Antiatherogenic effect of Caps HT2, a herbal Ayurvedic medicine formulation.

Summary

The antiatherogenic effect of a herbal formulation, Caps HT2, was evaluated as antioxidant, anticoagulant, platelet antiaggregatory, lipoprotein lipase releasing, anti-inflammatory and hypolipidaemic activity in rats. The formulation contained the methanolic extracts of selected parts of plants, Commiphora mukul, Allium sativum, Plumbago indica, Semecarpus anacardium, Hemidesmus indicus, Terminalia arjuna, Tinospora cordifolia, Withania somnifera and Ocimum sanctum. The formulation, Caps HT2 was found to scavenge superoxide and hydroxyl radicals; the I[C.sub.50] required being 55.0 and 610.0 [micro]g/ml respectively. The lipid peroxidation was found inhibited (50%) by 48.5 [micro]g/ml of Caps HT2. The intravenous administration of the formulation (5 mg/kg) delayed the plasma recalcification time in rabbits and enhanced the release of lipoprotein lipase enzyme significantly (p < 0.001). The formulation also inhibited ADP induced platelet aggregation in vitro, which was comparable to commercial heparin. The anti-inflammatory action of the formulation was significant (p < 0.001) with acute and chronic inflammations induced by carrageenan and formalin respectively in rats.

The hypolipidaemic effect of Caps HT2 was significant (p < 0.001) with the administration of the formulation, in diet-induced hyperlipidaemia of rats for a period of 30 days. Oral administration of the formulation, Caps HT2 (100, 200, 300 and 400 mg/kg) significantly raised HDL cholesterol levels. The atherogenic index and the reduction in body weight were significant indicating the effectiveness against hyperlipidaemia and obesity. All these results revealed the therapeutic potential of Caps HT2 against vascular intimal damage and atherogenesis leading to various types of cardiovascular problems.

Key words: Antiatherogenic, antioxidant, anticoagulant, platelet antiaggregation, anti-inflammatory, lipoprotein lipase, hypolipidemic, Commiphora mukul, Allium sativum, Plumbago indica, Semecarpus anacardium, Hemidesmus indicus, Terminalia arjuna, Tinospora cordifolia, Withania somnifera and Ocimum sanctum

* Introduction

Coronary heart diseases resulting from progressive atherosclerosis, remains the most common cause of death in our society. Atherosclerosis is primarily a lipid disorder affecting the arteries. Increased intracellular generation of reactive oxygen species has been proposed as a mechanism to tissue injury with a variety of pathological processes like ischaemia, inflammation, atherosclerosis and thrombosis (Mark et al. 1992). Lipids undergo peroxidative changes in the arterial wall, which eventually produce tissue injury. Increased free radicals can cause abnormal function of endothelial cells via reduced NO availability, and is believed to be an early event in atherogenesis (Ross, 1993). Diminished NO activity enhances the expression of transcription factor-nuclear kappa B (Barnes and Karin, 1997), which up-regulates the synthesis of inflammatory cytokines and adhesion molecules, leading to further complications and endothelial injury. Hence, compounds that can scavenge the excess of free radicals formed, inhibit their production, or protect membranes from peroxidation are of wide therapeutic value (Diaz et al. 1997).

Platelets, the major constituents of thrombus and a source of powerful vasoconstrictors, can cause vasospasm and enhance coagulation by diminished blood flow (Rodger, 1988). In atherosclerosis inflammation may also alter the integrity of the vascular endothelium and cause exposure to thrombogenic material in plaque with clot formation and reduction in coronary flow (Airaghi et al. 1995). The hypolipidaemic and anticoagulant agents are playing major roles in preventing cardiovascular diseases. Clinical trials and pathophysiological evidence support the use of aggressive therapy in patients with arteriosclerotic vascular disease and in those with several risk factors for the disease. Combination therapy with lipid lowering drugs is advisable, especially in patients with hypedipidaemia (La Rosa et al. 1990).

