Simultaneous Determination of 6 Components in Volatile Oil from Citrus medica by GC-QAMS / 中国药房

OBJECTIVE：To establish a metho d for simultaneous determinat ion of α-pinene，β-pinene，myrcene，limonene， γ-terpinene and α-terpineol in volatile oil from Citrus medica . METHODS ：GC-QAMS method was adopted. The determination was performed on Agilent DB- 17 capillary column. Flame ionization detector was adopted with nitrogen as carrier gas （purity 99.999％） at the flow rate of 0.5 mL/min. The flow rate of nitrogen （make-up gas ）was 25 mL/min，the flow rate of hydrogen was 30 mL/min， and the flow rate of air was 400 mL/min. The inlet temperature was 250 ℃，and the detector temperature was 280 ℃（programmed temperature），and injection volume was 0.5 μL with split ratio of 35 ∶ 1. Using limonene as internal reference ，relative correction factors of α-pinene，β-pinene，myrcene and γ-terpinene were calculated. The contents of 4 components in the samples were calculated according to the relative correction factors. The results were compared between QAMS and internal standard method （ISM，using dodecane as internal substance ，6 components to be determined ）. RESULTS ：The linear range of α-pinene，β-pinene， myrcene，limonene，γ-terpinene and α-terpineol were 0.030 5-0.213 8，0.066 6-0.466 0，0.021 8-0.152 3，0.765 2-5.356 4，0.387 3- 2.710 8，0.034 2-0.239 6 mg/mL（all r＞0.999）. The limits of detection were 4.82，7.89，4.01，4.54，5.53，2.47 µg/mL，and the limits of quantification were 15.34，25.91，13.69，15.70，18.68，8.36 µg/mL，respectively. RSDs of precision ，repeatability and stability tests （24 h）were all less than 2％. The average recovery rates were 96.08％，97.48％，100.90％，101.22％，100.54％， 95.84％（all RSD ＜3％，n＝6）. The average relative correction factors of α-pinene，β-pinene，myrcene and γ-terpinene were 0.997 8， 1.530 7，0.952 4 and 1.025 5 （all RSD ＜1％）. The contents of α-pinene，β-pinene，myrcene，limonene，γ-terpinene and α-terpineol by ISM were 3.296 9-20.994 1，11.300 6-39.440 9，3.684 2-11.291 4，174.857 8-511.611 8，0-285.127 3，0-48.858 6 mg/g， respectively. The contents of α-pinene，β-pinene，myrcene and γ-terpinene determined by QAMS were 3.296 8-20.994 0，11.300 3- 39.439 7，3.684 1-11.291 1，0-285.126 6 mg/g，respectively. Relative content errors of 4 components as α-pinene determined by 2 methods were all less than 1％ . CONCLUSIONS ： The Δ 基金项目：四川省科技厅应用基础研究项目（No.2021YJ0114）； established GC-QAMS method is simple ，rapid，accurate and 泸州市重点科技项目 [No.2011-S-31（3/3）]；合江县人民政府—西南医 科大学战略合作项目（No.2018-HJXNYD-2） repeatable，and can be used for quantitative analysis and ＊硕士研究生 。研究方向 ：中药制备工艺及质量评价 。电话： quality control of volatile oil from C. medica . 0830-3162291。E-mail：493359958@qq.com

Identification of Citrus medica var. sarcodactylis Infected with Citrus Huanglongbing / 中国实验方剂学杂志

