Impact of Extraction Methods on Polyphenol Contents and Antioxidant Activities of Melissa Officinalis and Evaluation of Their Antihypoxic Activities in Mice

The role of free radicals in causing many diseases has been well proven. These particles can destroy biomolecules. Antioxidants can prevent these harmful effects. Considering that plants are a source of natural antioxidants, research in this field is increasing. Hypoxia means the reduction of oxygen in the body tissues, which can lead to dysfunction of the body. Hypoxia causes a significant increase in reactive oxygen species, thus, antioxidants are considered anti-hypoxia. Melissa officinalis L. is a well-known medicinal plant of Lamiaceae. Leaves of this plant have been widely used for the treatment of cardiovascular and respiratory problems and as a memory enhancer. This investigation was carried out to examine the impact of extraction on total phenolic contents and antioxidant activities of M. officinalis aerial parts. In addition, the Antihypoxic activities of all extracts were evaluated in three models in mice.

In this experimental study, dried aerial parts were extracted by three different methods, i.e. maceration method, ultrasonic-assisted, and soxhlet-assisted extraction. Antioxidative capacity was assessed by utilizing DPPH free radicals scavenging and reducing power. The total phenolic and flavonoid contents were also determined. The protective effects of extract in the initial dose of 62.5-250 mg/kg were evaluated against hypoxia-induced lethality in mice by three experimental models of hypoxia, i.e. asphyctic, haemic, and circulatory. The latencies for death for mice in minutes were recorded. The Institutional Animal Ethical Committee of Mazandaran University of Medical Sciences approved the experimental protocol. In the asphyctic hypoxic model, phenytoin (50 mg/kg, i.p.) and in the next two tests, propranolol (20 and 30 mg/kg, i.p.) were used as the positive control. In all tests, Normal saline (0.5 ml, i.p.) was used as the negative control. Analysis of variance was performed followed by Newman-Keuls multiple comparisons (by GraphPad Prism 8) were used to determine the differences in means.

Meceration method and ultrasonic(P<0.0001) assisted extraction were the best methods for extraction of polyphenols. In DPPH radical scavenging activity, soxhlet-assisted extraction and extraction by the meceration method were more efficient than ultrasonic-assisted extraction(P>0.05). In the hemic model, none of the extracts showed any activity. Even the soxhlet-assisted extract at the dose of 250 mg/kg, even though it increased the death time by about one minute, could not cause a significant effect(P>0.05). In the circulatory model, none of the extracts showed any effect at the lowest tested dose i.e. 62.5 mg/kg but all the extracts showed a very potent activity at higher doses. All the extracts at a dose of 250 mg/kg showed a much stronger effect than propranolol 30 mg/kg. In the asphyctic model, all the extracts showed very good effects so we had to reduce the dosage many times. The extract obtained from the maceration method at a dose of 1.95 mg/kg and ultrasonic-assisted and soxhlet assisted extracts at the dose of 7.81 mg/kg showed the same activity as phenytoin(P>0.05).

The findings of the current study indicated that extraction methods significantly affect antioxidant capacities and total phenolic contents. The ultrasonic-assisted extraction and maceration method were the most suitable methods for extracting phenolic and flavonoid compounds from this plant. All extracts showed high antioxidant activities. All extracts showed very strong effects in the asphyctic model.