Although several chemicals and drugs are generally used against atherosclerosis and related heart diseases, the Indigenous drugs with a long descended heritage of traditional use are of supreme importance, to re-establish traditional claims with scientific interest. Clinical studies in India have consistently confirmed guggul (gum resin of Commiphora mukul) extracts improve lipid levels in humans (Malhotra et al. 1977). Guggulsterones are the active principles in the gum resin of Commiphora mukul (Dwivedi, 1996). Garlic (Allium sativum) is attributed to have antithrombotic, platelet antiaggregatory, hypolipidaemic, antimicrobial, diuretic and hypoglycaemic activities (Satyavati et al. 1976). The pharmacologically active constituents in Allium sativum are allyl sulphide, ajoene, allicin, etc (Dwivedi, 1996). Plumbagin, a known isolate from Plumbago species administered to hyperlipidaemic rabbits reduced serum cholesterol (Sharma et al. 1991). Feeding the extract of Semecarpus anacardium inhibited the progression of atherosclerotic lesion and promoted plaque regression and further helped in mobilization of lipids especially cholesterol from liver (Kurup et al. 1979). The active constituents of this plant are flavones and flavonoids (Arti et al. 1995)

Hemidesmus indicus, has been used as folklore medicine and as ingredient in Ayurvedic and Unani preparations against diseases of blood, inflammation etc. (Nayar, 1979). Hemidesmus indicus is rich in cardiac and pregnane glycosides (Deepak et al. 1997). Terminalia arjuna, was found to modify various risk factors like obesity, hypertension and diabetes mellitus without any known side effects (Dwivedi et al. 1994). The pharmacologically active constituents in Terminalia arjuna are cardiac glycosides, saponins, flavonoids, ellagic acid etc (Dwivedi, 1996; Kaur et al. 1997). Tinospora cordifolia, Withania somnifera and Ocimum sanctum were widely used in Ayurvedic and Unani preparations as tonic, vitaliser and remedy for metabolic disorders (Chopra et al. 1958; Nadkarni, 1976; Godhwani et al. 1988). Tinospora cordifolia contains eugenol as the active principle (Sen et al. 1992), Withania somnifera contains withanolides and steroidal lactones (Andallu and Radhika, 2000) and Ocimum sanctum contains fixed oils and fatty acids like linoleic and linolenic acid (Surender and Majumdar, 1999).

Generally the toxicity of indigenous drugs has largely been neglected as it is argued that these drugs are used in traditional clinical practices. But it has suggested that all natural products and active principles must be subjected to the same stringent toxicity studies as in the case of synthetic drugs (Grever et al. 1992). The acute (10,000 mg/kg) and sub acute toxicity (500 mg/kg, 1 month) studies conducted in rats in our laboratory, revealed that this formulation Caps HT2 has no toxic effects on heart, liver and kidney functioning as determined by estimations on serum biochemical parameters.

Scientists are in search for cardiac strengthening agents, blood vessel wall smoothening agents without allowing atheromatous plaques to gain around in and under the lining, agents that strengthen the heart muscle and keep the internal lining of the blood vessels intact. In these circumstances, the search for a harmless and clinically useful indigenous preparation for cardiovascular diseases was well warranted fulfilling these intentions.

Phytomedicine if combined with the preventive model of medical practice, could be among the most cost effective practical ways to shift the focus of modern cardiovascular disease treatment to prevention or cardio protection. Here we have tried to investigate the antiatherogenic effect of a herbal Ayurvedic formulation, Caps HT2, containing the extracts of selected plants, Commiphora mukul (Engl), Allium sativum (Linn), Plumbago indica (Linn), Semecarpus anacardium (Linn), Hemidesmus indicus (Linn), Terminalia arjuna (Bedd), Tinospora cordifolia (Wild), Withania somnifera (Dunn) and Ocimum sanctum (Linn). The formulation was screened for the antioxidant, anticoagulant, platelet aggregation inhibition, lipoprotein lipase releasing, anti-inflammatory and hypolipidaemic properties.

* Materials and Methods

Animals

Male Wistar albino rats (200-250 g) were inbred in the animal house of our institute. The animals were housed in well ventilated cages and fed with standard pellet diet (Lipton India Ltd.) and water ad libitum.