Objective::To analyze and compare different samples in many aspects to identify Citrus medica var. sarcodactylis infected with Huanglongbing(HLB) timely and accurately, in order to prevent and control the disease in time. Method::HLB was identified through character analysis, reverse transcription-polymerase chain reaction(RT-PCR), enzyme digestion reaction and Real-time PCR. Result::In terms of characters, there were typically variegated yellow leaves and relatively small fruit, even with deformity but without " red nose fruit" among C. medica var. sarcodactylis infected with HLB. All of these can be used as the basis for the preliminary identification of HLB in the fields. According to the RT-PCR test results and enzyme digestion reaction, when the primer was OI1/OI2c, there was specific band of 1 160 bp, which could be cut into 520 bp and 640 bp by Xba I enzyme. These results were consistent with the characters of other citrus plants infected with HLB. According to the Real-time PCR detection results, C. medica var. sarcodactylis infected with HLB had amplification curves and dissolved peaks, with the melting temperature was 82 ℃ and Ct between 24.6 to 28.2, while the normal plants were not amplified. Conclusion::Character analysis can be used to roughly distinguish HLB in the fields, but with a certain subjectivity. RT-PCR or Real-time PCR can be used to identify C. medica var.sarcodactylis infected with HLB in a timely and accurate manner, and qPCR detection is more sensitive and quantitative. Through the combination of character analysis and molecular identification, C. medica var.sarcodactylis infected with HLB can be determined more timely and accurately.

Comparative Analysis of the Components of Volatile Oil in Citrus medica from Different Producing Areas / 中国药房

OBJECTIVE：To compare th e difference of the components of volatile oil in Citrus medica from different producing areas. METHODS ：The volatile oil of C. medica from 10 different producing areas was extracted with steam distillation ，and the yield was calculated. The components of the volatile oil of C. medica from different producing areas were analyzed by GC-MS. The compounds were retrieved from NIST 14.L mass spectrum database and identified. Relative mass fraction of chemical component was determined by peak area normalization method. Cluster analysis of samples were performed by using SPSS 20.0 software. RESULTS：The yields of volatile oil of C. medica from 10 different producing areas were 0.10％-1.75％，among which sample from Qianwei county in Leshan city of Sichuan province was the highest （1.75％）. A total of 66 components were identified in the volatile oil of C. medica from different producing areas ，with a relative molecular weight of 126.20-392.66. The majority was C 10 and C 15 compounds；isomers with relative molecular weight of 136，154 took up the great proportion ，which were mainly cycloalkane monoterpenes. There were 12 common components in the volatile oil of C. medica from different areas ，which were limonene（24.90％），terpinene（14.71％），（－）-4 terpineol（2.88％），citral（2.33％），α-myrrhene（2.33％），geraniol（1.52％）， α-pinene（1.37％），trans bergamot olene （1.16％），isoterpinene（1.13％），methyl palmitate （1.12％），linalool（1.09％）and geranyl acetate（1.04％）according to relative mass fraction ；8 of them were monoterpenes ，2 were sesquiterpenes and 2 were esters. There were 4 categories of C. medica from different producing areas ，i.e. S 2，S4，S6 clustered into one ；S1，S3，S7，S8 clustered into one ； S5 and S 10 clustered into one ；S9 as one . CONCLUSIONS ： There are some difference of the components in volatile oil of medica from different producing areas ，and the content of the same component also has great difference in the volatile oil of C. medica from different producing areas.

Comparative transcriptomics analysis of Citrus medica var. sarcodactylis at different developmental stages / 中国中药杂志

In order to study the transcriptional differences of Citrus medica var. sarcodactylis at different developmental stages, we explored the genes regulating the biosynthesis of the effective components. In this study, Illumina Hiseq 4 000 high-throughput sequencing technology was used to sequence the transcriptome of C. medica var. sarcodactylis at different developmental stages, 121 235 unigenes were obtained with an average length of 2 434 bp, 3 379 different genes were obtained using DESeq screening, which mainly connected to biological processes such as signal transmission, biological regulation, and metabolic processes, and enriched in metabolic pathways such as starch, sucrose metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis. Further dynamic comparison of biosynthesis related genes of active ingredients: the expression levels of PAL, CHI, CYP75B1, ZDS, 4CL and FLS gradually increased as the fruit turned from green to yellow; the expressions of COMT, F3H and CYP73A increased at first and then decreased; CCR, HCT and HRP were down-regulated whereas up-regulated. This study provides references for further excavation of key genes in the biosynthesis of active components, as well as biopathway analysis of active components for C. medica var. sarcodactylis.

