Advances in Research of Identification of Cnidii Fructus and Its Adulterants

Weiwei HE, Yanxiu GUO

1. Hainan Vocational University of Science and Technology, Haikou 571126, China; 2. Gansu Medical College, Pingliang 744000, China

Abstract In this paper, the traditional identification and modern identification technology of Cnidii Fructus and its adulterant were reviewed, and their advantages, disadvantages and practicability were summarized, to provide a reference for the rapid and accurate identification and quality evaluation of Cnidii Fructus.

Key words Cnidii Fructus, Adulterant, Traditional identification, Modern identification technique

1 Introduction

Cnidii Fructus is the dried ripe fruit ofCnidiummonnieri(L.) Cuss. and has a long history of medicinal use.Shennong’sClassicofMateriaMedicatook it as top grade drug[1]. It has the effect of dispelling wind and dampness, killing insects and itching, warming the kidney and aphrodisiac, and is used for pudendal itch and vaginal discharge, eczema itching, dampness impediment low back pain, kidney deficiency impotence, uterine cold infertility[2]. Modern studies have shown that its main active components are coumarin compounds, volatile oils and carbohydrates, which have pharmacological effects such as anti-tumor, anti-cancer, antipruritic, anti-allergic, anti-mutagenic, anti-arrhythmia, inhibiting heart, dilating blood vessels, anti-inflammatory, anti-osteoporosis and local anesthesia[3-9].

With the enhancement of the research, development and utilization of Cnidii Fructus, its market demand has increased, resulting in confusion and adulteration in the market, which has seriously affected the quality and clinical safety of Cnidii Fructus. At present, there are many adulterants of Cnidii Fructus in the market, mainly the same genusCnidiumdauricum(Jacquin) Fischer &C. A. Meyer,CnidiumsalinumTurcz.,CnidiumjaponicumMiq.,Cnidiummonnieri(L.) Cuss. var.formosana(Yabe) Kitag, and plants of different genera in the same familyDaucuscarotaL.,Torilisscabra(Thunb.) DC.,Angelicabiserrata(Shan et Yuan) Yuan et Shan,FoeniculumvulgareMill.,AnethumgraveolensL.,ApiumgraveolensL.,CicutavirosaL.,CarumcarviL.,HeracleumscabridumFranch.,Saposhnikoviadivaricata(Turcz.) Schischk.,CuminumcyminumL., and dried fruit ofLepidiumapetalumWilldenow[10-16]. Therefore, we summarized the identification of Cnidii Fructus and its adulterants, especially the research progress of modern identification technology, in order to provide new ideas and new methods for the rapid, non-destructive and accurate identification of Cnidii Fructus.

2 Traditional identification methods of Cnidii Fructus and its adulterants

2.1 Character identificationCnidii Fructus is obviously different from its adulterant in plant morphology, fruit shape, size, color, longitudinal edge, oil tube, odor and other characteristics. Plants of the same genusC.dauricum(Jacquin) Fischer &C. A. Meyer,C.japonicumMiq., andC.salinumTurcz. have similar fruit shape and difficult to distinguish, so they can only be distinguished in plant morphology. The involucral bracts ofC.dauricum(Jacquin) Fischer &C. A. Meyer are long ovate-obovate with very wide membranous margins, while the involucral bracts ofC.monnieriare linear with only narrow membranous margins; involucral bracts ofC.japonicumMiq. andC.salinumTurcz. are not margined with fine lashes[17]. However, Cnidii Fructus sold in the market is the fruit without the original plant, so it is impossible to accurately identify its origin. In nature and taste, Cnidii Fructus is different fromCarumcarvi,LepidiumapetalumWilld.,Illiciumverum, andApiumgraveolensseeds. Cnidii Fructus tastes pungent and cool, and has a tingling sensation[10,18].Carumcarvitastes spicy[14].L.apetalumWilld. tastes slightly spicy[12].Illiciumverumtastes slightly sweet[12].Apiumgraveolensseeds is slightly bitter in taste and cool and pungent in nature[12,19-20], light taste[21], and slightly numbs tongue[11]. Cnidii Fructus can be distinguished from Angelicae Pubescentis Radix in shape and size. The fruit of Cnidii Fructus is elliptic, 2-4 mm long and about 2 mm in diameter. The fruit of Angelicae Pubescentis Radix is obovate-oblong, 5-10 mm in length and 5-9 mm in diameter.DaucuscarotaandTorilisscabra(Thunb.) DC. fruits have hair and Cnidii Fructus fruit is glabrous[10]. In summary Cnidii Fructus is a fruit and seed medicinal material, and it is difficult to distinguish it from adulterant in appearance and shape, especially when the fruit of the same genus is more similar in appearance and characteristics, and adulterant is added, it is even more difficult to distinguish it. Therefore, it is difficult to judge only by experience in the practical application process.

2.2 Microscopic identificationMicroscopic identification method is simple and objective, through observing the schizocarp transection of Cnidii Fructus and its adulterantsCarumcarviandApiumgraveolensseeds, their distinctive features are significant. Cnidii Fructus is semicircular, acute and pentagonal in shape, with thick mesocarp, abnormally prominent longitudinal ribs, a vascular bundle in the middle, bead-like endocarp cell wall[22], small endosperm, and fine calcium oxalate cluster crystals in each aleurone grain of endosperm cells[11,14,19,22]. Mesocarp cells ofCarumcarviare mostly compressed and irregular in shape, and endocarp cells vary in length[14].Apiumgraveolensseeds are semicircular and obtuse pentagonal[22], there are vascular bundles at the longitudinal edge of the mesocarp[11], the endosperm is large, and there are large calcium oxalate cluster crystals in the aleurone grains of the endosperm cells, which are obviously obtuse pentagonal[19,22].

