Corresponding author: Ani Dadayan (
Biologically active substances, the antioxidant and antimicrobial activity of aqueous, 70% ethylate and ethyl acetate extracts of fruits of maclura orange (Osage Orange), a representative of the
Based on the investigations performed, extracts of maclura orange can be recommended as a source of biologically active substances (
An ethanol extract of maclura fruits exhibits the antimicrobial activity against
Despite advances in synthetic pharmaceuticals, the interest in herbal medicine continues throughout the world. A fairly high adherence to treatment with plant components in different parts of the world is largely due to social and cultural peculiarities. For the population of low-income countries, herbal medicine is attractive for its relatively low cost, wider availability compared to drugs used by the official medicine (
Moreover, in the developing countries the number of certified doctors is small, while the number of practitioners of traditional methods of treatment is disproportionately higher (
Their pharmacological properties have not been completely determined and the antimicrobial potential and biochemical compositions of many plant species have not been analyzed yet (
In developing countries, traditional herbs have crucial significance for disease treatment and they aid to detect novel antibiotics. Maclura pomifera (Raf.) Osage orange, is a tree of the
It has been used as an insect repellent for many years. Pioneer researchers placed the ripe fruit of this tree in cupboards to repel roaches and other insects (Sand 1991, Brandies 1979). Past research indicates that there may be scientific justification for this well-popularized use. Karr and Coats (1991) found fragments of the fruit, as well as its hexane and methanol extracts, to be significantly repellent to the German cockroach Blattella germanica. The wood of the osage orange tree resisted termites and had antifungal properties. Antimicrobial activity has been found in extracts containing osajin or pomiferin, two of the major angular isoflavonoid components present in osage orange fruits.
The aim of the work is to study biologically active substances, antioxidant and antimicrobial activity of aqueous, 70% ethanol and ethyl acetate extracts of fruits of maclura orange, a representative of the
In the presented work we used aqueous, aqueous-ethanol, and ethyl acetate extracts of maclura orange. To obtain extracts, the dried raw material was ground in a ceramic mortar to a powdery state (particle size ≤1 мм), passed through a sieve with a hole diameter of 1 mm and 50 ml of extractant was poured into 1 g (precise weight) of the raw material, and extracted with reflux for 30 min at 50–70 °C. Then the contents of the flask were filtered through a paper filter, cooled and the volume of the extract was adjusted with extractant to 50 ml.
Determination of the amount of
In the investigated samples, the quantitative composition of vitamins P, C, B
The HPLC analysis was carried out at the Laboratory of Biologically Active Compounds Purification and Certification. It was performed with Aqueouss HPLC Alliance 2695 Separation Module HPLC. The analysis process was carried out with a ZORBAX Eclipse C18, 4.6 × 250 mm, 5 µm particle size column, volume injection of 10 µl and a flow rate of 1.0 ml/min. All analyses were performed with a mobile phase of Methanol: H2O:acetic acid of 50:48:2 ratios and detection at 254 nm.
Quantification of ascorbic acid content was performed on an Aqueous Alliance 2695 Separation Module HPLC system. Chromatographic separation was achieved on a ZORBAX Eclipse C18, 4.6 × 250 mm column through isocratic delivery of a mobile phase (Methanol:H2O (0.1% TFA) of 5:95 ratios) at a flow rate of 0.5 mL/min. UV absorbance was recorded at 254 nm.
HPLC analysis was performed with the Aqueous Alliance 2695 Separation Module HPLC system including a UV detector. For B12 quantification, the Phenomenax C18 column was used (10 μm, 3.9 × 300 mm) with an acetonitrile-aqueous (70:30) solvent separations with a flow rate of 0.8 ml per min. UV absorbance was recorded at 214 nm and 230 nm.
To determine the antioxidant activity of maclura orange, we studied the extracts (samples 1–3), taking into account the data obtained (Fig.
Results of chromatography of organic acids in aqueous extract of Osage Orange.
