DNA barcode reference library construction and genetic diversity and structure analysis of Amomum villosum Lour. (Zingiberaceae) populations in Guangdong Province

Background Amomum villosum Lour. is the plant that produces the famous traditional Chinese medicine Amomi Fructus. Frequent habitat destruction seriously threatens A. villosum germplasm resources. Genetic diversity is very important to the optimization of germplasm resources and population protection, but the range of inherited traits within A. villosum is unclear. In this study, we analyzed the genetic diversity and genetic structures of A. villosum populations in Guangdong and constructed a local reference DNA barcode library as a resource for conservation efforts. Methods DNA barcoding and Inter-Simple Sequence Repeat (ISSR) markers were used to investigate the population genetics of A. villosum. Five universal DNA barcodes were amplified and used in the construction of a DNA barcode reference library. Parameters including percentage of polymorphic sites (PPB), number of alleles (Na), effective number of alleles (Ne), Nei’s gene diversity index (H), and Shannon’s polymorphism information index (I) were calculated for the assessment of genetic diversity. Genetic structure was revealed by measuring Nei’s gene differentiation coefficient (Gst), total population genetic diversity (Ht), intra-group genetic diversity (Hs), and gene flow (Nm). Analysis of molecular variance (AMOVA), Mantel tests, unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, and principal co-ordinates (PCoA) analysis were used to elucidate the genetic differentiation and relationship among populations. Results A total of 531 sequences were obtained from the five DNA barcodes with no variable sites from any of the barcode sequences. A total of 66 ISSR bands were generated from A. villosum populations using the selected six ISSR primers; 56 bands, 84.85% for all the seven A. villosum populations were polymorphic. The A. villosum populations showed high genetic diversity (H = 0.3281, I = 0.4895), whereas the gene flow was weak (Nm = 0.6143). Gst (0.4487) and AMOVA analysis indicated that there is obvious genetic differentiation amongA. villosum populations and more genetic variations existed within each population. The genetic relationship of each population was relatively close as the genetic distances were between 0.0844 and 0.3347.

HOẠT TÍNH SINH HỌC CỦA DOXORUBICIN KHI KẾT HỢP VỚI CAO CHIẾT CỦA NĂM LOÀI HỌ GỪNG (ZINGIBERACEAE)

Thân rễ của các loài Zingiber officinale Roscoe (ZO), Zingiber zerumbet (L.) Roscoe ex Sm. (ZZ), Curcuma clovisii Škornič (CC), Curcuma zedoaria (Christm.) Roscoe (CZ) và quả của Amomum villosum Lour (AV) được sử dụng để khảo sát lý trích cao chiết bằng phương pháp ngâm dầm trong ba loại dung môi hữu cơ khác nhau dưới điều kiện tránh sáng tại nhiệt độ phòng. Các dung môi sử dụng bao gồm chloroform (TCM), ethyl aceate (AcOEt) và ethanol (EtOH) với các khoảng thời gian ngâm dầm khảo sát gồm 1, 7, 14 và 30 ngày. Trong đó, TCM và EtOH được đánh giá là hai dung môi thích hợp cho việc trích ly cao chiết. ZZ cho hiệu suất ly trích cao nhất (8%) so với các nguyên liệu còn lại. Hoạt tính kháng tế bào ung thư ruột kết HCT-116 và độc tính trên nguyên bào sợi da người HSF của các cao chiết được đánh giá bằng MTT - test. Kết quả cho thấy tại nồng độ IC25, khi kết hợp các cao chiết này với doxorubicin, đều có ảnh hưởng tích cực trên hoạt tính sinh học. Thành phần hóa học của các cao chiết được phân tích bằng phương pháp sắc ký lỏng phối khổ (LC/MS), sắc ký bản mỏng (TLC) và sắc ký cột.

