Quality Standard for Euonymi Grandiflorus Caulis and Folium

Qi CUI, Yuanlin KONG, Mingkang FENG, Riluo FENG, Luha AYI, Ying LI*, Chen CHEN, Wenbing LI, Yuan LIU*

1. College of Pharmacy, Southwest Minzu University, Chengdu 610041, China; 2. Sichuan Provincial Qiang-Yi Medicinal Resources Protection and Utilization Technology Engineering Laboratory, Chengdu 610225, China; 3. Tibetan Plateau Ethnic Medicinal Resources Protection and Utilization Key Laboratory of National Ethnic Affairs Commission of the People’s Republic of China, Chengdu 610225, China

Abstract [Objectives] To establish the quality standard for Euonymi Grandiflorus Caulis and Folium. [Methods] Qualitative identification was performed by methods of origin identification, microscopic identification and thin layer chromatography (TLC); the moisture, total ash, acid-insoluble ash, and extract were detected using the method in the Chinese Pharmacopoeia (2020 Edition), and the contents of quercetin and kaempferol were determined by High Performance Liquid Chromatography (HPLC). [Results] The leaves of Euonymi Grandiflorus were subleathery, narrow and long elliptic or narrow obovate with cyme, and capsules were often with narrow wing ridge. Characteristics of microscopic identification were significant. Calcium oxalate cluster crystals, cubic crystals, inlaid parenchyma cells, starch granules, stomata, and fiber bundles could be observed. A TLC method for identification of Euonymi Grandiflorus Caulis and Folium was established. The contents of water, total ash, acid-insoluble ash were not higher than 13%, 9%, 12%, respectively; the extract were no less were not higher than 13%, 9%, 12% and 17% in ten batches of samples, respectively; the sum of quercetin and kaempferol were no less than 0.20%.[Conclusions] The quality standard of Euonymi Grandiflorus Caulis and Folium were established through the research. This method is accurate, specific, and reproducible and can be used for the quality control of Euonymi Grandiflorus Caulis and Folium.

Key words Euonymi Grandiflorus Caulis and Folium, Quercetin, Kaempferol, Quality standard (draft)

1 Introduction

Euonymi Grandiflorus Caulis and Folium is the dry branches and leaves ofEuonymusgrandiflorusWall. of the Celastraceae family[1-2], also called black eucommia, tree eucommia, and golden silk eucommia[1]. There are records of Euonymi Grandiflorus Caulis and Folium inNationalCompendiumofChineseHerbalMedicine[2],DictionaryofMedicinalPlants[3], andDictionaryofChineseEthnicMedicines[4]. It is a commonly used Qiang people and Yi people in Sichuan Province for activating blood, relieving pain, stimulating main and collateral channels, and tonifying the kidney[5]. Physicians of Qiang people in Sichuan Province are good at the treatment of bone injuries and diseases. Euonymi Grandiflorus Caulis and Folium are the traditional Chinese medicinal materials in experiential prescriptions of Qiang medicine osteopathic ointment ointment and Yi medicine gout granules. Chen Aiwu[7]found that the addition of golden silk eucommia to different prescriptions can relieve various types of pain caused by bone hyperplasia. Yan Xinshun[8]used black eucommia in the prescription for the treatment of psoatic strain, and found that it had a significant effect on the treatment of low back pain due to chronic consumptive diseases. Euonymi Grandiflorus Caulis and Folium has significant clinical effects in Sichuan Qiang medicine and Yi medicine, and has abundant resources, but there is still no quality standard for it. In view of this, we studied the quality standard of medicinal materials in the aspects of original plant identification, morphological identification, microscopic identification, thin layer chromatography (TLC) identification, content determination, and routine item inspection, to provide experimental data for establishing the quality standard of Euonymi Grandiflorus Caulis and Folium and its rational utilization, as well as incorporating the medicinal material intoSichuanProvinceQiang,Yi,andMiaoMedicinalMaterialsStandard.

2 Materials

2.1 InstrumentsWaters 2695 Liquid Chromatograph, Axio scop5 Optical Microscope, RM225 Paraffin Microtome, ATY124 Electronic Balance, AUW220D Electronic Balance, and Skymen Ultrasonic Cleaner (Skymen Cleaning Equipment Shenzhen Co., Ltd.).

2.2 Reagents and drugsQuercetin reference substance (Chengdu ConBon Biotech Co., Ltd., batch No.:16010127); Kaempferol reference substance (Chengdu Preferred Biotechnology Co., Ltd., batch No.:17011305); silica G TLC plates; acetonitrile was of chromatographic grade; ethanol and other reagents were of analytical grade; water was ultrapure water.

2.3 Medicinal materialsTen batches of Euonymi Grandiflorus Caulis and Folium materials were identified by Professor Liu Yuan and associate professor Li Ying from Southwest Minzu University as dry branches and leaves ofEuonymusgrandiflorusWall. The sample information is shown in Table 1.

