant (Adesina, 1979), antinematodal (Vijayalakshmi et al., 1979), antifibrinolytic (Jain and Khanna, 1989), antibacterial (Olukoya et al., 1993), antihepatotoxi (Mishra, 1980; Chandan et al., 1991; Rawat et al., 1997), antihelmintic, febrifuge, antileprotic, antiscabby and antistress activites, antidiabetic activity (Pari et al. 2004), antifibrionolitic and anti-inflammatory activities (Hiruma-Lima, 2000), immunosuppressive activity (Mehrotra et al., 2002). Protective effect of Nigella sativa seed against carbon tetrachloride-induced liver damage, (Al-Ghamdi, M.S., 2003). Nigella sativa oil for prevention of chronic cyclosporine nephrotoxicity: an experimental model, (Uz, E. et al., 2008). Effect of Nigella sativa on oxidative stress and beta-cell damage in streptozotocin-induced diabetic rats, (Kanter, M. et al., 2004). The antioxidative and antihistaminic effect of Nigella sativa and its major constituent, thymoquinone on ethanol-induced gastric mucosal damage, Kanter, M. et al., 2006). Effect of volatile oil constituents of Nigella sativa on carbon tetrachloride-induced hepatotoxicity in mice: evidence for antioxidant effect of thymoquinone, (Mansour, M.A. et al., 2001). Nigella sativa protect against ischaemia reperfusion injury rat kidney, (Bayrak, O. et al., 2008). The role of black seed in the proliferation and biochemical marker levels of Hep-2 cells, (Hansen, J.T. et al., 2003). Effect of black cumin (Nigella sativa) on heart rate, some hematological values and pancreatic beta-cell damage in cadmium-treated rats, (Demir, H. et al., 2006).

The proposed study designed to investigate both hypolipidemic and cancer chemotherapeutic properties of Boerhaavia Diffusa roots extracts and Black caraway Oil. Cardiovascular diseases (CVD) are more killer then Cancer in the world. So, creates a need to develop a simple and cost-effective methodology for the isolation and extraction of ethanolic roots of B. diffusa (Bd) and Black Caraway Oil (BCO).

1. Phytochemical Screening of B. diffusa (Bd) and Black Caraway Oil (BCO) using HPLC. 2. To estimate the effect of ethanolic extract of B. diffusa (Bd) and Black Caraway Oil (BCO) in the suppression of experimental carcinogenesis in rat liver and their impact on different marker enzymes. Will be investigated carcinogen 7, 12-dimethylbenz [] anthracene (DMBA), which is known to induce both carcinogenesis and hypercholesterolemia/CVD, was used. 3. To correlate the efficacy of B. diffusa (Bd) and Black Caraway Oil (BCO) extracts in preventing the increase in plasma triglycerides (TG), total cholesterol (TC), VLDL-C, LDLC, small dense (sd)-LDL-C and large buoyant (lb)-LDL-C in hyperlipidemic rats.

4. In rats with DMBA induced carcinogenesis coupled with hypercholesterolemia, in addition of Boerhaavia Diffusa and Black caraway Oil mediated anticancer impacts, anticholesterol effects on plasma and lipoprotein lipids and HMG-CoA reductase activity will also be investigated. 5. The role of B. diffusa (Bd) and Black Caraway Oil (BCO) in the suppression of carcinogeninduced cancer at pre- and post-initiation and progression stages will also be examined histological in liver, kidney and pancreas. Thus, both hypercholesterolemia and carcinogenesis mediate a sustained free radical load (Antioxidant power), which could enhance lipid / lipoprotein peroxidation.

Animals White male albino rats, weighing about 150-180 g were purchased from Indian Veterinary Research Institute, Bareilly, India, were maintained to animal house under standard normal condition having natural photoperiod (12 hours light/dark cycle) at temperature 2510C and 50-60% humidity. Animal experimentation protocols conform to the Institutional Animal Ethics committees guidelines. The hairs on dorsal skin of the animals in the interscapular area was shaved 3 days before the commencement of the experiment and only those animals in the resting phase of the hair cycle were taken for the study, because resting hair retains carcinogens for a longer period. The rats were sacrificed at sixth hour of dark cycle at the peak of diurnal rhythm of HMG-CoA reductase (Edwards et al., 1977). Diet The rats were given pelleted rat chow and tap water ad libitum. Experimental rats from respective groups were administrated with Boerhaavia diffusa (Bd) and Black Caraway Oil (BCO). Both Bd (1mg/ 1ml methanol stock) and BCO (2ml/Kg b.w.orally) were solubilised in methanol. Rat chow was administered as through orally. Bd and BCO suspension was also administrated through orally in two equal doses of 2 ml/Kg b.w.orally.

Studies in rats For investigating the hypercholesterolemia, carcinogens and antioxidant effect of B. diffusa (Bd) and Black Caraway Oil (BCO), rats were divided in the following groups as described below: Group I - Normal Control (N-C) Fifteen rats were given a single dose of 0.9% NaCl, 5ml/Kg b.w.ip through intraperitoneal injection and were feed rat chow for 16 weeks. Group II - Infected Control (I-C) Fifteen rats in this group were given a single dose of 65 mg/Kg body weight of DMBA in Acetone though intraperitoneal injection and were feed rat chow for 16 weeks. Animals were infected in three weeks. Group III - Infected B. Diffusa Treated (I-BdT) DMBA induced fifteen rats in this group were given a topical application of B. diffusa extract dissolve in 1mg/1ml methanol and two equal doses (morning and evening) of 2 ml/Kg b.w. through orally and were feed rat chow for 16 weeks. Group IV - Infected Black Caraway Oil Treated (I-BCOT) DMBA induced fifteen rats in this group were given a topical application of Black Caraway Oil two equal doses (morning and evening) of 2ml/Kg b.w. through orally and were feed rat chow for 16 weeks.

