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Thirty patients with coronary artery disease (CAD) were administered garlic (study group) while another 30 patients received the placebo (control group). Various risk parameters were determined at 1.5 and 3 months of garlic administration. Garlic, administered in a daily dose of 2 x 2 capsules (each capsule containing ethyl acetate extract from 1 g peeled and crushed raw garlic), reduced significantly total serum cholesterol and triglycerides, and increased significantly HDL-cholesterol and fibrinolytic activity. There was no effect on the fibrinogen and glucose levels. In vitro effects of the garlic oil on platelet aggregation (PAg) and eicosanoid metabolism were examined; it inhibited PAg induced by several platelet agonists, and also platelet thromboxane formation. Two important paraffinic polysulphides - diallyl disulphide (DADS) and diallyl trisulphide (DATS) - derived from garlic and are usual constituents of garlic oil, showed antiplatelet activity, and also inhibited platelet thromboxane formation. In this respect DATS was more potent than DADS. The nature of inhibition of PAg by DATS was found to be reversible.

In traditional medicine, Ayurveda, several spices and herbs are held to possess medicinal properties. Earlier we have reported that extracts from several spices, including turmeric, inhibit platelet aggregation and modulate eicosanoid biosynthesis. Due to their eicosanoid-modulating property, it was suggested that the spices may serve to provide clues to drugs directed to arachidonic acid (AA) pathway enzymes as pharmacological targets. Curcumin, a major component of turmeric, inhibited platelet aggregation induced by arachidonate, adrenaline and collagen. This compound inhibited thromboxane B2 (TXB2) production from exogenous [14C] arachidonate in washed platelets with a concomitant increase in the formation of 12-lipoxygenase products. Moreover, curcumin inhibited the incorporation of [14C]AA into platelet phospholipids and inhibited the deacylation of AA-labelled phospholipids (liberation of free AA) on stimulation with calcium ionophore A23187. Curcumin's anti-inflammatory property may, in part, be explained by its effects on eicosanoid biosynthesis.

In continuation of our studies on the oil of cloves — a common kitchen spice and a drug for home medicine — we have isolated and identified two antiplatelet components, eugenol and acetyl eugenol. They inhibited arachidonate-, adrenaline- and collagen-induced platelet aggregation; they were more potent in inhibiting aggregation by the first two agonists. Their inhibitory effect was reversible. These components were antiaggregatory by a combination of at least two effects: (i) inhibition of platelet thromboxane formation, and (ii) increased formation of 12-lipoxygenase products (12-HPETE). Though the presence of plasma proteins would reduce the effective concentration of these substances due to binding, the relatively lower amounts of these components which inhibited arachidonate-induced aggregation when compared to their effects on thromboxane production was intriguing. The answer might partly lie in an increased formation of 12-HPETE facilitated by albumin which acts as a ‘conduit' to divert free arachadonic acid (AA) from the platelet cyclooxygenase (CO) to the lipoxygenase pathway (22). Based on their IC 50 values, it was found that both eugenol and acetyl eugenol were more potent than aspirin in inhibiting platelet aggregation induced by arachidonate, adrenaline and collagen. In arachidonate-induced aggregation eugenol was on a par with indomethacin. It was found that eugenol and acetyl eugenol when used in combination potentiated inhibition of platelet aggregation induced by arachidonate, adrenaline and collagen. This effect was, however, not evident from the metabolism of AA in platelets; when used in combination the two compounds produced an additive effect.

One of the features of inflammation is increased oxygenation of arachidonic acid which is metabolized by two enzymic pathways--the cyclooxygenase (CO) and the 5-lipoxygenase (5-LO)--leading to the production of prostaglandins and leukotrienes respectively. Amongst the CO products, PGE2 and amongst the 5-LO products, LTB4 are considered important mediators of inflammation. More than 200 potential drugs ranging from non-steroidal anti-inflammatory drugs, corticosteroids, gold salts, disease modifying anti-rheumatic drugs, methotrexate, cyclosporine are being tested. None of the drugs has been found safe; all are known to produce from mild to serious side-effects. Ginger is described in Ayurvedic and Tibb systems of medicine to be useful in inflammation and rheumatism. In all 56 patients (28 with rheumatoid arthritis, 18 with osteoarthritis and 10 with muscular discomfort) used powdered ginger against their afflictions. Amongst the arthritis patients more than three-quarters experienced, to varying degrees, relief in pain and swelling. All the patients with muscular discomfort experienced relief in pain. None of the patients reported adverse effects during the period of ginger consumption which ranged from 3 months to 2.5 years. It is suggested that at least one of the mechanisms by which ginger shows its ameliorative effects could be related to inhibition of prostaglandin and leukotriene biosynthesis, i.e. it works as a dual inhibitor of eicosanoid biosynthesis.

