Ph.D.Presentation1

8/2/2019 Ph.D.Presentation1

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Chemotherapeutic Properties ofBoerhaavia Diffusa and BlackCaraway Oil on DMBA-Induced Carcinogenesis and

Hypercholesterolemia in Animals

Supervision By Ph.D. Thesis Presentation

By

Dr. R.P. Thapliyal Subodh Kumar Chauhan

Department of Zoology & BiotechnologyFaculty of Life Science

H.N.B. Garhwal Central University, Srinagar

(Garhwal)


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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 inIndia 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 canattain heights of 40 and up to 70cm.


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Cancer is the second most common cause of death after cardiovascular diseases (CVD) inmost 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).


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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 disability-

adjusted 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).


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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 ofB. diffusa and Black Caraway Oilon DMBA-Induced carcinogenesis and hypercholesterolemia in animals which was not

studied earlier.


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The first pharmacological studies have demonstrated that the root ofpunarnava exhibits a wide range ofproperties: 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 ofNigella 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 ofNigella sativa on oxidative stress and beta-cell damage in

streptozotocin-induced diabetic rats, (Kanter, M. et al., 2004). The antioxidative and antihistaminic effect

ofNigella sativa and its major constituent, thymoquinone on ethanol-induced gastric mucosal damage,

Kanter, M. et al., 2006). Effect of volatile oil constituents ofNigella 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).


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The proposed study designed to investigate both hypolipidemic and cancer chemotherapeutic

properties ofBoerhaavia 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 ofB. diffusa (Bd) and Black Caraway Oil (BCO) using HPLC.

2. To estimate the effect of ethanolic extract ofB. 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 ofB. diffusa (Bd) and Black Caraway Oil (BCO) extracts in

preventing the increase in plasma triglycerides (TG), total cholesterol (TC), VLDL-C, LDL-

C, small dense (sd)-LDL-C and large buoyant (lb)-LDL-C in hyperlipidemic rats.


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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 ofB. diffusa (Bd) and Black Caraway Oil (BCO) in the suppression of carcinogen-

induced 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.


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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 atthe 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.


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Studies in rats

For investigating the hypercholesterolemia, carcinogens and antioxidant effect ofB. diffusa(Bd) and Black Caraway Oil (BCO), rats were divided in the following groups as describedbelow:

Group I - Normal Control (N-C)

Fifteen rats were given a single dose of 0.9% NaCl, 5ml/Kg b.w.ip through intraperitonealinjection 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 inAcetone though intraperitoneal injection and were feed rat chow for 16 weeks. Animals wereinfected in three weeks.

Group III - Infected B. Diffusa Treated (I-BdT)

DMBA induced fifteen rats in this group were given a topical application ofB. diffusa extractdissolve 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 CarawayOil two equal doses (morning and evening) of 2ml/Kg b.w. through orally and were feed ratchow for 16 weeks.


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Isolation and Aqueous ethanolic Extraction ofBoerhaavia diffusa roots (Bd)

The fresh roots ofBoerhaavia 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 ofB. 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.


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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.


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Determination of free radical scavenging activity (antioxidant capacity) ofB. 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 ofB. 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, 2-diphenyl-1-picryl hydrazyl (DPPH). The reaction was started by the addition of ethanolic solution ofB.

diffusa andBlack 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.


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Extraction ofB. diffusa (Bd)and Black Caraway Oil (BCO)The aqueous extract of B. diffusa was continued into dry solid by rotary vacuum

evaporator. The yield ofB. 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 ofB. 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).


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Antioxidative Activities ofB. diffusa (Bd) and Black Caraway Oil (BCO)

Antiradical activity or hydrogen donating ability ofB. 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; BlackCaraway 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.


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Fig. 2.2. Free radical scavenging activities ofB. diffusa (Bd) and Black Caraway Oil (BCO). Theantioxidant activities of the above compounds at the indicated concentrations were carried out asdescribed 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 triplicatedeterminations. 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.