Isolation and Aqueous ethanolic Extraction of Boerhaavia diffusa roots (Bd) The fresh roots of Boerhaavia diffusa linn. (Punarnava) were collected from the foothills of Dehradun (Uttarakhand) and its adjoining area and identified by Dr. R.L. Painuly at the Botany Department of HNBGU and the specimen was preserved in the herbarium voucher no. GUH20434. The plant material roots were washed, shad-dried avoiding sun drying due to the signature modification of the biochemical's, therein under the influence of UV radiation (Sun-rays) pulverized, and finally sieved. 100 gm roots were then subjected to soxhlet extraction using 70% hydro-alcoholic solvent (70% ethanol: 30% distilled water). The final extract was filtered and the remaining alcohol was allowed to evaporate. Black Caraway Oil parched market in Dehradun, which used as herbal medicine.

HPLC of extract of B. diffusa (Bd) and Black Caraway Oil (BCO) The technical details have been described by Simpson, C.F. (1978). Extraction of the constituents from Bd and BCO were carried out using C18 PrepSep mini columns followed by quantification of the recovered constituvents by HPLC on a reversed-phase muBondapack C18 analytical column, using an isocratic mobile phase of water: methanol: 2-propanol (50: 45: 5% v/v) at a flow rate of 1ml/min, detection was 254nm.

Analytical Procedures Collection of blood and packed erythrocytes At the end of the experiment treatment, overnight fasted rats in each group were anaesthetized and blood drawn from cardiac puncture. The blood from each rat in a given group was collected in heparinised tubes, mixed gently by inversion 2-3 times and incubated at 40C for 2-3 h. Plasma was separated from blood by centrifugation at 2,500 rpm for 30 min, aliquoted and stored at 40C or frozen at 200C for use in other experiments. Packed erythrocytes hemolysate was prepared as described by Lakshmi and Rajagopal (1998). Collection of different organs In addition to the blood, liver, kidney and pancrease from male rats were excised that kept into ice cold saline. Portion of organs was immediately fixed in 10% neutral formalin for histological studies. Preparation of liver, lung and kidney homogenate and post-mitochondrial supernatant At the end of the experiment, liver, lung and kidney from each rat were promptly excised and chilled in ice cold saline. After washing with saline, liver, lung and kidney were blotted and weighed. Each liver, lung and kidney were cut into pieces, mixed and 10 g of wet tissue was homogenized with 90 ml of chilled 0.1 M sodium phosphate buffer, pH 7.4, containing 1.17 % KCl in a waring blender. The volume of each homogenate was recorded and centrifuged at 1,000 rpm for 10 min at 40C. After centrifugation, a portion of each homogenate from liver, lung and kidney thus obtained was aliquoted and stored at 200C. The remaining portions of the liver, lung and kidney homogenates were centrifuged at12, 000 rpm for 20 min at 40C. The post-mitochondrial supernatant thus obtained was aliquoted and stored at 200C for future use.

Determination of free radical scavenging activity (antioxidant capacity) of B. diffusa and BCO The procedure of Mellors and Tappel (1966) as modified by Khanduja and Bhardwaj (2003) was used for determining the free radical scavenging activity of B. diffusa and Black Caraway Oil. The assay was carried out in a medium containing 40 mM tris buffer, pH 7.4 and 125 M ethanolic solution of 2, 2diphenyl-1-picryl hydrazyl (DPPH). The reaction was started by the addition of ethanolic solution of B. diffusa and Black Caraway Oil (25-200 M) in a total volume of 2 ml. The samples were mixed thoroughly and the absorbance was recorded in dark at 517 nm (27 20C) at 1 min time interval up to 10 min against absolute ethanol. Determination of plasma triglycerides Triglycerides were determined by using enzymatic kit. The method uses a modified Trinder color reaction to produce a fast, linear, end point reaction (Trinder, 1969). The intensity of the color produced after incubation is directly proportional to the concentration of the triglycerides in the plasma samples when measured at 500 nm in a Beckman DU 640 spectrophotometer. Triglycerides in plasma samples were calculated by using a triolein standard. Determination of plasma free fatty acid Free fatty acid in plasma was estimated as described by Duncombe (1963). The procedure of Folch et al. (1957) was used for extracting free fatty acid from plasma lipids. Determination of all other parameter (LDL, VLDL, HDL, Sd-LDL, Lb-LDL, HMG-CoA, GST, Catalase, SOD, XO, GPx) All parameter were estimated as described by slandered protocol.