Migraine is considered as a neurological disorder with little convincing evidence of the involvement of some vascular phenomenon. Recent understanding of the mechanisms behind migraine pain generation and perception have considerably helped the development of modern migraine drugs. Most migraine drugs in use, i.e., ergotamine and dihydroergotamine, iprazochrome, pizotifen and diazepam; and non-steroidal antiinflammatory drugs (i.e. aspirin, paracetamol, persantin, etc.) have side-effects and are prescribed with caution for a limited duration. Ginger is reported in Ayurvedic and Tibb systems of medicine to be useful in neurological disorders. It is proposed that administration of ginger may exert abortive and prophylactic effects in migraine headache without any side-effects.

A further re-examination of the seeds of Nyctanthes arbor-tristis has led to the isolation and identification of two new minor iridoid glucosides, arbortristoside D and E, together with the previously reported, arbortristoside B. Antiviral activity was observed in the crude alcoholic extract, n-butanol fraction and pure compounds.

In a placebo-controlled study the effect of ginger and fenugreek was examined on blood lipids, blood sugar, platelet aggregation, fibrinogen and fibrinolytic activity. The subjects included in this study were healthy individuals, patients with coronary artery disease (CAD), and patients with non-insulin-dependent diabetes mellitus (NIDDM) who either had CAD or were without CAD. In patients with CAD powdered ginger administered in a dose of 4 g daily for 3 months did not affect ADP- and epinephrine-induced platelet aggregation. Also, no change in the fibrinolytic activity and fibrinogen level was observed. However, a single dose of 10 g powdered ginger administered to CAD patients produced a significant reduction in platelet aggregation induced by the two agonists. Ginger did not affect the blood lipids and blood sugar. Fenugreek given in a dose of 2.5 g twice daily for 3 months to healthy individuals did not affect the blood lipids and blood sugar (fasting and post prandial). However, administered in the same daily dose for the same duration to CAD patients also with NIDDM, fenugreek decreased significantly the blood lipids (total cholesterol and triglycerides) without affecting the HDL-c. When administered in the same daily dose to NIDDM (non-CAD) patients (mild cases), fenugreek reduced significantly the blood sugar (fasting and post prandial). In severe NIDDM cases, blood sugar (both fasting and post prandial) was only slightly reduced. The changes were not significant. Fenugreek administration did not affect platelet aggregation, fibrinolytic activity and fibrinogen.

The overall goal of the project is to develop an advanced, clean coal biogasification (MicGAS) Process. The objectives of the research during FY 1993--94 were to: (1) enhance kinetics of methane production (biogasification, biomethanation) from Texas lignite (TxL) by the Mic-1 consortium isolated and developed at ARCTECH, (2) increase coal solids loading, (3) optimize medium composition, and (4) reduce retention time. A closer analysis of the results described here indicate that biomethanation of TxL at >5% solids loading is feasible through appropriate development of nutrient medium and further adaptation of the microorganisms involved in this process. Further understanding of the inhibitory factors and some biochemical manipulations to overcome those inhibitions will hasten the process considerably. Results are discussed on the following: products of biomethanation and enhance of methane production including: bacterial adaptation; effect of nutrient amendment substitutes; effects of solids loading; effect of initial pH of the culture medium; effect of hydrogen donors and carbon balance.

Maximum methane production was obtained in the experimental vials that contained 0.2% SNTM supplemented with 10 mM sodium citrate and 1% TxL (144 cc), while in the control vials CH{sub 4} production was only 58 cc. The conversion efficiency was 24%. This clearly shows citrate to be an important mediator for the formation of acetate (main precursor for CH{sub 4} formation) in the glyoxylate cycle, on the one hand, and as a sequestering agent, on the other. These results further indicate that citrate can, be successfully used as co-substrate for enhancement of the TxL biogasification process. The results obtained reconfirmed our hypothesis that the metals (such as Fe{sup 3+}, Mn{sup 2+}, Mg{sup 2+}, CO{sup 2+}, Zr{sup 2+}, etc., present in the coal structure) are chelated/sequestered by the addition of citrate. Mass balance calculations show that this increase in CH{sup 4} production is due to the biomethanation of TxL and not because of the chemical conversion of co-substrate(s) to CH{sub 4} (Table 1). The effect of sodium citrate on biomethanation of TXL from the first experiment ``Effect of co-substrate addition No. 1`` was reconfirmed in this experiment. The peak in acetate concentration (1317 ppm) on day 7 was followed by amore » rapid conversion of this precursor to CH{sub 4} (Figure 16). The VFA data obtained from both experiments (``Effect of co-substrate addition No. 1 and No. 2``) confirms the hypothesis that citrate and methanol can significantly enhance the biomethanation of TxL (Figure 17).« less