The New Zealand white rabbits (1.5-2 kg) were purchased from Veterniary College, Kerala Agricultural University, Mannuthy, Thrissur (India). The animals were caged in uniform hygenic conditions and fed with a control diet with wheat flour base plus the addition of milk powder, hydrogenated fat, butter, dried yeast, salt, sucrose and vitamins to produce the following nutrients in the given proportions; protein 20%, carbohydrate 65%, sucrose 3%, fat 5%, salt 4%, vitamins 1% and fibre 2% (Ritu et al. 1996).

Preparation of the herbal formulation, Caps HT2 All the plants used in the study were collected locally, identified and authenticated by Dr. Sasidharan, Taxonomist, Kerala Forest Research Institute, Peechi, India. The voucher specimens have stored in the herbarium of our institute, namely, Specimen No. 131/ACRH, 132/ACRH, 133/ACRH, 134/ACRH, 25/ACRH, 135/ACRH, 136/ACRH, 137/ACRH and 138/ACRH respectively for the following plants (family and plant parts used were in the parenthesis), Commiphora mukul, (Burceraceae, gum), Allium sativum (Liliaceae, bulb), Plumbago indica (Plumbaginaceae, stem), Semecarpus anacardium (Anacardiaceae, seed), Hemidesmus indicus (Asclepiadaceae, seed), Terminalia arjuna (Combretaceae, bark), Tinospora cordifolia (Menispermaceae, stem), Withania somnifera (Solanaceae, root) and Ocimum sanctum. (Labiateae, whole plant). The powdered plant parts (100 g) of each plant was taken mixed together and extracted twice with 70% MeOH by continuous stirring for 3 overnights and the extracted fraction was recovered by rotavaporization. The yield of the solvent free extract was 17% (w/w). The extract was resuspended in 2% gum acacia (w/v) and used for further studies under the technical name Caps HT2.

Antioxidant activity

* Superoxide radical scavenging activity: Superoxide radical scavenging activity was determined by the Nitrobluetetrazolium (NBT) reduction method (McCord and Fridovich, 1969). The reaction mixture contained EDTA (0.1 M) containing 0.0015% NaCN, riboflavin (0.12 mM), NBT (1.5 mM), various concentrations of Caps HT2 (10-500 [micro]g/ml) and phosphate buffer (M/15, pH 7.5) in a final volume of 3 ml. The tubes were uniformly illuminated under an incandescent lamp for 15 min and the optical density was measured at 530 nm before and after illumination. The percentage inhibition of superoxide generation was evaluated by comparing the absorbance values of the control and experimental tubes. Curcumin (1-100 [micro]g) was used as reference standard.

* Inhibition of lipid peroxide formation

Induction by [Fe.sup.2+]/ascorbate system: The reaction mixture contained rat liver homogenate (0.1 ml, 25% w/v) in tris HCl buffer (20 mM, pH 7.0), KCl (150 mM), ferrous ammonium sulphate (0.8 mM), ascorbic acid (0.3 mM) and various concentrations of Caps HT2 (10-500 [micro]g) in a final volume of 0.5 ml and was incubated for 1 h at 37 [degrees]C (Bishayee and Balasubramaniam, 1971). The lipid peroxide formation was measured by the method of Ohkawa et al. (1979). The incubated reaction mixture (0.4 ml) was treated with sodium dodecyl sulphate (0.2 ml, 8%), thiobarbituric acid (1.5 ml, 8%) and acetic acid (1.5 ml, 20%, pH 3.5). The total volume was made up to 4ml by adding distilled water and kept in a water bath maintained at 100 [degrees]C for 1 h. After cooling, 1ml of distilled water and 5ml of a mixture of n-butanol-pyridine (15:1) were added and shaken vigorously. The absorbance of the organic layer was measured at 560 nm after centrifugation. The percentage inhibition of lipid peroxide formation was determined by comparing the results of Caps HT2 treated and untreated samples. Curcumin was used as reference standard (1-100 [micro]g).