Extraction of B. diffusa (Bd) and Black Caraway Oil (BCO) The aqueous extract of B. diffusa was continued into dry solid by rotary vacuum evaporator. The yield of B. diffusa was 7.78%. The powder obtained was stored at 40C till further use. Whereas Black Caraway oil extracts from black cumin or Nigella sativa L. seeds was obtained from the local market (100% pure), which is marked as a medicine After both 1mg/1ml dilution in the methanol the extract was two equal doses of 2ml/Kg b.w.orally. HPLC extracts of B. diffusa (Bd) and Black Caraway Oil (BCO) The fraction was purified by HPLC have eleven peaks of B. diffusa (Bd) shows Boeravinone A1, B1, C2, D, E, F, Hypoxanthine, Punarnavoside and Urosolic acid as the major phytoconstituents and 4 main peaks found in Black Caraway Oil (BCO) in HPLC. Pharmacological active constituents of the Black Caraway Oil (BCO) as found as thymoquinone (TQ), dithymoquinone (DTQ), thymohydroquinone (THQ) and thymol (THY) (Ghosheh, O.A. et. al., 1999).

Antioxidative Activities of B. diffusa (Bd) and Black Caraway Oil (BCO) Antiradical activity or hydrogen donating ability of B. diffusa (Bd) and Black Caraway Oil (BCO) was measured by using DPPH (2,2-diphenyl,1-picrylhydrazyl), which reflects the antioxidative properties of these compounds. As shown in Fig. 2.2, the half quenching concentrations (IC50) were as follows: B. diffusa (Bd), 44.42 M; Black Caraway Oil (BCO), 38.11 M. These findings indicate that as compared to B. diffusa (Bd) 89% was found more efficient then Black Caraway Oil (BCO) 76% respectively. B. diffusa shows more free radical scavenging property then Black Caraway Oil.

Fig. 2.2. Free radical scavenging activities of B. diffusa (Bd) and Black Caraway Oil (BCO). The antioxidant activities of the above compounds at the indicated concentrations were carried out as described in methods. The assay is based on the reduction of 2, 2-diphenyl, 1-picrylhydrazyl (DPPH), which gives strong absorption maxima at 517nm. Values represent the mean of triplicate determinations. The average error in the data points in these assay were mean less than 3 %. The average absolute absorbance value of 100 % DPPH was 0.00786 0.00029.

TABLE 1 IMPACT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON HEMOGLOBIN IN DMBA-INDUCED RATS AFTER TREATMENT

Hemoglobin Group N-C (g/dl) 14.280.264* 11.420.012* (-20.03%)a 13.960.016* (+22.24%)a

I-C

I-BdT

I-BCOT

13.440.011*
(+17.69%)a

*Values

are mean SD from pooled blood or plasma of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001. Significantly different from I-C at ap<0.001.

TABLE 2 IMPACT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON PLASMA TOTAL LIPID, TRIGLYCERIDES, FREE FATTY ACID AND TOTAL CHOLESTEROL IN DMBA-INDUCED RATS AFTER TREATMENT

Group

Total lipid

Triglycerides

Free fatty acid 134.150.412 151.290.512 (+12.78 %)a 137.310.291 (-9.05%)b 134.150.212 (-11.33 %)a

Total cholesterol 88.662.46 154.683.86 (+74.46 %)a 110.622.88 (-28.48 %)a 102.211.76 (-33.92 %)a

N-C I-C

390.191.40* 510.362.23* (+30.79 %)a 483.221.56* (-5.32 %)b 486.311.12* (-4.71 %)b

54.221.17 114.612.43 (+111.38 %)a 66.241.92 (-42.20 %)a 62.681.86 (-45.3 %)a

I-BdT
I-BCOT

*Values

are mean (mg/dl) SD from pooled plasma of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap < 0.001. Significantly different from I-C at ap<0.001 and bp<0.05.

TABLE 3 IMPACT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON PLASMA VLDL-C, LDL-C, HDL-C, HDL2-C AND HDL3-C SUBFRACTIONS AND NON-HDL-C IN DMBA-INDUCED RATS AFTER 16 WEEKS OF TREATMENT

Parameters
VLDL- C

N-C 11.010.054*

I-C 21.440.242* (+94.73 %)a 111.220.265 (+105.75 %)a 18.040.043 (-13.93 %)a 5.120.012 (-27.68 %)a

I-BdT 13.190.165* (-38.48 %)a 62.420.302 (-43.87 %)a 31.010.106 (+71.89%)a 14.120.022 (+175.78 %)a

I-BCOT 13.260.214* (-38.15 %)a 57.960.192 (-47.88 %)a 26.910.176 (+49.17 %)a 11.160.014 (+ 117.97 %)a

LDL-C

54.060.131

HDL- C

20.960.071

HDL2- C

7.080.012

HDL3- C

13.880.042

13.320.043 (-4.03 %)c 136.643.89 (+99.07 %)a

18.960.071 (+42.34 %)a 79.612.52 (-41.74 %)a

17.960.072 (+34.83 %)a 75.303.11 (-44.89 %)a

Non-HDL- C

68.642.88

*Values

are mean (mg/dl) SD from pooled plasma of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001 and cp<0.02. Significantly different from I-C at ap<0.001.