Table 1 Information of samples

3 Methods and results

3.1 Identification study

3.1.1Original plant identification. The original plants of Euonymi Grandiflorus Caulis and Folium are shrubs or arbors, semi-evergreen, with height up to 8 m; leaves are subleathery, narrow and long elliptic or narrow obovate with cyme, 4-10 cm long, and 1-4 cm wide, apex is rounded or sharp, base is often tapered into a wedge shape, the margins are densely superficially serrated, and the lateral veins are dense; petiole is up to 1 cm long; loose cymes with 3-9 flowers, the peduncle is 3-6 cm long; small pedicels are about 1 cm long; bracteoles take on narrow line shape; flowers are yellowish-white, 4-numbered, larger, up to 1.5 cm in diameter; calyx are mostly connate; sepals are very short; the petals are nearly round, and there are chewing wrinkles in the center; stamens are attached to the disc-shaped protrusions at the four corners of the disc; ovary is quadrangular pyramid-shaped; capsules are nearly spherical, often with a narrow wing ring, persistent calyx is disc-shaped, up to 7 mm in diameter; seeds are oblong and black-red, aril is red, helmet-shaped, and covers the upper part of seed. The flowering period is June to July, and the fruiting period is September to October, as shown in Fig.1.

Note: A. whole plant; B. branches and leaves.

3.1.2Morphological identification. The Euonymi Grandiflorus Caulis and Folium has cylindrical branches with a diameter of 0.2-1.5 cm, the surface is gray-green to gray-brown or reddish brown, with vertical fine wrinkles; hard texture, not easy to break, flaky fibrous cross-section layer, white; leaves are often fallen off, the folds are broken, gray-green to brown-yellow; intact leaves are narrowly oblong or narrow obovate when flattened; slight odor, light taste, and slightly astringent, as shown in Fig.2.

Note: 1. branches; 2. Leaves.

3.1.3Microscopic identification. (i) Cross section of leaf. One row of upper epidermal cells, round, densely arranged, covered by cuticle; lower epidermal cells are round, closely arranged, and stomata are visible; collenchyma is visible inside the upper and lower epidermal cells; leaf is bifacial leaf, palisade tissue passes through the main vein; one vascular bundle in the main vein, collateral type, with pericyclic fibers on the upper and lower sides, and parenchyma cells containing calcium oxalate cluster crystals, forming crystal fibers, as shown in Fig.3.

Note: 1. upper epidermis; 2 and 10. collenchyma; 3. palisade tissue; 4. xylem; 5. spongy tissue; 6. phloem; 7. pericyclic fibers; 8. stomata; 9. calcium oxalate cluster crystals; 11.lower epidermis.

(ii) Powder. Powder is light grass green to brownish green; calcium oxalate cluster crystals are numerous, single scattered or embedded in epidermis or parenchyma cells, round, different sizes, 16-72 μm in diameter, with blunt edges and corners; calcium oxalate cubic crystals are rare, irregular shape; starch granules are many, the single granule is round, the umbilical point is chevron, dot-shaped or crack-shaped with diameter of 15-132 μm; compound granules are few and consist of 2 granules; stomata are rare, indefinite, with 4 to 6 (8) subsidiary cells; pericyclic fibers are mostly bundled or scattered individually, with a diameter of 16-153 μm and a wall thickness of 4-62 μm, as shown in Fig.4.

Note: 1. calcium oxalate clusters; 2.inlaid parenchyma cells; 3. calcium oxalate cubic crystals; 4. starch grains; 5. epidermal cells and stomata; 6. fiber bundles.

3.1.4TLC qualitative identification. Took 1.0 g of medicinal powder (screened with a No.2 sieve), added 10 mL of ethyl acetate, ultrasonically treated for 30 min, filtered, evaporated the filtrate to dryness, then added 0.5 mL of ethyl acetate to the residue to dissolve it, and used it as the test solution. At the same time, took 1.0 g of powder of Euonymi Grandiflorus Caulis and Folium reference substance, and prepared the reference drug solution using the same method. Pipetted 2-5 μL of each of the above two solutions using the TLC method in General Rules 0502 ofChinesePharmacopoeia(2020Edition) Volume IV, dotted the solutions on the same silica gel G thin layer plate separately, used cyclohexane-ethyl acetate (6∶1.5) as the developing solvent, developed, took out, and air dried. Then, sprayed with 10% sulfuric acid ethanol solution and heated at 105 ℃ until the spots became clear. The results showed that the test product showed spots of the same color at the position corresponding to the chromatogram of the reference drug, and the image was clear (Fig.5).

Note: 1. Reference drug, 2-11. Ten batches of medicinal materials.

3.2 Determination of moisture, ash and extract contentsThe moisture, total ash, acid insoluble ash and extract contents were separately determined using the method in General Rules 0832, General Rules 2302, General Rules 2302, and General Rules 2201 ofChinesePharmacopoeia(2020Edition) Volume IV. The Results are shown in Table 2.

3.3 Determination of quercetin and kaempferol contents

The quercetin and kaempferol contents were determined with reference to literature[10-12].

