Abstract: Cold press technique is a traditional method to obtain oil. The cold-pressing procedure involves neither heat nor chemical treatments; therefore, cold press technique has low oil yield. The cold pressed herbal material residue still contains some oil after cold press. In this study, the oil that is remained in the cold pressed aniseed was extracted with hexane; and it was analyzed to determine physicochemical properties and quality parameters. It was found that the aniseed after cold press process still contained 10% oil. The values of other analysis parameters were 2.1 mgKOH/g for free fatty acid (FFA) and 7.6 meq02/kg for peroxide. Cold pressed aniseed oil values were determined as 2.1 mgKOH/g for FFA and 4.5 meq02/kg for peroxide, respectively. In addition, fatty acid composition was analyzed, and it was found that both types of oil had same fatty acid compositions. The main fatty acids were oleic, linoleic and palmitic acids.
Abstract: Goat dairy products are gaining popularity worldwide. In developing countries, but also in many marginal regions of the Mediterranean area, goats represent a great part of the economy and ensure food security. In fact, these small ruminants are able to convert efficiently poor weedy plants and small trees into traditional products of high nutritional quality, showing great resilience to different climatic and environmental conditions. In developed countries, goat milk is appreciated for the presence of health-promoting compounds, bioactive compounds such as conjugated linoleic acids, oligosaccharides, sphingolipids and polyammines. This paper focuses on the recent advances in literature on the nutritional properties of goat milk and on innovative techniques to improve its quality as to become a promising functional food. The environmental sustainability of different methodologies of production has also been examined. Goat milk is valued today as a food of high nutritional value and functional properties as well as small environmental footprint. It is widely consumed in many countries due to high nutritional value, lower allergenic potential, and better digestibility when compared to bovine milk, that makes this product suitable for infants, elderly or sensitive patients. The main differences in chemical composition between a cow and goat milk rely on fat globules that in goat milk are smaller and in fatty acids that present a smaller chain length, while protein, fat, and lactose concentration are comparable. Milk nutritional properties have demonstrated to be strongly influenced by animal diet, genotype, and welfare, but also by season and production systems. Furthermore, there is a growing interest in the dairy industry in goat milk for its relatively high concentration of prebiotics and a good amount of probiotics, which have recently gained importance for their therapeutic potential. Therefore, goat milk is studied as a promising matrix to develop innovative functional foods. In addition to the economic and nutritional value, goat milk is considered a sustainable product for its small environmental footprint, as they require relatively little water and land, and less medical treatments, compared to cow, these characteristics make its production naturally vocated to organic farming. Organic goat milk production has becoming more and more interesting both for farmers and consumers as it can answer to several concerns like environment protection, animal welfare and economical sustainment of rural populations living in marginal lands. These evidences make goat milk an ancient food with novel properties and advantages to be valorized and exploited.
Abstract: 400 one-day-old male broiler chicks (Ross-308) randomly divided to 2 main groups, 1st main group (GA) was feeding basal diet with medium chain fatty acid (MCFA) at rate of 0.15% and divided to four subgroups, 3 subgroups vaccinated with different routes with Newcastle Disease Virus (NDV) and non-vaccinated group. The 2nd main group (GB) feeding basal diet without MCFA and divided the same as 1st main group. The parameters used in this study included: ND antibody titers at 1, 10, 21, 28, 35 and 42 days of age and values of CD4 and CD8 at 1, 20, 30 and 42 days of age. This experiment detected increase in ND antibodies titers in (G1, G2, G3) groups were fed on basal diet MCFA comparing to groups were fed without adding MCFA (G5, G6, G7) and control groups (G4, G8). The results of cellular immune response (CD4 and CD8) T-cells in broiler chicks indicated that there was obviously significant relationship between dietary Fatty Acid (FA) versus the diet without FA on the level of CD4 parameter, for the entire experimental period. The effect of different ages was statistically significant in creating different values of CD4 level, whereas the CD4 level decreases markedly with age. However, analyzing the data of different vaccination methods, oculonasal method of vaccination led to the highest value of CD4 compared with the oral, S/C and control groups. There were statistical differences in CD8 values due to supplementation of FA versus the basal diet and due to the effect of different age periods. As for the age effect, the CD8 value at 20 days of age was significantly higher than at 42 and 30 days.