When garlic cloves are chopped or crushed several dialkyl thiosulfinates are rapidly formed by the action of the enzyme alliin lyase or alliinase (EC 4.4.1.4) on S(+)-alkyl-L-cysteine sulfoxides. Allicin (diallyl thiosulfinate or allyl 2-propene thiosulfinate) is the dominant thiosulfinate released. A variety of sulfur containing compounds are formed from allicin and other thiosulfinates depending on the way in which garlic is handled. One such compound identified recently is ajoene which has been reported to possess antithrombotic properties. We present here data on the antiplatelet properties of ajoene together with its effects on the metabolism of arachidonic acid (AA) in intact platelets. Thus, ajoene was found to inhibit platelet aggregation induced by AA, adrenaline, collagen, adenosine diphosphate (ADP) and calcium ionophore A23187; the nature of the inhibition was irreversible. In washed platelets stimulated by labelled arachidonate, ajoene inhibited the formation of thromboxane A2; 12-lipoxygenase product(s) were reduced at higher ajoene concentrations. This garlic-derived substance inhibited the incorporation of labelled AA into platelet phospholipids at higher concentration. In labelled platelets, on stimulation with either calcium ionophore A23187 or collagen, reduced amounts of thromboxane and 12-HETE (12-hydroxyeicosatetraenoic acid) were produced in ajoene-treated platelets compared to control platelets. This substance had no effect on the deacylation of platelet phospholipids. The results suggest that at least one of the mechanisms by which ajoene shows antiplatelet effects could be related to altered metabolism of AA.

Laboratory and bench scale reactor research carried out during the report period confirms the feasibility of biomethanation of Texas lignite (TxL) and some other low-rank coals to methane by specifically developed unique anaerobic microbial consortia. The data obtained demonstrates specificity of a particular microbial consortium to a given lignite. Development of a suitable microbial consortium is the key to the success of the process. The Mic-1 consortium was developed to tolerate higher coal loadings of 1 and 5% TxL in comparison to initial loadings of 0.01% and 0.1% TxL. Moreover, the reaction period was reduced from 60 days to 14 to 21 days. The cost of the culture medium for bioconversion was reduced by studying the effect of different growth factors on the biomethanation capability of Mic-1 consortium. Four different bench scale bioreactor configurations, namely Rotating Biological Contactor (RBC), Upflow Fluidized Bed Reactor (UFBR), Trickle Bed Reactor (TBR), and Continuously Stirred Tank Reactor (CSTR) were evaluated for scale up studies. Preliminary results indicated highest biomethanation of TxL by the Mic-1 consortium in the CSTR, and lowest in the trickle bed reactor. However, highest methane production and process efficiency were obtained in the RBC.

In continuation of our studies with the oil of cloves--a common kitchen spice and a crude drug for home medicine--we have isolated yet another active component identified as acetyl eugenol (AE); the earlier reported active component being eugenol. The isolated material (IM) was found to be a potent platelet inhibitor; IM abolished arachidonate (AA)-induced aggregation at ca. 12 microM, a concentration needed to abolish the second phase of adrenaline-induced aggregation. Chemically synthesized acetyl eugenol showed similar effects on AA- and adrenaline-induced aggregation. A dose-dependent inhibition of collagen-induced aggregation was also observed. AE did not inhibit either calcium ionophore A23187- or thrombin-induced aggregation. Studies on aggregation and ATP release were done using whole blood (WB). AA-induced aggregation in WB was abolished at 3 micrograms/ml (14.6 microM) which persisted even after doubling the concentration of AA. ATP release was inhibited. Inhibition of aggregation appeared to be mediated by a combination of two effects: reduced formation of thromboxane and increased generation of 12-lipoxygenase product (12-HPETE). These effects were observed by exposing washed platelets to (14C)AA or by stimulating AA-labelled platelets with ionophore A23187. Acetyl eugenol inhibited (14C)TxB2 formation in AA-labelled platelets on stimulation with thrombin. AE showed no effect on the incorporation of AA into platelet phospholipids.

Age and sex related platelet biosynthesis of prostaglandins, thromboxane B2, prostaglandin endoperoxides, HHT and HETE was studied in normal human subjects and in acute myocardial infarction (AMI) patients (all males over 50 yr). The following results were obtained. 1. No significant difference was observed in the platelet biosynthesis of prostaglandins (F2α, E2, D2 ), TxB2 and HHT in normal subjects when values of these arachidonic acid metabolites were compared for different age groups and sex. 2. HETE formation in females was significantly more (p < 0.001) than that in males. 3. No significant difference was observed in the prostaglandin endoperoxide level (PGG2, PGH2 + PGF2α) amongst different age groups and sex in the normal subjects and this was so when compared with AMI patients. 4. Significantly less TxB2 was produced by platelets from the AMI patients compared to normal males over 50 years. 5. In AMI patients, platelets produced significantly more HHT (p < 0.005) and PGF2α (p < 0.02) compared to normal males over 50 years. 6. A significantly reduced (p < 0.005) formation of HETE in the AMI patients compared to normal males (over 50 yr) was observed. A comparison of ratios between HHT, HETE and TxB2 for normal males (over 50 yr) and AMI patients was made for their possible use in diagnostic purposes. These results have been discussed in the light of the existing knowledge about the aggregation and antiaggregation properties of various AA metabolites.

Surface response methodology was used to study the effects of three variables, viz. total reducing sugars (116–222 g l −1 ), yeast inoculum concentration (10–30%) and supplemented inorganic nitrogen as (NH 4 ) 2 SO 4 (1–3 g l −1 ) on the production of ethanol. The complete and incomplete factorial designs (3 3 ) were compared through the residual analysis of variance. Since there was no statistically significant difference at the 5% level, on the basis of cost-efficiency and space limitations, it was recommended to use incomplete factorial design constituting 12 treatments with three repetitions in central point totalling 15 experiments which control the batch alcoholic fermentation.