* Hydroxyl radical scavenging activity: Hydroxyl radical scavenging was measured by studying the competition between deoxyribose and the extract for hydroxyl radicals generated from the [Fe.sup.3+]/Ascorbate /EDTA/[H.sub.2][O.sub.2] system (Elizabeth and Rao, 1990). The hydroxyl radicals attack deoxyribose, which eventually results in TBARS formation. The reaction mixture contained deoxyribose (2.8 mM), Fe[Cl.sub.3] (0.1 mM), EDTA (0.1 mM), [H.sub.2][O.sub.2] (1 mM), ascorbate (0.1 mM), K[H.sub.2]P[O.sub.4]KOH buffer (20 mM, pH 7.4) and various concentrations of Caps HT2 (10-500 [micro]g/ml) in a final volume of 1ml. The reaction mixture was incubated for 1 h at 37 [degrees]C. Deoxyribose degradation was measured as TBARS by the method of Ohkawa et al. (1979) and percentage inhibition was calculated. Curcumin (1-100 [micro]g) was used as reference standard.

Anticoagulant activity by plasma racalcification method

Blood was collected from normal rabbits through the ear vein in EDTA (0.1M) added tubes. The plasma was separated by centrifugation (1000 rpm x 5 min). 200 [micro]l of M/100 Ca[Cl.sub.2] was added to 100 [micro]l of the plasma prewarmed at 37 [degrees]C. The time taken for the formation of a firm clot was noted immediately using a stopwatch (Quick, 1940). Similarly the plasma recalcification time was noted 10 min after the intravenous administration of heparin (1 mg/kg) and the formulation, Caps HT2 (5 mg/kg).

Platelet antiaggragation activity--ADP induced

Platelet rich plasma (PRP) was prepared by centrifugation (1000 rpm x 5 min) of blood collected from normal aspirin free blood bank donors. 1.5 ml of acid citrate dextrose (ACD) was used as anticoagulant for every 8.5 ml of blood. PRP was taken into siliconized glass cuvettes. Platelet poor plasma (PPP) collected by centrifugation (3000 rpm x 5 min) was kept as reference. The cuvettes were incubated at 37 [degrees]C for 5 min. The aggregation was initiated by adding 20 [micro]l of ADP (10 [micro]M) to 1ml of PRP. The aggregation was recorded for 5 min at 600 nm. The effect of different concentrations (50-250 [micro]g) of Caps HT2 was studied by incubation with PRP at 37 [degrees]C for 5 min before the addition of

ADP. Commercial heparin (20 [micro]g/ml) was used as reference standard (Subramaniam and Satyanarayana, 1989).

Lipoprotein lipase releasing activity

The Lipoprotein lipase releasing activity of the drug was determined by the method of Korn (1962). Blood was collected from normal rabbits through the ear vein in EDTA (0.1 M) added tubes. The plasma collected by centrifugation (3000 rpm x 5 min) was used as the enzyme source. The human lipoid serum (TG < 400 mg/dl) was used as the substrate. 0.1 ml of substrate, 0.1-0.4 ml of enzyme, 0.4 ml of 20% albumin (pH 8.5) and 0.1 ml of [(N[H.sub.4]).sub.2]S[O.sub.4] were mixed at low temperature and made up to a final volume of 1 ml. The mixture was incubated at 37 [degrees]C and the aliquots were taken into tubes containing 0.1 ml of 1 N [H.sub.2]S[O.sub.4] at intervals of 0, 1/2, 1, 1 1/2 hrs. The samples treated with 0.1 ml of sodium periodate (0.05 M) and 0.1 ml of sodium arsenate (0.05 M) was kept in boiling water bath for 30 min. After adding 9 ml of chromotropic acid, the volume was adjusted to 10 ml and the optical density was measured after cooling, at 570 nm. The assay was standardised with glycerol solution of known molarity and the glycerol liberated was calculated. The same procedure was repeated after the administration of Caps HT2 (5 mg/kg) for a period of 10 min. The glycerol liberated was calculated and compared with normal untreated group.

Antiinflammatory activity

* Carrageenan induced pedal edema in rats: The rats were divided into groups of 6 each. Acute inflammation was induced by sub plantar injection of 0.1 ml of freshly prepared 1% suspension of carrageenan in normal saline in the right hind paw of rats and paw thickness was measured using vernier calipers before carrageenan injection (0 h) and at intervals of 1 h, up to 4 hrs (Winter et al. 1962). The animals were premedicated with vehicle, the formulation, Caps HT2 (300 and 500 mg/kg) or acetyl salicylic acid (standard, 100 mg/kg) orally 1 h prior to carrageenan injection. Mean increase in paw thickness was measured and percentage inhibition was calculated.

