(I) Polysaccharide composition and activity The hot-water extract of Ashwagandha contains glucan and proteoglycan, and Miyazoki (1985) showed by gas chromatography analysis that the extract mainly contains β-(1→3) glucan with β-(1→6) side chain, and a small amount of α-(1→4) and α-(1→6) glucan. Currently, it is believed that the main anticancer activity comes from β-(1→3) glucan with β-(1→6) side chain, which is soluble in alkali but insoluble in acid. The main chain group of polysaccharides is important for anticancer activity. The anticancer activity of ashwagandha glucan is due to the side chain β-(1→3) glucan on the main chain C-6 for two reasons: firstly, the anticancer activity is directly proportional to the content of β-(1→3) glucan side chain in the fraction; secondly, the anticancer activity of the fraction does not change after α-amylase digests α-(1→4)-glucan, which indicates that α-(1→4)-glucan does not have anticancer activity. In addition, it was demonstrated that hot-water extract plus purified dextran CF-1 (90% sugar content measured by the copper complex method) had anticancer activity equivalent to that of the hot-water extract, suggesting that the hot-water extract does not necessarily need to be highly purified.
Multiple studies have shown that among the active components of ashwagandha polysaccharides, the main chain can have β-(1→3)-, β-(1→4)-, β-(1→6) glucans, with β-(1→3) glucan being the most important. The side chains are β-(1→6)-, β-(1→3) glucan. The neutral fraction of the extract mainly contained α-(1→4)- and β-(1→3)-glucan of the C-6 branch of the side chain; the acidic fraction mainly contained β-(1→3)-glucan. Similarly, Yunzhi glycopeptide mainly contained main chain β-(1→3)- and β-(1→4)- as well as side chain β-(1→6)-glucan, so that oral administration also inhibited S-180 tumors in mice.
(II) Polysaccharide conformation and activity Ashwagandha polysaccharides are known to have two conformations, natural and helical. The dextran in the substrate is of the natural conformational type, and the chemical fractions obtained by different extraction methods are somewhat different. Glucans with different conformations have different activities and Ohno (1986) demonstrated that some of the natural type can be converted to the helical type during extraction and purification (after dialysis with urea). Unlike the conversion of ambrosia glycans (Pachyma) to U-ambrosia (the latter of which has cancer activity), both conformations of Ashwagandha have anticancer activity. During hot water or alkaline extraction of P. ashwagandha, the polypeptide bond may be broken, resulting in a conformational shift that affects its anticancer activity.
(C) Extraction method and activity Ashwagandha polysaccharides are mainly extracted from the fruiting bodies, solid mycelium and liquid mycelium, and Ohno proved that the glucan in the fruiting bodies of ashwagandha exists in the cell wall, so it is necessary to bring out the internal glucan, and Ohno speculated that the extract of ashwagandha enters into the gastrointestinal tract, and then it will be released by enzymes, and then it will show its activity as an anti-cancer active structure. Hishida reported that ashwagandha extract was ineffective when administered intraperitoneally and showed anticancer effects when administered orally, which seems to support Ohno's hypothesis. However, Ohno et al. (1984) gave extracts obtained by six different methods intraperitoneally to mice inoculated with S-180 tumors at doses of 40, 400, and 4000 μg/(only day) x 10 days in four groups, resulting in greater than 90% tumor inhibition. The highest anticancer activity was found in the water-soluble CF-7 fraction extracted from thermoalkali, the structure of which is C-6 branched with side chains of β-(1→3) glucan. The hot water extract contained a large amount of α-(1→4)-glucan, while the cold and hot alkali extracts contained a large amount of β-glucan. Therefore, the anticancer activity of the hot base extract was higher.Kuroda (1983) reported that the hot water extract of Ashwagandha was also active, and it was hypothesized that the active structure was the C-3 side chain β-(1→6)-glucan.
Consolidation of several studies shows that ashwagandha seeds contain about 8% of mycopolysaccharides, and that its hot-water, cold-base, and hot-base extracts have essentially the same primary structure. From the point of view of intestinal absorption, the small molecular weight is easier to pass through the intestinal mucosa, while the molecular weight of the hot alkaline extract is smaller.
The most commonly used extraction method is hot water immersion, but also ethanol and dilute alkali extraction. The same batch of raw materials, extracted with different solutions, the resulting polysaccharide composition is different. Dilute alkali extraction, in order to reduce the degradation of polysaccharides often add sodium borohydride or potassium borohydride, polysaccharide extraction rate is higher than the hot water method, but due to the viscosity of pectin and large, should be filtered while hot. The polysaccharide extracted with ethanol has less impurities and is easy to filter. Hot water extract must be centrifuged or filtered, the liquid is concentrated to a certain volume under reduced pressure, add 95% cold ethanol, so that the final concentration of ethanol up to 70% ~ 80%, you can get the polysaccharide precipitation, and then washed by acetone ether, dried polysaccharides crude products, which is soluble in water brown to white powder. Guo Qian et al. (1998) use bagasse solid culture of mycelium extract polysaccharides, polysaccharides extracted from the substrate than a short cycle and low cost, to be introduced:
1. Process Solid culture (containing mycelium) → boiling water extraction → filtration → concentration → alcohol precipitation → centrifugation → vacuum drying → weighing.
2. Orthogonal design The four factors of water addition times, extraction time, concentration specific gravity and alcohol precipitation concentration were selected as the factors to be examined, and each of them was taken at three levels (Table 4-8) to carry out the orthogonal test of L9 (34), and the rate of alcohol precipitation was used as the index to examine and screen the optimal process conditions, and the dosage of mycelium was 100 grams each time.
Table 4-8 Experimental factor levels
3. Analysis of results The results showed that the multiplicity of water addition (Factor A) had a significant effect on polysaccharide yield, the alcohol precipitation concentration (Factor D) had an effect on polysaccharide yield, while the extraction time (Factor B) and the concentration specific gravity (Factor C) had a weak effect on the polysaccharide yield in the three selected levels. However, for the extraction time (factor B) in production, the extraction time suitable for the production process should be selected, and for the specific gravity of concentration (factor C), although it is the least significant factor among the four factors, the amount of ethanol used in the alcohol precipitation process increases dramatically with the decrease of the specific gravity of concentration in order to achieve the proper concentration of alcohol precipitation. Therefore, under the premise of the production process permits, a higher concentration specific gravity should be selected (Table 4-9).
4. Conclusion According to the results of orthogonal design experiments, the best parameters for the extraction process of polysaccharides from the mycelium of Ashwagandha were determined as follows: the multiplicity of water addition was 25 times, and the ratio of the amount of water added was 3:2:4 according to the results of the preexperimentation; the concentration of alcohol precipitation was 80%; the specific gravity of concentration was 1.035; the extraction time could be selected as suitable for the production of the three parameters of 6, 8, and 10 hours, and, generally, the shorter extraction time should be selected, and the higher concentration specific gravity should be selected as the production process permits. Choose shorter leaching time, which can save energy and shorten the process cycle, the ratio of leaching time is 1:2:3 according to the results of the preexperiment.
Table 4-9 L9 (34) orthogonal design experimental data