The composition, pharmacological effects, related mechanisms and drug delivery of alkaloids from Corydalis yanhusuo.

Corydalis yanhusuo W. T. Wang, also known as yanhusuo, yuanhu, yanhu and xuanhu, is one of the herb components of many Chinese Traditional Medicine prescriptions such as Jin Ling Zi San and Yuanhu-Zhitong priscription. C. yanhusuo was traditionally used to relieve pain and motivate blood and Qi circulation. Now there has been growing interest in pharmacological effects of alkaloids, the main bioactive components of C. yanhusuo. Eighty-four alkaloids isolated from C. yanhusuo are its important bioactive components and can be characterized into protoberberine alkaloids, aporphine alkaloids, opiate alkaloids and others and proper extraction or co-administration methods modulate their contents and efficacy. Alkaloids from C. yanhusuo have various pharmacological effects on the nervous system, cardiovascular system, cancer and others through multiple molecular mechanisms such as modulating neurotransmitters, ion channels, gut microbiota, HPA axis and signaling pathways and are potential treatments for many diseases. Plenty of novel drug delivery methods such as autologous red blood cells, self-microemulsifying drug delivery systems, nanoparticles and others have also been investigated to better exert the effects of alkaloids from C. yanhusuo. This review summarized the alkaloid components of C. yanhusuo, their pharmacological effects and mechanisms, and methods of drug delivery to lay a foundation for future investigations.

Mechanistic analysis for the origin of diverse diterpenes in Tripterygium wilfordii.

Tripterygium wilfordii is a valuable medicinal plant rich in biologically active diterpenoids, but there are few studies on the origins of these diterpenoids in its secondary metabolism. Here, we identified three regions containing tandemly duplicated diterpene synthase genes on chromosomes (Chr) 17 and 21 of T. wilfordii and obtained 11 diterpene synthases with different functions. We further revealed that these diterpene synthases underwent duplication and rearrangement at approximately 2.3-23.7 million years ago (MYA) by whole-genome triplication (WGT), transposon mediation, and tandem duplication, followed by functional divergence. We first demonstrated that four key amino acids in the sequences of TwCPS3, TwCPS5, and TwCPS6 were altered during evolution, leading to their functional divergence and the formation of diterpene secondary metabolites. Then, we demonstrated that the functional divergence of three TwKSLs was driven by mutations in two key amino acids. Finally, we discovered the mechanisms of evolution and pseudogenization of miltiradiene synthases in T. wilfordii and elucidated that the new function in TwMS1/2 from the terpene synthase (TPS)-b subfamily was caused by progressive changes in multiple amino acids after the WGT event. Our results provide key evidence for the formation of diverse diterpenoids during the evolution of secondary metabolites in T. wilfordii.

Key Glycosyltransferase Genes of Panax notoginseng: Identification and Engineering Yeast Construction of Rare Ginsenosides.

Panax notoginseng is one of the most famous valuable medical plants in China, and its broad application in clinical treatment has an inseparable relationship with the active molecules, ginsenosides. Ginsenosides are glycoside compounds that have varied structures for the diverse sugar chain. Although extensive work has been done, there are still unknown steps in the biosynthetic pathway of ginsenosides. Here, we screened candidate glycosyltransferase genes based on the previous genome and transcriptome data of P. notoginseng and cloned the full length of 27 UGT genes successfully. Among them, we found that PnUGT33 could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides by extending the sugar chain. We further analyzed the enzymatic kinetics and predicted the catalytic mechanism of PnUGT33 by simulating molecular docking. After that, we reconstructed the biosynthetic pathway of rare ginsenoside Rg3 and gypenoside LXXV in yeast. By combining the Golden Gate method and overexpressing the UDPG biosynthetic genes, we further improved the yield of engineering yeast strain. Finally, the shake-flask culture yield of Rg3 reached 51 mg/L and the fed-batch fermentation yield of gypenoside LXXV reached 94.5 mg/L, which was the first and highest record.

Probing the functions of friedelane-type triterpene cyclases from four celastrol-producing plants.

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

[Research progress in synthetic biology of active compounds in Chinese medicinal plants].