* Formalin induced paw edema: Formalin (0.1 ml, 2%) was injected into the subplantar area of right hind paw of ether-anaesthetised rats (Chau, 1989). The formulation, Caps HT2 was given orally 1 h before formalin injection and continued for 7 consecutive days. The degree of inhibition was measured and the percentage inhibition was calculated.

Hypolipidemic effects

The animals were divided into 7 groups A, B, C, D, E, F and G of 6 each. The group A was maintained as normal. The groups B, C, D, E, F and G received atherogenic diet (HFD) comprising wheat flower base plus milk powder, dried egg yolk, hydrogenated fat, butter, dried yeast, salt, sucrose and vitamin mixture to produce the following nutrients in the given proportions: protein 15%, carbohydrate 60%, sucrose 3%, fat 15%, salts 4%, vitamins 1% and fibre 2%. In addition, cholesterol powder (400 mg/kg) dissolved in (5 ml) coconut oil was administered daily by gastric intubations (Ritu et al. 1996). Among these the group B was treated as control (HFD alone) and the group C received standard drug lovastatin (5 mg/kg). The Caps HT2 was administered into the groups D, E, F and G as 100, 200, 300 and 400 mg/kg daily by oral intubations, for a period of 30 days.

At the end of the experimental period all the animals were fasted overnight and sacrificed. The total cholesterol (Duncan et al. 1982) triglycerides (NCEP, 1995), phospholipids (Connerty et al. 1961) and HDL cholesterol (Haris et al. 1996) were estimated in the serum using commercially available standard kits. LDL cholesterol was calculated by using Friedwald's formula (Friedwald, 1972); LDL cholesterol (mg/dl) = Total cholesterol - (HDL cholesterol + Triglycerides/5).

The results were analyzed using student's t-test (Gupta, 1978) and the values are expressed as the mean value of [+ or -] SD with value of p < 0.05 considered as significant.

* Results

Caps HT2 was found to scavenge the superoxide generated by photoreduction of riboflavin. The concentration for 50% scavenging of superoxide was found 55.0 [micro]g/ml and 6.3 [micro]g/ml for Caps HT2 and curcumin respectively (Table 1).

The generation of lipid peroxides by [Fe.sup.2+]/ascorbate in rat liver homogenate was found inhibited by the addition of the formulation. The concentration for 50% inhibition was 48.5 [micro]g/ml and the curcumin needed was only 2.7 [micro]g/ml (Table 1).

Degradation of deoxyribose mediated by hydroxyl radicals generated by the [Fe.sup.3+]/ascorbate/EDTA/[H.sub.2][O.sub.2] system was also found inhibited by the formulation Caps HT2. The concentration needed for 50% inhibition was 610.0 [micro]g/ml and 8.9 [micro]g/ml for Caps HT2 and curcumin (Table 1) respectively.

Normal plasma recalcification time noticed was 58 [+ or -] 2 Sec in rabbits. Administration of heparin delayed the recalcification time to 142 [+ or -] 4 Sec (Table 2) and administration of the Caps HT2 delayed to 139 [+ or -] 5 Sec; which is much greater than the recalcification time of normal plasma. Compared to heparin this drug is more or less equally effective.

Addition of ADP to a suspension of washed human platelets caused a marked decrease in optical density at 600nm indicating the aggregation of platelets. The aggregation effect was greater at 37 [degrees]C compared to that at room temperature. The formulation, Caps HT2 (50-150 [micro]g/ml) interestingly inhibited platelet aggregation (Fig. 1). Greater inhibition of aggregation was noticed with increased concentrations.

[FIGURE 1 OMITTED]

The drug treated animals for a period of 10 min showed increased production of glycerol as an index of the greater release and activity of enzyme from the arterial intima. The glycerol liberated in the Caps HT2 treated animals was found to be 10.9 mg% where as in the normal animals it was only 3.6% (Table 3).