TABLE 4 IMPACT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON PLASMA LDL, SMALL DENSE AND LARGE BUOYANT LDL SUBPOPULATION IN DMBA-INDUCED RATS AFTER TREATMENT
Parameters LDL-C LDL- apoB Sd-LDL-C % LDL-C Sd-LDL- apoB % LDL-apoB Lb-LDL-C N-C 54.060.131* 122.340.821 16.430.052 30.400.321 39.520.612 32.350.832 36.970.046 I-C 111.120.265* (+105.55 140.860.422 (+15.14 %)a 60.400.086 (+267.62 %)a 54.351.26 (+78.78 %)a 72.331.019 (+83.02 %)a 51.351.62 (+58.73 %)a 50.390.144 (+37.68 %)a 45.360.962 (-36.16 %)a 68.621.10 (-15.80 %)a 48.720.835 (-26.85 %)a %)a I-BdT 62.420.302* (-43.83 126.060.824 (-10.51 %)b 19.270.061 (-68.09 %)a 30.880.624 (-43.18 %)a 41.380.616 (-44.17 %)a 32.040.605 (-37.60 %)a 43.580.022 (-13.51 %)a 69.830.943 (+53.95 %)a 82.741.12 (+20.58 %)a 65.640.859 (+34.73 %)a %)a I-BCOT 57.960.192* (-47.84 %)a 129.420.624 (-8.12 %)c 17.250.044 (-71.44 %)a 29.770.462 (-45.22 %)a 38.731.180 (-46.45 %)a 29.930.531 (-41.71 %)a 40.640.081 (-19.35 %)a 70.121.129 (+54.58 %)a 82.460.424 (+20.17 %)a 63.720.825 (+30.79 %)a

% LDL-C
Lb-LDL-apoB % LDL-apoB

71.051.213
81.501.181 66.600.926

N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001. Significantly different from I-C at ap<0.001, bp<0.01 and cp<0.02

TABLE 5 EFFECT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON THE RATIOS OF LDL-C/HDL-C, HDLC/TC, Sd-LDL-C/HDL-C AND Lb-LDL/HDL-C IN DMBA-INDUCED RATS AFTER TREATMENT

Group

Ratio N-C LDL-C/HDL-C 2.700.021* I-C 6.160.034* (+128.15 %)a 0.1160.001 (-48.67 %)b 3.350.029 (+308.54 %)a 2.790.029 (+50.81 %)a I-BdT 2.010.024* (-67.37 %)a 0.2800.075 (+141.37 %)b 0.6210.004 (-81.46 %)a 1.400.010 (-49.82 %)a I-BCOT 2.150.019* (-65.15 %)a 0.2530.034 (+118.10 %)d 0.6410.003 (-80.86 %)a 1.510.014 (-45.88 %)a

HDL-C/TC

0.2260.030

Sd-LDL-C/HDL-C Lb-LDL/HDL-C

0.8200.006 1.850.019

For

the calculation of ratios, data is taken from Table 3, 4 and 5. are mean SD from pooled plasma of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001 and bp<0.01. Significantly different from I-C at ap<0.001, bp<0.05 and dp<0.01.
*Values

TABLE 6 EFFECT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON LIVER, LUNG AND KIDNEY TRIGLYCERIDES, TOTAL CHOLESTEROL AND FREE FATTY ACID CONTENT IN DMBA-INDUCED RATS AFTER TREATMENT
Liver Lung Kidney

Group

Total Triglycerides cholesterol (mg /100mg (mg /100mg protein) protein) 0.6820.001* 0.8660.006* (+26.98%)a 2.720.066 4.960.025 (+82.35%)a

Free fatty acid (mg /mg protein) 21.240.566 28.960.231 (+36.35%)a

Total Triglycerides cholesterol (mg /100mg (mg /100mg protein) protein) 0.5840.003 0.6790.005 (+16.27%)a 2.500.012 3.210.043 (+28.40%)a

Free fatty acid (mg /mg protein) 12.820.143 13.020.04 (+1.56%)e

Total Triglycerides cholesterol (mg /100mg (mg /100mg protein) protein) 0.5590.005 0.6970.005 (+24.68%)a 2.260.003 2.820.005 (+24.78%)a

Free fatty acid (mg /mg protein) 8.210.081 12.430.11 2 (+51.40%)a 9.580.065 (-22.93%)a

N-C I-C

I-BdT

0.7580.004* (-12.47%)a

3.690.022 (-25.60%)a

23.500.112 (-18.85%)a

0.6210.004 (-8.54%)c

2.530.045 (-21.18%)a

11.960.652 (-8.14%)a

0.5650.003 (-18.94%)a

2.460.005 (-12.76%)a

I-BCOT

0.8240.004* (-4.85%)a

3.260.021 (-34.27%)a

24.860.093 (-14.16%)b

0.5960.001 (-12.22%)c

2.660.056 (-17.13%)a

11.990.096 (-7.91%)a

0.5960.003 (-14.49%)a

2.410.004 (-14.54%)a

10.480.14 2 (-15.69%)a

are mean SD from homogenate of pooled liver, pooled lung or pooled kidney 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001. Significantly different from N-C at ap<0.001 and ep not significant. Significantly different from I-C at ap<0.001, bp<0.05 and cp<0.02.
*Values

TABLE 7 IN VIVO MODULATION OF HEPATIC HMG-CoA REDUCTASE ACTIVITY IN DMBA-INDUCED RATS TREATED WITH B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) FOR 16 WEEKS OF TREATMENT

Group

HMG-CoA reductase activity

6.550.082* 3.110.024* (-52.52 %)a 4.820.067* (+54.98 %)a 3.960.036* (+27.33 %)a

N-C

I-C

I-BdT I-BCOT

are mean SD from homogenate of pooled liver of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001. Significantly different from I-C at ap<0.001.
*Values

TABLE 8 ANTIOXIDANT IMPACT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON PLASMA TOTAL ANTIOXIDANTS, CONJUGATED DIENE, LIPID HYDROPEROXIDE AND MALONDIALDEHYDE CONTENT IN DMBA-INDUCED RATS AFTER TREATMENT

