Comprehensive Investigation of the Differences of the Roots of Wild and Cultivated Mirabilis himalaica (Edgew) Heim Based on Macroscopic and Microscopic Identification Using HPLC Fingerprint

Mirabilishimalaica (Edgew) Heim (MH) is an important Tibetan medicine with demonstrated medicinal efficacy and promising developmental value. A previous study of MH was limited to vague morphological and microscopic descriptions, restricting its clinical application and further development as a medicine. The goal of this study was to comprehensively characterize wild and cultivated products of MH using macroscopic and microscopic identification using HPLC fingerprint. The results revealed that the cultivated and wild MH exhibited differences in macroscopic and microscopic characteristics and chemical components. This analysis can facilitate the establishment of a more comprehensive quality evaluation method for MH. These results provide the basis for clinical applications and the improvement of quality standards of MH as a step towards modernization of Tibetan medicine.


Introduction
e Qinghai-Tibet Plateau has a unique geographical environment, with high altitude, strong ultraviolet rays, thin air, a large temperature difference between day and night, and many unique species. Many of these unique species have been used as components of Tibetan medicine due to their bioactive effects. However, current Tibetan medicine quality standards have disadvantages, such as backward evaluation technology, a single evaluation component, cumbersome evaluation methods, and incomplete evaluation systems, which seriously restrict development and clinical use. e application of modern scientific tools to investigate these species can provide insight into available medicinal plant germplasm resources and facilitate their eventual development into modern medical products. Tibetan medicine has a long history [1][2][3][4][5][6]. AshaGanaha is originally from Mirabilis himalaica (Edgew) Heim radix (MH), and its dried root is popular in Tibetan medicine (TM), a medicine that is used to treat nephritis, edema, gonorrhoea, lumbago, arthralgia, uterine cancer, and bladder stones [7,8]. e components of MH include glycolipids, rotenoids, phenylpropionate derivatives, steroids, glucosides, organic acids, lignin, and anthraquinones [8][9][10][11]. Modern pharmacological studies have shown antifertility effects of MH seed extract in mice [12]. e MH components mirabijalone E, 9-O-methyl-4-hydroxyboeravinone B, boeravinone C, and boeravinone H were isolated from the radix and exhibited toxicity to A549 and HeLa cells [13]. Additionally, (E)-3-(4hydroxy-2-methoxyphenyl)-propenoic acid 4-hydroxy-3-methoxyphenyl ester was found to have inhibitory activities on tumour growth in vivo using xenografts of HepG2 cells [14]. Due to these important pharmacological effects, wild MH resources have been exploited [15]. e plant can be cultivated, but little is known about how the chemical composition and the quality of MH cultivars might differ from those of wild plants. erefore, comprehensive identification methods are urgently needed to allow for the accurate identification of wild and cultivated MH. is is an important step required to ensure the safety of clinical medication and will provide scientific support for the future development and utilization of MH.
Tibetan medicine is an important part of traditional Chinese medicine (TCM), and the classic medicinal identification methods used for TCM are also applicable to Tibetan medicine. Morphological identification is a classical and frequently applied method of identification of TCM, using "observing," "touching," "tasting," and "smelling" to identify plant materials [16]. Previous studies reported significant differences in morphological characteristics between cultivated and wild products of Glycyrrhiza uralensis, Gentiana macrophylla, and other medicinal plants, with correlations between their morphological characteristics and their effective components [17][18][19]. However, there has been little morphological analysis to compare cultivated and wild MH.
In addition to direct morphological observation of plants, the internal structures of plants can be analysed using microscopy, which provides more detailed information that can be used to accurately identify medicinal plants from multiple origins or with similar characteristics. For example, microscopy could distinguish differences in the tissue structure and the degree of development of cultivated and wild-grown plant samples of Salvia miltiorrhiza Bge and Fritillaria taipaiensis P. Y. Li [20][21][22]. However, the microscopic characteristics of MH have not been described. e authenticity, purity, and quality of plants used for TCM can be evaluated based on physicochemical identification using physical or biochemical analysis methods. High-performance liquid chromatography (HPLC) fingerprint is an effective method that facilitate quality control of traditional medicine materials. HPLC fingerprint studies also revealed significant differences in the characteristics of the effective components of wild and cultivated Bletilla striata ( unb) Reich. F. and Polygalae Radix [23,24]. HPLC fingerprint techniques could be developed for quality control in MH cultivation and utilization.
In this study, wild and cultivated MH were evaluated based on macroscopic and microscopic studies using HPLC fingerprint. Our results provide scientific evidence for the further development and utilization of MH for clinical application and offer a reference to guide the modernization of other Tibetan medicines.  (Table 1). e dry roots of MH were identified by Prof. Yonghong Yan (Beijing University of Chinese Medicine, Beijing, China).

