Medicinal Mushrooms Cordyceps as a New Source of Bioactive Compounds and Their Complexation With Silver Ions

Three samples of strain MFTCCB022 Ophiocordyceps sinensis CS₄ were tested: the first sample was cultured on maize (1), the second was cultured on chickpea (2), and the third sample was cultured on millet (3). Mushroom samples were cultured and processed to a final powder form by drying at 40 °C in an APT Line dryer and ground in an SM-100 mill. They were stored at 8 °C.

Six extracts were prepared from Ophiocordyceps sinensis samples by refluxing (R) and maceration (M). Three sample extracts were obtained by reflux (1R, 2R, 3R), and three extracts were prepared by maceration (1M, 2M, 3M). We chose reflux and maceration in order to get as much as possible in comparison with the literature studied. In both cases of R/M preparation, 5.0 g of powdered mushroom material and 50 mL of a 1:1 ethanol:water mixture were used. This different cultivation was the cause of their different activities (antioxidant and antimicrobial activities). Publications state that millet is a very good choice of substrate for cultivation. It has no distinct taste or smell and allows the full expression of secondary metabolites. Ophiocordyceps does not grow as fast on millet as on other cereals, but the quality of the final product is higher [1]. The prepared extracts were already visually different. Extracts 2R and 3R were clear, while the rest were cloudy. The extracts also differed from each other in their consistency. Samples 1R and 2R obtained by reflux were solid, while the other samples were oily [2,3,4]. These changes were caused by the decreasing ability of the extracts to scavenge DPPH radicals based on their decreasing concentration. However, samples 3R and 3M did not show this trend, which implies that they did not demonstrate antioxidant activity.

The antioxidant activity of the samples was compared based on their IC₅₀ values. The extract of sample 2, prepared by both reflux and maceration, showed the highest antioxidant activity. The difference between the antioxidant activities of this sample depending on its preparation was minimal. A more pronounced difference depending on the method of obtaining the extract was observed for sample 1. It showed approximately 1.3 times higher antioxidant activity after processing by maceration with an IC₅₀ value of 7.20 mg.mL⁻¹ compared to its preparation by reflux. A similar procedure was used in a study [4] in which an ethanolic extract of Ageratum plants showed antioxidant activity, determined by the DPPH radical scavenging method, with ascorbic acid as the standard for comparison. By comparing the IC₅₀ values of the extracts after the addition of AgNO₃, we can state that the highest antioxidant activity with an IC₅₀ value of 4.99 mg.mL⁻¹ was shown by the 2M sample processed by maceration. On the other hand, this sample treated with reflux 2R showed the lowest antioxidant activity, while its IC₅₀ value was up to two times higher compared to sample 2M. In the case of sample 1, from the point of view of the method of its preparation, no significant difference in its activity was noted; however, the 3M sample had 1.3 times higher antioxidant activity compared to the 3R sample. From a comparison of the antioxidant activity of extracts without AgNO₃ and extracts with the addition of 10.1 mmol.L⁻¹ AgNO₃, it can be concluded that there is no significant change in the antioxidant activity of sample 1R processed by reflux, while when this sample was processed by maceration, there was approximately 1.3 times decrease in its antioxidant activity after complexation with silver ions. In sample 2R prepared by reflux, there was also a significant decrease in its antioxidant activity after the addition of AgNO₃, and its IC₅₀ value increased more than 1.6 times with the addition of silver nitrate. However, in the case of its processing by maceration, on the contrary, after complexation with silver ions, its antioxidant activity increases with a decrease in IC₅₀ from the value of 6.33 mg.mL⁻¹ to the value of 4.99 mg.mL⁻¹. Other methods for measuring antioxidant activity will be used in the near future.

The results showed that when testing the extract of sample 3 without the addition of AgNO₃, it did not show any antioxidant activity regardless of the method of its preparation; after the addition of AgNO₃, we can observe that by complexing with silver, antioxidant activity was detected in sample 3, which was more pronounced when the sample was processed by maceration compared to reflux. From the point of view of the preparation method of the extract samples, we can conclude that the extracts processed by maceration showed better antioxidant activity than the extracts prepared by reflux. The author of the study [5], points out the relationship between maceration and extraction yield from Chinese medicinal herbs. The work shows that the swelling of the tissue of herbs by maceration increases the extraction yield of the chemical marker of the subsequent extraction because water-soluble substances are dissolved in the water during maceration, which increases the availability of the marker, which is extracted with a mixture of ethanol and water [5].

When determining the antimicrobial activity by the disc diffusion method, the effects of Ophiocordyceps sinensis extracts 1–3 alone before the addition of AgNO₃, as well as the extracts with the addition of AgNO₃, were evaluated. Gentamycin (G) at a concentration of 30 mg.mL⁻¹ was used as a positive control, and distilled water was used as a negative control. No antimicrobial activity was observed when the extracts alone were tested on Staphylococcus aureus and Escherichia coli strains. The pure silver nitrate solution inhibited the growth of the Escherichia coli strain but did not effect on the Staphylococcus aureus strain. The antibacterial activity of the extracts became apparent only after complexation with silver ions.

The highest antimicrobial activity on the Escherichia coli strain was exhibited by sample 1R (prepared by reflux), which had an average value of relative inhibition zone (RIZD) of 131.5 % (Fig. 1). Consequently, this sample prepared by maceration 1M along with 2R and 3R samples prepared by reflux showed 1.05-fold lower antimicrobial activity compared to the 1R sample. Samples 2M and 3M, whose mean %RIZD was 87.5%, showed the lowest activity among the extracts tested.

Figure 1.

The use of UV/Vis spectrophotometry showed that after adding AgNO₃ to the extracts, an increase in absorbance was observed in the 415-nm region (Fig. 2), which indicates the formation of complexes between the extracts and AgNO₃. The region from 400 to 500 nm represents the wavelength range with maximum absorption for silver nanoparticles [6].

Figure 2.

Gradually heating the extracts increased the absorbance value at a wavelength of 415 nm. According to the Lambert–Beer law, an increasing concentration causes an increasing absorbance, and this represents an increase in the ability of the extracts to form complexes with AgNO₃.

The extract samples were heated in a water bath (80 °C) for 20, 40, and 100 minutes. After heating, the UV/Vis spectra were measured in the wavelength interval from 200 to 900 nm. The most significant increase in absorbance at 415 nm was observed after 100 minutes of heating for samples 1R and 1M. At all time intervals, the 1M and 3M samples that were prepared by maceration had higher absorbance values compared to the samples prepared by reflux. We assume that the formation of complexes with AgNO₃ was avoided. The exception was sample 1M, which had a significantly lower absorbance maximum even after 100 minutes heating compared to sample 1R. Sample 2R, at all three heating times, entered complexation with AgNO₃ most readily. Sample 2M exhibited the lowest absorbance value even after 100 minutes of heating.