Group

Total antioxidants

Conjugated diene

Lipid hydroperoxide

MDA

N-C

48.370.056*

9.310.011

2.510.032

3.420.081

I-C

36.890.121* (-23.73 %)a 46.240.034* (+25.34 %)a 42.100.020* (+14.12 %)b

14.360.016 (+54.24 %)a 12.460.014 (-13.23 %)a 13.210.022 (-8.00 %)b

3.810.015 (+51.79 %)a 3.120.006 (-18.11 %)a 3.200.024 (-16.01 %)a

5.910.116 (+72.80 %) a 4.820.044 (-18.44 %)a 4.990.062 (-15.57 %)a

I-BdT

I-BCOT

*Values

are mean (mole/dl) SD from pooled plasma of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001. Significantly different from I-C at ap<0.001 and bp<0.05.

TABLE 9 EFFECT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON LIVER, LUNG AND KIDNEY CONJUGATED DIENE, LIPID HYDRPEROXIDE AND MALONDIALDEHYDE CONTENT IN DMBA-INDUCED RATS AFTER TREATMENT

Liver Group Conjugated diene Lipid hydroperoxide

Lung Lipid hydroperoxide

Kidney Lipid hydroperoxide

MDA

Conjugated diene

MDA

Conjugated diene

MDA

N-C

6.120.021*

1.4980.001

3.610.014

2.520.054

0.5260.02

3.010.024

2.210.011

0.4460.004

4.280.06 0 5.840.02 0 (+36.45% )a 5.320.03 1 (-8.90%)a 5.160.01 0 (11.64%)c

I-C

8.180.015* (+33.66%)a

2.2120.001 (+47.66%)a

4.610.084 (+27.70%)a

3.240.034 (+28.54%)a

0.7540.003 (+43.35%)a

3.990.016 (+32.56%)a

3.100.025 (+47.27%)a

0.7020.012 (+57.39%)a

I-BdT

6.420.054* (-21.51%)a

1.9630.002 (-11.26%)a

3.580.066 (-22.34%)a

2.800.035 (-13.58%)a

0.5650.003 (-25.07%)a

3.200.026 (-19.80%)a

2.580.024 (-16.77%)a

0.4850.004 (-30.91%)a

I-BCOT

6.500.011* (-20.54%)a

1.9780.022 (-10.58%)a

4.240.011 (-8.03%)d

2.740.034 (-15.43%)d

0.5850.002 (-22.41%)a

3.170.044 (-20.55%)a

2.590.020 (-16.45%)a

0.5100.006 (-27.35%)a

*Values

are mean (nmole/mg protein) SD from homogenate of pooled liver, pooled lung or pooled kidney 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001. Significantly different from I-C at ap<0.001 and cp<0.02.

TABLE 10 B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON PLASMA, LIVER, LUNG AND KIDNEY XANTHINE OXIDASE ACTIVITY IN DMBA-INDUCED RATS AFTER TREATMENT

Xanthine oxidase activity

Group
Plasma (U/ml) N-C I-C 6.320.021* 10.040.126* (+58.86%)a 7.110.116* (-29.18%)a 7.660.152* (-23.70%)a Liver (U/mg protein) 0.6120.0021 0.8500.0025 (+38.89%)a 0.6360.0016 (-25.18%)a 0.7120.0012 (-16.23%)a Lung (U/mg protein) 0.3920.0011 0.5750.0010 (+46.68%)a 0.5260.0012 (-8.52%)a 0.5220.0021 (-9.22%)a

Kidney (U/mg protein) 0.1790.0011 0.4560.0011 (+154.75%)a 0.2900.0042 (-36.40%)a 0.2880.0060 (-36.84%)a

I-BdT

I-BCOT

are mean SD homogenate of pooled liver, pooled lung or pooled kidney of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001. Significantly different from I-C at ap<0.001.
*Values

TABLE 11 IMPACT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON LIVER, LUNG AND KIDNEY CATALASE AND SUPEROXIDE DISMUTASE ACTIVITIES IN DMBA-INDUCED RATS AFTER TREATMENT

Liver Catalase (U/mg protein) Superoxide dismutase (U/mg protein) 0.8540.002 0.6540.002 (-23.42 %)a 0.7870.003 (+20.34 %)a 0.7700.005 (+17.74 %)a

Lung Catalase (U/mg protein) Superoxide dismutase (U/mg protein) 2.110.006 1.8830.004 (-10.76 %)a 2.1400.004 (+13.65 %)a 2.1560.004 (+14.50%)a

Kidney Catalase (U/mg protein) 3.450.154 2.230.052 (-35.36 %)a Superoxide dismutase (U/mg protein) 0.9320.004 0.8020.002 (-13.95 %)b

Group

N-C
I-C I-BdT I-BCOT

4.680.142* 3.320.223* (-29.06 %)a 4.220.121* (+27.11 %)a 4.120.022* (+24.10 %)a

2.510.062 2.270.033 (-9.56 %)b 2.400.054 (+5.73 %)b 2.540.022 (+11.89 %)a

3.500.042 0.9240.002 (+56.95 %)a (+15.21 %)a 3.560.166 0.9350.005 (+59.64 %)a (+16.58 %)a

are mean SD from PMS fraction of pooled liver, pooled lung or pooled kidney of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001 and bp<0.01. Significantly different from I-C at ap<0.001 and bp<0.05.
*Values