Effects of labetalol on the arachidonic acid metabolism in washed human blood platelets, and in lung and aorta of the rat was studied. Effects of this drug on the aggregation induced by usual aggregation agents, and on the prostacyclin generating capacity of rat aorta as assessed by its antiaggregation effect were also studied. The following results were obtained. 1. Reduced generation of thromboxane and HHT in platelets was observed at 1 mM cone. of the drug. 2. This drug inhibited ADP-, collagen-, and epinephrine-induced aggregation in a dose dependent manner. 3. Arachidonate-induced aggregation was only slightly inhibited at 1 mM cone. of labetalol. 4. Aorta and lung synthesized more prostacyclin in its presence from exogenous labelled arachidonate. 5. Aorta synthesized more prostacyclin from its endogenous AA pool in the presence of labetalol as assessed by inhibition of platelet aggregation.

An ethereal extract of omum (Trachyspermum ammi; Hindustani: ajwan)--a frequently consumed spice--was found to inhibit platelet aggregation induced by arachidonic acid (AA), epinephrine and collagen; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by omum could be explained by its effect on platelet thromboxane production as suggested by the following experimental observation. (i) Omum reduced TxB2 formation in intact platelet preparations from added arachidonate, and (ii) it reduced the formation of TxB2 from AA-labelled platelets after stimulation with Ca2+-ionophore A23187 by a direct action on cyclooxygenase as it did not affect the release of AA from labelled platelets. An increased formation of lipoxygenase-derived products from exogenous AA in omum-treated platelets was apparently due to redirection of AA from cyclooxygenase to the lipoxygenase pathway.

The effect of some saturated and unsaturated fatty acids pre-incubated with human washed platelets was examined on the platelet utilization of (1-14C) arachidonic acid. The platelets were incubated with the radioactive arachidonic acid for periods of either 10 min or 30 sec. The following results were obtained. Conversion of arachidonic acid to thromboxane B2 was increased during 10 min in the presence of oleic, alpha-linolenic and lauric acids. Increased conversion of 14C-arachidonic acid to hydroxy fatty acids (HHT and HETE) was observed in the presence of stearic acid (10 min incubation) and lauric acid (30 sec incubation). Their synthesis, however, was decreased in presence of gamma-linolenic acid (10 min incubation). Endoperoxide generation from arachidonate was reduced by alpha-linolenic acid but increased by stearic acid. The conversion to PGE2 was not altered by these acids during 10 min incubation with arachidonic acid. However, in presence of gamma-linolenic, lauric and stearic acids conversion of arachidonic acid into PGE2 during 30 sec incubation was increased. Under these conditions, production of PGF2 alpha and PGD2 remained unchanged.

Eleven TLC solvent systems were evaluated for the separation of prostaglandins F 2α , E 2 and D 2 , thromboxane B 2 , 12-OH-5,8,10-heptadecatrienoic acid (HHT), 12-OH-5,8,10,14-eicosatetraenoic acid (HETE), and arachidonic acid (AA) from each other. A three-solvent development TLC procedure, which can conveniently be used for routine resolution of mixtures of these metabolites on a single TLC plate, is reported. The method was used for quantification of the metabolites formed from 14 C-labeled AA by washed human platelets.

The effects of several cis and two trans fatty acids (elaidic and linoelaidic acids) - sodium salt and also in free acid form - on ADP-induced platelet aggregation were studied. The effects of the cis and trans fatty acids (sod. salt) on collagen-induced aggregation were also examined. Besides the effects of several cis fatty acids, including dihomo-γ-linolenic acid, on arachidonic acid induced aggregation were examined. The results indicate that unsaturated fatty acids inhibit platelet aggregation induced by ADP and collagen. The unsaturated fatty acids, however, with most blood samples did not show any antiaggregation effect on arachidonic acid-induced aggregation. While showing an antiaggregation effect on collagen-induced aggregation and failing to do so on arachidonic acid-induced aggregation, this difference in the behaviour of the unsaturated fatty acids in the two aggregations can be explained on the basis of binding of exogenous test fatty acids to plasma proteins. The mechanism(s) by which unsaturated fatty acids, in general, may exert their antiaggregation effect is discussed.

Narcissiflorine, narcissiflorinine and narcissifloridine, three new saponins, have been isolated from the ethanolic extract of Anemone narcissiflora (Ranunculaceae). The structural elucidation of narcissiflorine, narcissiflorinine and narcissifloridine has showed them to be [α- l -arabinofuranosyl-(1 → 4)-β- d -glucuronopyranosyl-(1→3)]- 3β-hydroxy-olean-12-en-28-oic acid, [α,- l -arabinofuranosyl-(1→2)-α- l -rhamnopyranosyl-(1→4)-β- d - glucuronypyranosyl(1→3)]-3-β-hydroxy-olean-12-en-28-oic acid and [α- l -arabinofuranosyl-(1→2)-α- l - rhamnopyranosyl-(1→4)-β- d -glucopyranosyl-(1→3)]-3-β-hydroxy-olean-12-en-28-oic acid, respectively.

