Analysis of quality difference of different specifications and different origins of Citri Grandis Exocarpium / 中国药房

OBJECTIVE To study the quality difference of different specifications of Citri Grandis Exocarpium from different origins，and to provide reference for the orderly development of Citri Grandis Exocarpium industry. METHODS Different specifications [ Citrus grandis ‘Tomentos’young fruit ，Citrus grandis （L.）Osbeck young fruits ，exocarp] of 93 batches of Citri Grandis Exocarpium medicinal materials （decoction pieces ）from different origins [ Citrus grandis ‘Tomentosa’or Citrus grandis （L.）Osbeck] were taken as samples. The contents of naringin and rhoifolin in samples were determined by HPLC. Through pheatmap parameters of R language ，heatmap was drawn for the contents of naringin and rhoifolin according to origins and specifications （young fruit and exocarp ）. RESULTS Of 93 batches of samples ，the contents of naringin and rhoifolin were 16.52-214.64 and 1.03-10.96 mg/g，respectively. Among different specifications ，the contents of naringin and rhoifolin in the young fruit were the highest （their average contents were 108.96 and 6.30 mg/g respectively ）. Heatmap analysis of R language content showed that the contents of naringin and rhoifolin in Citri Grandis Exocarpium from origin of C. grandis ‘Tomentosa’were generally higher than those from origin of C. grandis （L.）Osbeck. Of different specifications of Citri Grandis Exocarpium from origins，the contents of naringin and rhoifolin were higher in KTP young fruit relatively. CONCLUSIONS The quality of Citri Grandis Exocarpium from origin of C. grandis ‘Tomentosa’with the young fruit as specification is the best.

Optimization of Determination Methods for Scopoletin from Caulis Erycibes / 广州中医药大学学报

Objective To optimize the high performance liquid chromatography (HPLC) method for determining the content of scopoletin from Caulis Erycibes. Methods Methanol-25% HCl ( v/v, 4 : 1) solvent was used to extract scopoletin. HPLC method was performed on Waters XBridge Shield RP18 column (4.6 mm × 250 mm, 5μm) with the mobile phase consisting of acetonitrile ( A) and 0.16% ( v/v) acetic acid ( B) solution by gradient elution. The detection wavelength was 298 nm and the flow rate was set at 1.0 mL/min. Results The linear range of scopoletin from Caulis Erycibes was 2.83-118 μg/mL, and the recovery rate was 99.47% ( sR=1.07%). Conclusion The optimized method is simple, specific and accurate, and can provide reference for content determination of scopoletin in Caulis Erycibes.

Quality Evaluation of Cortex Ilicis Rotundae Medicinal Materials / 广州中医药大学学报

Objective To investigate and evaluate the quality of Cortex Ilicis Rotundae medicinal materials in Chinese herbal medicine market. Methods Twenty-two batches of Cortex Ilicis Rotundae commercial medicinal materials were identified and analyzed by characteristic identification, microscopic identification, thin layer chromatography ( TLC) and extractives determination, and the contents of syringin and pedunculoside were detected by high performance liquid chromatography (HPLC) according to the Chinese Pharmacopoeia published in 2010. And then the quality of the medicinal material of Cortex Ilicis Rotundae was evaluated comprehensively. Results Of the 22 batches of Cortex Ilicis Rotundae medicinal materials, 2 batches were adulterated with fake Cortex Ilicis Rotundae, one batch was inferior and was mixed with counterfeits, and the quality of 12 batches was also poor. Conclusion At present, the quality of Cortex Ilicis Rotundae medicinal materials in Chinese herbal medicine market varies greatly, and adulterants and the inferior are common.

Quality Evaluation of Medicinal Materials of Radix et Caulis Ilicis Asprellae from Pingyuan Planting Base / 广州中医药大学学报