Mecicinal plants boast abundant natural compounds with significant pharmacological activity, and such compounds, featuring diversified and complex structures, can be used for research and development of drugs. At present, these natural compounds are directly extracted from herbs which, however, suffer from damaged wild resources and shortage of planting resources attributing to the increasing demand. Moreover, the low content in medicinal plants and complex structures are another challenge to the research and development of drugs. Heterologous synthesis with synthetic biology methods is a solution that has attracted wide attention. Synthetic bio-logy for the production of natural active compounds in Chinese medicinal plants involves the exploration of key enzymes in compound bio-synthetic pathways from plants, analysis of enzyme functions and mechanisms, and reconstruction and optimization of biosynthetic pathways in microorganisms for efficient synthesis of compounds. This study briefed the development process of synthetic biology and the biosynthetic pathways of terpenoids, alkaloids, and flavonoids, and summarized the related strategies of synthetic biology such as the reconstruction and optimization of metabolic pathways, regulation of fermentation process, and strain improvement, and the latest applications of heterogeneous synthetic biology in the production of natural compounds from Chinese medicinals. This study is expected to serve as a reference for the efficient production of terpenoids, alkaloids, flavonoids, and other active compounds from Chinese medicinal plants with strategies of synthetic biology.

The chromosome-level reference genome assembly for Panax notoginseng and insights into ginsenoside biosynthesis.

Panax notoginseng, a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng, with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax. Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng. The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng.

Genome of Tripterygium wilfordii and identification of cytochrome P450 involved in triptolide biosynthesis.

Triptolide is a trace natural product of Tripterygium wilfordii. It has antitumor activities, particularly against pancreatic cancer cells. Identification of genes and elucidation of the biosynthetic pathway leading to triptolide are the prerequisite for heterologous bioproduction. Here, we report a reference-grade genome of T. wilfordii with a contig N50 of 4.36 Mb. We show that copy numbers of triptolide biosynthetic pathway genes are impacted by a recent whole-genome triplication event. We further integrate genomic, transcriptomic, and metabolomic data to map a gene-to-metabolite network. This leads to the identification of a cytochrome P450 (CYP728B70) that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis. We think the genomic resource and the candidate genes reported here set the foundation to fully reveal triptolide biosynthetic pathway and consequently the heterologous bioproduction.

Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.

The gibberellin 13-oxidase that specifically converts gibberellin A9 to A20 in Tripterygium wilfordii is a 2-oxoglutarate-dependent dioxygenase.

MAIN CONCLUSION: A novel GA13-oxidase ofTripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase. It specifically catalyzes the conversion of GA9to GA20, but not GA4to GA1. Gibberellins (GAs) play essential roles in plant growth and development. Previous characterization of GA20- and GA3-oxidases yielded a large number of genetic elements that can interconvert different GAs. However, enzymes that catalyze the 13-hydroxylation step are rarely identified. Here, we report that the GA13-oxidase of Tripterygium wilfordii, TwGA13ox, is a 2-oxoglutarate-dependent dioxygenase instead of reported cytochrome P450 oxygenases, among 376 differential proteins in comparative proteomics. Phylogenetic analysis showed that the enzyme resides in its own independent branch in the DOXC class. Unexpectedly, it specifically catalyzes the conversion of GA9 to GA20, but not GA4 to GA1. Contrary to the previous research, TwGA13ox transcriptional expression was upregulated ~ 146 times by exogenous application of methyl jasmonate (MeJA). RNAi targeting of TwGA13ox in T. wilfordii led to an 89.9% decrease of triptolide, a diterpenoid epoxide with extensive anti-inflammatory and anti-tumor properties. In subsequent MeJA supplementation experiments, triptolide production increased 13.4-times. TwGA13ox displayed root-specific expression. Our results provide a new GA13-oxidase from plants and elucidate the metabolic associations within the diterpenoid biosynthetic pathway (GAs, triptolide) at the genetic level.

A specific UDP-glucosyltransferase catalyzes the formation of triptophenolide glucoside from Tripterygium wilfordii Hook. f.