Dietary fatty acids affect the synthesis and thus ultimate availability of prostaglandin precursors. During processing of edible fats a significant portion of the naturally occurring cis fatty acids is changed to the respective trans isomers. Trans fatty acids could have a wide variety of effects including their effect on prostaglandin synthesis. A comparative study on the effect of cis (oleic and linoleic) fatty acids and their corresponding trans isomers (elaidic and linoelaidic acids) on the in vitro prostaglandin biosynthesis in human blood platelets was made. Linoleic acid (0.5, 1.0 mM) showed the same effect on the various arachidonic acid (AA) metabolites as reported earlier by us, that is, increased generation of prostaglandins and thromboxanes and reduced amounts of HHT and HETE (26). Linoelaidic acid at these two concentrations had the same effect but to a lesser extent. Both linoleic and linoelaidic acids at 0.25 mM concentration reduced TxB2 formation. Oleic and elaidic acids (0.5, 1.0 mM) decreased the formation of TxB2 though not significantly. With oleic acid, the results in respect of other AA metabolites were similar to those reported by us earlier (26). MDA was reduced significantly in the presence of linoleic, linoelaidic and oleic acids; cis isomers being more effective. In the presence of linoleic and linoelaidic acids, prostaglandin endoperoxides were reduced significantly; there was no change in the presence of oleic and elaidic acids. Thus fatty acids seem to affect the platelet cyclo-oxygenase activity; this being more pronounced in the case of cis fatty acids compared to their trans isomers. Reduction in cyclo-oxygenase activity also depends upon the number of double bonds in the acids.

Oxygenation of arachidonic acid is increased in inflamed tissues. In this condition products of two enzymic pathways--the cyclooxygenase and the 5-lipoxygenase producing respectively prostaglandins and leukotrienes--are elevated. Of the cyclooxygenase products, PGE2 and of the lipoxygenase products, LTB4 are the strongest candidates for mediating inflammation. Non-steroidal anti-inflammatory drugs which inhibit the cyclooxygenase, and corticosteroids are used to treat such disorders. Both types of drugs produce adverse side-effects on prolonged use. Ginger is reported in Ayurvedic and Tibb systems of medicine to be useful in rheumatic disorders. Seven patients suffering from such disorders reported relief in pain and associated symptoms on ginger administration.

Effect of furosemide at various concentrations (0.05-4 mM) was examined on the in vitro biosynthesis of prostaglandins in human platelets. At lower furosemide concentrations (0.05 and 0.1 mM) no effect was observed. At 1 and 2 mM concentrations, PGE2 significantly (P less than 0.01) increased. At 3 and 4 mM concentrations PGE2 increased though not significantly probably because of the small number of samples (n=5). A decrease in PGD2 formation was noted at 1-4 mM furosemide concentrations, though significantly only at 3 mM conc. At 1 and 2 mM concentrations, TxB2 and HHT increased whereas at 3 and 4 mM concentrations these metabolites were decreased. These effects were, however, not significant. No effect was observed on endoperoxide generation at 1 and 2 mM conc. Furosemide at 1 and 2 mM concentrations inhibited ADP- and arachidonic acid induced platelet aggregation. An increased platelet formation of PGE2 in the presence of furosemide may point to the fact that this drug shows its effect mainly on the formation of PGE2 which has been found to exert a profound effect on renal blood flow and thus ameliorates some forms of hypertension.

Vitamin E (alpha-tocopherol) and tocopherol acetate produced a slightly increased amount of thromboxane in treated compared to untreated platelets. In tocopherol acetate-treated platelets significantly more lipoxygenase products were produced. alpha-tocopherol induced an increased, but not significant, production of thromboxane B2 during blood clotting. alpha-tocopherol was not found to affect platelet phospholipase activity as determined by its effect on the release of labelled arachidonic acid from platelet phospholipids by challenging the platelets with calcium ionophore A23,187. alpha-tocopherol potentiated the incorporation of labelled arachidonate in the platelet phospholipids. Inspite of having no effect on the arachidonic acid cascade in platelets, alpha-tocopherol inhibited aggregation induced by several aggregating agents including A23,187. Inhibition of aggregation may be explained by the ability of alpha-tocopherol to inhibit intracellular mobilization of sequestered calcium from the dense tubular system to the cytoplasm.

The ultrastructure of three strains of an obligate psychrophilic yeast, Candida gelida, has been examined by thin sectioning. Polysaccharide capsules were present whether the cultures were grown in glucose or glucose-free media. The budding of two of the strains was clearly of the Rhodotorula type whereas the third appeared to bud occasionally with the bud wall forming a continuation of the parent cell wall as in Saccharomyces. Endoplasmic reticulum running parallel to the nuclear membrane was a common feature of C. gelida. Vacuoles with and without granular contents were numerous in some cells; these are possibly reserves of lipid. Lomasomes and plasmalemmasomes were observed in association with the plasmalemma.