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Group

Hemoglobin

(g/dl)

N-C 14.280.264*

I-C11.420.012*(-20.03%)a

I-BdT13.960.016*(+22.24%)a

I-BCOT13.440.011*(+17.69%)a

TABLE 1

IMPACT OFB. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT)ON HEMOGLOBIN IN DMBA-INDUCED

RATS AFTER TREATMENT

*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 of2ml/Kg b.w.orally and I-BCOT, giventhrough orally in two equal doses of 2ml/Kg

b.w.orally for 16 weeks.

Significantly different from N-C at ap


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Group Total lipidTriglycerides

Free fatty acid

Total

cholesterol

N-C390.191.40* 54.221.17 134.150.412 88.662.46

I-C510.362.23*(+30.79 %)a

114.612.43(+111.38 %)a

151.290.512(+12.78 %)a

154.683.86(+74.46 %)a

I-BdT

483.221.56*(-5.32 %)b

66.241.92(-42.20 %)a

137.310.291(-9.05%)b

110.622.88(-28.48 %)a

I-BCOT486.311.12*

(-4.71 %)b

62.681.86(-45.3 %)a

134.150.212(-11.33 %)a

102.211.76(-33.92 %)a

TABLE 2

IMPACT OFB. 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

*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, giventhrough orally in two equal doses of 2ml/Kg


Significantly different from N-C at ap < 0.001.

Significantly different from I-C at ap


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

VLDL-C 11.010.054* 21.440.242*(+94.73 %)a

13.190.165*

(-38.48 %)a

13.260.214*

(-38.15 %)a

LDL-C 54.060.131 111.220.265(+105.75 %)a

62.420.302(-43.87 %)a

57.960.192(-47.88 %)a

HDL- C 20.960.071 18.040.043(-13.93 %)a

31.010.106(+71.89%)a

26.910.176(+49.17 %)a

HDL2- C 7.080.0125.120.012(-27.68 %)a

14.120.022(+175.78 %)a

11.160.014(+ 117.97 %)a

HDL3- C 13.880.04213.320.043(-4.03 %)c

18.960.071(+42.34 %)a

17.960.072(+34.83 %)a

Non-HDL- C 68.642.88 136.643.89(+99.07 %)a

79.612.52(-41.74 %)a

75.303.11(-44.89 %)a

TABLE 3

IMPACT OFB. 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

*Values are mean (mg/dl) SD from pooled plasma of 15 rats in each group.






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

LDL-C 54.060.131* 111.120.265*

(+105.55 %)a

62.420.302*(-43.83 %)a

57.960.192*(-47.84 %)a

LDL- apoB 122.340.821 140.860.422(+15.14 %)a

126.060.824(-10.51 %)b

129.420.624(-8.12 %)c

Sd-LDL-C 16.430.052 60.400.086(+267.62 %)a

19.270.061(-68.09 %)a

17.250.044(-71.44 %)a

% LDL-C 30.400.321 54.351.26(+78.78 %)a

30.880.624(-43.18 %)a

29.770.462(-45.22 %)a

Sd-LDL- apoB 39.520.612 72.331.019(+83.02 %)a

41.380.616(-44.17 %)a

38.731.180(-46.45 %)a

% LDL-apoB 32.350.832 51.351.62(+58.73 %)a

32.040.605(-37.60 %)a

29.930.531(-41.71 %)a

Lb-LDL-C 36.970.046 50.390.144(+37.68 %)a

43.580.022(-13.51 %)a

40.640.081(-19.35 %)a

% LDL-C 71.051.213 45.360.962(-36.16 %)a

69.830.943(+53.95 %)a

70.121.129(+54.58 %)a

Lb-LDL-apoB 81.501.181 68.621.10(-15.80 %)a

82.741.12(+20.58 %)a

82.460.424(+20.17 %)a

% LDL-apoB 66.600.926 48.720.835(-26.85 %)a

65.640.859(+34.73 %)a

63.720.825(+30.79 %)a

TABLE 4

IMPACT OFB. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT)ON PLASMALDL, SMALL DENSE AND