Abstract: The swordfish (Xiphias gladius) is a large oceanic fish of high commercial value, which is widely distributed in waters of the world’s oceans. They are considered to be an important source of high quality proteins, vitamins and essential fatty acids, although only half of the population follows the recommendation of nutritionists to consume fish at least twice a week. Swordfish is consumed worldwide because of its low fat content and high protein content. It is generally sold as fresh, frozen, and as pieces or slices. The aim of this study was to evaluate the effect of salting and frying on the composition of the water-soluble vitamins (B2, B3, B9 and B12) and fat-soluble vitamins (A, D, and E) of swordfish. Three loins of swordfish from Pacific Ocean were analyzed. All the fishes had a weight between 50 and 70 kg and were transported to the laboratory frozen (-18 ºC). Before the processing, they were defrosted at 4 ºC. Each loin was sliced and salted in brine. After cleaning the slices, they were divided into portions (10×2 cm) and fried in olive oil. The identification and quantification of vitamins were carried out by high-performance liquid chromatography (HPLC), using methanol and 0.010% trifluoroacetic acid as mobile phases at a flow-rate of 0.7 mL min-1. The UV-Vis detector was used for the detection of the water- and fat-soluble vitamins (A and D), as well as the fluorescence detector for the detection of the vitamin E. During salting, water and fat-soluble vitamin contents remained constant, observing an evident decrease in the values of vitamin B2. The diffusion of salt into the interior of the pieces and the loss of constitution water that occur during this stage would be related to this significant decrease. In general, after frying water-soluble and fat-soluble vitamins showed a great thermolability with high percentages of retention with values among 50–100%. Vitamin B3 is the one that exhibited higher percentages of retention with values close to 100%. However, vitamin B9 presented the highest losses with a percentage of retention of less than 20%.
Abstract: Grape seeds are waste products of wineries and often referred to as an important agricultural and industrial waste product with the potential to be used in pharmaceutical, food, and cosmetic applications. In this study, grape seed oil from traditional Ionian varieties was examined for the determination of the quality and the characteristics of each variety. Initially, the fatty acid methyl ester (FAME) profiles were analyzed using Gas Chromatography-Mass Spectrometry, after transesterification. Furthermore, other quality parameters of the grape seed oils were determined by Spectroscopy techniques, UV-Vis and Raman included. Moreover, the antioxidant capacity of the oil was measured by 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assays and their antioxidant capacity expressed in Trolox equivalents. K and ΔΚ indices were measured in 232, 268, 270 nm, as an oil quality index. The results indicate that the air-dried grape seed total oil content ranged from 5.26 to 8.77% w/w, which is in accordance with the other grape seed varieties tested in similar studies. The composition of grape seed oil is predominated with linoleic and oleic fatty acids, with the linoleic fatty acid ranging from 53.68 to 69.95% and both the linoleic and oleic fatty acids totaling 78-82% of FAMEs, which is analogous to the fatty acid composition of safflower oil. The antioxidant assays ABTS and DPPH scored high, exhibiting that the oils have potential in the cosmetic and culinary businesses. Above that, our results demonstrate that Ionian grape seed oils have prospects that can go further than cosmetic or culinary use, into the pharmaceuticals industry. Finally, the reclamation of grape seeds from wineries waste stream is in accordance with the bio-economy strategic framework and contributes to environmental protection.
Abstract: Fish is a kind of food that contains many nutritions, one of those is the long chain of unsaturated fatty acids as omega-3 and omega-6 fatty acids and essential amino acid in enough amount for the necessity of our body. Like pelagic fish that found in the sea of Maluku. This research was done to identify fatty acids and amino acids composition in Moonfish (M. maculata) using transesterification reaction steps and Gas Chromatograph-Mass Spectrophotometer (GC-MS) and High-Performance Liquid Chromatography (HPLC). The result showed that fatty acids composition in Moonfish (M. maculata) contained tridecanoic acid (2.84%); palmitoleic acid (2.65%); palmitic acid (35.24%); oleic acid (6.2%); stearic acid (14.20%); and 5,8,11,14-eicosatetraenoic acid (1.29%) and 12 amino acids composition that consist of 7 essential amino acids, were leucine, isoleucine, valine, phenylalanine, methionine, lysine, and histidine, and also 5 non-essential amino acid, were tyrosine, glycine, alanine, glutamic acid, and arginine.Thus, these fishes can be used by the people to complete the necessity of essential fatty acid and amino acid.