Aqueous extracts of onion, garlic and ginger were found to inhibit aggregation induced by ADP, epinephrine, collagen and arachidonate in a dose-dependent manner in vitro. In the case of onion and garlic extracts relatively much higher volumes were need to bring about even a modest inhibition (by ca. 13-18%) of thromboxane synthesis in washed platelets from labelled AA. On the other hand a good correlation was found between the amounts of ginger extract needed to inhibit platelet aggregation and those to inhibit platelet thromboxane synthesis. Ginger extract reduced also platelet prostaglandin-endoperoxides. A dose-related inhibition of platelet thromboxane- and prostaglandin (PGF2 alpha, PGE2 and PGD2) synthesis was affected by ginger extract. Extracts of onion, garlic and ginger inhibited biosynthesis of prostacyclin in rat aorta from labelled AA. Ginger extract mildly inhibited the synthesis of prostacyclin from endogenous pool of AA in rat aorta; the other two extracts were without effect.

The effects of four beta-adrenoceptor blocking agents on platelet aggregation, formation of thromboxane from exogenous arachidonic acid (AA) in platelets, on AA incorporation in platelet phospholipids, and on platelet phospholipase activity were studied. Of the four drugs pindolol inhibited thromboxane formation in a dose-related (0.25–1.0 mM) manner apparently by exerting its influence at the cyclooxygenase (CO) level. This drug also inhibited aggregation induced by AA, collagen, epinephrine and ADP. Atenolol and metoprolol though not showing inhibition of AA-induced aggregation did inhibit collagen- and ADP-induced aggregation; metoprolol reversed ADP-induced aggregation, and abolished second phase of epinephrine-induced aggregation. Timolol did not inhibit aggregation induced by all the aggregating agents. Atenolol inhibited (by ca. 1096) TxB 2 formation in platelets from exagenous as well as endogenous AA at rather high concentrations (1.0 mM). Metoprolol and Tmolol did not do so. The observations reported here can be best explained by taking into account the membrane stabilizing effects and lipophilic properties of the drugs.

Aqueous extract of garlic inhibited aggregation induced by ADP, collagen, arachidonate (AA), epinephrine and calcium ionophore A23187 in a dose-dependent manner. In an attempt to clarify the mechanism of inhibition of aggregation, metabolism of arachidonic acid in platelets was examined in the presence of garlic extract. It was found that: garlic reduced the formation of thromboxane from exogenous AA; garlic inhibited the phospholipase activity; garlic inhibited the formation of thromboxane and lipoxygenase products formed in platelets prelabelled with AA; and garlic inhibited the incorporation of arachidonate into platelet phospholipids. These effects may explain, in part, inhibition of platelet aggregation. Further, since garlic was also effective in inhibiting aggregation induced by calcium ionophore A23187 it may be suggested that the antiaggregation effect may be related to intraplatelet mobilization of calcium. Inhibition of epinephrine-induced aggregation by garlic extract may suggest that it may be inhibiting uptake of calcium into platelets thereby lowering cytosolic calcium concentrations. Thus garlic appears to be in possession of components which might exert their effects at various stages involved in the process of platelet aggregation.

The effects of some saturated (lauric, palmitic and stearic) an unsaturated (linoleic, gamma-linolenic, alpha-linolenic and oleic) fatty acids at 0.1. 0.25 and 0.5 mM concentrations on the in vitro metabolization of (1-14 C) arachidonic acid by washed human blood platelets have been studied. Effects of these fatty acids were studied with intact as well as lysed platelet preparations. With intact platelet preparations it was found that (i) all unsaturated fatty acids enhanced the biosynthesis of TxB2, PGE2, PGD2 and PGF2 alpha, (ii) unsaturated fatty acids reduced the formation of HHT and HETE with the exception of oleic acid which showed very little effect, (iii) unsaturated fatty acids reduced the formation of MDA, whereas palmitic and stearic acids increased its formation and (iv) all unsaturated fatty acids reduced the synthesis of prostaglandin endoperoxides. These results support our previous observations where effects of fatty acids were examined at higher concentrations (10). At 0.1 mM FA concentration, inconsistent results were obtained. With lysed platelet preparations all cyclooxygenase products were reduced in presence of unsaturated fatty acids, whereas HETE formation was reduced only in presence of linoleic and gamma-linolenic acids. Electron micrographs of washed platelet suspensions were obtained with untreated platelet preparations and platelet preparations treated with 0.25 and 0.5 mM linoleic acid concentrations. The results are discussed in the light of a possible soap-like effect of FA salt on platelets.

