Antioxidant and Antimicrobial Activities of Phenolic Components and Organic Acids from Camellia Oleifera Cake

State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, Jiangxi Province, 330047, China; hujielun@ncu.edu.cn (J.H.); family581624@163.com (D.Z.); spnie@ncu.edu.cn (S.N.) * Correspondence: myxie@ncu.edu.cn; Tel.: +86-791-83969009 # These authors contribute equally to this work. Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 7 June 2018 doi:10.20944/preprints201806.0122.v1


Introduction
Currently, more and more natural products have been found to have many benefits for the human body [1,2] and there is a great interest in finding antioxidants from natural sources to prevent oxidative stress and minimize oxidative damage to living cells [3].Oxidative damage is involved in the pathogenesis of various diseases such as atherosclerosis, cancer, diabetes mellitus and reperfusion disorder [4].
Antioxidants have been widely studied because of their applications in the food industry as food preservatives and in medication as treatments against diseases that are caused by oxidative stress [5].Accordingly, considerable attention has been paid to the evaluation of the antioxidant activity of naturally occurring substances in recent years [6,7].In addition, there also has been considerable interest in natural products from plants with antimicrobial activities for controlling toxin-producing microorganisms because the use of antibiotics is no longer desirable due to concerns about bacterial resistance [8].Increased awareness of the potential problems associated with antibiotics stimulates research efforts to carry out novel approaches to the development of new antimicrobial compounds.
Polyphenols, the secondary metabolites that have an important role in reducing oxidative stress, are reported to exhibit antioxidant activities [9,10] and act as reducing agents, hydrogen donors, singlet oxygen quenchers and metal chelating agents [11].Extracts of herbs and plant materials rich in phenolics are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food.There are many examples [12].Phenolic compounds are also reported to have antibacterial properties [13,14].In addition, organic acids are widely distributed in fruits, vegetables, herbs and plants.The content of organic acids in food could influence their nutrition, antioxidant and antimicrobial properties [15].
The seeds of Camellia oleifera contain numerous valuable compounds and nutritious oils, which are frequently used in oriental countries, especially in Southeast Asia.After the oil refining process, defatted seeds are always used as a detergent or organic fertilizer with low economic value.The remainder of seeds after oil extract is called the C. oleifera cake.There are several million tons of C. oleifera cakes that are discarded every year in China [8].However, the C. oleifera cake contains large amounts of active compounds.Hence, the extraction of phenolic compounds and organic acids from C. oleifera cake will be valuable.
To the authors' knowledge, there are limited published reports on phenolic components of C. oleifera cake and their activities.Similarly, few researches have been done to understand the organic acid compositions of C. oleifera cake.Due to the lack of such information, the phenolic components and organic acids of C. oleifera cake along with their antioxidant and antimicrobial potentials were investigated.

Materials and reagents
The seeds of C. oleifera were collected in Yichun County and the cakes were

Preparation of phenolic components and organic acids
The powders of C. oleifera cake (500 g) were defatted with petroleum ether in a round-bottom flask for 3 h.After that, the organic solvent was volatilized using a rotary evaporator (RE-52A, Yarong Bio-instrument Co., Shanghai, China), and the defatted powders were obtained.

Extraction and purification of phenolic component
Extraction and purification procedure of phenolic components was performed as follow: the defatted powders were extracted with 500 mL L -1 methanol [matters to solvent ratio (g mL -1 ) = 1:15] in a 50 °C water bath for 2 h.The mixtures were filtered using Whatman NO.1 filter paper.The filtrates were then concentrated by a rotary evaporator at 55 °C.The concentrated filtrates were passed through AB-8 macro-reticular resin column (80 x 20 mm, Sigma-Aldrich, Shanghai, China) and eluted with 850, 700, 500, and 300 mL L -1 ethanol, successively according to the method of Singleton & Rossi (1965) [16] .The 300 mL L -1 ethanol eluate, which was found to contain the primary phenolic components, was considered as the phenolic component-rich fraction.This fraction was then evaporated to dryness by a rotary evaporator at 55 °C.A minimum volume of distilled water was added to this fraction and the resulting solution was freeze-dried in a Freezone 12Plus lyophilizer (Labconco, USA).The procedure above was repeated several times to obtain sufficient sample.