Objective To evaluate the quality of Radix et Caulis Ilicis Asprellae from Pingyuan planting base and Chinese herbal medicine market. Methods The water- and alcohol-soluble extracts from 19 batches of Radix et Caulis Ilicis Asprellae medicinal materials were detected according to Appendix ⅨH, ⅩA of the Chinese Pharmacopoeia ( 2010 edition). And the quality of the medicinal materials was evaluated by microscopic identification technology according to the method for Radix et Caulis Ilicis Asprellae recorded in Guangdong Provincial Chinese Medicine Standard, and then thin layer chromatography ( TLC) was optimized to establish the high performance liquid chromatography (HPLC) fingerprint. The HPLC was performed on Waters XBridgeTM C18 column (250 mm × 4.6 mm, 5μm) with acetonitrile(A)-0.2% (v/v) phosphorus acid (B) as the mobile phase by gradient elution, flow rate was 1.0 mL/min, and detection wavelength was 220 nm. Results The results of sample characters, TLC and microscopic identification showed that the samples of Radix et Caulis Ilicis Asprellae in Chinese herbal medicine markets were certified products, but stems and roots were blended. Seven common peaks were showed by HPLC and confirmed by similarity analytical software. The similarity of 15 batches of planting base samples was all above 0.9. Of 19 batches of the commercial samples, the similarity of 11 batches was above 0.9. The alcohol-soluble extract contents were in the range of 64.55 mg/g to 186.18 mg/g. Conclusion The medicinal materials of Radix et Caulis Ilicis Asprellae from Chinese herbal medicine market are certified products, but the qualities vary greatly for the blending of stems and roots and inadequate growth years. The quality of materials from planting base is better. The established method is helpful for the quality evaluation and control of Radix et Caulis Ilicis Asprellae.

Quality Evaluation of Lignum Dalbergiae Odoriferae Medicinal Materials / 广州中医药大学学报

Objective To evaluate the quality of medicinal materials of Lignum Dalbergiae Odoriferae in Chinese herbal medicine market. Methods Eighteen batches of commercial medicinal materials of Lignum Dalbergiae Odoriferae were identified and analyzed by macroscopical identification, thin layer chromatography (TLC), and volatile oil assay according to the Chinese Pharmacopoeia published in 2010. Visible spectrometry was used to determine the content of total flavonoids from the qualified samples. And the gas chromatography was applied to evaluate the content of nerolidol from volatile oils of the qualified samples. Results Only 33.3%of the samples met the standard of Chinese Pharmacopoeia. The total flavonoid content of the qualified samples was in the range of 21.6-29.0 mg/g. The content of nerolidol from volatile oils of the qualified samples was in the range of 294-574 mg/g. Conclusion At present, the quality of medicinal materials of Lignum Dalbergiae Odoriferae in Chinese herbal medicine market and in clinic varies greatly, and adulterants and inferior are common. The contents of chemical components in different batches of samples are significant different.

Studies on Quality Standard of Radix Toddaliae Asiaticae / 广州中医药大学学报

Objective To establish the quality standard of Radix Toddaliae Asiaticae. Methods Thin layer chromatography ( TLC) and high performance liquid chromatography ( HPLC) were used to identify and determine chloride nitidine and toddalolactone in Radix Toddaliae Asiaticae. The moisture and total ash contents were detected according to the methods recorded in appendix of Chinese Pharmacopeia (2010 edition) . Results Toddalolactone and chloride nitidine were detectable by TLC, the spots were clear and the dissociation was good. The established HPLC method was simple and accurate. The linear ranges of toddalolactone and chloride nitidine in Radix Toddaliae Asiaticae were 2.84~42.6 μg/mL and 25.6~385 μg/mL, and their recovery rates were 99.2 % ( RSD=1.12%) and 100 % ( RSD=0.71%) , respectively. The content of moisture was in the range of 75.8~98.9 mg/g and that of total ash was in the range of 12.4~33.6 mg/g. Conclusion The developed method is specific and accurate, and can provide useful reference for establishing quality standard of Radix Toddaliae Asiaticae.

Study on Callus Induction in Explant of Aquiliaria sinensis Root / 广州中医药大学学报

Objective To study the conditions of callus induction with the roots of Aquilaria sinensis as explants. Methods Two sources of roots of Aquiliaria sinensis were selected as the explants. The effects of sterilization methods and the combination of different concentrations of phytohormones on callus induction were evaluated. Results When Aquiliaria sinensis root seedling was sterilized in 0.01mg/mL HgCl2 solution for 3 minutes, the sterilized effect was the best. The optimal callus induction medium was MS+0.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) +0.1 mg/L 6-benzylaminopurine (6-BA). Aseptic Aquiliaria sinensis root seedling cultivated in callus induction medium containing MS+1.0 mg/L naphthalene acetic acid ( NAA) +0.8 mg/L 6-BA achieved the highest callus induction rate. Conclusion Callus can be induced from two sources of Aquilaria sinensis roots. The induction rate of callus is lower when the explant root seedling is cultivated using 2,4-D alone as inducer, and is increased when used together with 6-BA.