Name | Retention time | Area | % Area | Height | Conc. mg/mL | |
---|---|---|---|---|---|---|
1 | 3,601 | 128642 | 0.18 | 21803 | ||
2 | Oxalic acid | 4,117 | 540825 | 0.74 | 34876 | 0.134 |
3 | 5,353 | 819280 | 1.12 | 64471 | ||
4 | Malic acid | 5,861 | 221155 | 0.30 | 15699 | 0.63 |
5 | 6,353 | 7178674 | 9.81 | 506320 | ||
6 | Succinic acid | 11,996 | 3003186 | 4.10 | 99935 | 12.93 |
7 | 13,763 | 61311921 | 8376 | 1822144 |
Results of chromatography of organic acids in aqueous extract of Osage Orange.
For this purpose, 8 ml of freshly distilled boiled water of room temperature, 1 ml of 20% sulfuric acid solution, 1 ml of 0.05
For the control experiment about 0.05 g (precise weight) of quercetin (FS 42-1290-79) was dissolved in 40 ml of ethyl alcohol, transferred to a 100 ml measuring flask, adjusted with alcohol to the mark and stirred. 8 ml of freshly boiled and cooled distilled aqueous, 1 ml of 20% sulfuric acid solution, 1 ml of 0.05
The calculation of the antioxidant activity index (
The total amount of
The screening of antimicrobial activity of extracts was carried out using the micromethod of diffusion in agar. It was designed at the Department of Microbiology, Virology and Immunology of Ivano-Frankivsk National Medical University (Kutsyk 2004).
To estimate the activity of extracts, test cultures of the following strains were used:
Test cultures were provided by the Microbial Depository Center of the SPC “Armbiotechnology”. 1 ml of the medium contained 107 bacteria. The cultivation was carried out in a bacteriological box on nutrient agar. Bacterial suspensions were prepared in sterile water and then added to a liquid nutrient medium. Agar was transferred into Petri dishes until solidification, then small disks (0.3 ml) of filter paper soaked with extract were placed on nutrient agar using sterile needles. After drying at room temperature for 30–40 min, the dishes were transferred to a thermostat for 24–48 h at 25–27 °C. At the end of incubation, the zones of inhibition of bacterial growth were measured.
The actual material was processed using the method of variation statistics with the calculation of the average arithmetic and its standard error, the reliability of the comparable values was estimated according to Student, Wilkinson, Mann Whitney criteria; the probability level was determined as p ≤ 0.05 using the Image Tool 2.0 (UTHSCSA ImageTool 2.0, The University of Texas Health Science Center in San Antonio, 1995–1996) and MS Excel (State Pharmacopeia 2015).
The qualitative and quantitative contents of biologically active substances, antioxidant and antimicrobial activity of aqueous, 70% ethanol and ethyl acetate extracts of fruits of maclura orange, a representative of the
Results of chromatography of organic acids in aqueous extract of Osage Orange.
Name | Retention time | Area | % Area | Height | Conc. mg/mL | |
---|---|---|---|---|---|---|
1 | 3,587 | 115940 | 0.19 | 16097 | ||
2 | Oxalic acid | 4,143 | 1727685 | 2.81 | 142915 | 0.23 |
3 | 5,032 | 680874 | 1.11 | 71449 | ||
4 | Malic acid | 5,976 | 6194818 | 10.08 | 578497 | 17.56 |
5 | 6,358 | 2885196 | 4.69 | 133464 | ||
6 | 12,932 | 49877120 | 81.13 | 950770 |
Results of chromatography of organic acids in ethanol extract of Osage Orange.
Results of chromatography of organic acids in ethyl acetate extract of Osage Orange.
Results of quantification and identification of flavonoids in aqueous extract of Osage Orange.
At the first stage, a quantitative analysis of
Figs
Figs
Results of quantification and identification of flavonoids in ethanol extract of Osage Orange.
Results of quantification and identification of flavonoids in ethyl acetate extract of Osage Orange.