Tripterygium wilfordii Hook. f. is a perennial woody vine member of the Celastraceae family. As a traditional Chinese medicine, it contains complex chemical components and exerts various pharmacological activities. In the present study, we identified a glucosyltransferase, TwUGT1, that can catalyze the synthesis of an abietane-type diterpene glucoside, namely, triptophenolide14-O-beta-D-glucopyranoside, and investigated the pharmacological activity of triptophenolide glucoside in diverse cancer cells. Triptophenolide glucoside exhibited significant inhibitory effects on U87-MG, U251, C6, MCF-7, HeLa, K562, and RBL-2H3 cells as determined by pharmacological analysis. The triptophenolide glucoside content of T. wilfordii was analyzed using Agilent Technologies 6490 Triple Quad LC/MS. The glucosyltransferase TwUGT1 belongs to subfamily 88 and group E in family 1. Molecular docking and site-directed mutagenesis of TwUGT1 revealed that the His30, Asp132, Phe134, Thr154, Ala370, Leu376, Gly382, His387, Glu395 and Gln412 residues play crucial roles in the catalytic activity of triptophenolide 14-O-glucosyltransferase. In addition, TwUGT1 was also capable of glucosylating phenolic hydroxyl groups, such as those in liquiritigenin, pinocembrin, 4-methylumbelliferone, phloretin, and rhapontigenin.

Biogenic Synthesis and Spatial Distribution of Endogenous Phytohormones and Ginsenosides Provide Insights on Their Intrinsic Relevance in Panax ginseng.

Ginseng, the root and rhizome of Panax ginseng C. A. Mey., is a well-known and valuable traditional Chinese medicine. The pharmacological activities of ginseng are mainly attributed to the presence of ginsenosides, which are considered to be critical biomarkers for evaluating ginseng quality. The biosynthesis of triterpenes, which serve defensive functions in plants, is regulated by endogenous phytohormones that play key roles in growth and defense of plant populations. However, the role of major hormones that are closely related to secondary metabolism pathways in P. ginseng is poorly understood. To gain insight into their potential correlation, we performed a spatial synthesis analysis and studied the distribution of endogenous phytohormones and ginsenosides in different tissue regions of the entire P. ginseng plant. Gibberellins are growth hormones that accumulate in the fiber root. In contrast, abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA), which are considered stress hormones, were predominantly found in the leaf and leaf peduncle. We observed a tissue-specific distribution of phytohormones consistent with the expression of genes involved in hormone biosynthesis that influenced ginsenoside synthesis and distribution. The aim of this study was to investigate the role of different endogenous phytohormones on triterpene metabolites in ginseng innate immunity.

Identification and functional characterization of diterpene synthases for triptolide biosynthesis from Tripterygium wilfordii.

Tripterygium wilfordii, which has long been used as a medicinal plant, exhibits impressive and effective anti-inflammatory, immunosuppressive and anti-tumor activities. The main active ingredients are diterpenoids and triterpenoids, such as triptolide and celastrol, respectively. A major challenge to harnessing these natural products is that they are found in very low amounts in planta. Access has been further limited by the lack of knowledge regarding their underlying biosynthetic pathways, particularly for the abeo-abietane tri-epoxide lactone triptolide. Here suspension cell cultures of T. wilfordii were found to produce triptolide in an inducible fashion, with feeding studies indicating that miltiradiene is the relevant abietane olefin precursor. Subsequently, transcriptome data were used to identify eight putative (di)terpene synthases that were then characterized for their potential involvement in triptolide biosynthesis. This included not only biochemical studies which revealed the expected presence of class II diterpene cyclases that produce the intermediate copalyl diphosphate (CPP), along with the more surprising finding of an atypical class I (di)terpene synthase that acts on CPP to produce the abietane olefin miltiradiene, but also their subcellular localization and, critically, genetic analysis. In particular, RNA interference targeting either both of the CPP synthases, TwTPS7v2 and TwTPS9v2, or the subsequently acting miltiradiene synthase, TwTPS27v2, led to decreased production of triptolide. Importantly, these results then both confirm that miltiradiene is the relevant precursor and the relevance of the identified diterpene synthases, enabling future studies of the biosynthesis of this important bioactive natural product.

Cloning and functional analysis of two sterol-C24-methyltransferase 1 (SMT1) genes from Paris polyphylla.

