A Preliminary Study on the Quality Standard of Raphanus sativus L. Produced in Guizhou Province

Feng BAO Pengfei XIA Wei CHEN Wen YANG Shimei ZHAO Kaibin LI

Abstract ［Objectives］ This study was conducted to analyze the Raphanus sativus L. medicinal materials from different origins and batches in Guizhou Province to explore its quality standards.

［Methods］The plant morphology, material shape and microscopic characteristics of R. sativus were identified; and qualitative identification was performed by the TLC method.

［Results］ The plant morphology, material shape and microscopic characteristics of the R. sativus medicinal materials were described; and the TLC identification method of the R. sativus medicinal materials was established, with clear spots, good separation, and good specificity.

［Conclusions］The method is accurate, simple, highly sensitive, interference-free, has good reproducibility, and can be used as a data support for the formulation of quality standards for R. sativus medicinal materials.

Key words Raphanus sativus L.; Medicinal materials; Quality standard; Determination

Received: December 12, 2020  Accepted: February 21, 2021

Supported by Science and Technology Innovation Platform Construction Project of Anshun City (ASKP［2017］03); Anshun Traditional Chinese Medicine (Ethnic Medicine) Inspection Technology Innovation Talent Team (ASKC［2018］09).

Feng BAO(1963-), male, P. R. China, pharmacist-in-charge, devoted to research about drug inspection and quality standards.

*Corresponding author. E-mail: 826735176@qq.com.

Raphanus sativus L. was first published in "Supplements to Compendium of Materia Medica", saying: "When cutting R. sativus, the remaining ones are left in the ground, thin and fleshless, old and stringy, like a skeleton, and thus named Kuluobo. It can ventilate the lung qi, and can be used for relieving gas poisoning." Compendium of Materia Medica records: "R. sativus is commonly cultivated today. It is planted in June, harvested for the aboveground part in autumn, and dug for roots in winter. The plants bolt highly and show small purple flowers in late spring, and siliques are formed in early summer. The seeds are as big as marijuana seeds, with varying lengths and yellow-red colors. The large leaves are like turnips, and the thin ones have fine pubescence. The roots are red or white, and their shapes are long or round." Compilation of Medical Records (Yizong Huibian) records its use for treating jaundice, which is also recorded in later pharmacy books.

In Guizhou, the ancestors of the Miao and Buyi peoples had the habit of using R. sativus to treat gastric diseases for a long time. On this basis, they developed Xianrenzhang Weikang Capsules and other patent medicines, which are widely used national medicines in our province, and are included in the 2003 edition of Quality Standards for Traditional Chinese Medicines and Ethnic Medicines in Guizhou Province. The original standard has few inspection items and lacks characteristic identification and quality control indexes, and the local standards of R. sativus medicinal materials need to be improved and perfected. Up to now, there are no detailed and systematic reports on the quality standards of R. sativus. Therefore, to study and improve the quality standards of the medicinal material and to provide plans and data support for the formulation of more reasonable, scientific, comprehensive and controllable statutory medicinal material standards have clear practical significance.

Materials and Methods

Experiental materials

Instruments

Mettler AB265 electronic balance (Switzerland  METTLER TOLEDO); LEIKCA DM500 microscope and LAS EZ imaging system (Leica, Germany); DHG-9041A electric heating constant temperature drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.); ultrasonic cleaner (type: YM-060S; manufacturer: Shenzhen Fangao Microelectronics Co., Ltd.); dark-box three-purpose ultraviolet analyzer (type: ZF-7; manufacturer: Shanghai Jiapeng Technology Co., Ltd.).

Materials

The R. sativus medicinal materials were collected by our research team from different producing areas in Guizhou. The specific information is shown in Table 1. The materials were identified by Professor Wei from the Department of Medicinal Plant Cultivation and Identification, College of Pharmacy, Guizhou University of Traditional Chinese Medicine, as the dry roots of cruciferous plant R. sativus after fruiting. Acetone, glacial acetic acid, n-hexane, ethyl acetate, and ether were all analytically pure, and β-sitosterol reference substance (batch number: 110851-201909 for thin layer chromatography identification) was provided by the National Institutes for Food and Drug Control. Silica gel G pre-coated plates were produced by Branch of Qingdao Ocean Chemical Plant.

Experimental methods

Preparation of test solution

A certain amount of the product powder (1 g) was weighed and added with 30 ml of ethanol and 30 ml of petroleum ether, followed by ultrasonic treatment for 10 min. The extraction system was filtered, and the petroleum ether was discarded. The residues were added with 10 ml of acetone and ultrasonically treated for 30 min, and filtration was performed, giving the subsequent filtrate as the test solution.

Preparation of reference solution

A certain amount of β-sitosterol reference substance was weighed and prepared into a 0.1 mg/ml solution with acetone as the reference solution.

Results and Analysis

Plant morphology［1-3］

R. sativus is a biennial or annual herb, 30-100 cm high. The taproot is fleshy, oblong, spherical or conical, with green, white or red skin. The stems are branched, glabrous, slightly pruinose. The basal leaves and lower stem leaves are large and pinnate, 8-10 cm long, 3-5 cm wide, and have an ovoid apical lobe, and 4-6 pairs of lateral lobes, which are oblong and have blunt teeth, sparsely coarse hairs. The upper leaves are oblong, serrated or nearly entire. The racemes are nape or axillary; sepals are oblong; the flowers have 4 petals, which are white, purple or pink, 1.5-2.0 cm in diameter, obovate, 1.0-1.5 mm long, and have purple stripes, and a claw of 5 mm long at the lower part; each flower have 6 stamens, 4 long and 2 short, and 1 pistil, which is subulate; and the stigma is columnar. The siliques are cylindrical, 3-6 cm long, constricted between the seeds to form a spongy septum, and have a beak at the apex of 1 to 1.5 mm long. Each silique accommodates 1 to 6 seeds, which are ovoid, slightly flat, about 3 mm long, red brown with fine netting. The flowering period is from April to May, and fruiting period is from May to June. The flower and root pictures are shown in Fig. 1.