Extraction and purification of organic acids
The defatted powders were extracted with deionized water [matters to solvent ratio (g mL -1 ) = 1:10] in a 70 °C water bath for 2 h.After that, the mixtures were filtered using Whatman NO.1 filter paper.The filtrates were evaporated by a rotary evaporator at 55 °C.Then, the concentrated filtrates were passed through HPDl00 macro-reticular resin column (80 x 20 mm, Sigma-Aldrich, Shanghai, China) and eluted with distilled water and 950 mL L -1 ethanol in H2O, successively.The mL L -1 ethanol eluate was evaporated to dryness by a rotary evaporator at 55 °C.A minimum volume of distilled water was added to this dried eluate, and then the resulting solution was freeze-dried in the Freezone 12Plus lyophilizer.The procedure above was repeated several times to obtain sufficient sample.
The obtained phenolic components and organic acids were then re-dissolved in distilled water to measure their total phenolic content, total organic acids content, Total phenolic content was analyzed using the Folin-Ciocalteu reagent according to the method described by Singleton & Rossi (1965) [16] with some modifications.0.2 mL of the sample (1 g L -1 dissolved in distilled water) were added to a conical flask and mixed with 1 mL of Folin-Ciocalteu reagent and 46 mL of distilled water.The mixture was allowed to react for 3 min, and 3 mL of aqueous solution of Na2CO3 (75 g L -1 ) was added.Then the mixture was shaken vigorously.
At the end of incubation for 2 h with intermittent shaking at room temperature, the absorbance of each mixture was measured at 760 nm on a TU-1900 spectrophotometer (PGeneral).The same procedure was also applied to the standard solutions of gallic acid.Total phenolic content was expressed as micrograms of gallic acid equivalents per kilogram of dry sample.The total phenolic content determination was repeated three times.

Identification of phenolic compounds
The phenolic compounds were analyzed using an Agilent 1200 HPLC-MS system containing of a surveyor auto-sampling system, interfaced to a 6430 ion-trap mass spectrometer via an electrospray ion source.HPLC was carried out on the liquid Nitrogen (>99.99%) and He (>99.99%) were used as sheath and damping gas, respectively.

Determination of organic acids
Analysis was performed on a Waters liquid chromatograph system (UK6 injector and 515 HPLC pump, Waters, Milford, MA, USA), equipped with a Waters C18 column (4.6 mm x 250 mm, 5 μm, Waters, Milford, MA, USA), a guard column (10 x 4 mm, Waters, Milford, MA, USA), and a Waters 2996 PDA detector connected to a Waters software.A constant elution system consisted of phosphate buffer solution (20 mmol L -1 KH2PO4, pH = 2.7) (A) and methanol (B).The separation was achieved using the following program: 0-30 min, 5% B. The flow rate was 0.5 mL min -1 and the system was operated at room temperature when the sample solution (10 μL) was injected in each run.The PDA detector was set at a wavelength of 214 nm.The hydroxyl radical scavenging ability was measured using a modified method of Halliwell et al [17].A reaction mixture was prepared by adding 0.1 mL EDTA (1 mM), 0.01 mL of FeCl3 (10 mM), 0.1 mL of H2O2 (10 mM), 0.36 mL of deoxyribose (10 mM), 1.0 ml of samples (0.1-10 g L -1 ) dissolved in distilled water, 0.33 mL of phosphate buffer (50 mM, pH 7.4) and 0.1 mL of ascorbic acid (1 mM) in sequence.
The mixture was then incubated at 37 °C for 1 h.1.0 mL of the incubated mixture was mixed with 1.0 mL of 100 mL L -1 trichloroacetic acid and 1.0 mL of 5 mL L -1 thiobarbituric acid (in 25 mM NaOH containing 250 μL L -1 butylated hydroxyl anisole) to develop the pink chromogen measured at 532 nm.The hydroxyl radical-scavenging activity of the samples was reported as the percentage of inhibition of deoxyribose degradation and was calculated according to the following equation: where A0 was the absorbance of the control (blank, without samples) and At was the absorbance in the presence of the samples.All of the tests were carried out in triplicate and IC50 values were expressed as means ± standard deviation (SD).
Ascorbic acid was used as a positive control.

