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 Moraceae genus growing in the Ararat region of Armenia have been studied. It is found that plant extracts are rich in a wide range of pharmacologically active substances, in particular, tannins, flavonoids, organic acids, vitamins, anthocyanins, micro- and microelements, etc.

Based on the investigations performed, extracts of maclura orange can be recommended as a source of biologically active substances (BAS), as well as a preventive and corrective agent in deficiency or imbalance of macro- and microelements in tissue and cellular structures in various pathological processes in the body.

An ethanol extract of maclura fruits exhibits the antimicrobial activity against Bacillus subtilis 1820, E. Coli 5002, Serratia marcescens 5251 and Staphylococcus aureus ATCC-6538 strains. Maclura extracts can be used as an environmentally friendly source of antioxidants against early aging.

Biologically active substances, antioxidant, tannins, flavonoids, organic acids, vitamins, antimicrobial activity

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 (Heinrich et al. 2004).

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 (Jamison et al. 2006; Jenssen et al. 2006). Herbal medicine remains a popular method also in developed countries (Russian National Standard 1995; Russian National Standard 1999; Ates et al. 2003). The interest of the population in states with a high level of income is largely due to the influence of mass media promoting alternative methods of treatment as more natural, accordingly, safer (Russian National Standard 1994). The search for medicinal substances in nature is still relevant today (Heinrich et al. 2004; Jenssen et al. 2006).

Their pharmacological properties have not been completely determined and the antimicrobial potential and biochemical compositions of many plant species have not been analyzed yet (Heinrich et al. 2004). World Health Organization (WHO) has predicted increasing antimicrobial resistance as a major threat for the public health for the 21^st century. In order to prevent spreading of antibiotic resistant infections, scientists have been conducting intensive researches to determine new antimicrobial agents. One way to prevent antibiotic resistance of microorganisms is using new compounds that are not based on existing antimicrobial agents (Jamison et al. 2006; Jenssen et al. 2006). In the last three decades, antimicrobial activity-related experiments have been applied by using plant extracts (Ates et al. 2003).

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 Moraceae or mulberry family. There are antimicrobial researches on M. pomifera in the literature and results are promising.

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 Moraceae genus growing in the Ararat region of Armenia.

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 BAS (the total amount of extractives, flavonoids, tannins, carboxylic acids, vitamins) that had the highest physiological and therapeutic activity in the samples analyzed was carried out according to generally accepted procedures (Russian National Standard 1994, 1995, 1999).

In the investigated samples, the quantitative composition of vitamins P, C, B₁₂ and organic acids was also studied by the HPLC.

Determination of the antioxidant activity of maclura orange extracts

To determine the antioxidant activity of maclura orange, we studied the extracts (samples 1–3), taking into account the data obtained (Fig. 1, Table 1),

Table 1.

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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

Figure 1. 

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 N potassium permanganate solution were poured into a glass with a 50 ml volume. The whole was stirred and titrated with extracts of maclura orange (samples 1–3) from microburette (with 1 ml volume with 0.01 ml graduation) until the pink color disappeared.

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 N potassium permanganate solution were poured into a titration glass with a 50 ml volume, stirred and titrated with a quercetin solution from a microburette (with 1 ml volume with 0.01 ml graduation) until the pink color disappeared. 1 ml of 0.05 N potassium permanganate solution corresponds to 0.25 mg of quercetin.

The calculation of the antioxidant activity index (AOA), which corresponds to the BAS concentration of a reducing nature in terms of quercetin (in mg/g) was carried out according to the formula B = C_kxV_kxV_o/V_xxm, where B is the concentration of BAS of the reducing nature of the object under study, consumed for titration of 1 ml of 0.05 N potassium permanganate solution, mg/ml (0.5 mg/ml); V_k is the volume of quercetin solution consumed for titration of 1 ml of 0.05 N potassium permanganate solution, ml (1.4 ml); Vо is the volume of the test solution, ml (50 ml); Vx is the volume of the solution of the test object consumed for titration of 1 ml of 0.05 N potassium permanganate solution, ml (0.4 ml); m is the mass of the precise weight of the object under study, g (1 g).

The total amount of BAS of a reducing nature was determined in terms of quercetin in 1 g of the preparation.

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 Moraceae genus growing in the Ararat region of Armenia have been studied. The results of the study are presented in Figs 1–4 and Tables 1, 2.

Figure 2. 

Results of chromatography of organic acids in ethanol extract of Osage Orange.

Figure 3. 

Results of chromatography of organic acids in ethyl acetate extract of Osage Orange.

Figure 4. 

Results of quantification and identification of flavonoids in aqueous extract of Osage Orange.

At the first stage, a quantitative analysis of BAS by HPLC and titration methods was performed. As a result, it was established that the composition of extracts differed depending on the type of extractant. Quantitative indices of BAS are shown in Figs 1–9.

Figs 1–3 show the results of chromatography of organic acids in aqueous, ethanol and ethyl acetate extracts of Osage Orange.

Figs 4–6 show the results of quantification and identification of certain flavonoids in aqueous, ethanol and ethyl acetate extracts of Osage Orange.

Figure 5. 

Results of quantification and identification of flavonoids in ethanol extract of Osage Orange.

Figure 6. 

Results of quantification and identification of flavonoids in ethyl acetate extract of Osage Orange.

Figs 7–9 show the results of vitamin C determination in aqueous, ethanol and ethyl acetate extracts of Osage Orange.

Figure 7. 

Quantitative content of vitamin C in aqueous extract of Osage Orange.

Figure 8. 

Quantitative content of vitamin C in ethanol extract of Osage Orange.

Figure 9. 

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:

$B=\frac{0,5·1,4·100}{1,95·1}=35,89\mathrm{mg}/\mathrm{g}$

In the case of 70% ethanol extract:

$B=\frac{0,5·1,4·100}{0,55·1}=127,27\mathrm{mg}/\mathrm{g}$

In the case of ethyl acetate extract:

$B=\frac{0,5·1,4·100}{0,48·1}=145,83\mathrm{mg}/\mathrm{g}$

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 BAS together with easily oxidized functional groups (for example, -SH, (CH₃)₂CH-), that relatively faster bind free radicals formed in living organisms.

The results of HPLC and quantitative analysis of substances with regenerative properties are presented in Table 10.

As can be seen from the data presented in the Table 10, flavonoids, tannins, vitamins C, P, carboxylic acids and other biologically active substances were found in the extracts obtained from the fruits of maclura orange growing in Armenia (Yerevan, Nor Nork). Experimental studies show that aqueous and ethanol extracts of maclura are distinguished for high content of the above mentioned biologically active substances.

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. 10. We found that maclura orange contained compounds that could inhibit the growth of microorganisms Bacillus subtilis 1820, E. Coli 5002, Serratia marcescens 5251 and Staphylococcus aureus ATCC-6538. The presence of compounds with antimicrobial properties in extracts depended on the nature of the solvent.

Figure 10. 

Evaluation of the antimicrobial activity of Osage Orange extracts on strains of Bacillus subtilis 1820, E. Coli 5002, Serratia marcescens 5251 and Staphylococcus aureus ATCC-6538.

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 Bacillus subtilis 1820, E. coli 5002, Serratia marcescens 5251 and Staphylococcus aureus ATCC-6538 causing the growth inhibition zone of 1.1–1.3 cm. Ethyl acetate extract specifically inhibited only the Serratia marcescens 5251 strain, causing the growth inhibition zone of 1.1 cm. and the aqueous extract had no effect on the studied strains.

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.