In-vitro Antibacterial Activity of Herba Taraxaci Extract on Escherichia coli / 广州中医药大学学报

Objective To evaluate the inhibitory activity of Herba Taraxaci extract on Escherichia coli DH5α (E. coli DH5α) and to investigate proteomic response of E. coli. Methods Medicinal powder of Herba Taraxaci was extracted with the solvents of different polarity ( n-hexane, ethyl acetate, distilled water) , and then the obtained 8 different extracts were subjected to thin layer chromatography ( TLC) analysis. Microdilution method was performed to detect the minimum inhibitory concentration ( MIC) of different extracts and the growth curves were described. The protein expression profiles of E . coli treated with the extracts were analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis ( SDS-PAGE) and two dimensional electrophoresis (2-DE) . Results Water decoction of Herba Taraxaci could obviously suppress the growth of E. coli with a MIC of 1.95 mg/mL. The different extractions exhibited no antibacterial activity except ethyl acetate phase 3 with a MIC of 0.13 mg/mL, which was equal to 19.23 mg/mL of crude drugs. The results of TLC analysis showed that chlorogenic acid was undetectable in n-hexane extract and ethyl acetate phase 1 extract, and ethyl acetate phase 2 and 3 extracts showed obviously increased spots. The results of SDS-PAGE and 2-DE showed that water decoction of Herba Taraxaci had inhibitory effect on the expression of functional protein. The results of 2-DE showed that after treatment with ethyl acetate phase 3 at the concentration of 2 × MIC for 21 hours, the amount of protein spots were 92 less than those of the blank control group, the spots of E. coli DH5α soluble protein with expression amount down-regulated doubly were 24, and those with expression amount up-regulated doubly were 19. Ethyl acetate phase 3 extract had an effect on down-regulating the protein expression of E. coli DH5α soluble protein pH3-10, and water decoction of Herba Taraxaci had inhibitory effect on E. coli DH5αprotein expression. Conclusion Herba Taraxaci has significant antibacterial activity on E. coli DH5α, and the water-soluble fraction of chlorogenic acid and caffeic acid might be the active components. The possible antibacterial mechanism may be related with the regulation of bacterial protein expression.

Experimental Study of Anti-inflammation of Durian Peel Extract / 广州中医药大学学报

Objective To investigate the anti-inflammatory effects and mechanism of durian peel extracts (DPE). Methods The in vivo anti-inflammation effects of DPE were examined by carrageenin-induced mice paw edema test and allergic contact dermatitis test induced by 2, 4-DNFB. And the in vitro anti-inflammation effects of DPE were evaluated with methyl thiazolyl tetrazolium ( MTT) assay in RAW 264.7 cell model of inflammation induced by lipopolysaccharide (LPS). Results The results of animal experiments showed that DPE groups could markedly relieve mice paw edema induced by carrageenin ( P<0.01 or P<0.001 compared with blank group). DPE could effectively inhibit the allergic contact dermatitis induced by 2, 4-DNFB in mice, showing good dose-effect relationship. The results of in-vitro test showed that DPE at the given concentrations had no influence on RAW 264.7 cell proliferation. Tumor necrosis factor alpha ( TNF-α) , interleukin 6 ( IL-6) , interleukin 1 beta (IL-1β), nitric oxide (NO) and nuclear factor kappaB (NF-кВ ) were observably inhibited, and anti-inflammatory cytokine IL-10 was enhanced by 25 and 50 mg/L of DPE. Conclusion DPE exert potential anti-inflammation effect, and the mechanism might be related to its inhibition of NF-кВsignal pathway.