Traditional Application of Citri Sarcodactylis Fructus / 中国实验方剂学杂志

Citri Sarcodactylis Fructus, a traditional Chinese medicine with dual-purpose of drug and food, has a long history of cultivation and application, showing high medicinal, dietetic, ornamental and economic values. Many modern researches have been conducted on the extraction technology, chemical constituents, quality standards and biological activities, but the herbal textual researches were relatively insufficient. The related records of Citri Sarcodactylis Fructus in the ancient Chinese medicinal classics were ambiguous and confusing. By referring to ancient Chinese medicinal classics and modern books, the textual researches of Citri Sarcodactylis Fructus, including the names, plant morphology, origin, medicinal properties, efficiency and application were systematically reviewed and analyzed in this paper, in order to provide the basis for the confirmation of original source of Citri Sarcodactylis Fructus. The results showed that Citri Sarcodactylis Fructus was usually recorded as ‘Xiangyuan’ ‘Juyuan’ ‘Ganyuan’ ‘Xiangyuan’ ‘Foshougan’ etc. in ancient books. After the Ming and Qing dynasties, it became clear that ‘Xiangyuan’ was equivalent to ‘Juyuan’, but different from ‘Foshougan’. Therefore, these two species were recorded separately. From the ancient times to the present day, Citri Sarcodactylis Fructus was always mainly distributed in south of the Yangtze River, such as Fujian, Guangdong, Guangxi, and Jiangxi provinces. Ancient records and modern reports basically have the same understanding on properties, efficacies and indications of Citri Sarcodactylis Fructus, considering it was warm in nature with the effectiveness of soothing liver, relieving pain, eliminating dampness and resolving phlegm. ‘Baking it into powder on new tiles’ and ‘steaming’ were the traditional processing methods. The description of ‘The longer of storage period, the better of the effect’ was occasionally recorded. These results provide theoretical references for the further study, resource development and comprehensive application of Citri Sarcodactylis Fructus.

Phytochemical analysis and evaluation antibacterial activity of Citrus medica peel and juice growing in Kurdistan/Iraq.

Citrus medica is an important medicinal plant of the family Rutaceae Qualitative and quantitative screening were performed for evaluation bioactive constituents in Citrus medica fruit. The antibacterial activity of the ethyl acetate and ethanol 80% peel extracts and juice of Citrus medica was assessed by agar well diffusion assay, minimum inhibitory concentration (MIC) value determination by 96 well serial dilution against five gram positive and two gram negative bacteria, while minimum bactericidal concentration (MBC) evaluated by plate cultures. Qualitative investigation shows the presence of carbohydrates, flavonoids, phenols, tannins, steroids, cardioactive glycosides in peel and juice, while saponins, terpenoids, and anthraquinones absent in all fraction of Citrus medica. On quantitative screening peel contain highest quantity tannin as compared to juice while juice contain higher quantity phenol and Citrus medica fruits contain high quantity phenol than flavonoids. The juice of Citrus medica exhibited largest inhibition zone (12mm) at (100mg/ml) and recorded the lowest MIC and MBC value (1.5625mg/ml & 3.125mg/ml) against Staphylococcus auricularis. The ethyl acetate extract of peel revealed antibacterial activity against four bacterial strain while ethanol 80% only against two bacteria. Peel extracts exhibited largest inhibition zone (10 & 22mm) at (100mg/ml) and recorded the lowest MIC value (25mg/ml & 12.5mg/ml) and MBC (50 mg/ml) against Escherichia coli.

Estrogenic activity of Citrus medica L. leaves growing in Egypt.

