3.1 Effects of plant topping on rhizome yield and functional components of Polygonatum cyrtonema
The rhizome yield and polysaccharide, total saponins, total flavonoids, and total polyphenols contents of topping and non-topping of Polygonatum samples are shown in Table 1. From the data in Table 1, topping Polygonatum cyrtonema has a significant impact on the fresh weight of the rhizome per plant and the yield per unit area. Furthermore, it was found that the rhizome weight gain coefficient can more accurately reflect the effect of topping on the rhizome weight gain of Polygonatum cyrtonema. These data showed that the topping of Polygonatum cyrtonema during the flowering period can significantly increase the yield of underground rhizomes. Picking off some flowers reduces the pulling force of flowers and fruits on carbon assimilates, while the pulling force of tubers on carbon assimilates increases sharply, which promotes the continuous flow of carbon assimilates to tubers 21,22. On the other hand, it may be possible to reduce the nutrient consumption of the aerial part of Polygonatum cyrtonema by topping so that the underground rhizome can distribute more nutrients and promote its expansion. The wound-inducing effect after the topping causes the strong compensatory growth of Polygonatum cyrtonema23,24, thereby driving the accumulation of underground rhizome biomass.
The output of Chinese herbal medicines is related to its economic value, and the content of the medicinal components is a decisive factor for the quality of Chinese herbal medicines. Polygonatum cyrtonema contains polysaccharides, saponins, flavonoids, and other chemical components. The underground rhizomes of Polygonatum cyrtonema are similar to bamboo whips, Aboveground parts grow on the current year rhizomes(Fig. 1). Therefore, the effect of topping on the quality of underground rhizomes was determined, and the functional components of the rhizomes of the current year correspond to the plants with and without topping. Polysaccharide is one of the main active components of Polygonatum cyrtonema 25, and it is commonly used to identify the quality of the medicinal material. With and without topping, there is no significant difference in polysaccharide content, and both meet the requirement of "2020 Pharmacopoeia" i.e., polysaccharides should not be less than 7.0%. Like polysaccharides, saponins are also the active ingredients and present in high content in Polygonatum cyrtonema26,27, and showed anti-inflammatory, hypoglycemic, intestinal flora regulation, antidepressant, and other pharmacological activities28,29. From the data in Table 1, the saponin content of the rhizome of topping was 37.60 mg/g, which was significantly higher (P < 0.01) than that of the rhizome without topping (32.53 mg/g). Topping can significantly increase the content of total saponins, which could promote the synthesis of saponins and terpenoid skeletons after removing the tops of Polygonum cyrtonema 30.
Interestingly, the content of total polyphenols and total flavonoids in Polygonatum rhizomes of topping (7.51 mg/g, 1.08 mg/g) were lower than those without topping (7.94 mg/g, 1.42 mg/g). Light exposure should increase the content and activity of flavonoid synthases such as phenylalanine ammonia-lyase, chalcone isomerase, flavanone-3-hydroxylase, and flavonol synthase 31. Light played a major role in increasing the flavonoids content in plants. After topping, the aboveground plants receive less light, thus decreasing the content of flavonoids in Polygonatum cyrtonema. Most flavonoids contain phenolic hydroxyl group, an important part of polyphenols. The decrease in the flavonoids content after topping is also one of the reasons for the decrease in the polyphenols content. These metabolites may be synthesized in the stems and then transferred to the roots or directly synthesized in the roots. The rhizomes' physiological and secondary metabolic mechanisms in response to light are still unclear, and further research is needed. Although the content of flavonoids and polyphenols in Rhizoma Polygonatis rhizomes decreased after decapitation; however, saponins, the main active ingredient usually used to evaluate its quality, increased significantly in Polygonatum cyrtonema. As a result, the content and yield of polysaccharides increased significantly. However, further research needs to be conducted to determine the regulatory mechanism of the distribution of nutrients in the aboveground and underground parts of Polygonatum cyrtonema to accumulate to the roots.
3.2 Functional components of stems, leaves, and flowers of Polygonatum
The contents of polysaccharides, total saponins, total phenols, total flavonoids, and proteins in each sample are shown in Table 2. The data in Table 2 showed that the rhizomes (PCR, the whole rhizomes), stems (PCS), leaves (PCL), and flowers (PCF) of Polygonatum cyrtonema contain functional components such as polysaccharides, total saponins, total phenols, total flavonoids, and proteins; however, their concentrations were significantly different. For instance, the polysaccharide content in PCR was the highest (10.47%), followed by leaves (5.99%) and flowers (4.76%), while the polysaccharide content in the stem was the lowest (3.65%). The content of total saponins in flowers (36.68mg/g) is very close to the rhizomes (39.09mg/g) content, and there is no significant difference between the two contents. The content of total saponins in stems and leaves is lower and represents only 1/7 of that in flowers (~ 1/5). The contents of total phenols and total flavonoids in different samples were consistent, all of which were in the order of PCF > PCL > PCS > PCR, which was inconsistent with the study of Zhao et al.32. The contents of total phenols and flavonoids in flowers, leaves, and stems were significantly (P < 0.01) higher than those in rhizomes, especially the polyphenols content in flowers was much higher than that in rhizomes. The results were consistent with Zhang et al.12, which could be related to the higher content of anthocyanins in flowers. The protein content in the rhizome was the lowest (3.08%), which was only half of the protein content in the stem. The protein content in the leaf and flower was higher, 13.81% and 11.64%, respectively, and about 3.7–4.5 times in the rhizome. These data showed that the non-medicinal parts such as stems, leaves, and flowers of Polygonati contain more bioactive compounds than the rhizomes. The content of total phenols, total flavonoids, and protein in the leaves is high, and it has a good potential to be developed into functional food and skincare products.