LARGE BUOYANT LDL SUBPOPULATION IN DMBA-INDUCED RATS AFTER TREATMENT






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Ratio

Group

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

LDL-C/HDL-C 2.700.021* 6.160.034*

(+128.15 %)a

2.010.024*(-67.37 %)a

2.150.019*(-65.15 %)a

HDL-C/TC 0.2260.030 0.1160.001(-48.67 %)b

0.2800.075(+141.37 %)b

0.2530.034(+118.10 %)d

Sd-LDL-C/HDL-C 0.8200.006 3.350.029(+308.54 %)a

0.6210.004(-81.46 %)a

0.6410.003(-80.86 %)a

Lb-LDL/HDL-C 1.850.019 2.790.029(+50.81 %)a

1.400.010(-49.82 %)a

1.510.014(-45.88 %)a

TABLE 5

EFFECT OFB. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON THE RATIOS OF LDL-C/HDL-C, HDL-

C/TC, Sd-LDL-C/HDL-C AND Lb-LDL/HDL-C IN DMBA-INDUCED RATS AFTER TREATMENT

For the calculation of ratios, data is taken from Table 3, 4 and 5. *Values are mean SD from pooled plasma of 15 rats in each group.






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Group

Liver Lung Kidney

Triglycerides

(mg /100mg

protein)

Totalcholesterol

(mg /100mg

protein)

Free fattyacid

(mg /mg

protein)

Triglycerides

(mg /100mg

protein)

Totalcholesterol

(mg /100mg

protein)

Free fattyacid

(mg /mg

protein)

Triglycerides

(mg /100mg

protein)

Totalcholesterol

(mg /100mg

protein)

Free fattyacid

(mg /mg

protein)

N-C0.6820.001* 2.720.066 21.240.566 0.5840.003 2.500.012 12.820.143 0.5590.005 2.260.003 8.210.081

I-C0.8660.006*(+26.98%)a

4.960.025(+82.35%)a

28.960.231(+36.35%)a

0.6790.005(+16.27%)a

3.210.043(+28.40%)a

13.020.04(+1.56%)e

0.6970.005(+24.68%)a

2.820.005(+24.78%)a

12.430.112

(+51.40%)a

I-BdT0.7580.004*(-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

9.580.065(-22.93%)a

I-BCOT0.8240.004*

(-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

10.480.142

(-15.69%)a

TABLE 6

EFFECT OFB. 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

*Values 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


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Group HMG-CoA reductase activity

N-C6.550.082*

I-C

3.110.024*(-52.52 %)a

I-BdT4.820.067*(+54.98 %)a

I-BCOT3.960.036*(+27.33 %)a

TABLE 7

IN VIVO MODULATION OF HEPATIC HMG-CoA REDUCTASE ACTIVITYIN DMBA-INDUCED RATS TREATED

WITHB. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) FOR 16 WEEKS OF TREATMENT

*Values 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, giventhrough orally in two equal dosesof 2ml/Kg b.w.orally for 16 weeks.



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Group Total antioxidants Conjugated diene Lipid hydroperoxide MDA

N-C 48.370.056* 9.310.011 2.510.032 3.420.081

I-C36.890.121*(-23.73 %)a

14.360.016(+54.24 %)a

3.810.015(+51.79 %)a

5.910.116(+72.80 %) a

I-BdT 46.24

0.034*

(+25.34 %)a

12.46

0.014(-13.23 %)a

3.12

0.006(-18.11 %)a

4.82

0.044(-18.44 %)a

I-BCOT42.100.020*(+14.12 %)b

13.210.022(-8.00 %)b

3.200.024(-16.01 %)a

4.990.062(-15.57 %)a

TABLE 8

ANTIOXIDANT IMPACT OFB. 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

*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 equaldoses of 2ml/Kg b.w.orally and I-BCOT, giventhrough orally in two equal doses

of 2ml/Kg b.w.orally for 16 weeks.