The estrogenic activity of petroleum ether extract of Citrus medica L. leaves as well as the chemical constituents responsible for the biological activity was studied. The petroleum ether extract proved to retain high estrogenic activity in immature female rats. The extract was saponified and its components (saponifiable part 23% and the unsaponifiable matter 77%) were identified using GC/MS technique. The extract proved to be safe (LD50< 2g/kg.bw). Oral administration of petroleum ether extract of C. medica in ovariectomized immature female Wistar rats for 7 days in a dose of 400 mg/kg resulted in significant increase in the uterine weight (g) (1.7±0.11) when compared with ovariectomized control rats (1.3±0.07). GC/MS analysis of both saponifiable and unsaponifiable matters revealed the presence of thirty three components (28 hydrocarbons and 5 sterols) in the unsaponifiable fraction, the major hydrocarbon was n- Heneicosane (16.7%) while the major sterol was β-sitosterol (4.03%) and 15 components in the saponifiable matter it's major component was hexadecanoic acid (19.93%). As a conclusion petroleum ether extract of Citrus medica L. leaves possess a significant estrogenic activity.

Study on Chemical Constituents of Citrus mediea var. sarcodactylis from Sichuan Province (Ⅱ) / 中药新药与临床药理

Objective To investigate the chemical constituents of Citrus medica var. sareodactylis from Sichuan province, and to provide evidence for the development and utilization and quality evaluation of the medicinal material.Methods Various chromatographic techniques were used to purify the components of this herb. Compounds were identi-fied by their physical characteristics and spectral feature. Results Seven compounds were isolated from Citrus medica var. sarcodactylis, and they were identified as sibiricol (Ⅵ), 7-methylesculetin (Ⅶ), bergapten (Ⅷ), sigmasteryl acetate(Ⅸ), 5-methoxyfurfural(Ⅹ), limonin(Ⅺ), daucosterol (Ⅻ). Conclusion Compound Ⅵ, Ⅶ and Ⅸ are isolated from plants of Rutaceae for the first time.

Study on Quality Standards of Citrus Medica / 中国中医药信息杂志

Objective To establish systematic and integrated quality standard for Citrus medica. Methods To make clear the ancient applicated history and the present situation of the research of Citrus medica. The breed resources citrus medica and its medicinal present situation were investigated, and some chemical constituents from citrus medica were extracted and separated. Qualitative and quantitative methods were employed on citrus medica by HPLC methods. Results Changes in the distribution of the two variety resources of citrus medica were discovered, and six components were separated and identificated. The identificated characteristics of the two variety was found in the HPLC fingerprint for the first time. Some methods such as character identification, thin-layer identification, check of water and ash, determination of useful ingredients and the content of extracturm were established to evaluate quality standard for citrus medica. Conclusion The research on quality control standards of the citrus media is systematic and integrated, and these methods are scientific and accurate, which are suggested to the country to adopt.

Factors Affecting on the Survival Rate for Shoot-tip Micro-grafting of Citrus medica L.var.sarcodactylis Swingle / 广州中医药大学学报

Objective To lay a foundation for the cultivation of disease-free plantlet of Citrus medica L.var.sarcodactylis Swingle(CMS) by discussion of the factors affecting survival rate for micro-grafted shoot-tip of CMS.Methods With citrus limon seedlings cultured in the dark as the rootstock,shoot-tip of CMS was grafted to the inverse T-shaped cut in the cotyledon of rootstocks.Then an investigation was carried out on the factors which affected the survival rate for micrografted shoot-tip of CMS.The factors included age of rootstocks,scion size,culture medium with different concentrations of phytohormones,and rootstocks with or without cotyledon.Results The survival rate for micro-grafted shoot-tip of CMS was closely related to the factors mentioned above.A higher survival rate was obtained when CMS shoot-tip with 4 pieces of leaf primordia was grafted to the inverse "T" shaped cut in the cotyledon of parental stock(citrus limon seedlings) which was cultured in the dark for 14 days,and when the citrus limon seedlings were cultivated in MT-based medium with ?-naphthylacetic acid 0.1 mg?L-1 added.Conclusion The optimized micro-grafting conditions for CMS shoot-tips improve the propagation of micro-grafted plantlet and the survival rate of micro-grafted shoot-tip of CMS.