Chemical Constituents of Functional Food Amomum villosum to Combat Human Diseases

Background: Amomum villosum is an important medicinal and edible plant owing to its valuable compositions of volatile and non-volatile compounds. Objective : In order to reveal the chemical constituents and medicinal values of A. villosum against various diseases. Methods : A critical and comprehensive data mining from 2004 to 2021 was performed based on CNKI, PubMed, and google. Results: 120 Compounds have been reported collectively in seeds, peels, and rhizomes of A. villosum, 10 compounds were common however the content of bornyl acetate was maximum in seeds and peels. Similarly, Amomi fructus of China and Vietnam showed 59 different types of volatile compounds while 13 were common, the round large black fruits (1.56% bornyl acetate and 4.05% volatile oil) and long small red fruits type (1.64% bornyl acetate and 3.04% volatile oil) from China showed good quality and high medicinal values. In addition, Amomi fructus manifested 30~40 kinds of nonvolatile compounds, i.e., phenolic, polysaccharides, saponins, flavonoid, vanillic acid, catechin, epicatechin, polydatin, isoquercitrin, quercitrin, glycosides, inorganic and organic acids. Conclusion: A. villosum is widely used as anti-inflammatory, gastrointestinal protection, anti-obesity, anticancer, antioxidant, analgesic, bacteriostasis, antidiabetic and anti-COVID-19 etc. So, it is manifested that the fruit, flowers, roots, stems, and leaves of A. villosum can be used as a medicine, especially fruit (Amomi fructus=Sharen) as the best functional food for the COVID-19 treatment and gastrointestinal protection as well as prevention of other diseases.

Research on the reproductive biological characteristics of Amomum villosum Lour. and Amomum longiligulare T. L. Wu

Objective To explore the influence of biological characteristics on the yield of Amomum villosum Lour. and Amomum longiligulare T. L. Wu, to find an effective pollen viability evaluation method and storage method to solve the problem of the low yield of Amomum plants. Methods Five germplasm of Amomum plants were used to investigate the effects of the phenological phase, pollen viability, and stigma receptivity on natural and artificial fruit set. Results Amomum longiligulare T. L. Wu showed late flowering, and its natural pollination rate is higher than that of Amomum villosum Lour. In all germplasm, the artificial pollination rate and fruit setting rate are more than 3 times higher than that under natural conditions. Fruits begin to drop seven days after successful pollination, and the fruit drop is basically stable after one month. The hybridization verification showed that TTC method was simpler and more accurate than in vitro germination method. Optimal storage conditions for pollen are 4°C and high humidity. After 36 h of storage, pollen can still be used for artificial cross-pollination or as hybrid parents. Conclusion The special biological characteristics are the fundamental reason for the low natural pollination rate of Amomum plants. The accurate measurement method of Amomum plants pollen is the TTC method, and storage at 4°C and high humidity can increase the yield, which was six times that of the natural yield.

A Comparison of Two Monoterpenoid Synthases Reveals Molecular Mechanisms Associated With the Difference of Bioactive Monoterpenoids Between Amomum villosum and Amomum longiligulare

The fruits of Amomum villosum and Amomum longiligulare are both used medicinally as Fructus Amomi the famous traditional Chinese medicine, however, the medicinal quality of A. villosum is better than that of A. longiligulare. Volatile terpenoids in the seeds, especially bornyl acetate and borneol, are the medicinal components of Fructus Amomi. The volatile terpenoids and transcriptome of developing seeds of A. villosum and A. longiligulare were compared in this study. The result revealed that the bornyl acetate and borneol contents were higher in A. villosum than in A. longiligulare. Additionally, six terpenoid synthase genes (AlTPS1–AlTPS6) were screened from the transcriptome of A. longiligulare, and AlTPS2 and AlTPS3 were found to share 98 and 83% identity with AvTPS2 and AvBPPS (bornyl diphosphate synthase) from A. villosum, respectively. BPPS is the key enzyme for the biosynthesis of borneol and bornyl acetate. Biochemical assays improved that AlTPS2 had an identical function to AvTPS2 as linalool synthase; however, AlTPS3 produced camphene as the major product and bornyl diphosphate (BPP) as the secondary product, whereas AvBPPS produced BPP as its major product. There was only one different amino acid between AlTPS3 (A496) and AvBPPS (G495) in their conserved motifs, and the site-directed mutation of A496G in DTE motif of AlTPS3 changed the major product from camphene to BPP. Molecular docking suggests that A496G mutation narrows the camphene-binding pocket and decreases the BPP-binding energy, thus increases the product BPP selectivity of enzyme. In addition, the expression level of AvBPPS was significantly higher than that of AlTPS3 in seeds, which was consistent with the related-metabolites contents. This study provides insight into the TPS-related molecular bases for the biosynthesis and accumulation differences of the bioactive terpenoids between A. villosum and A. longiligulare. BPPS, the key gene involved in the biosynthesis of the active compound, was identified as a target gene that could be applied for the quality-related identification and breeding of Fructus Amomi.

