Extraction, Physicochemical Properties, Anti-Aging, And Antioxidant Activities Of Polysaccharides From Industrial Hemp Residues Part 1
Jul 10, 2023
Abstract: A large amount of hemp polysaccharides remain in industrial hemp residues (IHR) after cannabidiol extraction, resulting in the waste of resources. Therefore, the systematic study of hemp polysaccharides is beneficial to the development of IHR in the future. In this study, the extraction of industrial hemp residue polysaccharides (IHRPs) was optimized by a single-factor experiment and orthogonal experimental design. The optimum heating extraction conditions were extraction temperature 98 ◦C, solid–liquid ratio 1:10, extraction time 1 h, number of successive extractions at 2, and pH at 4. The extraction ratio and the polysaccharide content were 20.12 ± 0.55% and 12.35 ± 0.26% at the conditions, respectively. Besides, the best alcohol precipitation conditions were pumping with 2 L/h, stirring continuously, and ice-water bath for 4 h. The crude IHRPs were further purified by column chromatography and the polysaccharide/protein contents of purified IHRPs were 34.44% and 1.61%. IHRPs were mainly made up of ten monosaccharides and some non-sugar components including organic acids, flavonoids, steroids, and glycosides. The FT-IR demonstrated the polysaccharide skeleton of IHRPs. Moreover, the DPPH and ABTS scavenging rates of IHRPs were 76.00% and 99.05% at the concentrations of 1 mg/mL. IHRPs could promote the epidermal cells proliferation and healing of cell scratches. Meanwhile, IHRPs could promote the expression of anti-aging-related genes. Overall, IHRPs could be a desirable natural source of antioxidants and anti-aging products in many aspects.
Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and can scavenge reactive oxygen species, prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.
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Keywords: industrial hemp; polysaccharide; extraction; antioxidant activity; anti-aging activity
1. Introduction
Hemp (Cannabis sativa L.), an annual herb of the Cannabis family, has been used for thousands of years [1]. The tetrahydrocannabinol (THC) content of industrial hemp is less than 0.3 wt%, which is different from medical marijuana and recreational marijuana [2,3]. Therefore, industrial hemp could be legally cultivated on a large scale. The chemical composition of industrial hemp is very complex, including cannabinoids, terpenoids, alkaloids, and flavonoids [4]. The most bioactive compounds are cannabinoids that include more than 130 terpene phenolic compounds which mainly accumulate in the flower tops [5]. The major cannabinoids are THC, cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabidivarin (CBDV), cannabidiol (CBT), and cannabicyclol (CBL) [6]. Among them, THC, a psychoactive compound restricted in different countries, and CBD are the most studied cannabinoids for their significant bioactivities [7], such as neuroprotective, antiepileptic, anticancer, and immunoregulatory activities [8]. The existence of these compounds increases the importance of industrial hemp. At present, industrial hemp has been widely used in the fields of daily chemicals, materials, energy, and medicine [9]. The current treatment of industrial hemp residues (IHR) is directly discarding them, which not only pollutes the environment but also causes a waste of resources [10].
Polysaccharide is a polymer that consists of exceeding 10 monosaccharides [11]. It is one of the most essential biological macromolecules. In recent years, many articles have reported that the natural polysaccharides from plants have good biological activities, including anticancer [12], anti-inflammatory [13], antioxidant [14], immunomodulatory [15], hypoglycemic [16], and antiviral activities [17]. The molecular composition and spatial structure of polysaccharides generally have significant effects on their properties and activities [18]. With the development of separation, purification, and structural analysis technology of polysaccharides, it is generally accepted that polysaccharides have great application values in drug, cosmetics, and food fields [19]. The content of polysaccharides from industrial hemp residues is about 10%–15%. Hemp polysaccharides have many excellent physicochemical properties because they contain lots of hydrophilic hydroxyls, such as strong water-absorption capability, emulsifying properties, and good film-forming ability. Meanwhile, hemp polysaccharides have moisture retention, antibacterial, and no-toxic effects. Therefore, they have extensive application prospects in the cosmetics field. The fermented products of hemp polysaccharides (oligosaccharides or some sugar alcohols), which are reported to have good antioxidant, anti-aging, and anti-fatigue activities, have been used to develop functional drinks [20]. The composition of hemp polysaccharides is relatively complex and it ranges from monosaccharides to 5 × 104 Da polysaccharides.