Anti-inflammatory effect of Caps HT2 against carrageenan induced acute inflammation is shown in the Table 4. The formulation significantly reduced paw thickness (p < 0.001) as compared to that of control rats. The paw oedema was highest at 2hrs after carrageenan administration and is followed by a gradual decline (Fig. 2).

[FIGURE 2 OMITTED]

The results of the formalin induced paw oedema showed that formulation was also significantly effective in chronic inflammation (Table 5). There was 42.7% reduction in paw oedema when the formulation was administered at a dose of 300 mg/kg) and 38.3% decrease by the higher dose (500 mg/kg) administration (Fig. 3). The anti-inflammatory efficacy of Caps HT2 was comparable to that of the standard acetyl salicylic acid (aspirin). In acute as well as chronic inflammations, 300 mg/kg dose administration showed greater efficacy.

[FIGURE 3 OMITTED]

Experimental hyperlipidaemia in rats was associated with an increase in serum lipid profile. Treatment with the formulation, Caps HT2 significantly changed the lipid parameters (Table 6). Administration of the formulation for a period of 30 days was associated with a highly significant decline in total cholesterol, LDL and VLDL cholesterol, triglycerides and phospholipids with the administration of the two different concentrations (300 and 400 mg/kg). A highly significant increase in the HDL cholesterol was noticed by the administration of all the four different concentrations (Table 6). The atherogenic index as well as the decrease in body weight was highly significant.

* Discussion

Traditional Ayurvedic Herbal Medicines have been used against several types of ailments such as insanity, fever, dysentery, loss of appetite, neurological disorders and cardiac troubles (Raghunath and Mitra, 1982). The present investigation has explored the use of selected plants for preventing coronary artery diseases.

The vascular endothelium is the principal site of action of cardiovascular risk factors and early atherogenesis. The imbalance between prooxidants and antioxidants in the development of atherosclerosis has prompted the investigation of antioxidants as possible therapy (Ross, 1993). The screening of antioxidant activity of Caps HT2 has revealed its capacity to scavenge the superoxide and hydroxyl radicals at low concentrations. The process of atherogenesis as initiated by peroxidation of lipids in low-density lipoproteins (Diaz et al. 1997), was also found inhibited by very low concentrations of Caps HT2.

The antiplatelet therapy constitutes one of the best available tools for ameliorating the mechanisms related to atherogenesis (Cimmniello and Toschi, 1999) and this herbal formulation Caps HT2 interestingly inhibited platelet aggregation. The plasma recalcification time was also delayed significantly by the intravenous administration of the formulation.

Lipoprotein lipase has been reported in the post heparin plasma of rabbits and has a major role in the transport and metabolism of triglycerides of exogenous origin (Korn, 1962). It is the key enzyme, which regulates the disposal of lipid fuels in the body (Barbara, 1998). The glycerol liberated by the action of lipoprotein lipase enzyme in the formulation treated rabbits was found 3 times greater than the normal untreated ones. Caps HT2 exhibited an enhancing role of releasing and activating the enzyme, resulting in the metabolic degradation of lipids.

Atherosclerosis is also an inflammatory disease and does not result simply from the accumulation of lipids. If we can selectively modify the harmful components of inflammation in the arteries and leave protective measures intact, we may create new avenues for the diagnosis and management of cardiovascular diseases (Ross, 1999). The significant inhibition of acute and chronic inflammations induced by carrageenan and formalin by the formulation, could be a collective anti-inflammatory effect of individual plants of the formulation, especially Withania somnifera, Ocimum sanctum and Hemidesmus indicus, as already been reported (Sahini and Srivastava, 1995; Singh and Majumdar, 1997; Alam and Gomes, 1998).

The risk of coronary heart disease (CHD) can also be lowered by treatment reducing the plasma cholesterol concentrations. Recent published studies have added to the evidence for a prethrombotic state in hyperlipidaemia. The consequence of plaque disruption in a coronary artery will depend partly upon the magnitude of the thrombotic response to this event. This is the rational for the antiplatelet and anticoagulant therapy in patients with CHD. Lipid lowering therapy may also be beneficial in this respect by reversing changes in the clotting pathway, fibrinolytic system and in blood platelets from hyperlipidaemic patients (George, 1995). Reduction in blood lipids/cholesterol especially from LDL fraction could be used as a means of arresting the development of atherosclerosis (Frank et al. 1990).