The biosynthetic pathways of phytosterols and steroidal saponins are located in two adjacent branches which share cycloartenol as substrate. The rate-limiting enzyme S-adenosyl-L-methionine-sterol-C24-methyltransferase 1 (SMT1) facilitates the metabolic flux toward phytosterols. It catalyzes the methylation of the cycloartenol in the side chain of the C24-alkyl group, to generate 24(28)-methylene cycloartenol. In this study, we obtained two full-length sequences of SMT1 genes from Pari polyphylla, designated PpSMT1-1 and PpSMT1-2. The full-length cDNA of PpSMT1-1 was 1369 bp long with an open reading frame (ORF) of 1038 bp, while the PpSMT1-2 had a length of 1222 bp, with a 1005 bp ORF. Bioinformatics analysis confirmed that the two cloned SMTs belong to the SMT1 family. The predicted function was further validated by performing in vitro enzymatic reactions, and the results showed that PpSMT1-1 encodes a cycloartenol-C24-methyltransferase, which catalyzes the conversion of cycloartenol to 24-methylene cycloartenol, whereas PpSMT1-2 lacked this catalytic activity. The tissue expression patterns of the two SMTs revealed differential expression in different organs of Paris polyphylla plants of different developmental stage and age. These results lay the foundation for detailed genetic studies of the biosynthetic pathways of steroid compounds, which constitute the main class of active substances found in P. polyphylla.

[Cloning and protein expression analysis of monoterpene synthase gene TwMS in Tripterygium wilfordii].

In this study, we cloned a monoterpene synthases, TwMS from Tripterygium wilfordii suspension cells. TwMS gene contained a 1 797 bp open reading frame (ORF), encoding a polypeptide of 579 amino acids, which deduced isoelectric point (pI) was 6.10 and the calculated molecular weight was 69.75 kDa. Bioinformation analysis showed that the sequence of TwMS was consistent with the feature of monoterpene synthases. Differential expression analysis revealed that the relative expression level of TwMS increased significantly after being induced by methyl jasmonate (MeJA). The highest expression level occurred at 24 h. TwMS protein was successfully expressed in Escherichia coli BL21 (DE3), which laid the foundation for identifying the function of T. wilfordii monoterpene synthases.

[Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii].

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

Functional characterization of NES and GES responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool in Tripterygium wilfordii.

Triptolide and celastrol, two principal bioactive compounds in Tripterygium wilfordii, are produced from geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate ((E,E)-FPP) through terpenoid biosynthesis pathway. However, little is known about T. wilfordii terpene synthases which could competitively utilize GGPP and (E,E)-FPP as substrates, producing C15 and C20 tertiary alcohols. Here we firstly cloned the genes encoding nerolidol synthase (NES) and geranyllinalool synthases (GES1, GES2), which are responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool. In vitro characterization of recombinant TwNES and TwGES1 revealed both were functional enzymes that could catalyze the conversion of (E,E)-FPP and GGPP to (E)-nerolidol and (E,E)-geranyllinalool, which were consistent with the results of yeast fermentation. Biochemical characterization revealed TwNES and TwGES1 had strong dependency for Mg2+, Km and Kcat/Km values of TwNES for (E,E)-FPP were 12.700 µM and 0.029 s-1/µM, and TwGES1 for GGPP were 2.039 µM and 0.019 s-1/µM. Real-time PCR analysis showed the expression levels of NES and GES1 increased by several fold in the suspension cells treated with alamethicin, indicating TwNES and TwGES1 are likely to utilize GGPP and (E,E)-FPP to generate tertiary alcohols as precursor of plant volatiles, which play important roles in the ecological interactions between T. wilfordii and other organisms.

Functional characterization of ent-copalyl diphosphate synthase, kaurene synthase and kaurene oxidase in the Salvia miltiorrhiza gibberellin biosynthetic pathway.