Abstract: In this article reduced nutritional value of the rabbits’ meat at 1, 1 dimethylhydrazine experimental toxicosis is shown. The assay was performed on liquid chromatograph SHIMADZU LC-20 Prominence (Japan) with fluorometric and spectrophotometric detector. This research has revealed that samples of rabbit meat of the experimental group had significant differences from the control group:in amino acids concentration from 1.2% to 9.1%; vitamin concentration from 11.2% to 60.5%, macro – minerals concentration from 17.4% to 78.1% and saturated fatty acids concentration from 17,1% to 34.5%, respectively. The decrease in the chemical composition of rabbits’ meat at 1,1 dimethylhydrazine toxicosis may be due to changes in the internal processes associated with impaired metabolic homeostasis of animals.
Abstract: The authors discuss the collecting abilities of desorbable species (DS) of saturated fatty acids. The DS species of the reagent are understood as species capable of moving from the surface of the mineral particle to the bubble at the moment of the rupture of the interlayer of liquid separating these objects of interaction. DS species of carboxylic acids (molecules and ionic-molecular complexes) have the ability to spread over the surface of the bubble. The rate of their spreading at pH 7 and 10 over the water surface is determined. The collectibility criterion of saturated fatty acids is proposed. The values of forces exerted by the spreading DS species of reagents on liquid in the interlayer and the liquid flow rate from the interlayer are determined.
Abstract: A study was conducted to evaluate the effects of diets containing different levels of lysine and canola oil on growth performance and fatty acid composition of meat of broilers chicks. 240-day old Ross broiler chicks were used in a 3×2 factorial arrangement with canola oil (1, 3, and 5%) and lysine (recommended, and 25% more than recommended by Ross broiler manual) in completely randomized design with four replicates and 10 birds per each. The experimental diets were iso-caloric and iso-nitrogenous. Feed intake and body weight gain were recorded at the end of starter (10 d), grower (24 d) and finisher (42 d) periods, and feed conversion ratio was calculated. The results showed that the weight gain of chickens fed diets containing 5% canola oil were greater than those of birds fed on other diets (P
Abstract: Microalgae are tiny photosynthetic plants. Nowadays, microalgae are being used as nutrient-dense foods and sources of fine chemicals. They have significant amounts of lipid, carotenoids, vitamins, protein, minerals, chlorophyll, and pigments. Oil extraction from algae is a hotly debated topic currently because introducing an efficient method could decrease the process cost. This can determine the sustainability of algae-based foods. Scientific research works show that solvent extraction using chloroform/methanol (2:1) mixture is one of the efficient methods for oil extraction from algal cells, but both methanol and chloroform are toxic solvents, and therefore, the extracted oil will not be suitable for food application. In this paper, the effect of two food grade solvents (hexane and hexane/ isopropanol) on oil extraction yield from microalgae Dunaliella sp. was investigated and the results were compared with chloroform/methanol (2:1) extraction yield. It was observed that the oil extraction yield using hexane, hexane/isopropanol (3:2) and chloroform/methanol (2:1) mixture were 5.4, 13.93, and 17.5 (% w/w, dry basis), respectively. The fatty acid profile derived from GC illustrated that the palmitic (36.62%), oleic (18.62%), and stearic acids (19.08%) form the main portion of fatty acid composition of microalgae Dunalliela sp. oil. It was concluded that, the addition of isopropanol as polar solvent could increase the extraction yield significantly. Isopropanol solves cell wall phospholipids and enhances the release of intercellular lipids, which improves accessing of hexane to fatty acids.