Superoxide radical-scavenging activity
This activity was measured using nitro blue tetrazolium (NBT) reagent as described by Sabu & Kuttan [18].The method is based on generation of superoxide radical O2¯ by auto-oxidation of hydroxylamine hydrochloride in the presence of NBT, which gets reduced to nitrite.Nitrite in the presence of EDTA gives a color that was measured at 560 nm.Test solutions of sample (0.1-10 g L -1 ) were taken in a test tube.To this, reaction mixture consisting of 1 mL of (50 mM) sodium carbonate, 0.4 mL of (24 mM) NBT and 0.2 mL of 0.1 mM EDTA solutions were added to the test tube and immediate reading was taken at 560 nm.0.4 mL of (1 mM) of where A0 was the absorbance of the control (blank, without samples) and At was the absorbance in the presence of the samples.All the tests were performed in triplicate and IC50 values were obtained.Ascorbic acid was used as a positive control.and left to stand for 30 min in the dark.The absorbance of the supernatant obtained after centrifugation was then measured at 517 nm.
The DPPH radical scavenging ability was calculated using the following equation: where Ac is the absorbance of DPPH solution without sample (2 mL DPPH + 3 mL of 950 mL L -1 ethanol); Ai is the absorbance of the test sample mixed with DPPH solution (1 mL sample + 2 mL DPPH + 2 mL of 950 mL L -1 ethanol) and Aj is the absorbance of the sample without DPPH solution (1 mL sample + 4 mL of 950 mL L -1 ethanol).All the tests were performed in triplicate and IC50 values were obtained.
Ascorbic acid was used as a positive control.

Inhibition of lipid peroxidation
This test was conducted using the method of Zhang et al [20] with some modifications.Briefly, an equal volume of egg yolk was added to 0.1 mol L -1 phosphate buffered saline (PBS, pH 7.45).The mixture was stirred magnetically for 10 min and then diluted with 24 volumes of PBS.The yolk homogenate (1 mL), samples (0.5 mL, 0.1-10 g L -1 ), PBS (1 mL) and 25 mmol L -1 FeSO4 (1 mL) were mixed in a tube and shaken at 37 °C for 15 min.The reaction was stopped by the addition of trichloroacetic acid and the mixture was centrifuged.Then 1 mL of 8 g L -1 thiobarbituric acid solution was added to 3 mL of the supernatant.This solution was heated at 10 °C for 10 min, after which its absorbance at 532 nm was measured.
The ability to inhibit lipid peroxidation was calculated as follows: where B0 is the absorbance of the control and B is the absorbance in the presence of samples.All the tests were performed in triplicate and IC50 values were obtained.
Ascorbic acid was used as a positive control.After that, a loop of each microorganism was inoculated into cool liquid medium.
They were then stored under aerobic conditions.The three bacteria were cultivated 24 h at 37 o C, while the three fungi were cultivated 48 h at 30 o C which was further adjusted to obtain a suitable turbidity for further use [21].After cultivation, average counts of bacteria Staphylococcus aureus, Escherichia coli and Bacillus subtilis were 3.5 x 10 6 , 8.6 x 10 5 , 5.4 x 10 6 colony forming units (cfu) mL -1 , and for fungi Mucor racemosus, Aspergillus oryzae, and Rhizopus stolonifer were 1.5 x 10 6 , 7.4 x 10 6 , and 2.9 x 10 6 (cfu mL -1 ), respectively.Cfu mL -1 density of cultures was confirmed after distribution into assay plates by the following procedure.All bacterial and fungi stock cultures were serially diluted 10-fold in a series of tubes, and plated by inoculating 100 µL onto medium plates which were incubated, and then colonies were counted to calculate density of the bacterial and fungi stock cultures.

Determination of inhibitory effect
The inhibition zone method was firstly employed to determine the antimicrobial activity of the samples [22].Twenty milliliters of medium were poured into each Petri dish.After the medium had solidified, 100 µL of suspension of the test microorganisms were smeared onto it.The wells were made, and the samples (100 or 200 mg L -1 ) were added to these wells (20 µL) and the same volume (20 µL) of gentamicin (1 g L -1 ) was used as a positive control.Deionized water was used as negative control.The inoculated plates were incubated and the diameter (mm) of the inhibition zone expressed as an average of the maximum diameter in three different directions was measured after the bacteria were cultivated 24 h at 37 o C and the fungi were cultivated 48 h at 30 o C.