It is possible that the beneficial cardiovascular effects of Caps HT2 may be related to its antioxidant, anticoagulant, hypolipidaemic, platelet antiaggregation and lipoprotein lipase releasing properties. All the selected plants of the formulation were found to possess hypolipidaemic/hypocholesterolemic properties (Angshula et al. 1994; Arti et al. 1995; Ram, 1996; Shaila et al. 1997; Bopanna et al. 1997; Malhorta et al. 1997; Stanley et al. 1999; Andallu and Radhika 2000; Augusti et al. 2001). Allium sativum was known for antioxidant, fibrinolytic and platelet aggregation inhibition activities (Augusti et al. 2001). Commiphora mukul was used as a slimming agent against obesity and for elevating the level of good (HDL) cholesterol (Seth, 1996).

In the present study, Caps HT2 resulted a highly significant reduction in total cholesterol, LDL cholesterol, triglycerides and phospholipids with a concomitant rise in HDL cholesterol. The mechanism of hypotriglyceridaemic effect has also been shown to be partly due to the stimulation of lipoprotein lipase activity. The percentage decrease in body weight observed between normal and treated groups was indicative of efficacy against obesity. These results demonstrate the effectiveness of Caps HT2 as an antiatherogenic agent preventing coronary artery diseases.

Table 1. Effect of the formulation on free
radical generation.

Test          I[C.sub.50] value ([micro]g/ml)
material

              Superoxide   Hydroxyl   Lipid
                           radical    peroxide

Formulation   55.00        610.00     48.50
Curcumin       6.32          2.71      8.93

The values are average of triplicate tubes.

Table 2. Anticoagulant activity of the formulation.

Sample                  Plasma recalcification time
                        (in seconds)

Normal rabbit blood      58 [+ or -] 2
Heparin (1 mg/kg)       145 [+ or -] 4
Formulation (5 mg/kg)   139 [+ or -] 5

The values are mean [+  or -] SD of 6 animals/group.

Table 3. Lipoprotein lipase releasing activity of the
formulation in rabbits.

Groups                  Glycerol liberated (mg/dl/hr)

Normal rabbits           3.7  [+ or -] 0.14
Formulation (5 mg/kg)   10.88 [+ or -] 0.2
Heparin (1 mg/kg)       12.7  [+ or -] 1.7

The values are mean [+  or -] SD of 6 animals/group.

Table 4. Anti-inflammatory activity of the formulation
in rats (Carrageenan induced).

Groups        Dose        Increase in paw          % inhi-
              (mg/kg      thickness after          bition
              body wt.)   7 days (cm)

Control       --          0.130 [+ or -] 0.01      --
Aspirin       100         0.082 [+ or -] 0.006 *   38.5
Formulation   300         0.061 [+ or -] 0.005 *   53.8
Formulation   500         0.081 [+ or -] 0.005 *   38.6

The values are mean [+  or -] SD of 6 animals/group, * p < 0.001.

Table 5. Anti-inflammatory activity of the formulation
in rats (Formalin induced).

Groups        Dose        Increase in paw        % inhi-
              (mg/kg      thickness after        bition
              body wt.)   7 days (cm)

Control       --          0.47 [+ or -] 0.04     --
Aspirin       100         0.29 [+ or -] 0.03 *   38.3
Formulation   300         0.27 [+ or -] 0.02 *   42.7
Formulation   500         0.29 [+ or -] 0.03 *   38.3

The values are mean [+  or -] SD of 6 animals/group, * p < 0.001.

Table 6. Effect of the formulation on serum lipid profile of rats fed
on HFD for a period of 30 days.