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Group

Liver Lung Kidney

Conjugated dieneLipid

hydroperoxideMDA Conjugated diene

Lipid

hydroperoxideMDA Conjugated diene

Lipid

hydroperoxideMDA

N-C6.120.021* 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

I-C8.180.015*(+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

5.840.020

(+36.45%

)a

I-BdT6.420.054*(-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

5.320.031

(-8.90%)

a

I-BCOT6.500.011*(-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

5.160.010

(-

11.64%)c

TABLE 9

EFFECT OFB. DIFFUSA (I-BdT) AND BLACK CARAWAY OIL (I-BCOT) ON LIVER, LUNG AND KIDNEY CONJUGATED

DIENE, LIPID HYDRPEROXIDE AND MALONDIALDEHYDECONTENT IN DMBA-INDUCED RATS AFTER TREATMENT

*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.



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Group

Xanthine oxidase activity

Plasma

(U/ml)

Liver

(U/mg protein)

Lung

(U/mg protein)

Kidney(U/mg

protein)

N-C 6.320.021* 0.6120.0021 0.3920.0011 0.1790.0011

I-C10.040.126*(+58.86%)a

0.8500.0025(+38.89%)a

0.5750.0010(+46.68%)a

0.4560.0011(+154.75%)a

I-BdT

7.110.116*(-29.18%)a

0.6360.0016(-25.18%)a

0.5260.0012(-8.52%)a

0.2900.0042(-36.40%)a

I-BCOT7.660.152*(-23.70%)a

0.7120.0012(-16.23%)a

0.5220.0021(-9.22%)a

0.2880.0060(-36.84%)a

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

*

Values 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





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Group

Liver Lung Kidney

Catalase

(U/mg protein)

Superoxide

dismutase

(U/mg protein)

Catalase

(U/mg protein)

Superoxide

dismutase

(U/mg protein)

Catalase

(U/mg

protein)

Superoxidedismutase

(U/mg

protein)

N-C4.680.142* 0.8540.002 2.510.062 2.110.006 3.450.154 0.9320.004

I-C

3.320.223*(-29.06 %)a

0.6540.002(-23.42 %)a

2.270.033(-9.56 %)b

1.8830.004(-10.76 %)a

2.230.052(-35.36 %)a

0.8020.002(-13.95 %)b

I-BdT

4.220.121*(+27.11 %)a

0.7870.003(+20.34 %)a

2.400.054(+5.73 %)b

2.1400.004(+13.65 %)a

3.500.042(+56.95 %)a

0.9240.002(+15.21 %)a

I-BCOT

4.120.022*(+24.10 %)a

0.7700.005(+17.74 %)a

2.540.022(+11.89 %)a

2.1560.004(+14.50%)a

3.560.166(+59.64 %)a

0.9350.005(+16.58 %)a

TABLE 11

IMPACT OFB. DIFFUSA(I-BdT) AND BLACK CARAWAY OIL (I-BCOT)ON LIVER, LUNG AND KIDNEY CATALASE AND

SUPEROXIDE DISMUTASE ACTIVITIESIN DMBA-INDUCED RATS AFTER TREATMENT

*Values 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.