Abstract: In Brazil, most soils are acidic and low in essential nutrients required for the growth and development of plants, making fertilizers essential for agriculture. As the biggest producer of soy in the world and a major producer of coffee, sugar cane and citrus fruits, Brazil is a large consumer of phosphate. Brazilian’s phosphate ores are predominantly from igneous rocks showing a complex mineralogy, associated with carbonites and oxides, typically iron, silicon and barium. The adopted industrial concentration circuit for this type of ore is a mix between magnetic separation (both low and high field) to remove the magnetic fraction and a froth flotation circuit composed by a reverse flotation of apatite (barite’s flotation) followed by direct flotation circuit (rougher, cleaner and scavenger circuit). Since the 70’s fatty acids obtained from vegetable oils are widely used as lower-cost collectors in apatite froth flotation. This is a very effective approach to the apatite family of minerals, being that this type of collector is both selective and efficient (high recovery). This paper presents Jatropha curcas L. oil (JCO) as a renewable and sustainable source of fatty acids with high selectivity in froth flotation of apatite. JCO is considerably rich in fatty acids such as linoleic, oleic and palmitic acid. The experimental campaign involved 216 tests using a modified Hallimond tube and two different minerals (apatite and quartz). In order to be used as a collector, the oil was saponified. The results found were compared with the synthetic collector, Fotigam 5806 produced by Clariant, which is composed mainly by soy oil. JCO showed the highest selectivity for apatite flotation with cold saponification at pH 8 and concentration of 2.5 mg/L. In this case, the mineral recovery was around 95%.
Abstract: The number of natural or organic product consumers is increased in recent years and this healthy demand pushes to increase usage of healthy meat. At the same time, consumers pay more attention on the healthy fat, especially on unsaturated fatty acids. These long chain carbohydrates reduce heart diseases, improve memory and eye sight and activate the immune system. One of the important issues to be solved for our Mongolia’s food security is to provide healthy, fresh, widely available and cheap meat for the population. Thus, an importance of the Selenge breed meat production is increasing in order to supply the quality meat food security since the Selenge breed cattle are rapidly multiplied, beneficial in term of income, the same quality as Mongolian breed, and well digested for human body. We researched the lipid, unsaturated and saturated fatty acid contents of meat of Selenge breed younger cattle by their muscle types. Result of our research reveals that 11 saturated fatty acids are detected. For the content of palmitic acid among saturated fatty acids, 23.61% was in the sirloin meat, 24.01% was in the round and chuck meat, and 24.83% was in the short loin meat.
Abstract: A field study was conducted to evaluate the efficacy of the sunflower (Helianthus annuus L.) for phytoremediation of contaminated soils. The experiment was performed on an agricultural field contaminated by the Non-Ferrous-Metal Works near Plovdiv, Bulgaria. Field experiments with a randomized, complete block design with five treatments (control, compost amendments added at 20 and 40 t/daa, and vemicompost amendments added at 20 and 40 t/daa) were carried out. The accumulation of heavy metals in the sunflower plant and the quality of the sunflower oil (heavy metals and fatty acid composition) were determined. The tested organic amendments significantly influenced the uptake of Pb, Zn and Cd by the sunflower plant. The incorporation of 40 t/decare of compost and 20 t/decare of vermicompost to the soil led to an increase in the ability of the sunflower to take up and accumulate Cd, Pb and Zn. Sunflower can be subjected to the accumulators of Pb, Zn and Cd and can be successfully used for phytoremediation of contaminated soils with heavy metals. The 40 t/daa compost treatment led to a decrease in heavy metal content in sunflower oil to below the regulated limits. Oil content and fatty acids composition were affected by compost and vermicompost amendment treatments. Adding compost and vermicompost increased the oil content in the seeds. Adding organic amendments increased the content of stearic, palmitoleic and oleic acids, and reduced the content of palmitic and gadoleic acids in sunflower oil. The possibility of further industrial processing of seeds to oil and use of the obtained oil will make sunflowers economically interesting crops for farmers of phytoremediation technology.
Abstract: The identification of lipid and soluble sugar components in flour samples of different cultivars belonging to common oat species (Avena sativa L.) was performed: spring oat, winter oat and hulless oat. Fatty acids were extracted from flour samples with n-hexane, and derivatized into volatile methyl esters, using TMSH (trimethylsulfonium hydroxide in methanol). Soluble sugars were then extracted from defatted and dried samples of oat flour with 96% ethanol, and further derivatized into corresponding TMS-oximes, using hydroxylamine hydrochloride solution and BSTFA (N,O-bis-(trimethylsilyl)-trifluoroacetamide). The hexane and ethanol extracts of each oat cultivar were analyzed using GC-MS system. Lipid and simple sugar compositions are very similar in all samples of investigated cultivars. Chemometric tool was applied to numeric values of automatically integrated surface areas of detected lipid and simple sugar components in their corresponding derivatized forms. Hierarchical cluster analysis shows a very high similarity between the investigated flour samples of oat cultivars, according to the fatty acid content (0.9955). Moderate similarity was observed according to the content of soluble sugars (0.50). These preliminary results support the idea of establishing methods for oat flour authentication, and provide the means for distinguishing oat flour samples, regardless of the variety, from flour samples made of other cereal species, just by lipid and simple sugar profile analysis.