Groups                     Total Cholesterol       HDL Cholesterol
                           (mg/dl)                 (mg/dl)

A - Normal                 60.7 [+ or -] 5.4       33.3 [+ or -] 2.9
[degrees]B - HFD           86.4 [+ or -] 8.2 *     30.0 [+ or -] 2.2
[sup.*]C - HFD+L           73.4 [+ or -] 5.7 ***   38.1 [+ or -] 3.0
[sup.*]D - HFD+[F.sub.1]   77.9 [+ or -] 4.2       38.6 [+ or -] 2.8 *
[sup.*]E - HFD+[F.sub.2]   77.5 [+ or -] 3.9       40.8 [+ or -] 3.6 *
[sup.*]F - HFD+[F.sub.3]   65.6 [+ or -] 5.1 *     40.3 [+ or -] 3.6 *
[sup.*]G - HFD+[F.sub.4]   65.8 [+ or -] 4.2 *     39.1 [+ or -] 2.4 *

Groups                     LDL Cholesterol       Atherogenic index
                           (mg/dl)

A - Normal                 18.4 [+ or -] 0.9     0.85 [+ or -] 0.05
[degrees]B - HFD           40.8 [+ or -] 2.7 *   1.87 [+ or -] 0.09
[sup.*]C - HFD+L           24.0 [+ or -] 2.0 *   0.91 [+ or -] 0.07 *
[sup.*]D - HFD+[F.sub.1]   22.7 [+ or -] 2.1 *   0.96 [+ or -] 0.08 *
[sup.*]E - HFD+[F.sub.2]   21.8 [+ or -] 1.2 *   0.87 [+ or -] 0.06 *
[sup.*]F - HFD+[F.sub.3]   20.1 [+ or -] 0.7 *   0.71 [+ or -] 0.05 *
[sup.*]G - HFD+[F.sub.4]   19.3 [+ or -] 1.1 *   0.71 [+ or -] 0.04 *

Groups                     Triglycerides         Phospholipids
                           (mg/dl)               (mg/dl)

A - Normal                 45.6 [+ or -] 4.1      81.7 [+ or -] 7.5
[degrees]B - HFD           76.5 [+ or -] 5.7 *   104.6 [+ or -] 9.7 *
[sup.*]C - HFD+L           54.9 [+ or -] 4.7 *    83.3 [+ or -] 5.3 *
[sup.*]D - HFD+[F.sub.1]   73.3 [+ or -] 4.2      86.7 [+ or -] 7.3 **
[sup.*]E - HFD+[F.sub.2]   68.9 [+ or -] 6.7      86.4 [+ or -] 6.50 **
[sup.*]F - HFD+[F.sub.3]   45.9 [+ or -] 4.7 *    75.6 [+ or -] 4.7 *
[sup.*]G - HFD+[F.sub.4]   42.4 [+ or -] 3.0 *    75.0 [+ or -] 3.2 *

Groups                     % Wt
                           increase

A - Normal                 15.1 [+ or -] 1.3
[degrees]B - HFD           33.33 [+ or -] 2.5
[sup.*]C - HFD+L           19.21 [+ or -] 1.5 *
[sup.*]D - HFD+[F.sub.1]   28.07 [+ or -] 1.9 **
[sup.*]E - HFD+[F.sub.2]   23.72 [+ or -] 1.9 *
[sup.*]F - HFD+[F.sub.3]   19.22 [+ or -] 1.5 *
[sup.*]G - HFD+[F.sub.4]   18.97 [+ or -] 1.3 *

L--Lovastatin (5 mg/kg), [F.sub.1]--Formulation
(100 mg/kg), [F.sub.2]--Formulation (200 mg/kg),
[F.sub.3]--Formulation (300 mg/kg),
[F.sub.4]--Formulation (400 mg/kg). [degrees]B
compared to A, [sup.*]C, [sup.*]D, [sup.*]E,
[sup.*]F and [sup.*]G compared to B, * P < 0.001,
** P < 0.005, *** P < 0.01.

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* Address

J. Padikkala, Amala Cancer Research Centre, Amala Nagar, Thrissur--680 553, Kerala, India

Tel./Fax: ++91 487 230 7868; e-mail: [e-mail]

N. K. Mary, B. H. Babu and J. Padikkala

Amala Cancer Research Centre, Thrissur, Kerala, India