Morphological Identification.
Appearance, cross-sections, odours, and tastes of different batches of MH were observed using the traditional morphological identification skills of "observing", "touching", "smelling", and "tasting".

Microscopic Identification.
Paraffin sections and temporary powder sections of cultivated and wild MH were prepared, and the characteristics of cultivated and wild products were observed by electron microscopy (Olympus DP71) using traditional methods.

Morphological Identification.
e characteristics of wild and cultivated MH were identified by morphological identification as the first step of identification and characterization. e colour of the surface of wild MH samples was yellow-brown, and the colour of the cultivated samples was pale yellow to pale brown. e wild MH root was yellowwhite with clear punctate fringes, and the cultivated root sample was grey-white to grey-brown with indistinct 2 Evidence-Based Complementary and Alternative Medicine punctate fringes. e root texture of wild MH was solid and resistant to breakage, with an intense earthy smell. e root of the cultivated plant was light in texture, easy to break, and had a faint earthy smell. e plant samples shared characteristics of pungency, astringency, and induction of a prickly feeling in the throat after chewing (Figure 1). Overall, there were differences in the characteristics between wild and cultivated species, allowing for the preliminary qualitative identification of wild and cultivated MH based on morphological identification. Due to the serious lack of wild resources, there are a large number of cultivated products of MH on the market. But the chemical composition and the quality of MH cultivars might differ from those of wild plants. And there were differences in the morphological characteristics between wild and cultivated species, and its incorrect use could result in serious consequences for patients. However, the morphological identification of the wild and cultivated MH cannot be achieved according to the current standards [27]. erefore, the morphological parts of MH were supplemented and improved in this section. An accurate morphological identification of MH could be achieved based on these characteristics, which could provide a basis for the identification of cultivated and wild MH and other TMs in morphological identification [13].

Microscopic Identification.
Microscopy can be used for more accurate identification of plant medicines. Transverse sections and powder and paraffin sections of cultivated and wild MH samples were analysed. e cork layer of the cultivated products appeared flat on the transverse section and that of the wild sample appeared rectangular or square. e cortex of the cultivated species was composed of 12-13 columns with parenchyma cells and that of the wild sample was composed of 8-10 columns and a loose arrangement. e transverse section of the cultivated MH contained three to four rounds of abnormal vascular bundles, fewer than the observed four to six rounds in the wild sample. e primary xylem of the normal vascular bundle of the cultivated was tetrarch and that of the wild sample was diarch. Large amounts of raphide and starch grain were observed in the parenchyma cells in both cultivated and wild samples ( Figure 2). However, there were no significant differences in the appearance of the powder samples of cultivated and wild products (Figure 3). e cultivated and wild species exhibited differences in the shape of the cork layer, the composition of the cortex, the distribution of abnormal vascular bundles, and the configuration of primary xylem. e cultivated and wild products could be distinguished based on these differences. ere is certain subjectivity in morphological identification. e qualitative identification of MH, which relies only on morphological identification without analysis of the internal structure, is not objective, and the microscopic features in the current MH quality standards are incomplete. Subsequently, the cultivated and wild MH were microscopically studied simultaneously for the first time, and there were differences in the shape of the cork layer, the composition of the cortex, the distribution of abnormal vascular bundles, and the configuration of primary xylem between them. e cork layer of the cultivated and the wild products appeared flat and rectangular or square. e cortex of the cultivated species was closely arranged and that of the wild sample was a loose arrangement. e abnormal vascular bundles of the cultivated MH were fewer than those of the wild sample. e primary xylem of the normal vascular bundle of the cultivated and the wild sample was tetrarch and diarch. ese differences provided effective data for the supplementation and improvement of microscopic characteristic determination and accurate qualitative identification.