Determination of minimum inhibitory concentration
Minimum inhibitory concentration (MIC) was determined by liquid medium dilution method [23].Two-fold serial dilutions of the samples with appropriate antibiotic control were prepared in liquid nutrient medium for bacteria and potato mediums for fungi [24].For liquid medium dilution test, 100 μL of stock cultures of bacteria or fungi were added to each tube (containing two-fold serial dilutions of sample at a final concentration of 7.8-4000 mg L -1 for bacteria and fungi) and incubated either at 37 o C for bacteria or at 30 o C for fungi for 24 h or 48 h.After the cultivation period, the turbidity was taken as the indication of growth and the lowest concentration which remained clear after microscopic evaluation was thus taken as the MIC, which was recorded as the mean concentration of triplicates.

Statistical analysis
All the experiments were done in triplicate.Statistical analysis was carried out using SPSS (version 16.0, Chicago, United States).The results were expressed as mean ± standard deviations and compared using the Tukey test at 5% confidence level.

Phenolic components and organic acids
Phenolics are aromatic secondary plant metabolites, and are widely spread throughout the plant kingdom [25].The purity of phenolic components obtained from C. oleifera cake is 94.1 ± 0.5% w/w. Figure 1 showed the HPLC chromatogram of the phenolic components.Under the LC-MS, the MS spectra of phenolic components from C. oleifera cake were acquired in the positive ion mode and Table 1 presented their most possible identification.Among the phenolics, there are 15 phenolic components have been detected (Figure 1, Table 1   Organic acids are important for their contribution to sensory attributes and nutrition value in fruits and plants [26].The purity of organic acids obtained from C. oleifera cake is 96.0 ± 0.3% w/w.Oxalic, citric, acetic, malic and succinic acids are found to be major organic acids in the C. oleifera cake (Figure 2).

Antioxidant activities
The importance of antioxidant constituents from plant materials is raising interest among scientists, food manufacturers, and consumers, as the trend of the future is moving towards functional food with specific beneficial effects [27].The phenolic compounds have been reported to have multiple biological effects, including antioxidant activity [28] reported that phenolic compounds in plants possess antioxidant activity, and may help protect cells against the oxidative damage caused by free radicals.The function of organic acids, in the reactive oxygen species scavenging, has also been demonstrated by other authors [29].In this study, antioxidant activity of phenolic compounds and organic acids were evaluated using four different assays.The IC50 values of different samples were summarized in Table 2.
Table 2. Half-inhibition (IC50) values of antioxidant activities of phenolic components and organic acids measured using hydroxyl radical-scavenging, superoxide radical-scavenging, DPPH radical scavenging and inhibition of lipid peroxidation assays.Superoxide anion plays an important role in the formation of reactive oxygen species such as hydrogen peroxide, hydroxyl radical, and singlet oxygen, which induces oxidative damage in lipids, protein, and DNA [31].In this study, the IC50 value for superoxide radical scavenging ability of phenolic components (304 ± 5 mg L -1 , Table 2) was higher than organic acids (671 ± 3 mg L -1 ) and ascorbic acid (IC50 = 304 ± 5 mg L -1 , p < 0.05).
The model of scavenging the stable DPPH radical is a widely used method of evaluating the free radical scavenging ability of natural compounds.The effect of antioxidants on DPPH radical scavenging was thought to be due to their hydrogen-donating ability [32].The DPPH scavenging activity of the phenolic components expressed in the term of IC50 was 103 ± 8 mg L -1 (Table 2), with the higher DPPH scavenging power than that of organic acids (184 ± 5 mg L -1 ) and ascorbic acid (205 ± 3 mg L -1 ) (p < 0.05).
There are 15 phenolic components have been detected in C. oleifera cake (Figure1, Table 1).It could be seen from the results above that these phenolic compounds in C. oleifera cake showed good antioxidant capacity for hydroxyl radical scavenging, superoxide radical scavenging, DPPH scavenging and lipid peroxidation inhibition.It was also reported that phenolic compounds have good radical-scavenging ability [34] and are also potent metal chelators and lipid peroxidation inhibitors [35], which was consistent with our results.In addition, different phenolic compounds may have various antioxidant capacities [36].
determined the inhibition of lipid oxidation of the phenolic compounds and found that catechin, quercetin and ellagic acid could have different antioxidant capacities, in a linoleic acid system.Meanwhile, the free radical-scavenging activity of them was also different.Thus, the phenolic compounds that we found in C. oleifera cake may also have different antioxidant capacities.
Other molecules, such as organic acids, are also present in a great amount in plants, fruits or vegetables.Previous studies attributed a direct action on free radicals scavenging and other antioxidant capacity to some organic acids [37].Besides, an effective antioxidant action of organic acids is well-known as their chelating action, which could inactivate reducing cations.In this study, oxalic, citric, acetic, malic and succinic acids are found to be major organic acids in the C. oleifera cake (Figure 2).
It was reported that citric acid and other hydroxycarboxylic acids such as tartaric, malic and isocitric acids also have antioxidant activity, for example, hydroxycarboxylic acids act as metal scavengers, and stimulate the decomposition of hydroperoxides by pathways that do not result in the formation of free radicals [38].These results was consistent ours.In addition, some other works confirmed that organic acids possess the biological activity both in reducing reactive oxygen species and in enhancing phenolics bio-availability [39].Thus, it could be supposed that the organic acids in C. oleifera cake might also enhance the antioxidant activity of the phenolic compounds in C. oleifera cake.