Abstract: In this study, total fatty acid composition of muscle
lipids of Cyprinus carpio L. living in Suğla Dam Lake, Altinapa Dam
Lake, Eğirdir Lake and Burdur Lake were determined using GC.
During this study, for the summer season of July was taken from each
region of the land and they were stored in deep-freeze set to -20
degrees until the analysis date. At the end of the analyses, 30
different fatty acids were found in the composition of Cyprinus
carpio L. which lives in different lakes. Cyprinus carpio Suğla Dam
Lake of polyunsaturated fatty acids (PUFAs), were higher than other
lakes. Cyprinus carpio L. was the highest in the major SFA palmitic
acid. Polyunsaturated fatty acids (PUFA) of carp, the most abundant
fish species in all lakes, were found to be higher than those of
saturated fatty acids (SFA) in all lakes. Palmitic acid was the major
SFA in all lakes. Oleic acid was identified as the major MUFA.
Docosahexaenoic acid (DHA) was the most abundant in all lakes. ω3
fatty acid composition was higher than the percentage of the
percentage ω6 fatty acids in all lake. ω3/ω6 rates of Cyprinus carpio
L. Suğla Dam Lake, Altinapa Dam Lake, Eğirdir Lake and Burdur
Lake, 2.12, 1.19, 2.15, 2.87, and 2.82, respectively. Docosahexaenoic
acid (DHA) was the major PUFA in Eğirdir and Burdur lakes,
whereas linoleic acid (LA) was the major PUFA in Altinapa and
Suğla Dam Lakes. It was shown that the fatty acid composition in the
muscle of carp was significantly influenced by different lakes.
Abstract: The main cause of Alzheimer disease (AD) was
believed to be mainly due to the accumulation of free radicals owing
to oxidative stress (OS) in brain tissue. The mechanism of the
neurotoxicity of Aluminum chloride (AlCl3) induced AD in
hippocampus Albino wister rat brain tissue, the curative & the
protective effects of Lipidium sativum group (LS) water extract were
assessed after 8 weeks by attenuated total reflection spectroscopy
ATR-IR and histologically by light microscope. ATR-IR results
revealed that the membrane phospholipid undergo free radical
attacks, mediated by AlCl3, primary affects the polyunsaturated fatty
acids indicated by the increased of the olefinic -C=CH sub-band area
around 3012 cm-1 from the curve fitting analysis. The narrowing in
the half band width (HBW) of the sνCH2 sub-band around 2852 cm-1
due to Al intoxication indicates the presence of trans form fatty acids
rather than gauch rotomer. The degradation of hydrocarbon chain to
shorter chain length, increasing in membrane fluidity, disorder, and
decreasing in lipid polarity in AlCl3 group indicated by the detected
changes in certain calculated area ratios compared to the control.
Administration of LS was greatly improved these parameters
compared to the AlCl3 group. Al influences the Aβ aggregation and
plaque formation, which in turn interferes to and disrupts the
membrane structure. The results also showed a marked increase in
the β-parallel and antiparallel structure, that characterize the Aβ
formation in Al-induced AD hippocampal brain tissue, indicated by
the detected increase in both amide I sub-bands around 1674, 1692
cm-1. This drastic increase in Aβ formation was greatly reduced in the
curative and protective groups compared to the AlCl3 group and
approached nearly the control values. These results supported too by
the light microscope. AlCl3 group showed significant marked
degenerative changes in hippocampal neurons. Most cells appeared
small, shrieked and deformed. Interestingly, the administration of LS
in curative and protective groups markedly decreases the amount of
degenerated cells compared to the non-treated group. In addition, the
intensity of congo red stained cells was decreased. Hippocampal
neurons looked more/or less similar to those of control. This study showed a promising therapeutic effect of Lipidium
sativum group (LS) on AD rat model that seriously overcome the
signs of oxidative stress on membrane lipid and restore the protein
misfolding.