TABLE 12 IMPACT OF B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON LIVER, LUNG AND KIDNEY TOTAL, FREE AND PROTEIN-BOUND -SULFHYDRYL CONTENTS OF GLUTATHIONE IN DMBA-INDUCED RATS AFTER TREATMENT

Liver Group Total-SH Free-SH

Lung

Kidney

Proteinbound-SH 64.521.10

Total-SH

Free-SH

Proteinbound-SH 56.110.558

Total-SH

Free-SH

Proteinbound-SH 50.300.401

N-C

76.821.21*

13.800.314

66.860.561

12.730.104

58.700.406

10.310.082

I-C

37.512.46* (-51.17%)a

12.520.116 (-9.27%)a

26.200. 462 (-59.39%)a

31.160.604 (-53.39%)a

10.610.071 (-16.65 %)a

22.310.627 (-60.26%)a

23.020.448 (-60.78%)a

9.040.026 (-12.32%)a

15.870.442 (-68.45%)a

I-BdT

72.421.62* (+93.07%)a

14.180.146 (+13.26%)b

60.080.564 (+129.31%)a

63.180.438 (+102.76%)a

16.130.104 (+52.03%)a

48.650.520 (+118.06)a

56.750.603 (+146.52%)a

13.180.054 (+45.80%) a

45.460.611 (+186.45%)a

I-BCOT

70.901.40* (+88.75%)a

13.600.262 (+8.63%)a

59.110.228 (+125.61%)a

52.181.45 (+67.46%)a

10.820.050 (+1.98%)b

51.670.142 (+131.60%)a

52.060.102 (+126.67%)a

12.580.064 (+39.16%)a

41.380.114 (+160.74%)a

*Values

are mean (nmole SH group/mg protein) SD homogenate of pooled liver, pooled lung or pooled kidney of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001. Significantly different from I-C at ap<0.001 and bp<0.05.

TABLE 13 B. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) MEDIATED EFFECT ON LIVER, LUNG AND KIDNEY GLUTATHIONE PEROXIDASE, GLUTATHIONE REDUCTASE AND GLUTATHIONE-S-TRANSFERASE ACTIVITIES IN DMBA-INDUCED RATS AFTER TREATMENT

Liver
Group

Lung

Kidney
Glutathione-S-

Glutathione peroxidase (U/ mg protein)

Glutathione reductase (U/ mg protein)

Glutathione -Stransferase (U/mg protein)# 135.221.01

Glutathione peroxidase (U/ mg protein )

Glutathione reductase (U/ mg protein )

transferase (U/mg protein)#

Glutathione peroxidase (U/ mg protein )

Glutathione reductase (U/ mg protein )

Glutathione -Stransferase (U/mg protein)# 93.621.06

N-C

58.461.06*

10.680.213

104.631.66

10.160.081

104.261.55

62.360.641

13.670.140

I-C

72.611.42* (+24.20%)a 55.221.08* (-23.95%)a 54.621.81* (-24.78%)a

8.220.218 (-23.03%) a 10.430.151 (+26.88%)a 10.630.616 (+29.32%)a

102.961.11 (-23.86%) a 123.481.22 (+19.93%)a 123.961.06 (+20.40%)a

117.481.59 (+12.28%) a 92.860.620 (-20.96 %)a 92.960.864 (-21.87%)a

7.880.112 (-22.44 %)a 11.690.081 (+48.35)a 10.880.022 (+38.07%)a

63.311.36 (-39.27 %)a 87.521.02

(+38.24 %)a

93.240.465 (+49.52 %) a 63.140.251 (-32.28%)a 62.980.452 (-32.45%)a

12.360.121

(-9.58%)a
15.230.169 (+23.22%)a

56.241.11 (-39.93%)a 84.261.41 (+49.82%)a 78.321.26 (+39.26%)a

I-BdT

I-BCOT

86.211.63 (+36.17%)a

14.990.08 (+21.28%)

are mean SD from homogenate of pooled liver, pooled lung or pooled kidney and PMS fraction of pooled liver, pooled lung or pooled kidney of 15 rats in each group. N-C, normal control; I-C, Infected control; I-BdT, through orally in two equal doses of 2ml/Kg b.w.orally and I-BCOT, given through orally in two equal doses of 2ml/Kg b.w.orally for 16 weeks. Significantly different from N-C at ap<0.001. Significantly different from N-C at ap<0.001. Significantly different from I-C at ap<0.001.
*Values

Fig.3. Histological and morphological studies of 16 weeks experimental rat liver. A. The normal histological section shows the well arranged cells and clear central vein. B. Section shows the complete destruction of hepatocytes degeneration of central vein, fatty degeneration and neutrophil distribution. Section shows the damage hepatocytes and various size vacuoles (bend arrow).

Fig.3.C. Histopathological liver changes are restored near to normal in the B. diffusa (I-BdT) treated group and D. The damage is recovered with the treatment of Black Caraway Oil (I-BCOT).

Fig.4. Histological and morphological studies of 16 weeks experimental rat kidney. A. The normal kidney section shows the well arranged cells. B. Carcinogenic group shows the enocytic vacuoles as characteristically seen in proximal tubules and the thicking of the glomerular basement with glomerulosclerosis (arrow).

Fig.4.C&D. The damage kidneys are recovered with the treatment of I-BdT and I-BCOT.