Table 2 Determination results of moisture, total ash, acid insoluble ash and extract %

3.3.1Chromatographic conditions. Waters C18chromatographic column was (4.6 mm × 250 mm, 5 μm); mobile phase acetonitrile-pure water was 49∶51; volume flow rate was 1 mL/min; column temperature was 25 ℃; the detection wavelength was 370 nm; the injection volume was 10 μL. The chromatogram is shown in Fig.6.

Note: 1. quercetin; 2. kaempferol.

3.3.2Preparation of test solution. Took about 0.5 g of the test product powder (screened with No.2 sieve), precisely weighed, and placed it in a conical flask with a stopper, precisely added 25 mL of methanol-hydrochloric acid solution (4∶1), weighed, put it in a water bath to reflux for 1 h, cooled down, and weighed again, made up the lost weight with methanol-hydrochloric acid solution (4∶1), shook up, filtered, then screened the supernatant with a 0.22 μm microporous membrane, and the subsequent filtrate was the test solution.

3.3.3Linear relationship test. Precisely weighed an appropriate amount of quercetin and kaempferol reference substance, added methanol-hydrochloric acid solution (4∶1) to dilute the volume to 10 mL, obtained a stock solution with a mass concentration of 0.2 mg/mL, stored in a refrigerator at 4 ℃ for later use. Precisely pipetted an appropriate amount to make multiple mass concentrations, and injected and measured under the chromatographic conditions in Section3.3.1. Taking the mass concentration of each component as the abscissa (X) and the peak area as the ordinate (Y) for regression, obtained the equations for quercetinY=3.557 8×107x-6.124 2×104(r=0.999 7), which had good linearity in the range of 0.01-0.16 mg/mL. Kaempferol regression equation wasY=3.603 6×107x-2.174 4×104(r=0.999 7), which had a good linearity in the range of 0.007 5-0.12 mg/mL.

3.3.4Precision test. Precisely pipetted 10 μL of the reference solution containing 0.040 0 mg/mL quercetin and 0.030 0 mg/mL kaempferol, injected samples 6 times under the chromatographic conditions in Section3.3.1, and theRSDvalues of the peak areas of quercetin and kaempferol were 0.75% and 0.56%, respectively, indicating that the instrument had good precision.

3.3.5Stability test. Precisely pipetted an appropriate amount of the test solution (D4), and injected samples at 0, 2, 4, 8, 12, and 24 h under the chromatographic conditions. TheRSDvalues of measured peak areas of quercetin and kaempferol were both 1.57%, indicating that the solution had good stability within 24 h.

3.3.6Reproducibility test. Precisely weighed 6 parts of medicinal material (D4) powder, each 0.5 g, and injected 10 μL under the chromatographic conditions in Section3.3.1. TheRSDvalues of quercetin and kaempferol were 1.69% and 1.63%, respectively, indicating the method had good reproducibility.

3.3.7Sample recovery test. Precisely weighed 6 parts of medicinal material (D4) powder, each 0.25 g, put in a stoppered conical flask, added 0.687 mg quercetin reference substance, 0.400 mg kaempferol reference substance, 25 mL methanol-hydrochloric acid solution (4∶1), prepared the test solution using the method in Section3.3.2, injected and measured it under the chromatographic conditions in Section3.3.1, calculated the sample recovery rate, the results are shown in Table 3.

Table 3 Results of sample recovery tests for various components (n=6)

3.3.8Determination of sample content. Took 10 batches of medicinal materials, prepared the test solution using the method in Section3.3.2, injected and measured under the chromatographic conditions in Section3.3.1, and calculated the content by external standard method. The results are shown in Table 4.

Table 4 Results of content determination of various components (%, n=3)

4 Discussion

(i) TLC identification. This experiment firstly investigated the developing systems of different ratios of cyclohexane-ethyl acetate (6∶1, 6∶1.5), and it found that the spots were clear and the separation was good at the ratio of 6∶1.5. Then, it investigated the silica gel G thin-layer plate and GF254thin-layer plate, and found that the G thin-layer plate exhibited complete separation of spots, the image was clear and the effect was ideal.

(ii) Limit of moisture, ash and extract contents. Combined with the relevant provisions of General Rules ofChinesePharmacopoeia(2020Edition) Volume IV and General Rules for the Verification of Medicinal Materials and Decoction Pieces, it is tentatively decided that the moisture content of Euonymi Grandiflorus Caulis and Folium should not exceed 13%, the total ash content should not exceed 9%, and the acid-insoluble ash content should not exceed 2%, and the extract should not be less than 17%.

(iii) Limit of quercetin and kaempferol contents. The contents of quercetin and kaempferol in Euonymi Grandiflorus Caulis and Folium were significantly different in 10 batches. Calculated at the dry product of Euonymi Grandiflorus Caulis and Folium, it is tentatively determined that the sum of quercetin and kaempferol should not be less than 0.20%.

5 Conclusions

This experiment fills in the blank of the quality standards for Euonymi Grandiflorus Caulis and Folium in Sichuan Qiang and Yi medicinal materials, and provides a reference for formulating the standards for Sichuan Qiang and Yi medicinal materials. When the active components quercetin and kaempferol are used as the indicator components, the test cost can be reduced, the accuracy can be increased, and the quality control feasibility can be improved.