Antimicrobial activities
Plants can possess antimicrobial natural products to protect themselves from microbial infection and deterioration [40].There are growing interests in using natural antimicrobial compounds, especially extracted from plants, for the preservation of foods.In addition, there are more consumers who tend to question the safety of synthetic additives and would prefer natural foodstuffs [41].
Antimicrobial activities of some spices in plant leaves, flowers, stems, roots, or fruits have been reported by scientists.The phenolic compounds and organic acids are very important antimicrobial compounds in plants [42].
Salmonella spp. is one of the major foodborne pathogens, and the widespread increase in the number of cases of salmonella infection in recent years has major social and economic consequences.Escherichia coli strains are also associated with adverse gastrointestinal incidents [43].The inhibition zone diameters of the phenolic components and organic acids from C. oleifera cake are shown in Figure 3, and the MICs are shown in Table 3.As shown in Figure 3, there is significant difference in the diameter of inhibition zone for phenolic components and organic acids (100 or 200 mg L -1 ) compared with deionized water (negative control, p < 0.05).It was indicated from Figure 3 that phenolic components and organic acids (100 or 200 mg L -1 ) could inhibit these three bacteria, and their inhibiting capacity was sometimes better than gentamicin (positive control, 1 g L -1 ).Inhibition zones of the phenolic components and organic acids for each assay on test bacteria showed an opposite relationship with MIC (Table 3).A lower MIC indicates a stronger antimicrobial activity.As shown in Table 3, MIC was at 125, 250 and 63 mg L -1 for phenolic components against bacteria Escherichia coli, Staphylococcus aureus and Bacillus subtilis, while it was 250, 63 and 125 mg L -1 for organic acids against the same bacteria.When against fungi, Rhizopus stolonifer was affected by the phenolic components and organic acids as the MICs were 500 and 1000 mg L -1 .In addition, phenolic components and organic acids could inhibit Aspergillus oryzae with MIC level of 1000 and 500 mg L -1 .Furthermore, MIC was 2000 and 1000 mg L -1 for phenolic components and organic acids, respectively against fungi Mucor racemosus.These results suggested that the phenolic components and organic acids had lower inhibitory activity against fugi than bacteria tested.
It is important to find food additives and preservatives for preventing food deterioration and extending the shelf life of perishable food ingredients.Many phenolic compounds are known to possess antimicrobial properties.In this work, there are 15 phenolic components found in C. oleifera cake (Figure 1, Table 1).It was also reported that ellagitannins could be one of the main phenolic components causing the inhibition against Salmonella [35], which was also one of our detected phenolic components in C. oleifera cake.In addition, different bacterial species exhibit different sensitivities towards phenolics, and the variations may come from the cell surface structures between Gram-negative and Gram-positive bacteria.In particular, the outer membrane of Gram-negative bacteria could be a preventive barrier against hydrophobic compounds [44].However, sometimes phenolics also showed activity against Gram-negative bacteria [35], which was consistent with our results.Helander et al. (1998) found some phenolic compounds could inhibit E. coli and Salmonella, and the inhibitory mechanism involved the disruptive action of these compounds on the outer membrane [44].The antimicrobial activity of phenolics may also be due to the glycosides in phenolics [45].Thus, the antimicrobial activities of the phenolic compounds from C. oleifera cake may also result from their glycosides.
Specific organic acids have also been advised to control microbial contamination and dissemination of foodborne pathogens in food production and processing [46].In this study, oxalic, citric, acetic, malic and succinic acids are found to be major organic acids in the C. oleifera cake (Figure 2).The antibacterial mechanisms for organic acids may depend on physiological status of the organism or the physicochemical characteristics of the external environment [47].Other toxicity mechanisms have also been proposed that organic acids may possess membrane uncoupling capacity [48].Furthermore, undissociated acids are considered to be the active form, because they can freely diffuse through the cell membrane and enter the cytoplasm [49].However, the toxic mechanisms of organic acids (either directly or indirectly) for foodborne pathogens have not been fully elucidated.This is in part due to complexities involved in organic acids, and factors such as chain length, side chain composition, and hydrophobicity could affect the antimicrobial activity of organic acids [50].This could be investigated in future study.
chromatograph system, equipped with a quaternary pump, a column temperature controller, and a photodiode array (PDA) detector.The analytical column temperature was kept at 35 °C.The sample was separated with a RP-C18 column (4.6 mm x 250 Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 7 June 2018 doi:10.20944/preprints201806.0122.v1mm, 5 μm) using 10 mL L -1 acetic acid in water (eluent A) and 5 mL L -1 acetic acid in water and acetonitrile (50:50, v/v; eluent B) under gradient conditions [from 10 to 25% B (15 min), 25 to 35% B (15 min), 35 to 40% B (5 min), 40 to 50% B (10 min), 50 to 100% B (25min), 100 to 10% B (10 min)].The mobile phase was at a flow rate of 0.3 mL min -1 within 80 min and the detector was employed to set at a wavelength of 280 nm.The injection volume was 20 μL.Peaks were assigned by their retention time.Source settings used for the ionization of phenolic compounds were: nebulizer gas pressure of 45.00 psi; dry gas flow rate of 11.00 L min -1 ; electrospray voltage of the ion source of 3000 V; capillary temperature of 350 °C; fragmentor of 120.0 V.
hydroxylamine hydrochloride was added to initiate the reaction.The reaction mixture was then incubated at 25 °C for 15 min and the reduction of NBT was measured at 560 nm.Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity.All the samples were treated in the similar manner, absorbance was recorded and the percentage of inhibition was calculated according to the following equation: % inhibition = (A0−At)/ A0 x 100(2)

