Can Ultrasonic Extraction Increase The Anti-influenza Activity of Aloe Vera?

1 Principle of ultrasonic extraction

Ultrasound is defined as a sound wave with a frequency greater than 20 khz, which exceeds the limit of human hearing detection. Ultrasound is a mechanical wave with a high energy density. Its sound energy output source is usually a vibrating object, which can cause the surrounding medium to vibrate and then transfer energy to other adjacent particles. When ultrasound passes through the medium, it causes the longitudinal displacement of the particles. These dense molecular effects cause cell wall damage and accelerate the mass transfer rate of effective substances in the medium, thereby achieving the purpose of improving the extraction rate. Ultrasonic extraction does not rely on a single mechanism of action, but works continuously or simultaneously through multiple physical mechanisms such as mechanical fragmentation, thermal effect, and cavitation effect. In the homogenate of solid-liquid mixture, the micro-beams and micro-turbulence generated by acoustic cavitation in the liquid medium will cause strong mechanical disturbances, thereby intensifying the collision between particles, which can easily lead to the decomposition and local rupture of some brittle materials. On the other hand, due to the reduction in particle size, the mass transfer rate of solid particles and the contact area between solid and liquid phases are increased, which are conducive to accelerating the dissolution of the contents in the sample matrix.

The cavitation effect is a unique and complex physical phenomenon caused by the propagation and vibration of ultrasound in liquid. It generally refers to the process of formation, expansion and rupture of cavitation bubbles in liquid. Simply put, when high-intensity ultrasound is applied, the attraction between medium molecules may exceed the critical value, thereby generating high shear stress in the liquid and subsequently forming cavitation bubbles. 1 Cavitation bubble is formed near the surface of the matrix. After undergoing continuous compression-rarefaction cycles, the cavitation bubble will rupture during the compression cycle and generate short-term heat energy, thereby forming high-speed microjet fluid on the surface of the matrix and generating strong shock waves. This process can make the surrounding local temperature as high as about 5,000 K, and the instantaneous pressure can reach 50~1000 atm. The high-pressure and high-temperature environment formed will destroy the cell wall of the plant matrix, thereby releasing intracellular substances into the solution. From the scanning electron microscope images of basil leaves taken by CHEMAT and others before and after oil extraction, it can be observed more vividly: before extraction, the glands on the leaves are smooth and full; after extraction, they begin to shrink, but the gland structure remains intact; and after ultrasonic-assisted extraction, the glands are completely broken and all their contents are released.

Aloe vera has gained wide popularity and has been commercialized as a beauty product, laxative, herbal medicine, and its antibacterial activity has been demonstrated. However, except for a few reports, the antiviral activity of aloe vera has not been well demonstrated. To date, it is common to extract aloe vera compounds using organic solvents, as the active ingredients can be effectively extracted in methanol. In this study, we used a 5-min ultrasonic extraction method to effectively extract aloin and aloe-emodin compounds from aloe vera in water. This rapid one-pot extraction process enhanced the extraction of flavonoids and phenolics in the ultrasonic water extract and enriched the aloin and aloe-emodin fractions. The extracts were tested for their anti-influenza activity, and the results showed that ultrasonic extraction enabled the water extract to exhibit excellent anti-influenza activity comparable to that of the methanol extract. The cytotoxicity of the water extract was zero compared to the methanol extract, which had high cytotoxicity. Spectrophotometric scanning of the extracts confirmed the enrichment of aloin and aloe-emodin peaks in the ultrasonic extract of aloe vera, indicating their handiwork behind the anti-influenza activity. The demonstrated techniques, if properly implicated, will lead to promising solutions in the pharmaceutical pursuit to combat influenza viruses.

To date, more than 200 bioactive chemicals have been found in aloe vera gel, and their bioactivity is more likely to be due to the synergistic effects of compounds rather than single compounds. Therefore, proper extraction of bioactive compounds is crucial to fully exploit all the active ingredients for their physiological and medicinal properties.

Aloe vera gel has been found to be effective against both Gram-positive and Gram-negative bacteria, as well as certain fungi and viruses. Inhibitory effects of Bacillus versicolor against human cytomegalovirus, herpes simplex virus type 2 (HSV-2), and poliovirus have been reported. Influenza is an acute respiratory infection caused by influenza viruses with a huge global impact. This virus has the potential to cause severe pandemics and economic losses. Its genome is highly variable with a high mutation rate, which makes it resistant to many drugs. Currently, synthetic antiviral drugs and methods (nucleoprotein inhibitors, neuraminidase inhibitors, ion channel blockers, and siRNA technology) have limited application due to the emergence of drug-resistant strains, high cost, and harmful side effects. Herbal medicines are low in cost, low in toxicity, and often have multi-target effects, which can not only act as antiviral drugs but also stimulate the immune system to fight viruses. Classically defined antiviral mechanisms of medicinal plants include inhibition of viral replication, blocking viral attachment, inactivation of viruses, and prevention of viral infection. The interest of investigators and customers in using these medicines has increased due to a preference for natural medicines and concerns about the toxic effects of synthetic materials.

Ultrasound is characterized by high frequency (18 kHz-1 MHz), small displacement (less than about 50 mm), moderate velocity (ms-1), steep transverse velocity gradient (up to 4,000 s-1), and very high acceleration (up to about 80,000 g). When the acoustic power input is high enough, ultrasound produces cavitation, allowing microbubbles to be generated at the nucleation site. The bubbles grow during the rarefaction phase of the sound wave and then collapse during the compression phase. During the collapse, strong shock waves pass through the medium. The entire process of nucleation, growth, and rupture of bubbles under the action of strong sound waves is called cavitation. In terms of extraction, extraction using ultrasound or ultrasonic membrane disruption has the advantages of significantly shortening the extraction time and increasing the extraction yield. The application of ultrasound destroys the cell wall structure and accelerates the diffusion of the cell membrane; therefore, the cells are lysed, thereby promoting the release of cell contents. Furthermore, it has been demonstrated that the enhancement of plant tissue hydration during ultrasound coincides with the disruption of plant tissue and leads to enhanced extraction during ultrasound. Therefore, the application of ultrasound technology in aloe extraction has good prospects.

The current work focuses on the extraction of rich bioactive compounds from aloe extract using ultrasound-based methods. The ultrasound extraction frequency was optimized to achieve the desired extraction effect. Ultrasonic extraction produced nanopackaging of active metabolites with enhanced solubility in water. For the first time, ultrasound technology was demonstrated to be effective in extracting aloin and rhubarb components that were the best extractors in organic solvents in water.