Effects of various concentrations (12.5-500 uM) of linoleic acid and docosahexaenoic acid (C22:6 omega 3)(sod. salt) were examined on the platelet metabolism of labelled arachidonate (AA) under two different incubation conditions. In the first platelets were pretreated with either fatty acid prior to incubation with labelled AA; and in the second incubation platelets were incubated with a mixture containing a fatty acid (linoleic acid or DHA) and labelled AA. At all concentrations the two fatty acids reduced the formation of TxB2. At lower concentrations (up to 200 uM) the fatty acids inhibited platelet cyclooxygenase as shown by a reduced formation of prostaglandins (PGs) and TxB2. At higher concentrations (400 and 500 uM), however, the fatty acids behaved differently. Although TxB2 formation was reduced, there was observed an increased formation of PGs. In DHA pretreated platelets only PGE2 increased (to double control values). Platelets pretreated with linoleic acid produced increased amounts of all PGs (PGF2 alpha, PGE2, PGD2), and this effect was greatest for PGE2 which increased by 5-6 fold of control values. DHA showed a dose-dependent inhibition of platelet aggregation induced by arachidonate, epinephrine and collagen.

The effects of onion and ginger consumption on platelet thromboxane production were examined. Volunteers, all Danish women, consumed either 70 g raw onion or 5 g raw ginger daily for a period of 7 days. Each participant in each (onion or ginger) group served as her own control. TxB2 determination was made in serum obtained after blood clotting. The following are the results. TxB2 (pmol/ml serum): (i) onion group--before consumption 910 +/- 327, after consumption 1005 +/- 713 (Mean +/- SD, N = 5); (ii) ginger consumption 782 +/- 482, after consumption 498 +/- 164 (Mean +/- SD, N = 7).

The effects of some vasodilatory (dipyridamole, nifedipine, verapamil) and antihypertensive (hydralazine, propranolol) drugs on the metabolism of exogenous arachidonic acid (AA), and on the production of thromboxane from endogenous AA stores were examined. In a coupled system of platelets and rat aorta, dipyridamole (100 microM), nifedipine (300 microM) and hydralazine (250 microM) potentiated the formation of 6-keto-F1 alpha from exogenous AA. Hydralazine in this system reduced the formation of TxB2. At higher concentrations the drugs (dipyridamole 500 microM; hydralazine 1 mM, verapamil 2mM; propranolol 2mM) were effective in reducing the formation of TxB2. Moreover, the drugs at lower concentrations produced reduced amounts of TxB2 in clotting blood. A dose-dependent inhibition of platelet aggregation induced by ADP, epinephrine, collagen and arachidonate was observed with these drugs. The results demonstrate that some of the beneficial effects of the drugs in the cardiovascular system can be explained on the basis of their effects on platelet aggregation and on AA metabolism.

Evaluation of several thin-layer chromatographic procedures for the separation of various labeled arachidonic acid metabolites (including 6-keto-prostaglandin F1 alpha) produced in the biological system is described. Manual scanning and autoradiography of the plates developed by two-dimensional thin-layer chromatography was also done for locating the radioactivities due to arachidonic acid metabolites other than thromboxane B2 and the classical prostaglandins (PGF2 alpha, PGE2, and PGD2).

Effects of some vasodilating (dipyridamole, nifedipine and verapamil) and antihypertensive (propranolol, hydralazine) drugs on arachidonic acid metabolism in isolated rat aorta and lung have been studied. Dipyridamole significantly increased the formation of PGI2 in aorta and lung. Nifedipine and verapamil decreased the formation of PGI2 in aorta, these drugs though significantly increased the formation of PGI2 in lung. Nifedipine showed no appreciable effect on the generation of TxA2 in rat aorta but in lung both nifedipine and verapamil reduced TxA2 formation though significantly only in the latter case. Dipyridamole showed no effect. The beneficial effect of dipyridamole, seems, at least in part, to be due to its ability to enhance the production of PGI2 both in the aorta and lung, and probably in other tissues as well. Nifedipine and verapamil may show their antianginal effect by a combined effect of enhanced PGI2 and reduced TxA2 formation in lung. In lung, whereas hydralazine reduced the formation of both PGI2 and TxA2, propranolol increased the formation of PGI2. Hydralazine reduced the formation of TxA2 and increased PGI2 formation in aorta. The effect of the drugs on the ability of rat aorta to inhibit collagen induced platelet aggregation of human blood platelets was also examined.

Oil of cloves (OC) was found to be a potent inhibitor of platelet aggregation induced by arachidonic acid (AA), collagen and epinephrine; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by OC seems to be mediated through a reduced formation of thromboxane as indicated by the following experimental evidence. (i) OC inhibited TxB2 formation in intact as well as lysed platelet preparations from added arachidonate, and (ii) it inhibited the formation of TxB2 from AA-labelled platelets after activation with Ca2+-ionophore A23187. The formation of lipoxygenase derived products was dependent on the concentration of OC used; at its lower concentration their amounts increased but this was found to be reversed at higher concentrations. At all concentrations thromboxane was decreased with a concomitant increase in unused AA.