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Group

Liver Lung Kidney

Total-SH Free-SH Protein-bound-SH



N-C 76.821.21* 13.800.314 64.521.10 66.860.561 12.730.104 56.110.558 58.700.406 10.310.082 50.300.401

I-C37.512.46*(-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-BCOT70.901.40*(+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

TABLE 12

IMPACT OFB. 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

*Values are mean (nmole SH group/mg protein) SD homogenate of pooled liver, pooled lung or pooled kidney of 15 rats ineach 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,



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Group

LiverLung

Kidney

Glutathioneperoxidase

(U/ mg

protein)

Glutathionereductase

(U/ mg

protein)

Glutathione-S-

transferase

(U/mg

protein)#


(U/ mg

protein )


(U/ mg

protein )

Glutathione-S-transferase

(U/mg

protein)#


(U/ mg

protein )


(U/ mg

protein )

Glutathione-S-

transferase

(U/mg

protein)#

N-C 58.461.06* 10.680.213 135.221.01 104.631.66 10.160.081 104.261.55 62.360.641 13.670.140 93.621.06

I-C 72.611.42*(+24.20%)a

8.220.218(-23.03%)a

102.961.11(-23.86%)a

117.481.59(+12.28%) a

7.880.112(-22.44 %)a

63.311.36(-39.27 %)a

93.240.465(+49.52 %) a

12.360.121(-9.58%)a

56.241.11(-39.93%)a

I-BdT 55.221.08*(-23.95%)a

10.430.151(+26.88%)a

123.481.22(+19.93%)a

92.860.620(-20.96 %)a

11.690.081(+48.35)a

87.521.02(+38.24 %)a

63.140.251(-32.28%)a

15.230.169(+23.22%)a

84.261.41(+49.82%)a

I-BCOT 54.621.81*(-24.78%)a

10.630.616(+29.32%)a

123.961.06(+20.40%)a

92.960.864(-21.87%)a

10.880.022(+38.07%)a

86.211.63(+36.17%)a

62.980.452(-32.45%)a

14.990.08(+21.28%)

78.321.26(+39.26%)a

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

.*

Values are mean SD from homogenate of pooled liver, pooled lung or pooled kidney and PMS fraction of pooled liver, pooled lungor 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,



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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).

A B

C D

Fig.3.C. Histopathological liver changes are restored near to normal in theB. diffusa (I-BdT) treated group and D. The

damage is recovered with the treatment of Black Caraway Oil (I-BCOT).


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A B

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).

C D

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


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A B

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 welldefined and defect in cell membrane. Necrosis of the cells is very clear.C D

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

Histopathological changes.


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DISCUSSION:

Hypercholesterolemia is firmly established as a primary risk factor for atherosclerotic

cardiovascular disease. In this present investigation we tried to examine the effect ofB.

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.


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They quench free radicals in cell membranes and protect them against lipid peroxidation.

The higher antioxidant potency ofB. diffusa (Bd)as compared to Black Caraway Oil

(BCO).

The reduction in the free radical quenching efficiency ofB. 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).


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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 ofB. 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 bothB. 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.


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Consistent with these findings, LDL-C/HDL-C, HDL-C/TC, sd-LDL-C/HDL-C and lb-LDL-

C/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 withB. 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.


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Our results show a significant increase in plasma, liver, lung and kidney XO activity of DMBA-

Induced 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 activitiesB. 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 (I-

BdT) and Black Caraway Oil (I-BCOT).

Our results show a decline in GSH (Table 12), Gred and GST activities and an increased GPxactivity 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 theextent of damage


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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 oxidativestress, 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 ofB. diffusa supplementation and BlackCaraway Oil similarly. However, cholesterol content of more antioxidative HDL3 was not affected during ofB.

diffusa treatment. This may be due to markedly increased production of oxidants and significantly diminished

antioxidant defense including a decline in total plasma antioxidants.


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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 dietaryB. 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.


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Based on strong hypolipidemic and antioxidant properties ofB. diffusa (I-BdT) and Black Caraway Oil (I-

BCOT), 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 ofB. 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.


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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 withB. diffusa

and Black Caraway Oil, significantly reversed/restoredthe 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.


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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 (I-

BCOT) 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 ofB. 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 onB. 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.


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*Thank You*

Ph.D.Presentation1

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