There are few reports about the separation and extraction of hemp polysaccharides. Hillestad et al. isolated two glycoproteins from hemp. The linkage structure of the glucoside chain and the connection between the chain and protein were analyzed by methylation and Smith degradation [21]. J.W. Groce et al. measured the carbohydrate content of hemp grown in the United States, Thailand, and Vietnam respectively. It was pointed out that large differences existed in the type and content of compounds between the cultivars from different areas [22]. Zheng et al. analyzed the monosaccharide composition and structure of HSP0 and HSP0.2, two components of hemp seed polysaccharide, by HPLC and FT-IR. It was found that HSP0 and HSP0.2 both contained sulfate radicals [23]. Bi et al. obtained two hemp seed polysaccharides HS1 and HS2 by gel filtration chromatography. The main monosaccharides were D-arabinose, D-xylose, D-mannose, D-galactose, and D-glucose, and their molar ratios were 0.12, 0.09, 0.15, 0.11, and 0.12, respectively [24]. Guo et al. prepared polysaccharides from industrial hemp leaves and studied the effects of ultraviolet light, temperature, and pH on their antibacterial activity. It was found that the minimum bactericidal concentration and minimum inhibition concentration of the polysaccharides against S. aureus were 6.25 and 3.125 mg/mL, respectively. Additionally, the antibacterial activity was the highest when the pH was 7.
In this study, the IHR after cannabidiol extraction was used as the raw material for polysaccharides extraction. The extraction and purification process of industrial hemp residue polysaccharides (IHRPs) was optimized. Then the structure and composition of IHRPs were analyzed. Besides, the ABTS and DPPH scavenging ability of IHRPs was evaluated. Its anti-aging activity was also studied at the cellular level. It is hoped that IHRPs could be widely used in cosmetics and functional food fields.
2. Results and Discussion
2.1. Single Factor Experiments of Extraction for IHRPs
2.1.1. Effects of Extraction Methods on Polysaccharide Extraction
The effects of percolation extraction, heating extraction, and ultrasonic-assisted extraction on polysaccharide extraction are shown in Figure 1a. It could be seen that the polysaccharide content of percolation extraction was close to 0%, which indicated that nearly no polysaccharide was extracted and the extract mainly consisted of pigment and other water-soluble components. The results were due to the percolation extraction being very gentle and the cell wall of IHR not being broken. Thus, the polysaccharides could not be extracted from cells, which caused the polysaccharide content and extraction ratio of percolation extraction to be very low. The extraction ratio of heating extraction was higher than that of ultrasonic-assisted extraction. However, the polysaccharide content of heating extraction (11.27%) was lower than that of ultrasonic-assisted extraction (13.81%). Heating extraction is one of the most commonly used extraction methods because of the process's simplicity and low cost. However, the time of heating extraction is relatively long and the extraction ratio is not high. Extraction temperature generally has a great influence on the extraction ratio. The higher extraction ratio of heating extraction in this work was because the heating extraction was performed at 98 ◦C while the ultrasonic-assisted extraction was performed at 60 ◦C. Ultrasonic-assisted extraction could shorten the extraction time because the cavitation effect and mechanical effect could destroy cell walls, improve the penetration of the solvent, and accelerate the transfer of polysaccharides to the solvent [25]. Thus, the polysaccharide content of ultrasonic-assisted extraction was higher than that of heating extraction. The heating extraction was chosen for the following experiments given process simplicity and industrialization.
2.1.2. Effects of Extraction Temperature on Polysaccharide Extraction
The effects of extraction temperature on polysaccharide extraction were shown in Figure 1b when the solid–liquid ratio was 1:10, extraction time was 1 h, the number of successive extractions was 2, and pH was 7. The extraction ratio and polysaccharide content both increased with the increase in temperature. When the temperature was 98 ◦C, the highest extraction ratio and polysaccharide content were 14.99% and 12.03%, respectively.
