Quantification Of Chlorogenic Acid Research Articles

Phytochemical analysis of Silybum marianum flowers: Quantitative analysis of natural compounds and molecular docking application

Secondary metabolites in plants, identifying, quantifying, and determining the biological activity of plants enables the use of plants in different fields such as pharmacology, food, and cosmetics. Different chromatographic methods such as GC-MS/MS (volatile compounds, fatty acid) and LC-MS/MS (phenolic compounds) are used to identify and quantify these secondary metabolites. Silybum marianum is a member of the Asteraceae family and grows naturally. It is known among the public by names such as Thistle, Virgin Mary Thorn, and Milky Kengel. In this study, S. marianum hexane extract was analyzed by GC-MS/MS, and the methanol-chloroform (1:1 v/v) extract was analyzed by LC-MS/MS. Palmitic acid methyl ester (17.96%), linoleic acid methyl ester (14.20%), and sesquicineole (10.22%) were determined by GC-MS/MS. Moreover, LC-MS/MS analysis resulted in the quantification of chlorogenic acid (250.171 µg/g extract), salicylic acid (234.95 µg/g extract), isoquercitrin (210.65 µg/g extract), and rutin (102.05 µg/g extract). According to the analysis results, palmitic acid and chlorogenic acid were detected as the main components of fatty acid and phenolic compound respectively. Molecular docking was applied to determine their interaction with the urease enzyme. Palmitic acid and chlorogenic acid interaction with urease were calculated as a MolDock score of -104.63, and -113.21, with binding energies of -3.70, and -6.50 kcal/mol respectively. According to the results, chlorogenic acid may be a urease enzyme inhibitor.

Quantification of chlorogenic acid in Pluchea indica L. stem ethanolic extracts and its antioxidant activity

Abstract Chlorogenic acid (CA) is an important phenolic acid antioxidant. It is found in Pluchea indica L. (Asteraceae). However, it has only been extensively studied in the leaves, while studies on the stems have not been reported. This study aimed to identify and measure the levels of CA in the stem extract of P. indica. The extract was also determined for its antioxidant activities. In the course of the work, P. indica stems powder was extracted using the ultrasonic-assisted extraction (UAE) technique employing 50%-ethanol as solvent directly and sequentially. The extract was then measured for total phenolic content (TPC) and CA content using RP-HPLC. Meanwhile, antioxidant activities were determined by the DPPH, ABTS, and reducing power (RP) methods. TPC in the sequential and the direct of P. indica stems ethanol extracts were 1.4694±0.0228 and 1.9314±0.0318 mgGAE/g DW, respectively. We found that the CA content of 50%-ethanol extract of P. indica stems from sequential extraction (0.2045±0.0128%, w/w) was higher than 50%-ethanol extract from direct extraction (0.1984±0.0113%, w/w). The two extracts demonstrated good antioxidant capacity, while the ethyl acetate and n-hexane extracts did not. Identifying of other antioxidants phenolics using other extracting methods still needs further study.

Chemical profiling and biological activities of Opopanax hispidus extracts: A comparative insight on conventional and green extraction technologies

The plant species Opopanax hispidus is known in traditional Turkish medicine and nutrition. In addition to all the advantages that are known by the traditions of old generations, O. hispidus has been insufficiently examined, and its potential could be used in treatment in modern medicine and diet. In this paper, O. hispidus extracts obtained by conventional (maceration (MAC), and Soxhlet (SOX)) and contemporary (accelerated solvent extraction (ASE), homogenizer assisted extraction (HAE), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), supercritical fluid extraction (SFE)) methods were examined in terms of their chemical composition and biological potential. The extract obtained by HAE extraction had the highest content of total phenolics and flavonoids (36.98 mg GAE/g and 15.04 mg RE/g, respectively). The highest antioxidant activity was recorded for HAE and ASE extracts. O. hispidus extracts achieved inhibitory activity against acetylcholinesterase (4.12–6.44 mg GALAE/g), tyrosinase (44.78–76.72 mg KAE/g), α-amylase (0.35–0.46 mmol ACAE/g), and α-glucosidase (1.67–1.77 mmol ACAE/g) enzymes. LC-MS technique was used for the quantitative analysis of the extracts, allowing the quantification of chlorogenic acid, neochlorogenic acid, and p-coumaric acid, rutin, luteolin, and kaempferol 3-O-glucoside as the most abundant phenolic components. Results suggested that the green extraction techniques for obtaining extracts that are rich in bioactive compounds have advantages compared to conventional extraction. Therefore, these extracts can be used as antioxidants, neuroprotective, antityrosinase, and antidiabetic agents while respecting the principles of sustainable development and green chemistry.