Ethyl Acetate Fraction of Amomum villosum var. xanthioides Attenuates Hepatic Endoplasmic Reticulum Stress-Induced Non-Alcoholic Steatohepatitis via Improvement of Antioxidant Capacities

Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), affects 25% of the global population. Despite the prevalence of NAFLD worldwide, effective therapeutics are currently lacking. Amomum villosum var. xanthioides (Wall. ex Baker) T.L.Wu & S.J.Chen (AX) is a medicinal herb traditionally used for treating digestive tract disorders in countries across Asia. We aimed to examine the pharmacological effects of the ethyl acetate fraction of AX (AXEF) against tunicamycin (TM)-induced ER stress in a NASH mouse model using C57/BL6J male mice. Following TM injections (2 mg/kg), the mice were orally administrated AXEF (12.5, 25, or 50 mg/kg), silymarin (50 mg/kg), or distilled water daily for 5 days, and the outcomes for fatty liver, inflammation, and oxidative stress were measured in serum or liver tissue levels. AXEF drastically attenuated hepatic ER stress-induced NASH as indicated by decreases in lipid droplet accumulations, serum liver enzymes, hepatic inflammations, and cell death signals in the hepatic tissue and/or serum levels. Interestingly, AXEF showed potent antioxidant effects by quenching reactive oxidative stress and its final product lipid peroxide in the hepatic tissue, specifically an increase in metallothionein (MT). To confirm the underlying actions of AXEF, we observed that AXEF increases MT1 gene promoter activities in the physiological levels. Collectively, AXEF showed antioxidant properties on TM-induced ER stress in a NASH mice model through the improvement of MTs.

Purification, Structural Characterization and Immunomodulatory Effects of Polysaccharides from Amomum villosum Lour. on RAW 264.7 Macrophages

Amomum Villosum Lour. (A. villosum) is a folk medicine that has been used for more than 1300 years. However, study of the polysaccharides of A. villosum is seriously neglected. The objectives of this study are to explore the structural characteristics of polysaccharides from A. villosum (AVPs) and their effects on immune cells. In this study, the acidic polysaccharides (AVPG-1 and AVPG-2) were isolated from AVPs and purified via anion exchange and gel filtration chromatography. The structural characteristics of the polysaccharides were characterized by methylation, HPSEC-MALLS-RID, HPLC, FT-IR, SEM, GC-MS and NMR techniques. AVPG-1 with a molecular weight of 514 kDa had the backbone of → 4)-α-d-Glcp-(1 → 3,4)-β-d-Glcp-(1 → 4)-α-d-Glcp-(1 →. AVPG-2 with a higher molecular weight (14800 kDa) comprised a backbone of → 4)-α-d-Glcp-(1 → 3,6)-β-d-Galp-(1 → 4)-α-d-Glcp-(1 →. RAW 264.7 cells were used to investigate the potential effect of AVPG-1 and AVPG-2 on macrophages, and lipopolysaccharide (LPS) was used as a positive control. The results from bioassays showed that AVPG-2 exhibited stronger immunomodulatory activity than AVPG-1. AVPG-2 significantly induced nitric oxide (NO) production as well as the release of interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α), and upregulated phagocytic capacities of RAW 264.7 cells. Real-time PCR analysis revealed that AVPG-2 was able to turn the polarization of macrophages to the M1 direction. These results suggested that AVPs could be explored as potential immunomodulatory agents of the functional foods or complementary medicine.