Detection of Huanglongbing Pathogen in Citrus medica L. var. sarcodactylis (Noot.) Swingle by Polymerase Chain Reaction Analysis / 广州中医药大学学报

[Objective] To establish an effective way for the detection of Huanglongbing pathogen in Citrus medica L. var. sarcodactylis (Noot. ) Swingle by polymerase chain reaction (PCR) analysis, and to provide evidence for the early diagnosis of the culture of pathogen-free plantlets and for the prevention and control of diseases. [ Methods ] The incidence of Huanglongbing disease was investigated in the main producing area of Citrus medica L. var. sarcodactylis (Noot.) Swingle. By using the special primers designed by the DNA sequence of Citrus Huanglongbing pathogen, PCR amplification of DNA from the samples was conducted. [Results] Symptoms similar to the Citrus Huanglongbing disease occurred in Citrus medica L. var. sarcodactylis (Noot.) Swingle growing in Zhaoqing, Guangdong. The results of PCR analysis showed that a special DNA fragment of 400bp had been detected in the affected plants of Citrus medica L. var. sarcodactylis (Noot. ) Swingle, but it was not found in the healthy plants, indicating that the symptoms were caused by the Huanglongbing pathogen. [ Conclusion ] PCR analysis is an effective way for the detection of Huanglongbing pathogen in Citrus medica L. var. sarcodactylis ( Noot. ) Swingle.

RAPD analysis of genetic polymorphism and molecular identification of Citrus medica cv. sarcodactylis from Jinhua / 中草药

Object To analyse genetic polymorphism in DNA level of three species and determine them in molecular level by using of the optimization of Citrus medica cv. sarcodactylis (Noot.) Swingle RAPD reaction.Methods RAPD reactions were performed under the following conditions:template DNA 10～100 ng/?L, 3.0～5.0 mmol/L MgCl 2, 200 ?mol/L dNTP,0.2 ?mol/L 10 mer Primer, lunit Taq Polymerase/25 ?L reaction volume.Results Twelve efficient and stable primers were selected from 38 primers. Phenograms were constructed by using genetic distance UPGMA method. Based on the results of the RAPD analysis and on a review of morphological characterisitic, C. medica cv. sarcodactylis species BAIHUAQINGPI could be used as BAIHUABAIPI in three species, there were some special amplified bands in every RAPD reactions from different species. Conclusion This method could serve as an easy and highly efficient method for C. medica cv. sarcodactylis genetic polymorphism and molecular identification of species.

Vitrified cryopreservation and regeneration of shoot-tips for Citrus medica var.sarcodactylis / 中草药

Objective To provide a new approach of preservation for germplasm resources of Citrus medica var.sarcodactylis(CMS).Methods A micrografting test was made on CMS shoot-tips after vitrified cryopreservation,resulting in living shoot-tip.Results First the CMS shoot-tip is inoculated in medium consisting of MS,5%DMSO,and 5%sucrose for 48 h preincubation.Then the shoot-tips were cut off 3—4 mm long and fore-treated by 60%PVS_2 at room temperature(25℃) for 30 min.After that,they were treated by PVS_2 at 0℃for another 80 min and conserved in liquid nitrogen for 24 h.Next the shoot-tips were defrosted by aqueous bath at 40℃and cleaned twice at room temperature(25℃) by MT minimal medium with additive 1.2 mol/L sucrose,10 min once.Finally data collected recorded a higher survival rate of 81.25%by TTC inspectation and a better regeneration rate of 52.94%if the CMS shoot-tips had been grafted on darkly-cultured parental stock,growing,and differentiating normally afterwards.The plantlets detected by polymerase chain reaction(PCR) showed that the pathogens of Huanglong disease had been removed.Conclusion Therefore a conclusion is made that the approach of vitrified cryopreservation may be applied to the preservation of CMS germplasm resources.