Identification and Quantification of Chlorogenic Acids from the Root Bark of Acanthopanax gracilistylus by UHPLC-Q-Exactive Orbitrap Mass Spectrometry.

The purpose of this study is to identify and quantify the chlorogenic acids (CGAs) from the root bark of Acanthopanax gracilistylus, which is conventionally regarded as a tonic in folk Chinese Traditional medicine. The effective methods for identification and quantification analysis of CGAs were developed based on ultra high performance liquid chromatography-Q-exactive orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) in parallel reaction monitoring (PRM) and selected reaction monitoring (SIM), which showed high sensitivity and resolution for screening and quantifying compounds. The root bark of A. gracilistylus was extracted under ultrasonication with 70% methanol. Ultimately, a for total of 70 CGAs, 64 of these were tentatively identified for the first time. Moreover, a methodological study of seven kinds of CGAs was carried out. The proposed procedure was optimized and validated in terms of selectivity, linearity of analytical curves (r2 > 0.990), accuracy (recovery range from 96.7 to 105%), and repeatability (relative standard deviation <5%). Then it was applied to determine the content of the CGAs in A. gracilistylus roots from 66 of different batches. The total CGAs was quantified in a range between 2.150 and 33.51 mg/g, which could be considered as excellent source of natural bioactive compound. The result was extremely useful for understanding the bioactive substance and quality control of A. gracilistylus in depth.

Quantification of Chlorogenic Acid and Vanillin from Coffee Peel Extract and its Effect on α-Amylase Activity, Immunoregulation, Mitochondrial Oxidative Stress, and Tumor Suppressor Gene Expression Levels in H2O2-Induced Human Mesenchymal Stem Cells.

Background: Polyphenols and flavonoid-rich foods help in arresting reactive oxygen species development and protecting DNA from oxidative damage. Coffee peel (CP) preparations are consumed as beverages, and their total polyphenol or flavonoid content and their effect on oxidative stress–induced human mesenchymal stem cells (hMSCs) are poorly understood. Method: We prepared hot water extracts of CP (CPE) and quantified the amount of total polyphenol and flavonoid using HPLC analysis. In addition, CPE have been studied for their α-amylase inhibitory effect and beneficial effects in oxidative stress–induced hMSCs. Results: The obtained results show that the availability of chlorogenic acid, vanillin, and salicylic acid levels in CPE is more favorable for enhancing cell growth, nuclear integrity, and mitochondrial efficiency which is confirmed by propidium iodide staining and JC-1 staining. CPE treatment to hMSCs for 48 h reduced oxidative stress by decreasing mRNA expression levels of LPO and NOX-4 and in increasing antioxidant CYP1A, GSH, GSK-3β, and GPX mRNA expressions. Decreased pro-inflammatory (TNF-α, NF-κβ, IL-1β, TLR-4) and increased tumor suppressor genes (except Bcl-2) such as Cdkn2A, p53 expressions have been observed. Conclusions: The availability of CGA in CPs effectively reduced mitochondrial oxidative stress, reduced pro-inflammatory cytokines, and increased tumor suppressor genes.

Anticancer Effects of the Corchorus olitorius Aqueous Extract and Its Bioactive Compounds on Human Cancer Cell Lines.