2.3 Physical and chemical identificationZhou Lushanetal.[23]applied the water test method to identify Cnidii Fructus andApiumgraveolensseeds. Cnidii Fructus floats on the water surface, while theApiumgraveolensseeds sink. Ultraviolet spectroscopy was used to identify Cnidii Fructus andApiumgraveolensseeds, which have different absorption wavelengths to achieve the purpose of identification[24-25]. Cnidii Fructus can be distinguished fromApiumgraveolensseeds by the detection of osthole by TLC[11,19,24]. Zhu Jinhuaetal.[19,22]separately took 2 g of Cnidii Fructus andApiumgraveolensseeds powder, added 20 mL of ethanol, heated and refluxed for 30 min, filtered, took several drops of the filtrate on a white porcelain plate, and observed under an ultraviolet lamp (365) nm. Cnidii Fructus showed blue fluorescent yellow light, andApiumgraveolensseeds shows bright blue fluorescence; took another 2 mL of filtrate, add the same amount of 3% sodium oxide solution, heated for 5 min, cooled, and then added 1-2 drops of newly prepared diazo-p-nitroaniline test solution, Cnidii Fructus was cherry red,Apiumgraveolensseeds were light brown. Ding Shaochunetal.[15]used thin layer chromatography and ultraviolet spectroscopy to distinguish Cnidii Fructus,Anethumgraveolens, and Cicuta virosa. If Cnidii Fructus is adulterated withAnethumgraveolens, Cicuta virosa andApiumgraveolensseeds, the distinguishing characteristics of Cnidii Fructus still exist by thin layer chromatography, and the three cannot be distinguished. Qin Lupingetal.[26]used polyacrylamide gel electrophoresis to analyze Cnidii Fructus,C.dauricum(Jacquin) Fischer &C. A. Meyer,Daucuscarotaseeds, andTorilisscabra(Thunb.) DC. seeds and found that the number and position of electrophoresis bands were significantly different, so they could be distinguished.

3 Modern identification techniques for Cnidii Fructus and its adulterants

Zhu Lietal.[16]used HS-SPME-GC-MS method to analyze the volatile components of Cnidii Fructus and its adulterantCuminumcyminum, and found that there were obvious differences in the volatile components between them. Based on high-resolution melting curve (HRM) analysis method, Zhang Mengtingetal.[27]achieved simple and rapid identification of four spicesFoeniculumvulgareMill.,CuminumcyminumL.,Carumcarvi,AnethumgraveolensL. and their adulterants Cnidii Fructus andSaposhnikoviadivaricata. Wu Yutian identified Salviae Miltiorrhizae Radix Et Rhizoma, Isatidis Radix and Cnidii Fructus by convolution transform and information visualization of near-infrared spectroscopy of traditional Chinese medicines, and the classification results were consistent with those of traditional plant taxonomy. Guo Lihengetal.[11]identified Cnidii Fructus andApiumgraveolensseeds by HPLC.

4 Conclusions and prospects

At present, there are many adulterants of Cnidii Fructus in the market, and they are fruit medicinal materials with similar characters, so it is difficult to identify them, and the identification methods are mainly traditional empirical identification, which is subjective and objective. Zhong Yulanetal.[28]used near-infrared spectroscopy combined with discriminant analysis to rapidly identify the quality ofPanaxnotoginsengpowder and its adulterants. Wu Xiyuetal.[29]established aZanthoxylumbungeanumMaxim. powder and its adulterants by applying near-infrared technology and using discriminant partial least squares (DPLS) and support vector machine (SVM) qualitative identification model. Gong Jiantingetal.[30]achieved the rapid and accurate identification of Curcumae Radix, Curcumae Rhizoma, Curcumae Longae Rhizoma, and Wenyujin Rhizoma Concisum by collecting the odor fingerprints of four kinds ofCurcumaL. by electronic nose combined with the discriminant model established by XGBoost. Zhang Danchunetal.[31]applied ITS2 and psbA-trnH sequences to identifyPulsatillachinensisand adulterants (Potentillachinensis,Potentilladiscolor,Polycarpaeacorymbosa) for DNA barcode molecules, confirmed the universality of bar code inPulsatillachinensisand adulterants, which provides a basis for their quality evaluation. Near-infrared technology has the advantages of rapid and non-destructive detection means, low detection cost, no need to treat samples and fast speed, and is widely used in the identification of origin, quality evaluation and identification of mixed products of traditional Chinese medicine[32-33]. Electronic nose is an electronic system that imitates biological nose. It has the characteristics of simple operation, fast detection and accurate identification. It has been widely used in food quality detection, environmental quality control, grain storage and identification, medicine and health and other fields. In recent years, it has also been used to evaluate the authenticity of traditional Chinese medicine, with great potential[34-36]. DNA barcode molecular identification technology has unique advantages in the identification of original species of medicinal plants. It has the advantages of accurate and rapid identification, good repeatability and stability, strong practicability and clear judgment criteria. It can realize the accuracy, digitization and standardization of the identification of Chinese medicinal materials, and has developed rapidly in the identification of Chinese medicinal materials[37-38]. Therefore, the above techniques can be used to establish a rapid and non-destructive identification method and model for Cnidii Fructus and its adulterant, which can provide a reference for its quality evaluation and a supplement for its identification and detection means.