Fig.5. Histological and Morphological studies of 16 weeks experimental rat pancreas. A. Pancreatic section of normal rat shows cells with well preserved cytoplasm and nucleus. B. In the pancreatic section of DMBA-Induced rats, the cells are irregular, not well defined and defect in cell membrane. Necrosis of the cells is very clear.

Fig.5. C&D. B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) treatment were improved restored the altered pancreatic Histopathological changes.

DISCUSSION:

Hypercholesterolemia is firmly established as a primary risk factor for atherosclerotic cardiovascular disease. In this present investigation we tried to examine the effect of B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) supplementation on overall proatherogenic actions of DMBA-Induced carcinogenesis we found that in the white albino rats DMBA-Induced inflated oxidative stress hypercholesterolemia and carcinogenesis when compared with normal control rats.

The DMBA-Induced extensive proatherogenic changes, that occurred in rats, were reflected on a variety of parameters, such as, hemoglobin, plasma and lipoprotein lipids including cholesterol and apoB content of LDL and its subfractions and antioxidant enzymes. Supplementation of infected rats with B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) for treatments, significantly reduced the overall oxidative burden and effectively the above altered parameters.

They quench free radicals in cell membranes and protect them against lipid peroxidation. The higher antioxidant potency of B. diffusa (Bd) as compared to Black Caraway Oil (BCO).

The reduction in the free radical quenching efficiency of B. diffusa (44.42 M) in comparison to BCO (38.11 M). B. diffusa was found more efficient scavenger of peroxyradical then Black Caraway Oil. (Fig 2.2)

Consistent with the results from infected rats (Table 1), rats DMBA-Induced also exhibited significant decrease hemoglobin. Both B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) were equally effective in increasing (18-22 %) the hemoglobin, the results demonstrate that a DMBA-Induced a severe hyperlipidemia in I-C rats as compared to normal control (NC).

A much higher increase in plasma TG (111 % and atherogenic non-HDL-C (99 % vs. 45 %) levels contributed to the increased degree of hyperlipidemia in I-C rats. On the other hand, in I-C rats, atherogenic HDL-C was decreased by only 14 %, in comparison to a decline of 49 % in I-BCOT, indicating a very mild dyslipidemia in I-C rats. Highly increased plasma TG, TC, VLDL-C, LDL-C and atherogenic non-HDL-C concentrations in I-C rats were significantly reduced to a level close to their counterparts in N-C rats, In complete contrast to infected rats, B. diffusa treatment not only blocked the minimal decline of 14 % seen in HDL-C level of I-C rats but increased it to a level, 21 % higher than normal control value. On the other hand, after treatment of B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) treatment, the cholesterol content of more antioxidative small, dense HDL3 was increased, from a reduced value of only 4 % in I-C rats, to 42 % and 35 % higher than normal control HDL3 value. These results demonstrate that both B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) exert a potent effect in normalizing the high levels of TG as well as atherogenic non-HDL-C in DMBA-induced hyperlipidemia in rats.

Consistent with these findings, LDL-C/HDL-C, HDL-C/TC, sd-LDL-C/HDL-C and lb-LDLC/HDL-C ratios were markedly altered (+ 128 %, - 49 %, + 308 % and + 51 %, respectively, vs. corresponding ratio values in N-C), in I-C rats (Table 5), while treatment with B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) fully normalized these altered ratios to a level, 20 % to 25 % better than their counterparts in N-C, indicating an excellent normalization of highly atherogenic sd-LDL-C and LDL-C.

The significant increase in plasma, liver, lung and kidney lipid levels is consistent with a significant increase (52 %) in liver HMG-CoA reductase activity of I-C rats (Table 7). Treatment of I-C rats with dietary B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) was associated with a significant decrease in hepatic HMG-CoA reductase activity, which was restored to 55 % and 52 % of N-C value, thus, providing a mechanism for the reduction of plasma and tissue lipids.

Our results show a significant increase in plasma, liver, lung and kidney XO activity of DMBAInduced rats. B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) administration significantly blocked this increase in XO activity and reduced it to a level close to corresponding normal control values. A similar decrease in hepatic CAT and SOD activities B. diffusa and Black Caraway Oil feeding of I-C rats significantly reduced the FFA and lipid peroxidation products, and increased the CAT and SOD activities in liver, lung and kidney, and reversed these parameters to near normal levels (Table 11). These results indicate a potent free radical scavenging property of both B. diffusa (IBdT) and Black Caraway Oil (I-BCOT). Our results show a decline in GSH (Table 12), Gred and GST activities and an increased GPx activity in liver, lung and kidney of I-C rats (Table 13). In addition, since GSH also acts as substrate and co-substrate in essential enzymatic reactions of Gpx and GST, reduction of GST activity may also be due to decreased levels of GSH in tissues. Histological examination of livers, kidneys and pancreas from rats treated with DMBA treatment with revealed damage to the cells, particularly the nuclei. The degree of irregularity of the nucleus was reported to be related to the degree of malignancy of the cells or to the extent of damage