Corchorus olitorius is a common, leafy vegetable locally known as “Saluyot” in the Philippines. Several studies have reported on its various pharmacological properties, such as antioxidant, anti-inflammatory, analgesic, and anticancer properties. However, little is known about its effects on angiogenesis. This study aimed to evaluate the anticancer properties, such as the antiproliferative, anti-angiogenic, and antitumor activities, of the C. olitorius aqueous extract (CO) and its bioactive compounds, chlorogenic acid (CGA) and isoquercetin (IQ), against human melanoma (A-375), gastric cancer (AGS), and pancreatic cancer (SUIT-2), using in vitro and in ovo biological assays. The detection and quantification of CGA and IQ in CO were achieved using LC-MS/MS analysis. The antiproliferative, anti-angiogenic, and antitumor activities of CO, CGA, and IQ against A-375, AGS, and SUIT-2 cancer cell lines were evaluated using MTT and CAM assays. CGA and IQ were confirmed to be present in CO. CO, CGA, and IQ significantly inhibited the proliferation of A-375, AGS, and SUIT-2 cancer cells in a dose-dependent manner after 48 h of treatment. Tumor angiogenesis (hemoglobin levels) of A-375 and AGS tumors was significantly inhibited by CO, CGA, IQ, and a CGA–IQ combination. The growth of implanted A-375 and AGS tumors was significantly reduced by CO, CGA, IQ, and a CGA–IQ combination, as measured in tumor weight. Our investigation provides new evidence to show that CO has promising anticancer effects on various types of human cancer cells. CO and its compounds are potential nutraceutical products that could be used for cancer treatment.

POTENCIAL FUNCIONAL Y NUTRACÉUTICO DE HOJAS DE CAFÉ INJERTADO (Coffea spp.) DURANTE UN CICLO ANUAL

Compuestos fenólicos como el ácido clorogénico, cafeína, azúcares, vitaminas, minerales y sustancias volátiles que son componentes principales del café (Coffea spp.) tienen funciones biológicas, calificadas como nutracéuticas e insecticidas. En al menos una etapa fenológica, las hojas de café tienen mayores posibilidades de utilizarse con propósitos comerciales. El objetivo de este estudio fue analizar el área foliar, contenido de clorofila a, b y total, cafeína, ácido clorogénico (5-CGA), azúcares y macro y micronutrimentos en hojas de café arabica (Coffea arabica) var. Caturra rojo injertado sobre café robusta (Coffea canephora) var. Romex durante un ciclo anual. Con los datos obtenidos se realizó un análisis de varianza (ANDEVA) y las medias fueron comparadas por la prueba de Tukey (p≤0.05) para un experimento factorial. El muestreo de 10 árboles fue completamente al azar, las muestras se corrieron por triplicado. Los valores mayores se presentaron como sigue: área foliar en la etapa de floración y fructificación; clorofila a, b y total durante la etapa vegetativa; cafeína en la etapa de floración; 5-CGA se obtuvo durante la etapa vegetativa, en la cual también se observaron las concentraciones mayores de glucosa y fructosa. La concentración de macronutrimentos, excepto Mg, fue mayor durante la etapa de fructificación, mientras que las de Fe y Zn fueron mayores durante la floración y Mn y B en fructificación. Los macro y micronutrimentos se acumularon durante la etapa de fructificación. Las hojas de café pueden ser funcionales y nutracéuticos durante las etapas vegetativa y fructificación.

Quantification of chlorogenic acid, rosmarinic acid, and caffeic acid contents in selected Thai medicinal plants using RP-HPLC-DAD

The chlorogenic acid, rosmarinic acid, and caffeic acid contents in 100 selected plants were determined using reversed phase high performance liquid chromatography equipped with diode array detector. The optimum condition was 0.2% phosphoric acid in water (solvent A) and methanol (solvent B) as the mobile phase, which was set at 45% B for 20 minutes at a flow rate of 1.2 mL/min. The column temperature was maintained at 30 oC and the detection wavelength was 325 nm. Among 100 selected plants, 39.64% contained all 3 compounds, 40.54% contained 2 compounds, 14.41% contained only 1 compound, and 5.41% could not detect any of the 3 compounds. The highest contents of chlorogenic acid, rosmarinic acid, and caffeic acid were found in Lonicera japonica flowering buds, Melissa officinalis leaves, and Coffea canephora seeds at the concentration of 9.900 ± 0.004, 19.908 ± 0.171, and 1.233 ± 0.003 g/100 g of dried plant, respectively.