Figs
Quantitative content of vitamin C in aqueous extract of Osage Orange.
Quantitative content of vitamin C in ethanol extract of Osage Orange.
Quantitative content of vitamin C in ethyl acetate extract of Osage Orange.
Taking into account the rich composition of the extracts, the next stage was study of the antioxidant activity. 0.05% Solution of quercetin was used as a control. In accordance with the results obtained during titration, in the case of quercetin (1.4 ml), the consumption of maclura extracts was respectively 1.95, 0.55 and 0.48 ml, which indicates that extract of maclura orange can be considered as an efficient preventive agent against the antioxidant aging of the body.
The amount of antioxidant substances was determined using the following formula:
In the case of aqueous extract:
In the case of 70% ethanol extract:
In the case of ethyl acetate extract:
Studies have shown that ethanol and ethyl acetate extracts are richer in the above mentioned biologically active substances and have more pronounced antioxidant activity.
The presence of high antioxidant activity in extracts can be explained by the fact that the investigated extracts contained multifunctional
Results of chromatography of organic acids in ethyl acetate extract of Osage Orange.
Name | Retention time | Area | % Area | Height | Conc. mg/mL | |
---|---|---|---|---|---|---|
1 | Oxalic acid | 4,152 | 4157399 | 75.01 | 379271 | 1.029 |
2 | 6,345 | 60470 | 1.09 | 5413 | ||
3 | 7,185 | 83573 | 1.51 | 8100 | ||
4 | 7,529 | 719133 | 12.98 | 42597 | ||
5 | 7,750 | 80286 | 1.45 | 9223 | ||
6 | 13,679 | 441279 | 7.96 | 11583 |
Results of quantification and identification of flavonoids in aqueous extract of Osage Orange.
Name | Retention time | Area | % Area | Height | Conc. mg/mL | |
---|---|---|---|---|---|---|
1 | Tannic acid | 3,005 | 23515994 | 76.14 | 2609491 | 3,016 |
2 | 3,338 | 5060321 | 16.38 | 459709 | ||
3 | 3,672 | 356487 | 1.15 | 33379 | ||
4 | 5,529 | 1954134 | 6.33 | 193737 |
Results of quantification and identification of flavonoids in ethanol extract of Osage Orange.
Name | Retention time | Area | % Area | Height | Conc. mg/mL | |
---|---|---|---|---|---|---|
1 | 2,976 | 22533959 | 46.97 | 2573678 | ||
2 | 3,311 | 5766580 | 12.02 | 530493 | ||
3 | 4,862 | 435700 | 0.91 | 32132 | ||
4 | 5,434 | 2673574 | 5.57 | 223008 | ||
5 | Quercetin | 10,875 | 16569058 | 34.53 | 841168 | 0.885 |
Results of quantification and identification of flavonoids in ethyl acetate extract of Osage Orange.
Name | Retention time | Area | % Area | Height | Conc. mg/mL | |
---|---|---|---|---|---|---|
1 | 2,957 | 145294 | 0.66 | 18258 | ||
2 | 3,203 | 82294 | 0.37 | 7876 | ||
3 | 3,355 | 78481 | 0.36 | 9281 | ||
4 | 3,591 | 12516 | 0.06 | 2007 | ||
5 | 4,084 | 90515 | 0.41 | 7968 | ||
6 | 4,769 | 411845 | 1.87 | 25032 | ||
7 | 9,620 | 7982247 | 36.18 | 344978 | ||
8 | 10,480 | 6897562 | 31.27 | 347672 | ||
9 | Quercetin | 10,865 | 6360149 | 28.83 | 331168 | 0.340 |
Quantitative content of vitamin C in aqueous extract of Osage Orange.