2.1.3. Effects of Solid–Liquid Ratio on Polysaccharide Extraction
When the extraction temperature was 98 ◦C, extraction time was 1 h, the number of successive extractions was 2, and pH was 7, the effects of solid–liquid ratio on polysaccharide extraction were displayed in Figure 1c. The extraction ratio increased gradually with solid–liquid ratio from 1:6 to 1:10, and then became smooth. However, the polysaccharide content was nearly not affected by solid–liquid ratio. Besides, the solution could just submerge the raw material when the solid–liquid ratio was 1:5 because of the low density of plant leaves. Therefore, the solid–liquid ratio of 1:6 was studied primarily.
2.1.4. Effects of pH on Polysaccharide Extraction
When the extraction temperature was 98 ◦C, extraction time was 1 h, number of successive extractions was 2, and solid–liquid ratio was 1:10, the effects of pH on polysaccharide extraction were displayed in Figure 1d. When the solution was acidized or alkalified gradually, the extraction ratio was improved to some extent. However, the polysaccharide content had a decreasing trend with the increase in pH, which indicated that the acidic condition was beneficial to the extraction of IHRPs.
2.1.5. Effects of Extraction Time on Polysaccharide Extraction
When the extraction temperature was 98 ◦C, pH was 7, number of successive extractions was 2, and solid–liquid ratio was 1:10, the effects of extraction time on polysaccharide extraction were displayed in Figure 1e. The extraction ratio decreased lightly when the extraction time exceeded 1 h. Furthermore, the polysaccharide content was nearly not influenced by the different extraction times.
2.1.6. Effects of Number of Successive Extractions on Polysaccharide Extraction
When the extraction temperature was 98 ◦C, extraction time was 1 h, pH was 7, and solid–liquid ratio was 1:10, the effects of several successive extractions on polysaccharide extraction were shown in Figure 1f. The extraction ratio dropped to 5.11% when the raw material was extracted the second time. Besides, the polysaccharide content was very low when the raw material was extracted the third time.
2.2. Orthogonal Experimental Design of Extraction for IHRPs
It could be seen that extraction temperature, solid–liquid ratio, number of successive extractions, and pH had a great influence on polysaccharide extraction based on the single-factor experiments. Therefore, the orthogonal experiments were carried out to further optimize the extraction conditions. As shown in Table 1, the order of effect of individual factors on polysaccharide extraction was: extraction temperature > several successive extractions> solid–liquid ratio > pH. Group 5 (Solid–liquid ratio was 1:8, number of successive extractions was 3, extraction temperature was 80 ◦C, and pH was 4) had the highest polysaccharide content (12.79%) while group 9 (Solid–liquid ratio was 1:10, number of successive extractions was 2, extraction temperature was 98 ◦C, and pH was 4) had the second highest polysaccharide content (12.26%). Considering the energy consumption and operability, the comparison validation between groups 5 and 9 was conducted to determine the most appropriate extraction conditions. As shown in Table 2, the verified experiments showed that the polysaccharide content between groups 5 and 9 was very close. Thus, to save energy consumption, the extraction conditions of group 9 were selected to extract IHRPs for its use in the following experiments: solid–liquid ratio was 1:10, extraction temperature was 98 ◦C, number of successive extractions was 2, and pH was 4.
2.3. Screen of IHRPs Alcohol Precipitation Conditions
The IHRPs extraction solution was obtained according to the extraction conditions optimized by 2.2. The effects of different alcohol precipitation conditions were displayed in Table 3. Compared with one-time addition and stirring for 0.5 h, pumping and stirring continuously could further improve the polysaccharide content. However, the effect of the cooling rate on polysaccharide content was not obvious. Therefore, the alcohol precipitation conditions of group 4 were chosen for the preparation of IHRPs.