Comprehensive identification and quantification of chlorogenic acids in sweet cherry by tandem mass spectrometry techniques

This paper deals with an extensive analytical approach, which uses two complementary tandem mass spectrometry techniques, to characterize the chlorogenic acids (CGAs) present in a typical Italian sweet cherry variety (cv Ferrovia). Sixteen monoacyl-quinic acids and esters, five diacyl-quinic acids, three caffeoyl-quinic acids glycosides, and two caffeic acid hexoses were detected by HPLC-MSn analyses (MSn up to MS4), among which four methyl coumaroyl quinate and three methyl caffeoyl quinate isomers were tentatively identified in sweet cherries for the first time. HPLC-MS/MS analyses through multiple reaction monitoring (MRM) experiments showed that trans-3-O-caffeoylquinic acid, cis-3-O-coumaroylquinic acid, trans-3-O-coumaroylquinic acid, trans-5-O-caffeoylquinic acid, and methyl-3-O-caffeoyl quinate were the main CGAs in the mature berries of cv Ferrovia. Considering that CGAs can have several health benefits depending on their amount but also on their structural features, the results of this study provide new insight into the knowledge of the quali-quantitative profile of these phytochemicals in a widespread fruit such as sweet cherry.

Microwave assisted extraction for the determination of chlorogenic acid in Flos Lonicerae by direct analysis in real time mass spectrometry (DART-MS)

Flos Lonicerae was an important Chinese medicine. In this research, a microwave assisted extraction method was applied for the extraction of chlorogenic acid from Flos Lonicerae. The operating conditions were optimized using a Box–Behnken design test. Under the optimal conditions, the extraction yield of chlorogenic acid reached 32.52 ± 1.31 mg/g. Next, a direct analysis in real time mass spectrometry (DART-MS) method was utilized to quantify of chlorogenic acid in Flos Lonicerae extracts. The primary parameters were optimized to obtain maximum signal intensity. In the detection process of the actual samples, the results obtained by DART–MS are consistent with those obtained by HPLC method with short detection time and acceptable repeatability and precision (<15%). In addition, the DART–MS/MS method has several advantages, such as speed, low cost and simplicity. Therefore, the DART-MS method is an efficient method that can be applied in the quantification of chlorogenic acid in Flos Lonicerae.

RAPID RESOLUTION LIQUID CHROMATOGRAPHY METHOD FOR DETERMINATION OF CHLOROGENIC ACID IN ECHINACEA EXTRACTS

This study presents a development and validation of a new, fast, efficient and cost effective reversed-phase rapid resolution liquid chromatography (RP-RRLC) method for determination of chlorogenic acid in echinacea extracts. The optimum separation with symmetrical peak shape and good index purity of the analyte was achieved on a Poroshell 120 EC-18 (50 mm x 3 mm; 2.7 µm) analytical column, mobile phase consisted of acetonitrile/(water with 1 % phosphoric acid), (10/90, V/V)in isocratic elution with flow rate of 1 mL/min and UV diode-array detection (UV-DAD) at 325 nm. The developed method was validated by testing specificity, selectivity, linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ). The calibration curve of chlorogenic acid followed Beer’s law within the range 28.97 ng - 362.19 ng (R2 = 0.9994). The LOD was 0.29 pg, while LOQ was 0.96pg. The intra-day precisions was evaluated for the retention time, peak area and peak height and the calculated values for relative standard deviations (RSD) were 0.21 %, 0.11 % and 0.22 %, respectively. The mean recoveries ranged from 98.75 to 104.63 % and RSD was less than 0.23 %. The developed method was successfully applied for identification and quantification of chlorogenic acid in three samples of echinacea extracts, taken from local pharmacies.