Abstract: Grains, including oats (Avena sativa L.), have been
recognized functional foods, because provide beneficial effect on the
health of the consumer and decrease the risk of various diseases. Oats
are good source of soluble fibre, essential amino acids, unsaturated
fatty acids, vitamins and minerals. Oat breeders have developed oat
varieties and improved yielding ability potential of oat varieties.
Therefore, the aim of investigation was to analyze the composition of
perspective oat varieties and breeding lines grains grown in different
conditions and evaluate functional properties. In the studied samples
content of protein, starch, β-glucans, total dietetic fibre, composition
of amino acids and vitamin E were determined. The results of
analysis showed that protein content depending of varieties ranged
9.70% to 17.30% total dietary fibre 13.66 g100g-1 to 30.17 g100g-1,
content of β-glucans 2.7 g100g-1 to 3.5 g100g-1, amount of
vitamin E (α-tocopherol) determined from 4 mgkg-1 to 9.9 mgkg-1.
The sums of essential amino acids in oat grain samples were
determined from 31.63 gkg-1 to 54.90 gkg-1. It is concluded that
amino acids composition of husked and naked oats grown in organic
or conventional conditions is close to optimal for human health.
Abstract: Several parameters are established in order to measure
biodiesel quality. One of them is the iodine value, which is an
important parameter that measures the total unsaturation within a
mixture of fatty acids. Limitation of unsaturated fatty acids is
necessary since warming of higher quantity of these ones ends in
either formation of deposits inside the motor or damage of lubricant.
Determination of iodine value by official procedure tends to be very
laborious, with high costs and toxicity of the reagents, this study uses
artificial neural network (ANN) in order to predict the iodine value
property as an alternative to these problems. The methodology of
development of networks used 13 esters of fatty acids in the input
with convergence algorithms of back propagation of back
propagation type were optimized in order to get an architecture of
prediction of iodine value. This study allowed us to demonstrate the
neural networks’ ability to learn the correlation between biodiesel
quality properties, in this caseiodine value, and the molecular
structures that make it up. The model developed in the study reached
a correlation coefficient (R) of 0.99 for both network validation and
network simulation, with Levenberg-Maquardt algorithm.
Abstract: Egg yolk oil is a natural source of bioactive
compounds such as unsaturated fatty acids, oil soluble vitamins,
pigments and others. Bioactive compound content in egg yolk oil
depends from its content in eggs, from which oil was extracted. Many
studies show that bioactive compound content in egg is correlated to
the content of these compounds in hen feed, but there is also an
opinion that hen housing systems also have influence on egg
chemical content. The aim of this study was to determine which
factor, laying hen housing system or hen diet, has a primary influence
on bioactive compound content in egg yolk oil. The egg yolk oil was
extracted from eggs obtained from 4 different hen housing systems:
cage, barn and two groups of free range. All hens were fed with
commercially produced compound feed except one group of free
range hens which get free diet – pastured hens. Extracted egg yolk
oils were analyzed for fatty acids, oil soluble vitamins and β-carotene
content. α-tocopherol, ergocalcipherol and polyunsaturated fatty acid
content in egg yolk oil was higher from eggs obtained from all
housing systems where hens were fed with commercial compound
feed. β-carotene and retinol content in egg yolk oils from free range
free diet eggs was significantly (p>0.05) higher that from other eggs
because hens have access to green forage. Hen physical activity in
free range housing systems decreases content of some bioactive
compound in egg yolk oil.
Abstract: Lipases constitute one of the most important groups of
industrial enzymes that catalyze the hydrolysis of triacylglycerol to
glycerol and fatty acids. Muscarinic antagonist relieves smooth
muscle spasm of the gastrointestinal tract and effect on the
cardiovascular system. In this research the effect of a muscarinic
antagonist on the lipase activity of Pseudomonas aeruginosa was
studied. Lineweaver–Burk plot showed that the drug inhibited the
enzyme by competitive inhibition. The IC50 value (0.16 mM) and Ki
(0.03 mM) of the drug revealed the drug bound to enzyme with high
affinity. Determination of enzyme activity in various pH and
temperature showed that the maximum activity of lipase was at pH 8
and 60oC both in presence and absence of the drug.