| Plant growth regulators(PGRs)are a kind of chemical substances that can be artificially extracted or synthesized to regulate plants growth,development and physiological function according to the structure,function and principle of plant hormones,and widely used in the production of Chinese medicinal materials(CMMs).PGRs play an important role in promoting the growth and yields of medicinal plants.Whether or not PGRs can be used depends on their effect on the effectiveness and safety of CMMs.Some studies indicated that the blindly use of 'Zhuanggenling' PGRs or PGR-containing fertilizers in the cultivation of CMMs significantly reduced the quality of CMMs,and resulted in the residues in CMMs and the cultivation environment,which might cause serious safety risks to human health.Here,based on the field investigation of the application of PGRs in genuine medicinal materials,we established the multiple residues detection technology for PGRs in CMMs.The technology was performed to detect the PGR residues in 480 samples from 34 kinds of CMMs.And then,we studied the effects of paclobutrazol(PP333)and brassinolide(BR)on the quality of Ophiopogon japonicus and Panax notoginseng respectively,which the application of PGR were popular with these two genuine medicinal materials during production.The results provide important bases for the scientific application,the formulation of PGRs limit standards,the risk assessment and supervision of PGRs in CMMs,the formulation of laws and regulations that restrict the use of PGRs.The main research contents and achievements are as follows:1.The application situation of PGRs in 9 genuine medicinal materials were carried out by field survey.The investigation found that PGRs or PGR-containing fertilizers were commonly used in the cultivation of rhizome medicinal materials.Through field investigation for 9 genuine medicinal materials in 12 counties and cities of 7 genuine producing areas including Sichuan,Yunnan,Shanxi,Gansu,Henan,Ningxia and Guangxi,we found PGRs were commonly used in rhizome medicinal materials including O.japonicus,P.notoginseng,Angelica sinensis,Codonopsis pilosula,Rehmannia glutinosa and Astragalus membranaceu.For example,PP333 has been widely sprayed for more than 15 years in the cultivation of O.japonicus,and BR has also been used in P.notoginseng for nearly 15 years.Notably,‘Zhuanggenling'PGRs or PGR-containing fertilizers were widely used in rhizome medicinal materials.In addition,‘Zhuanggenling' were mostly registered in the form of agricultural fertilizer,and their ingredients were not marked.The significant increase in production has made this type of agricultural fertilizer popular among growers,but this phenomenon has brought potential risks to human,and the quality and safety of TCMs could not be guaranteed.The improper use of PGRs or PGR-containing fertilizers have caused the quality change of the genuine medicinal materials.2.The multiple residues detection technology was established by HPLC-MS/MS for 23 kinds of PGRs in CMMs.It was found that PGR residues in CMMs were widespread after detecting 480 samples from 34 kinds of CMMs.The study established a fast,simple,sensitive and high-throughput multi-residue detection method that can simultaneously determine 23 kinds of PGRs and 12 kinds of pesticides in CMMs.The method was based on a simplified one-step extraction method and dilution pretreatment conbined with HPLC-MS/MS determination.It was applied to the PGR residues detection in 480 samples from 34 kinds of CMMs which collected from 11 CMM markets and 5 genuine producing areas.The results showed that a variety of PGRs were detected in all CMMs,especially 7-10 types of PGRs were detected in these rhizomes medicinal materials such as Ophiopogonis Radix,Notoginseng Radix et Rhizoma,Codonopsis Radix,Angelicae sinensis Radix,Rehmanniae Radix,Atractyodis macrocephalae Rhizome,Chuanxiong Rhizome and Panacis quinquefolia Radix.A total of 14 types of PGRs were detected in 480 batches of CMMs.Among them,atonik(73.75%),Sodium 4-nitrophenol(53.12%),chlormequat(40%)and uniconazole(39.58%)had higher detection rates.The most detected PGR types were up to 10 in Ophiopogonis Radix.The detection rate of PP333 was 100%and the high residual concentrations of PP333 in the most samples of Ophiopogonis Radix were found.In addition,14 kinds of agricultural chemicals which were commonly used in CMMs were tested,the result showed various PGRs were detected in samples registered as fertilizer.The above results indicated that PGRs were widely used in the cultivation of CMMs.3.It was found for the first time that the use of BR could change the ratio of the contents of saponins components in Notoginseng Radix et rhizoma,such as notoginsenoside R1,ginsenoside Rb1,ginsenoside Rd,ginsenoside Re and ginsenoside Rg1.During the cultivation process of P.notoginseng,BR was commonly sprayed to promote the rapid growth of P.notoginseng seedlings.The results showed that BR at the appropriate concentration could promote the growth,survival rate and yield of P.notoginseng.BR could significantly improve the accumulation of notoginsenoside R1;however,it showed no significant effect on the other four saponins components.After using BR,the ratio of various active ingredients of Notoginseng Radix et rhizome has been changed.It is unclear whether or not BR have an positive influence on the quality and efficacy of Notoginseng Radix et rhizome,because the efficacy of CMMs usually depends on the combined action of various active ingredients.Therefore,the scientific spraying of BR on P.notoginseng need be further explored.4.It was found for the first time that 25 saponins and flavonoid metabolites in Ophiopogonis Radix were significantly changed after using PP333.The content of Ophiopogonin D,Ophiopogonin D',Ophiopogonin Ra and Ophiopojaponin C were reduced observably.During the cultivation process of O.japonicus,PP333 was commonly sprayed to promote the production of Ophiopogonis Radix.This study systematic evaluated the effects of PP333 on the contents of 4 steroidal saponins and 5 flavonoids in Ophiopogonis Radix.The results showed that PP333 could significantly reduce the content of Ophiopogonin D,Ophiopogonin D',Ophiopogonin Ra,Ophiopojaponin C,Ophiopogon flavanone C,especially Ophiopogonin D which was decreased by 50.92%?79.09%.UPLC-ESI/Q-TOF-MS/MS based on metabolomics was employed to study the differential metabolites of Ophiopogonis Radix from different sources.The results showed that 25 saponins and flavonoid metabolites in Ophiopogonis Radix had significantly changed after using PP333.Among them,the content of 8 differential metabolites increased compared to the control,and the content of 17 differential metabolites including Ophiopogonin D,Ophiopogonin D' and Ophiopogonin C decreased compared to the control,which further confirmed that the use of PP333 could affect the accumulation of Ophiopogon saponins content.The analysis results showed that different levels of PP333 residue were detected in Ophiopogonis Radix,soil,and water samples,and the residues in some Ophiopogonis Radix samples exceeded the maximum residue limit of food stipulated in GB2763-2019 by more than 2 times.In conclusion,the negative regulation of PP333 on the effective components of Ophiopogonis Radix brings potential harm to the environment and human health.Therefore,we recommended that the application of PP333 should be restricted in the cultivation of O.japonicus. |
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