Chemical partitioning and antioxidant capacity of green coffee (Coffea arabica and Coffea canephora) of different geographical origin

Green coffee beans of Coffea arabica and Coffea canephora accessions from different geographical origin (Brazil, Colombia, Ethiopia, Honduras, Kenya, Mexico, Peru, Uganda and Vietnam) were extracted and the extracts analyzed by HPLC–ESI-MS/MS for the identification and quantification of chlorogenic acids and caffeine content. Principal component and cluster analyses were used to identify chemical patterns separating the different species and accessions based on their geographical origin. C. canephora showed always a higher caffeine content with respect to C. arabica, whereas the C. arabica accessions from Kenya showed a higher chlorogenic acids and a lower caffeine content. The antioxidant capacity of green coffee extracts was assayed by the reducing power and DPPH assays. The antioxidant capacity correlated with the chlorogenic acids content. The results show that the C. arabica from Kenya possesses the highest chlorogenic acids/caffeine ratio and, among the C. arabica accessions, the highest antioxidant capacity. Therefore, the C. arabica from Kenya is the most suitable green coffee source for nutraceutical applications because of its high antioxidant capacity and low caffeine content.

Determination of plasma concentration of five phenolic acid by LC-MS/MS and study of pharmacokinetics in rats after Mailuoning injection

To establish a LC-MS/MS method for quantification of chlorogenic acid, caffeic acid, 3,4-DCQA, ferulic acid and cinnamic acid in rats plasma and study its pharmacokinetics after administration of Mailuoning injection at a single dose to rats. Plasma samples were acidified with hydrochloric acid and extracted with ethyl acetate. The analytes were determined by LC-MS-MS using a ZOBAX SB C18 column with a mobile phase of methanol-water (containing 2 mmol x L(-1) ammonium acetic) (60:40)at a flow rate of 0.5 mL x min(-1) and detected using ESI with negative ionization mode. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 353.1/191.0 [M-H]- for chlorogenic acid, m/z 178.9/134.9 [M-H]- for caffeic acid, m/z 515.2/353.0 [M-H]-for 3,4-DCQA, m/z 193.0/133.9 [M-H]-for ferulic acid, m/z 146.9/102.9 [M-H]- for cinnamic acid and m/z 246.0/125.8 [M-H]- for tinidazole (IS). After administration of Mailuoning injection at a single dose to eight Sprague-Dawley rats, the concentrations of chlorogenic acid, caffeic acid, 3,4-DCQA, ferulic acid and cinnamic acid in plasma were determined by LC-MS/MS method. The main pharmacokinetics parameters of measured data were caluculated by using DASver 1.0 software. The linear concentration ranges of the calibration curves for chlorogenic acid, caffeic acid, 3,4-DCQA and cinnamic acid were 2.006-1,027 microg x L(-1) (r = 0.999 6), 1.953-1,000 microg x L(-1) (r = 0.999 7), 28.51-1.459 x 10(4) microg x L(-1) (r = 0.998 9), 1.836-940.0, g x L(-1) (r = 0.997 7) and 4.780-2,447 microg x L(-1) (r = 0.998 6) respectively. The inner and inter-days relative standard deviations were both less than 5.0%, indicating legitimate precise and accuracy to the requirement of biological sample analysis. For chlorogenic acid, the pharmacokinetic parameter t1/2, AUC0-t, and CL were (49.78 +/- 12.81) min, (123.55 +/- 14.82) mg x min x L(-1) and (0.004 3 +/- 0.000 5) L x min(-1), respectively. For caffeic acid, the pharmacokinetic parameter t1/2, AUC0-t, and CL were (36.65 +/- 10.59) min, (91.67 +/- 11.77) mg x min L(-1) and (0.005 7 +/- 0.000 7) L x min(-1), respectively. For 3,4-DCQA, the pharmacokinetic parameter t1/2, AUC0-t, and CL were (50.08 +/- 13.78) min, (278.34 +/- 31.82) mg x min x L-1 and (0.001 6 +/- 0.000 2) L x min(-1), respectively. For ferulic acid, the pharmacokinetic parameter t1/2, AUC0-t, and CL were (51.39 +/- 15.52) min, (34.72 +/- 4.67) mg x min x L(-1) and (0.000 4 +/- 0.0001) L x min(-1), respectively. For cinnamic acid, the pharmacokinetic parameter t1/2, AUCo-t, and CL were (74.42 +/- 18.32) min, (34.63 +/- 4.82) mg x min x L(-1) and (0.007 7 +/- 0.001 1) L x min-', respectively. The assay method is proved to be sensitive, accurate and convenient. It can be applied to the pharmacokinetic study of chlorogenic acid, caffeic acid, 3,4-DCQA, ferulic acid and cinnamic acid.