Shape of the medicinal materials［4］

The product is cylindrical or spheroidal, slightly flat, slightly distorted, varying in size, and the surface color varies widely, such as purple, red or gray-brown. The surface is shrunken, and has wavy texture and rootlets or rootlet marks, and horizontal arranged stripes also can be observed. There is a hollow stem base at the top. The body is light and tough, not easy to break, and the section is pale yellow and white, loose. It has faint odor and taste sweet. Fig. 2 shows the characteristic picture of the medicinal material.

Microscopic identification［5］

Cross section: The epidermis is composed of several rows of cells, and the hypodermis is composed of 1 to 2 rows of tangentially elongated suberized cells. The cortex is relatively broad, and contains polygonal stone cells with slightly thickened walls, and there are many small vascular bundles. There are 1 to 2 rows of polygonal cells at the junction of the cortex and the hypodermis, which are suberized and have obvious pore patterns (Fig. 3, Fig. 4).

Powder: The powder of the product is off-white to light yellowish brown. The vessels are mostly reticulated, scalariform or bordered pits, with a diameter of 20 to 45 μm, and the pits are relatively dense. The stone cells are round and polygonal, with slightly thickened walls, and sparse pits and colporates can be observed. The wood fibers are elongated or irregularly long fusiform, often curved, with sharp ends, and some of them are branched at one end. On the wood fibers, there are single diagonal pits or intersected cross-shaped pits, distributed sparsely. The cork cells are polygonal, with thin walls which are slightly lignified (Fig. 5).

TLC identification

According to thin layer chromatography (General rules 0502 in fourth part of the 2015 edition of Chinese Pharmacopoeia), 5 μl of each of the above two solutions was drew and used for thin layer chromatography with cyclohexane-ethyl acetate-glacial acetic acid (10∶3∶0.1) as the developing solvent through the steps of developing, taking out, drying and spraying with phosphomolybdic acid ethanol solution. In the chromatograms of the test substances, there were spots of the same color at the corresponding positions of the chromatogram of the reference substance (Fig. 6).

Determination of moisture, acid-insoluble ash and extract

Our research team tested 10 batches of R. sativus medicinal materials in accordance with the general rules in the fourth part of the 2015 edition of Chinese Pharmacopoeia (0832 moisture determination method (second method), 2302 ash determination method, 2201 hot dipping method), and the results are shown in Table 2.

Feng BAO et al. A Preliminary Study on the Quality Standard of Raphanus sativus L. Produced in Guizhou Province

Conclusions and Discussion

Radish is a common vegetable in our lives. It is rich in a variety of amino acids, vitamins, potassium, magnesium, zinc, manganese, mustard oil, glucose, calcium and other nutritional elements. It has the functions of invigorating the stomach and promoting digestion, detoxifying and reducing phlegm, promoting bile secretion, promoting intestinal motility, preventing scurvy, and resisting cancer［6-8］. R. sativus is a by-product of seeds harvested in the process of radish planting. It has the effects of ventilating the lung and resolving phlegm, promoting digestion, and inducing diuresis to alleviate edema, and is used for treating cough, swelling pain of stomach duct and abdomen, edema, indigestion and anorectic dysentery as an ethnic medicine in Guizhou［4］. R. sativus are obtained from different varieties of radishes, and their traits are also slightly different, and are mainly reflected in the color of the skin. Generally, the skin of R. sativus is yellowish-white or purple-brown. In this study, the microscopic characteristics and thin-layer

chromatograms of different batches of radish with different skin colors were compared, and no obvious differences were found. We established the microscopic identification and thin-layer identification methods of radish, measured the moisture, ash, and extract contents of 10 batches of medicinal materials, and formulated the quality standard plan for R. sativus systematically. This study provides relatively complete draft quality standards for the formulation of local standards for R. sativus.

References

［1］ Editorial board of flora of Guizhou. Flora of Guizhou Province［M］. Sichuan: Sichuan Ethnic Publishing House, 1989. (in Chinese)

［2］ Institute of Botany, Chinese Academy of Sciences. Illustrated book of higher plants in China［M］. Beijing: Science Press, 1972. (in Chinese)

［3］ Teaching and Research Section of Pharmacognosy, Department of Pharmacy, Second Military Medical University. Illustrated book of Chinese medicinal plants［Ｍ］. Shanghai: Education Press, 1960. (in Chinese)

［4］ Guizhou Medical Products Administration. Quality standards for chinese medicinal materials and ethnic medicinal materials in Guizhou Province (2003 edition)［S］. Guiyang: Guizhou Science and Technology Publishing House, 2003: 275. (in Chinese)

［5］ ZHANG L. Pharmacognostic identification of old radish root［J］.Strait Pharmaceutical Journal, 2017, (29): 32-34. (in Chinese)

［6］ ZHANG L, SONG SH, WANG WQ, et al. Comparison of nutrition components in different radish cultivars［J］. Northern Horticulture, 2010(20): 57-582. (in Chinese)

［7］ HUANG R. Treasure of vegetables: Radish［J］. Modern Vegetable, 2005(9): 153. (in Chinese)

［8］ YANG L. The health benefits of radish［J］. Nongxun Xinjishu, 2013(1): 43. (in Chinese)