2.4. Polysaccharide Yield and Chemical Composition of Purifified IHRPs
The crude IHRPs were decolorized and purified by activated carbon adsorption, membrane filtration, column chromatography, and sevage method, respectively. As shown in Figure 2, the treatment with 8% activated carbon adsorption had the best decolorizing effect, followed by the column chromatography treatment. Besides, the selvage method and membrane filtration had almost no decolorizing effect. Considering the industrialization and the simplicity of the process, column chromatography with anion exchange resin was used to purify IHRPs in this manuscript. Besides, as displayed in Table 4, the IHRPs’ weight was 15.26 g when the purification was conducted after the alcohol precipitation, while IHRPs’ weight was 12.74 g when the purification was conducted directly after heating extraction. However, the actual polysaccharide weight of the two processes was very close (4.69 g and 4.38 g). Therefore, to save costs, column chromatography purification could be carried out directly after heating extraction.
2.5. FT-IR Spectroscopy of IHRPs
Some structural characteristics of polysaccharides could be inferred based on the characteristic absorption peaks of FT-IR [26]. The FT-IR spectrum of IHRPs is shown in Figure 3 and the typical absorption of polysaccharide could be observed. The peak around 3378 cm−1 was attributed to -OH stretching vibration, indicating the existence of -OH [27]. The weak peak at 2932 cm−1 was C-H stretching vibration, including the vibration of -CH2 and -CH3 [28]. The strong peak at about 1600 cm−1 was related to the -C=O stretching vibration [29], and the peak around 1413 cm−1 was denoted for the C-O stretching vibration, indicating the existence of -COOH. Besides, the relatively strong peak at 1080 cm−1 very likely corresponds to the O-H bending vibration. The FT-IR spectrum of IHRPs displayed many characteristic absorption peaks of polysaccharides, demonstrating the polysaccharide skeleton of IHRPs.
2.6. Monosaccharide Composition Analysis of IHRPs
The bioactivity of polysaccharides is frequently related to monosaccharide compositions. In this study, the monosaccharide composition of IHRPs was acquired by ion chromatography as displayed in Figure 4. According to the monosaccharide standards, IHRPs were composed of fucose, arabinose, rhamnose, galactose, glucose, xylose, ribose, galacturonic acid, guluronic acid, and glucuronic acid. Their quality percentages were 1.33, 19.60, 10.41, 20.87, 27.42, 4.23, 3.12, 6.22, 0.28, and 2.37 respectively. It could be found that arabinose, rhamnose, galactose, and glucose were the main monosaccharides in IHRPs. Glucose was a dominant monosaccharide in IHRPs and the result was consistent with the previous study [30]. The content of uronic acid could have made polysaccharides negatively charged. This result had a significant influence on the bioactivities of polysaccharides [31]. Besides, the polysaccharide content of IHRPs was between 30% and 35%. The non-sugar components were mainly organic acids, flavonoids, steroids, and glycosides. Meanwhile, the content of each non-sugar component was relatively low.
2.7. Antioxidant Activity Study
The ABTS and DPPH free radical scavenging ability have been extensively studied to evaluate the antioxidant activity of polysaccharides [32]. The scavenging capacity of IHRPs against ABTS and DPPH is displayed in Table 5. As exhibited in Table 5, the scavenging rate of DPPH and ABTS both increased with the increase in IHRPs concentration. When the concentration of IHRPs was 1.0 mg/mL, the DPPH scavenging rate was 76.00% while the ABTS scavenging rate was 99.05%. After calculation, the EC50 values of ABTS and DPPH were determined as 0.34 and 0.47 mg/mL. To our knowledge, there is no report about the antioxidant activity of IHRPs. The antioxidant activity of the inflorescences from 12 Cannabis sativa L. monoecious cultivars was studied [33]. Among them, pineapple had the strongest scavenging activity against DPPH (EC50 = 60.00 µg/mL) while Kc-Virtus had the strongest scavenging capacity against ABTS (EC50 = 304.77 µg/mL). In terms of the antioxidant activity of cannabidiol-full spectrum oil, it was reported that its DPPH EC50 was 158.0 µg/mL [34]. Additionally, the DPPH EC50 of CBD was reported to be 0.937 mM [35]. The free radicals scavenging capacity of CBDV was also studied, i.e., the ABTS EC50 was 406.3 µM while the DPPH EC50 was 3736 µM [36]. The above results indicated that IHRPs showed great antioxidant activity
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