Identification and quantification of bioactive compounds in coffee brews by HPLC–DAD–MSn

Coffee is a beverage widely consumed worldwide because of its organoleptic characteristics and stimulating and beneficial health effects. This paper presents the identification and quantification of chlorogenic acids and derivatives, trigonelline, nicotinic acid, 5-hydroxymethylfurfural, theobromine, theophylline, and caffeine in brews of 10 roasted ground coffee and 4 soluble coffee determined by HPLC–DAD–MSn. Among the chlorogenic acids and derivatives, 17 chlorogenic acids, 5 chlorogenic acid lactones, 2 cinnamoyl-amino acid conjugates, and 2 free cinnamic acids were identified. This is the first study that reports the presence of caffeoylferuloylquinic acid isomers, and cinnamoyl-amino acid conjugates in soluble coffee brews. Regular roasted ground coffee brews showed higher contents of chlorogenic acids (4162mg/100g), chlorogenic acid lactones (779mg/100g), p-coumaric acid (2.4mg/100g), trigonelline (2044mg/100g), nicotinic acid (100.4mg/100g), caffeine (4565mg/100g), and theobromine (12.5mg/100g) than regular soluble coffee brews. Decaffeinated coffee brews presented higher levels of chlorogenic acids, chlorogenic acid lactones, caffeic acid, and trigonelline when compared to the regular ones. Despite the differences in the levels of these compounds, brews of roasted ground coffee and soluble coffee, both regular and decaffeinated, represent an important source of bioactive compounds for habitual coffee consumers.

UPLC and HPLC of caffeoyl esters in wild and cultivated Arctium lappa L.

Analytical methods including ultra-performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC) with photodiode array (PDA) detector were developed for the analysis of caffeoylquinic acid derivatives in seeds, leaves and roots of Arctium lappa L. Separation was performed on C18 column utilising 5% (v/v) acetic acid in water and acetonitrile at 330nm. Both methodologies were validated in terms of linearity, precision, and recovery. The results showed that the major advantages of UPLC, over HPLC were the fast analysis, narrow peaks, high sensitivity, and reduction of solvent consumption. Subsequently the methods were applied for the identification and quantification of chlorogenic acid (5-CQA) and 1,5-dicaffeoylquinic acid (1,5-DCQA) as main compounds in samples. The total phenolic content of samples ranged from 3.93 to 14.13g of 5-CQA equivalent/100g dry weight (DW). There was a significant variability from 89 to 571mg/100g for 5-CQA and 48 to 486mg/100g for 1,5-DCQA in dry material.

Quantification of Chlorogenic Acid and Hyperoside Directly from Crude Blueberry (Vaccinium angustifolium) Leaf Extract by NMR Spectroscopy Analysis: Single-Laboratory Validation

A single-laboratory-validated NMR spectroscopy method was established for determining the quantity of chlorogenic acid and hyperoside from crude extract material of blueberry leaves of the species Vaccinium angustifolium var. laevifolium House. The calibration curve of chlorogenic acid showed a highly linear regression, R = 0.99998. NMR spectroscopy identification and quantification of the constituents directly from the mixture, within the error of HPLC-diode array detector analysis, were determined as 7.53 mM chlorogenic acid (64.0 mg chlorogenic acid/g powdered leaf) and 0.77 mM hyperoside (8.58 mg hyperoside/g powdered leaf). The LOD was calculated to be 0.01 mM and the LOQ 0.01 mM by the 9 min and 13 s NMR spectroscopy experiment utilized. The assay showed no significant interference from different field strengths, extraction mesh size, gravimetric scale precision, NMR spectroscopy tube type, pulse program, amount of starting dry material, or day-to-day operation. The robustness of NMR spectroscopy as a means of definitively monitoring chlorogenic acid and hyperoside content directly from crude extracts was demonstrated by Youden statistical analysis.

Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts

Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.