The data in Table 3 showed the total amino acids, their content, and individual content in rhizomes (PCR), stems (PCS), leaves (PCL), and flowers (PCF). Polygonatum cyrtonema stems, leaves, and flowers are rich in amino acids, which are much higher than rhizomes. The total amino acid content in leaves is the highest (23.25%), about 3.16 times that in the rhizomes. The contents of valine, leucine, isoleucine, phenylalanine, and lysine in the stems, leaves, and flowers are all higher, especially in the leaves; the contents of these essential amino acids are above 1%. These amino acids provide better health care functions such as regulating blood sugar and improving immunity. The contents of umami amino acids such as aspartic acid and glutamic acid are higher in the leaves. These results indicated that stems (PCS), leaves (PCL), and flowers (PCF) contained more abundant amino acids than rhizomes.
3.3 Antioxidant activity
Antioxidants can be divided into synthetic antioxidants and natural antioxidants. With the research on the toxicology of synthetic antioxidants and the enhancement of people's health awareness, the development of natural antioxidants came to light. Most natural antioxidants are obtained from plants 33. Intake of natural antioxidants Many studies have shown that the natural antioxidant components contained in plants can effectively reduce the incidence of aging-related diseases such as cardiovascular disease, diabetes, cancer, etc. by scavenging free radicals34,35.
DPPH is a purple, very stable free radical, which is used to measure the ability of various antioxidants to scavenge free radicals. The scavenging effect on DPPH free radicals is shown in Fig. 2-A. Both leaves and flowers have high DPPH free radical scavenging ability and the scavenging rate increases with increasing concentration. Among them, flowers have the strongest scavenging ability to DPPH free radicals, and at 1000 µg/mL, the scavenging rate reaches 88.9%, respectively. This could be related to the high polyphenols and flavonoids concentration in the flowers. In the case of stems and leaves samples at 3000 µg/mL, the scavenging abilities of DPPH free radicals were 79.2% and 85.1%, respectively, which were higher than those of rhizomes (61.2%). This may be related to the fact that stems and leaves contain more flavonoids and polyphenols than rhizomes and can directly capture free radicals.
As shown in Fig. 2-B, the stems, leaves, and flowers of Polygonatum cyrtonema also showed comparatively stronger ·OH radicals scavenging ability than rhizomes. For example, at a concentration of 4000 µg/mL, the·OH radicals scavenging abilities of the stems, leaves, and flowers are very close, reaching more than 85%, while the scavenging power of rhizomes is only 43.6%. However, at lower concentrations (250–1000 µg/mL), the leaves showed stronger ·OH radical scavenging ability than stems and flowers. Moreover, the scavenging ability of flowers changed greatly with increasing concentration, indicating that the flower has a strong ability to scavenge ·OH radicals.
The scavenging effect on ABTS free radicals is shown in Fig. 2-C. Similar to the results of DPPH and ·OH free radical scavenging experiments, flowers and leaves showed higher scavenging ability to ABTS free radicals at a concentration of 1500 µg/mL. At this concentration, the ABTS clearance rates of leaf and flower extracts reached 88.4% and 89.3%, respectively, while the ABTS clearance rates of stems at this concentration are weaker (60.3%), like those of underground rhizomes.
The IC50 values of the stem, leaf, and flower extracts for scavenging DPPH, ·OH, and ABTS free radicals, and the FRAP total reducing power analysis results are shown in Table 4. The smaller the IC50, the stronger the free radical scavenging ability. It can be seen from Table 4 that for the IC50 of DPPH and ABTS, the scavenging effect of stems, leaves, and flowers is stronger than that of rhizomes (p < 0.01), but compared with stems (719.35, 862.39 µg/mL), leaves (301.86, 342.87 µg/mL) and flower (251.44, 264.61 µg/mL) showed stronger DPPH and ABTS free radical scavenging effects (p < 0.01). However, unlike DPPH, the IC50 of leaf against·OH (1069.93 µg/mL) was the smallest, indicating that leaves had a stronger ability to scavenge ·OH, followed by flowers and stems.
The reducing power is also an important indicator of measuring antioxidant activity. As shown in Table 4, the total reduction of FRAP scavenging power was similar to that of DPPH and ABTS, with higher reducing power for flowers (1343.65 µg/mL VC eq/gsample), leaves (1232.43 µg/mL VC eq/gsample), and stems (552.69 µg/mL VC eq/gsample), but both were significantly stronger (p < 0.01) than the reducing power of underground rhizomes.
The above results of DPPH, ·OH, ABTS, and FRAP showed that the stems, leaves, and flowers of Polygonatum cyrtonema have strong free radical scavenging ability and antioxidant activity. The inhibitory activity of the rhizomes was significantly lower than other samples (P < 0.01) ,especially ,the aboveground parts (leaves and flowers) showed high antioxidant effects related to the higher content of flavonoids and polyphenols. In addition, there are differences in the free radical scavenging ability of DPPH, ·OH, ABTS, and the antioxidant capacity of FRAP among the stems, leaves, and flowers of Polygonum cyrtonema, which could be related to the different mechanisms of different antioxidant methods. The antioxidant activities of stems, leaves, and flowers in the oxidation system were lower than Vc, which may be because the content of active antioxidant substances in the crude extracts of stems, leaves, and flowers are not high enough.