Diagnosis and Comprehensive Evaluation of Soil Fertility of Good Agricultural Practices Base for Spatholobus suberectus Dunn. in Pingyuan of Guangdong Province / 广州中医药大学学报

Objective To investigate the status of soil fertility of Good Agricultural Practices ( GAP) base for Spatholobus suberectus Dunn. (SSD) in Pingyuan county of Guangdong province, thus to provide reference for GAP research and the subsequent fertilization for SSD. Methods The deep layer and superficial layer of GAP soil were collected for the physiochemical detection and nutrient assay. Compared with the classification standard of the second national general soil investigation, single base soil fertility index was diagnosed and the comprehensive soil fertility was evaluated with modified Nemoro Index. Results The soil pH value and the contents of exchangeable calcium and magnesium were unbalanced, and the contents of macroelements of nitrogen and phosphonium, microelements, and organic matter were low. Therefore, the measures for improving the base soil fertility should be as follows: ( 1) soil amendments of bentonite, gypsum and slaked lime should be used to adjust the soil pH value; ( 2) each plant should be given 10 kg of slaked organic fertilizer as base fertilizer; ( 3) in the process of nurturing, some special micro-fertilizer solution should be used to treat the cut slips, and 5 kg of urea should be used for every 667 meter square of land; ( 4) besides compound fertilizer, every 667 meter square of land should be fertilized with 15 kg of ammonium dihydrogen phosphate for the supplement of nitrogen and phosphorus, and slaked lime and magnesium carbonate should be used for the supplement of soil moderate-quantity elements after transplantation. Conclusion The comprehensive fertility of Pingyuan GAP base for Spatholobus suberectus Dunn. is at low level, and should be improved in combination with GAP requirements.

Analysis of Fingerprints and Formononetin Content of Caulis Spatholobi from Different Habitats by High Performance Liquid Chromatography / 广州中医药大学学报

Objective To establish the fingerprints and formononetin content determination method for Caulis Spatholobi from different habitats by high performance liquid chromatography ( HPLC) , thus to control the quality of Caulis Spatholobi. Methods Reversed phase-high performance liquid chromatography (RP-HPLC) for fingerprint was performed on Feini Gen RedClassical AQ-C18 column ( 4.6 mm × 250 mm, 5 μm) with acetonitrile-0.1%acetic acid solution as the mobile phase by gradient elution, and the detection wavelength was 260 nm. High performance liquid chromatography-diode array detector ( HPLC-DAD) for the determination of formononetin content was performed on AcclaimTM 120-C18 column ( 4.6 mm × 250 mm, 5 μm) with acetonitrile-water solution by isocratic elution, the detection wavelength was 254 nm, the flow rate was 1.0 mL/min and the column temperature was 25℃. Results The standard fingerprint of Caulis Spatholobi was set up through the evaluation of the fingerprints of 24 batches of Caulis Spatholobi samples from different habitats. Thirteen common peaks were identified with reference to formononetin peak, and the content of formononetin was determined by HPLC-DAD method. The similarity of the fingerprints of Caulis Spatholobi from different habitats and their formononetin content had great differences. Conclusion The established method is simple, accurate, highly sensitive, and repeatable, and can be applied for the quality control of Caulis Spatholobi.

Transcriptomic Analysis and Systematic Mining of Genes Involved in Biosynthetic Pathway of Triterpenoid Saponins in Ilex Asprella / 世界科学技术-中医药现代化

This study was aimed to screen candidate genes involved in the triterpenoid saponins biosynthetic pathway of the Ilex asprella root. The Illumina platform was applied to perform transcriptomic sequencing of I. asprella root, followed by a series of bioinformatics analysis. The results showed that a total of 272 candidate unigenes were anno-tated to be involved in the biosynthetic pathway of terpenoid in the transcriptome of I. asprella root, including 72 u-nigenes for the upstream pathway and 26 unigenes for cyclization, oxidation and glycosylation in the downstream pathway. Phylogenetic analysis was carried out to further analyze the evolution relationship of some candidate uni-genes and their homologous genes. Two genes IaA S1 and IaA S2 were proved to be mixed amyrin synthases in yeast expression system. Moreover, IaA S1 was identified to one of the rare ASs with α-amyrin as the major product. It was concluded that a series of candidate genes, which might be involved in the biosynthetic pathway of triterpenoid saponins, were screened out from the transcriptome of I. asprella root. Further investigation of these candidate genes will provide insight into their actual functions in the triterpenoid saponins biosynthetic pathway in I. asprella.