Name | Retention time | Area | % Area | Height | Conc. mg/mL | |
---|---|---|---|---|---|---|
1 | 4,263 | 342839 | 0.76 | 50870 | ||
2 | 4,462 | 908973 | 2.01 | 58952 | ||
3 | 4,788 | 239527 | 0.53 | 31166 | ||
4 | Vitamin C | 4,997 | 10991905 | 24.30 | 1579617 | 0.27 |
5 | 5,276 | 1173897 | 2.59 | 162786 | ||
6 | 5,474 | 5774006 | 12.76 | 657596 | ||
7 | 5,711 | 303754 | 0.67 | 53656 | ||
8 | 5,966 | 206448 | 0.46 | 26028 | ||
9 | 6,130 | 194335 | 0.43 | 31603 | ||
10 | 6,262 | 609509 | 1.35 | 78206 | ||
11 | 7,512 | 22012404 | 48.66 | 2172692 | ||
12 | 8,741 | 2187446 | 4.84 | 213267 | ||
13 | 10,645 | 296205 | 0.65 | 27386 |
The results of HPLC and quantitative analysis of substances with regenerative properties are presented in Table
As can be seen from the data presented in the Table
At the next stage, we studied the antimicrobial activity. The extracts were evaluated using clinical strains of microorganisms characterized by varying degrees of resistance to modern antimicrobial drugs. The research results are presented in Fig.
Evaluation of the antimicrobial activity of Osage Orange extracts on strains of
I. Aqueous
II. Ethyl acetate
III. Ethanol
As can be seen from the figures, only 70% ethanol extract of maclura had the antimicrobial effect on the strains of
Quantitative content of vitamin C in ethanol extract of Osage Orange.
Name | Retention time | Area | % Area | Height | Conc. mg/mL | |
---|---|---|---|---|---|---|
1 | 4,155 | 343799 | 0.81 | 55852 | ||
2 | 4,276 | 198768 | 0.47 | 33680 | ||
3 | 4,525 | 442036 | 1.04 | 36387 | ||
4 | 4,644 | 223404 | 0.53 | 39602 | ||
5 | 4,830 | 367434 | 0.87 | 57371 | ||
6 | Vitamin C | 4,950 | 9797336 | 23.07 | 1282931 | 0.24 |
7 | 5,240 | 5336482 | 12.56 | 762387 | ||
8 | 5,308 | 4911080 | 11.56 | 904638 | ||
9 | 6,091 | 54602 | 0.13 | 13663 | ||
10 | 7,597 | 20796881 | 48.97 | 1076773 |
Quantitative content of vitamin C in ethyl acetate extract of Osage Orange.
Name | Retention time | Area | % Area | Height | Conc. mg/mL | |
---|---|---|---|---|---|---|
1 | 4,155 | 806177 | 53.92 | 140581 | ||
2 | 5,514 | 139621 | 9.34 | 21144 | ||
3 | 6,182 | 293835 | 19.65 | 24296 | ||
4 | 7,503 | 152459 | 10.20 | 13890 | ||
5 | 8,653 | 103081 | 6.89 | 7275 |
Results of HPLC and quantitative analysis of substances with regenerative properties.
|
Extracts | ||
---|---|---|---|
Aqueous | Ethanol | Ethyl acetate | |
Oxalic acid (mg/ml) | 0.134 | 0.23 | 1,029 |
Malic acid (mg/ml) | 0.63 | 17.56 | 0 |
Succinic acid (mg/ml) | 12.93 | 0 | 0 |
Tannic acid (mg/ml) | 3,016 | 0 | 0 |
Quercetin (mg/ml) | 0 | 0.885 | 0.34 |
Vitamin C (mg/ml) | 0.27 | 0.24 | 0 |
Antioxidants (mg/g) | 35.89 | 127,27 | 145,83 |
The aqueous, ethanol, ethyl acetate extracts of maclura were obtained, and quantitative analysis of a number of biologically active substances was carried out by HPLC.
The study of the antioxidant activity showed that ethyl acetate extract was richer in substances with antioxidant activity.
Evaluation of the antimicrobial activity of the extracts showed that 70% ethanol extract exhibited the greatest antibacterial activity.