Salvia miltiorrhiza Bunge is highly valued in traditional Chinese medicine for its roots and rhizomes. Its bioactive diterpenoid tanshinones have been reported to have many pharmaceutical activities, including antibacterial, anti-inflammatory, and anticancer properties. Previous studies found four different diterpenoid biosynthetic pathways from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) in S. miltiorrhiza. Here, we describe the functional characterization of ent-copalyl diphosphate synthase (SmCPSent), kaurene synthase (SmKS) and kaurene oxidase (SmKO) in the gibberellin (GA) biosynthetic pathway. SmCPSent catalyzes the cyclization of GGPP to ent-copalyl diphosphate (ent-CPP), which is converted to ent-kaurene by SmKS. Then, SmKO catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid. Our results show that the fused enzyme SmKS-SmCPSent increases ent-kaurene production by several fold compared with separate expression of SmCPSent and SmKS in yeast strains. In this study, we clarify the GA biosynthetic pathway from GGPP to ent-kaurenoic acid and provide a foundation for further characterization of the subsequent enzymes involved in this pathway. These insights may allow for better growth and the improved accumulation of bioactive tanshinones in S. miltiorrhiza through the regulation of the expression of these genes during developmental processes.

The MVA pathway genes expressions and accumulation of celastrol in Tripterygium wilfordii suspension cells in response to methyl jasmonate treatment.

Celastrol is an important bioactive triterpenoid in traditional Chinese medicinal plant, Tripterygium wilfordii. Methyl Jasmonate (MJ) is a common plant hormone which can regulate the secondary metabolism in higher plants. In this study, the mevalonate (MVA) pathway genes in T. wilfordii were firstly cloned. The suspension cells of T. wilfordii were elicited by MJ, and the expressions of MVA pathway genes were all enhanced in different levels ranging from 2.13 to 22.33 times of that at 0 h. The expressions were also enhanced compared with the CK group separately. The accumulation of celastrol in the suspension cells after the treatment was quantified and co-analyzed with the genes expression levels. The production of celastrol was significantly increased to 0.742 mg g(-1) after MJ treatment in 288 h which is consistent with the genes expressions. The results provide plenty of gene information for the biosynthesis of terpenoids in T. wilfordii and a viable way to improve the accumulation of celastrol in T. wilfordii suspension cells.

Cloning and functional characterization of an isopentenyl diphosphate isomerase gene from Tripterygium wilfordii.

Tripterygium wilfordii Hook.F. is one of the most valuable medicinal plants because it contains a large variety of active terpenoid compounds, including triptolide, celastrol, and wilforlide. All of the pharmacologically active secondary metabolites are synthesized from the 2-C-methyl-d-erythritol 4-phosphate and mevalonate pathway in the isoprenoid biosynthetic system. The key step in this pathway is the isomerization of dimethylallyl diphosphate and isopentenyl diphosphate, which is catalyzed by isopentenyl diphosphate isomerase (IPI). In the present study, a full-length cDNA encoding IPI (designate as TwIPI, GenBank accession no.KT279355) was cloned from a suspension of cultured cells from T. wilfordii. The full-length cDNA of TwIPI was 1,564 bp and encoded a polypeptide of 288 amino acids. The bioinformatics analysis showed that the deduced TwIPI sequence contained the TNTCCSHPL and WGEHELDY motif. The transcription level of the TwIPI in the suspension cells increased almost fivefold after treatment with methyl jasmonate as an elicitor. A functional color assay in Escherichia coli indicated that TwIPI could promote the accumulation of lycopene and encoded a functional protein.

Molecular identification of antelope horn by melting curve analysis.

Antelope horn is a valuable Chinese traditional medicine and widely used in clinic. However, with the deterioration of antelope's living environment and a lot of killing, the saiga population begins falling and in some places plummet. Since the increasing demand of this expensive and good bioactive medicine, the horn of artiodactyla animals is often used as the antelope horn. The adulterated or impostor not only cause damage to clinical medicine but also affect the antelope resources protection and sustainable development. Here, in order to establish a melting curve analysis (MCA) method to distinguish the antelope horn from other animal horns and identify the decoction pieces and Chinese patent medicine in a fast and easy way, animal horns and its decoction pieces, Chinese patent medicines were collected from the market and the DNA of all the collected samples were extracted. The melting curve of two universal fragments (COI and Cyt b) was scanned and Cyt b was selected as feasibility fragment for identifying authentic antelope horn from eight adulterant animal horns. After optimizing the condition for MCA, inspecting the precision and the replication of the method, a reference melting curve modern was established and we performed MCA on the antelope horns, fakes, and adulterants on a 1:1 mix, decoction pieces, and Chinese patent medicine. Thus, this study provides fast and easy methods so that MCA can detect the truth, fakes, and adulterations of antelope horns.