Overexpression of HMGR and DXR from Amomum villosum Lour . Affects the Biosynthesis of Terpenoids in Tobacco / 世界科学技术-中医药现代化

HMGR and DXR are key enzymes of terpenoids biosynthesis pathway. This study was aimed to discuss the effects of overexpression of HMGR and DXR from A momum villosum Lour. on the biosynthesis of terpenoids in transgenic tobacco. The real-time fluorescence quantitative PCR (RT-qPCR) was used to analyze the expression level of AvHMGR and AvDXR. Then, enzyme activities of HMGR and DXR were determined by spectrometer using the substrate-specific method. Different terpenoids were detected by GC-MS. The results showed that individual overex-pression of HMGR/DXR can inhibit the enzyme activities of HMGR and DXR but promote the biosynthesis of men-thene, neophytediene, cembrenene and sterol. The co-overexpression of HMGR and DXR had different enzyme activ-ities and can promote the biosynthesis of sterol and phytol, but inhibit the biosynthesis of neophytadiene. It was con-cluded that the overexpression of HMGR and DXR had diverse effects when regulating the biosynthesis of different terpenoids. This study provided the basis for using A vHMGR and A vDXR to regulate the metabolism of terpenoids.

Methyl Jasmonate Affects Metabolism and Gene Transcription of Volatile Terpenoids from Amomum villosum Lour / 世界科学技术-中医药现代化

This study was aimed to reveal the effects and molecular regulation mechanism of methyl jasmonate (Me-JA) on volatile terpenoids from Amomum villosum Lour. After the leaves and fruits of A momum villosum Lour. were treated with different concentrations of MeJA, the volatile terpenoids of fresh fruits from A . villosum Lour. were ex-tracted with microwave method and analyzed by GC-MS. Then, leaves and fruits treated with MeJA were sequenced by Illumina. The transcriptome data was analyzed by bioinformatic methods. The results showed that there were 20 and 33 volatile terpenoids detected in peels and seed groups, respectively. Contents of volatile terpenoids in peels and seed groups were both improved after 600 μmol·L-1 MeJA treating fruits for 24 h, such as bornyl acetate, cam-phor, borneol, and etc. While 200 μmol·L-1 MeJA treating different parts for 24 h can regulate the biosynthesis of some volatile terpenoids in peels differently. And 200 μmol·L-1 MeJA treating fruits can improve the content of ma-jor volatile terpenoids in seed groups. A total of 68 168 unigenes were obtained with de novo assembly, and 48 627 unigenes were annotated after comparison with public protein databases. Analysis of functional annotation against KEGG database showed that there were 208 unigenes closely related with metabolism of volatile terpenoids and 22 u-nigenes related with MYC2 transcription factors. It was concluded that MeJA can effectively regulate the metabolism of volatile terpenoids from A . villosum Lour. There were a lot of candidate genes related with the biosynthesis of volatile terpenoids obtained by analyzing the transcriptome data which also provided a large amount of data for the discovery and regulation of functional genes related with the biosynthesis of volatile terpenoids from A . villosum Lour.

Identification of Original Plants of Guanzhong (Dryopteris Species) by Chloroplast psbA-trnH Intergenic Region / 世界科学技术-中医药现代化

This article was aimed to study the species identification capability of psbA-trnH among Guanzhong herbs from Dryopteri. The nucleotide sequence information of psbA-trnH region was abstracted using standardized manners from 9 Dryopteri species (19 samples). The identification efficiency of psbA-trnH was analyzed based on TaxonGap among both tested materials (9 species, 19 samples) and tested materials plus GenBank data (17 species, 44 samples in total). The results showed that with the expanded species range, the discriminative efficiency of psbA-trnH de-clined from 100% (9/9) to 82.4% (14/17), while the proportion of within-specific heterogeneity larger than between-specific separability increased from 11.1% (1/9) to 52.9% (9/17). It was concluded that psbA-trnH can be recom-mended as the valuable barcode for homonym distinguishment among Guanzhong herbs, with attention of adequate sampling within species.

Differentiation on Original Plants of Xi-Huang-Cao through DNA Barcodes / 世界科学技术-中医药现代化

This study was aimed to identify original plants of Xi-Huang-Cao (XHC) through DNA barcodes. The nu-cleotide sequence information of rbcL, psbA-trnH, matK and ITS2 regions were abstracted using standardized man-ners from 41 samples (including Rabdosia serra, R. lophanthoides and R. lophanthoides var. graciliflora). Sequencing efficiency of each marker was calculated. Species identification capability was tested on the basis of TaxonGap. The results showed that sequence success rates were 100% for rbcL, 90.2% for psbA-trnH, 87.8% for ITS2, and 70.7%for matK. Three markers (rbcL, psbA-trnH and ITS2) were competent for species discrimination (not for subspecies). It was concluded that rbcL can be the preferred barcode for XHC because of its convenience and efficacy.