Physicochemical Identification.
Among the methods for the quality control of TMs and TCMs, HPLC is one of the most widely used methods. HPLC fingerprint of the active constituents of MH was established by optimizing the HPLC    Evidence-Based Complementary and Alternative Medicine detection conditions for the active ingredients of MH. e results showed nine peaks in both cultivated and wild products. Peaks 4, 5, 6, 7, 8, and 9 were identified as boeravinone C, boeravinone F, mirabijalone A, mirabijalone I, abronione, and mirabijalone H (Figure 4). e principal component analysis score map suggested that differences in the main active components of MH correlate to different growth patterns. e multiple analyses indicated relatively higher amounts of the main chemical components of wild MH than those in the cultivated sample. e HPLC fingerprint of MH was constructed for the first time, revealing differences in the types and contents of the main components of MH with different growth patterns (Figures 5 and   6). e HPLC fingerprint of MH established in this study allows for the qualitative and quantitative identification of MH with different quality and growth patterns.
It is not enough to rely on qualitative identification to evaluate the pharmacological and medicinal effects of medicinal materials, as the content and identification of active ingredients of medicinal materials are more important. In this study, HPLC fingerprint methods of MH analysis were established and optimized for the first time, which provided effective data for the identification and content determination of active ingredients and can be a reference for the study of the content of active ingredients in other Tibetan medicines.

Conclusions
Tibetan medicines are important traditional medicines with excellent clinical efficacy [28][29][30][31][32]. Among them, MH holds an important position. However, the clinical application and comprehensive exploitation of Tibetan medicine are limited due to a lack of systematic research. Moreover, as wild MH is going extinct, a quantity of cultivated products has appeared on the market, which may pose a huge threat to human health. For the first time, MH was analysed as a representative TM, and relatively comprehensive research on the Tibetan medicine was conducted based on macroscopic characteristics and microscopic characteristics using HPLC fingerprint. ese methods can be used to identify this medicinal plant, and this type of comprehensive analysis could be applied to characterize other Tibetan medicines. Our data provide a scientific basis for the evaluation of MH, which could facilitate its development as a medicine.
Moreover, to more accurately analyse the macroscopic and microscopic characteristics, determine the identification and content of active ingredients, and evaluate the different accumulated mechanisms of the active ingredients from diverse production areas and different growth patterns of Tibetan medicine, using MH as a reference, we will investigate the following: in microscopic identification, the microfeatures and micromorphological characteristics of MH will be analysed based on polarizing microscopy, stereo microscopy, and other identification technologies to greatly improve the understanding of the macroscopic and microscopic characteristics of MH. For physicochemical identification, ultraperformance liquid chromatography coupled with quadrupole time-of-flight-mass spectrometry, infrared ray-mass spectrometry, high-performance capillary electrophoresis-mass spectrometry, mass spectrometry imaging, and nuclear magnetic resonance will be employed to identify new components and explore the accumulation and structure of active MH components. In summary, we will apply the above technologies to perform a more comprehensive study on MH and provide better systematic data references for the quality standards and modernization of MH and other Tibetan medicines in the future.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare no conflicts of interest.