SUMMARY AND CONCLUSION

Several epidemiologic studies have established a strong and consistent link between DMBA-Inducing and increased cardiac morbidity and mortality. Other studies show that in addition to substantial increase in oxidative stress, certain other compounds of DMBA-Induced. Our results showed that due to sustained free radical, DMBA-Induced infected rats (155-182g) exhibited several proatherogenic actions, including a significant increase in atherogenic non-HDL-C, with a concomitant decline in the cholesterol concentrations of antiatherogenic HDL and its subfractions, HDL2 and HDL3. B. diffusa treatment of infected rats resulted in a significant increase hemoglobin levels, which were reversed to 20 % of respective control values of age-matched normal control (N-C). Our results show that in comparison to normal control (N-C), a modest and significant increase in plasma total lipid and atherogenic non-HDL-C levels in infected rats (I-C), which were significantly reversed to 42-98 % of normal control (N-C) value after treatment of B. diffusa supplementation and Black Caraway Oil similarly. However, cholesterol content of more antioxidative HDL3 was not affected during of B. diffusa treatment. This may be due to markedly increased production of oxidants and significantly diminished antioxidant defense including a decline in total plasma antioxidants.

Consistent with the increase in plasma TG, FFA and TC levels of I-C rats, a significant increase in these lipids of liver, lung and kidney were also observed. While, B. diffusa and Black Caraway Oil treatment of I-C rats was associated with a significant decreases in TG, FFA and TC of each tissue. These results demonstrate that sustained oxidative stress in I-C rats is also able to induce hyperlipidemia in the above tissues with a maximum increase in hepatic TC, while both B. diffusa and Black Caraway Oil effectively blocked these increases and restored the TG, FFA and TC levels of liver, lung and kidney close to corresponding normal control values. Treatment of I-C rats with dietary B. diffusa and Black Caraway Oil was associated with a significant decrease in plasma and tissue lipid levels as well as hepatic HMG-CoA reductase activity, which was restored to 55 % and 27 % of N-C value, which may provide a mechanism for the reduction of plasma and tissue lipids.

Based on strong hypolipidemic and antioxidant properties of B. diffusa (I-BdT) and Black Caraway Oil (IBCOT), we have investigated the anti-tumour activity of I-BdT and I-BCOT in experimental carcinogenesis of liver, kidney and pancreases. We have utilized the carcinogen, DMBA, which is known to induce hepatic carcinogenesis and hypercholesterolemia. As expected, administration of DMBA causes a significant increase in plasma triglycerides, total cholesterol, LDL-C and apo-B levels. Feeding of B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) to rats, during pre- and post-initiation stages, was associated with a significant decline in the above lipid parameters. Treatment with DMBA resulted in the formation of neoplastic nodules, which evident from the appearance of multiple tumours on greyish white patches on the liver of the carcinogen treated rats. Morphological and histological examination of liver, kidney and pancreases from rats treated with DMBA, revealed damage to the cells, particularly the nuclei. Consistent with morphological and histological examinations, elevated the carcinogen treated rats are indicative of the severity of liver, lung and kidney carcinogenesis.

It is well known that DMBA-Inducing is associated with a substantial increase in oxidative stress, oxidant stress may be increased owing to a higher production of ROS, which are controlled by antioxidant enzymes, SOD, catalase, Gpx, Gred and GSH. An impaired radical scavenger function has been linked to decreased/increased activity of enzymatic and nonenzymatic antioxidants. Our results show a significant decrease in the activities of antiperoxidative enzymes, CAT, SOD, Gred, GST as well as GSH, in liver, lung and kidney of DMBA-Induced rats, except Gpx activity which was significantly increased. Treatment of DMBA-Induced hyperlipidemic rats with B. diffusa and Black Caraway Oil, significantly reversed/restored the altered tissue activities of SOD, catalase, Gpx, Gred and GST including total, free and protein bound-SH contents of glutathione, to near normal control values, indicating an almost total alleviation of oxidative damage by these antioxidants. Both B. diffusa and Black Caraway Oil mediated a near normalization of peroxide levels and scavenging enzyme activities as well as GSH in liver, lung and kidney of infected rats, indicating a strong antilipid/lipoprotein peroxidative effect of these hypolipidemic and anticarcinogenic agents.

The combined results provide evidence that analysis of marker enzymes, GST, lipid peroxide and examination of gross morphology and histology suggested that B. diffusa (I-BdT) and Black Caraway Oil (IBCOT) feeding did offer a significant protection and did reduce the severity and extent of neoplastic transformation during both initiation and/or promotion in the liver and kidney of experimental carcinogenic rats. B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) treatment, in addition to its anti-cancer and antioxidant impacts, also exerted a strong hypocholesterolemic action, indicating a linkage between atherosclerosis and cancer. The dual chemotherapeutic properties of B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) in atherosclerosis and cancer are apparently mediated by reducing HMG-CoA reductase, thus limiting the availability of mevalonate-derived products required for cholesterol production and tumour growth. Daily use of B. diffusa (I-BdT) and Black Caraway Oil (I-BCOT) by normal population will prevent the occurrence of hypercholesterolemia and cardiovascular diseases as well as initiation and promotion of certain forms of cancer.

In conclusion, based on B. diffusa mediated multiple therapeutic benefits, described in the present study, daily intake of Black Caraway Oil as a dietary supplement by hypolipidemic /antiatherogenic /antihypercholesterolemic, antioxidant actions and anticarcinogenic may be useful in the prevention and treatment of DMBA-Induced hyperlipidemia/ and atherosclerosis. In addition, daily use of dietary B. diffusa and Black Caraway Oil will be efficacious, cost effective, no side effects and a good source of antihypercholesterolemic, antioxidant actions and anticarcinogenic.

*Thank You*