Qualitative Identification of Spatholobus suberectus by Near-infrared Spectroscopy / 世界科学技术-中医药现代化

This study was aimed to establish an identification method for Spatholobus suberectus and its adulterants by near-infrared spectroscopy (NIRS). Near-infrared diffuse reflection spectroscopy (NIRDRS) spectra of different S. suberectus and its adulterants were acquired by using OPUS INDENT analysis software. NIRDRS spectra clustering analysis model and identification model were established and verified. The results showed that S. suberectus from dif-ferent regions and its adulterants were identified successfully by clustering analysis model and identification model. It was concluded that Spatholobus suberectus and its adulterants can be identified rapidly and non-destructively by NIRS.

Dynamic Study on Soluble Sugar Accumulation and Sucrose Metabolizing Enzyme Activities During Fruit Development of Amomum villosum Lour / 世界科学技术-中医药现代化

This study was aimed to discuss the dynamic variation of soluble sugar contents, sucrose metabolizing en-zyme activities and gene expression quantities during the fruits development of A momum villosum, in order to pro-vide the basis of improvement of the fruit yield. Fresh fruits at three different development processes (30 DAF, 60 DAF, 90 DAF) were used to investigate changes of soluble sugar components and sucrose metabolizing enzyme activ-ities by HPLC and UV spectrophotometry. Combining with the high-throughput sequencing expression profile data of three fruit development period, the trends of three key enzymes gene expressed in sugar metabolism were analyzed. The results showed that the fruit sugar components were dominated by fructose, glucose and sucrose. The concentra-tion of hexose (fructose and glucose) gradually decreased in peel. But in seeds the concentration of hexose decreased at first and then increased. The content of sucrose and the net activities of sucrose synthase (synthesizing direction minus decomposing direction) in peel and seeds were gradually increased. The expression trends of key enzyme gene in sugar metabolism examined by RNA-seq quantification showed that sucrose phosphate synthase and sucrose syn-thase gene increased and then kept constant, but the invertase gene expression trend was gradually rising. Conse-quently, sucrose synthase was the key enzyme catalyzing sucrose synthesis and decomposition. The activity of sucrose synthase and sucrose contents in peel and seeds reached the highest peak in the end of fruit mature.

"Opinions on a New Cross-discipline ""Biological Chemistry of Chinese Herbal Germplasm Resources""" / 世界科学技术-中医药现代化

Biological chemistry of Chinese herbal germplasm resources (BCCHGR) is a new cross-discipline formed from rapid development of modern science and technology and its application in the area of Chinese herbal resources. BCCHGR was defined as probing and understanding biological processes like heredity, gene transcription, expression and metabolism of Chinese herbal germplasm, at the interface of biochemistry, molecular biology and chemistry, elu-cidating the nature of Chinese herbal germplasm using as TCM medicine as well as the forming mechanism thereof. In this paper, the scientific background, definition, significance and contents of BCCHGR were discussed to depict a preliminary picture of BCCHGR and arouse popular consideration and discussions.

Screening of Reference Genes for Real-time Fluorescence Quantitative PCR in Amomum villosum Lour / 广州中医药大学学报

Objective To identify the reliable reference genes for gene expression analysis of the pericarp and seed of Amomum villosum Lour. by using real-time fluorescence quantitative polymerase chain reaction ( qRT-PCR). Methods Using the fruits ( separated into peels and seeds) of A. villosum at three different developmental periods as the experimental material, 5 candidate reference genes (β-actin, EF-1α, GAPDH, PGK, TUA) with steady expression were screened out by the high throughout sequencing of transcriptome and expression profile data. The qRT-PCR technique was applied to study the expression levels of 5 candidate reference genes in different samples. The stability of the candidate reference genes were evaluated by GeNorm and NormFinder software. Results The 5 reference genes had different stabilities in the pericarp and seed of A. villosum Lour. at different development periods . The order of the steadiness of reference genes showed by GeNorm was EF-1α = TUA>PGK>GAPDH>β-actin. The results of NormFinder revealed that EF-1α was the most stable, followed by TUA, and the order of the other three genes was as same as the results of GeNorm. Conclusion EF-1αand TUA could be used as double reference genes for the normalization of gene expression in A. villosum fruits at different developmental periods by using qRT-PCR.