In vitro effects of an oily extract of onion were examined on the metabolism of arachidonic acid (AA) in human platelets. Onion was found to reduce the formation of thromboxane and lipoxygenase products from exogenous arachidonic acid in platelets; it did not inhibit the incorporation of AA into platelet phospholipids. While not affecting the platelet phospholipase activity it did reduce the formation of thromboxane B2 and lipoxygenase products in platelets that were prelabelled with arachidonic acid and then activated by A23187. This suggests that onion inhibits the formation of AA metabolites by exerting its effect at steps later than the liberation of AA. With concentrations of onion extract producing abolition of AA-induced aggregation, only partial inhibition of aggregation was observed with ADP- and epinephrine-induced aggregation. Onion did not inhibit A23187 induced aggregation. The results suggest that inhibition of platelet aggregation by onion is mediated largely by its effect on platelet thromboxane production.

Aqueous ginger extract was extracted in three organic solvents viz., n-hexane, chloroform and ethyl acetate with increasing polarity. The extracted materials from these solvents reduced platelet thromboxane formation from exogenous arachidonate (AA) and also inhibited platelet aggregation induced by AA, epinephrine, ADP and collagen; in this respect they were most effective against AA-induced aggregation. The extracted material in n-hexane was further resolved by thin-layer chromatography into various fractions some of which were effective in inhibiting platelet thromboxane formation and platelet aggregation. Aqueous ginger extract reduced the formation of TxB2 from AA-labelled platelets without showing effects on platelet phospholipase activity. Thromboxane formation in labelled platelets on activation with calcium ionophore A23187 was reduced by ginger components, isolated from two TLC bands, in a dose-dependent manner (10-100 ug/500 ml). At the higher dose lipoxygenase products were also reduced. Interestingly the incorporation of AA into platelet phospholipids increased in platelets treated with aqueous ginger extract.

Unsaturated fatty acids (FAs) (oleic, linoleic, α -linolenic, and γ -linolenic acids) were found to inhibit platelet thromboxane synthetase. This conclusion was based on the following experimental evidence. 1. Reduced formation of TxB 2 and increased formation of prostaglandins F 2α , E 2 and D 2 as seen in autoradiography of TLC plate. 2. Inhibition of ADP- and collagen-induced aggregation of PRP samples in the presence of the unsaturated FAs. 3. Reduced formation of TxB 2 determined by RIA in PRP samples pretreated with the unsaturated FAs followed by collagen-induced aggregation. 4. Reduced formation of MDA in the presence of the unsaturated FAs. 5. Increased formation of PGI 2 (determined as 6-keto-PGF 1α ), PGE 2 , PGF 2 a and PGD 2 (redirection effect) in the presence of the unsaturated FAs in a system consisting of washed human platelet preparation and a piece of rat aorta from labelled arachidonate. TxB 2 was reduced. 6. Aggregation experiment confirming above.

Effects of some vasodilating (oxyfedrine, dipyridamole, verapamil and nifedipine) and antihypertensive (propranolol, hydralazine, prazosin and dihydralazine) drugs on the human blood platelet biosynthesis of prostaglandins from labelled arachidonic acid have been examined in vitro. Malondialdehyde (MDA) production in platelets and their aggregation in presence of these drugs were also studied. All the drugs inhibited the generation of thromboxane A2 which correlated well with the reduced platelet formation of MDA in presence of several of these drugs. In case of seven of the eight drugs, separation of HHT from HETE could be made. Values of these metabolites - especially those of HHT were found to be reduced, thus supporting the MDA and TxA2 data. With several of these drugs, ADP-induced platelet aggregation was inhibited either completely or partially. The results show that the well-known completely or partially. The results show that the well-known effects of these drugs might well be augmented by their effect on the platelet metabolism of arachidonic acid.

Effects of various concentrations (12.5-50 microM) of eicosapentaenoic acid (EPA) on the metabolism of exogenous arachidonic acid (AA) in washed human platelets were examined under two different incubation conditions. First platelets were pretreated with EPA before exposing them to labelled AA. In the second incubation platelets were exposed to a mixture of EPA (12.5 and 25 microM) and labelled AA. At all concentrations EPA reduced thromboxane B2 (TxB2) formation, and at 25 and 50 microM this fatty acid reduced also prostaglandins (PGE2, PGF2 alpha). A reduced formation of PGs and TxB2 was confirmed by a decreased formation of PG-endoperoxides. While in EPA treated platelets less CO-(TxB2, HHT, PGs) and lipoxygenase (HETE) products were formed at 25 and 50 microM, more HETE seemed to be generated at lower EPA concentration (12.5 microM). Oleic acid reduced TxB2 formation at high concentration (100 microM). EPA showed a dose-dependent inhibition of platelet aggregation induced by arachidonate, epinephrine and collagen; it was most effective against AA-induced aggregation.

Effects of ascorbic acid at physiologically achieved concentrations were examined on the metabolism of exogenous dihomo-gamma -linolenic acid and arachidonic acid (AA) in washed human platelets, and of AA in rat aortic rings. In the presence of ascorbic acid an increased formation of PGF1 alpha, PGE1 and PGE2 was observed in platelets. Also this vitamin induced an increased production of prostacyclin (measured as its stable metabolite 6-keto-PGF1 alpha) from exogenously provided substrate in aortic rings. In addition, from endogenous stores of AA in aortic rings ascorbic acid induced an increased generation of prostacyclin as revealed by inhibition of ADP-induced platelet aggregation.