2. 6 .
Antimicrobial activity assayThe activities of different samples were tested against bacteria Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and fungi Mucor racemosus, Aspergillus oryzae, Rhizopus stolonifera.The bacteria and fungi were grown and maintained on nutrient agar medium slants and potato medium slants respectively when they were obtained from the Institute of Microbiology Chinese Academy of Sciences.Before inoculation, the liquid nutrient medium for bacteria and potato mediums for fungi were autoclaved at 121 o C in vertical heating pressure stream sterilizer (LDZX-50KB, Shenan Medical Instrument Works Co., Shanghai, China) for 20 min.

Figure 1 .
Figure 1.The HPLC chromatogram of the phenolic components.

Figure 2 .
Figure 2. Liquid chromatograms of standards and samples.(a)standard organic acids; value is expressed as mg L -1 and represents the concentration of sample that is required for 50% inhibition of hydroxyl radical, superoxide radical, DPPH radical, and lipid peroxidation.A lower IC50 value indicates a higher antioxidant activity.Each value in the table was obtained by calculating the average of three determinations ± standard deviation.b Mean values in the same column with different letters are significantly different (Tukey test, p < 0.05).Preprints (www.preprints.org)| NOT PEER-REVIEWED | Posted: 7 June 2018 doi:10.20944/preprints201806.0122.v1cAscorbic acid was used as a positive control.

Figure 3 .
Figure 3. Diameter of zone of inhibition.

Table 1 .
Identification of phenolic compounds from C. oleifera cake using their HPLC-DAD, LC-MS and LC-MS n data.aPeak numbers refer to HPLC chromatograms in Figure1.

Table 3 .
MICs a of antimicrobial activity for phenolic components and organic acids from C. oleifera cake against bacteria and fungi.The lowest concentration which the liquid medium remained clear after microscopic evaluation was taken as the minimum inhibitory concentration (MIC).It was recorded as the mean