Pharmacognostic Identification of Euphorbia maculata L. and Its Related Species

Jing HOU, Li LI, Xiaotong JING, Yuan LIANG, Shaoqi LUO, Yi CAI

Guangxi University of Chinese Medicine, Nanning 530001, China

Abstract [Objectives] To make pharmacognostic identification of Euphorbia maculata L. and its related species. [Methods] The classical pharmacognostic identification method was adopted. [Results] The four main medicinal materials are very similar, the fluff can be seen in E. maculata and Euphorbia thymifolia L., but not in Euphorbia prostrata Ait. and Euphorbia taihsiensis (Chaw et Koutnik) Oudejians; the tissue structure is basically the same; except for E. taihsiensis without non-glandular hairs, the powder has secretory cells, laticifers, cells, calcium oxalate crystals, fibers, vessels, and seed coat fragments. Through ultrasonic extraction with 80% ethanol, extraction with isobutanol, extending with chloroform∶ethyl acetate∶formic acid (5∶3∶0.3), developing color with 3% aluminum trichloride ethanol solution, under ultraviolet light (365 nm), the fluorescent spots of the same color appeared on the corresponding position of the chromatogram of the reference substance (quercetin, kaempferol). [Conclusions] It is not easy to distinguish the four medicinal materials by character identification and microscopic identification, while the thin-layer chromatography (TLC) is more reliable.

Key words Euphorbia maculata L., Euphorbia thymifolia L., Euphorbia prostrata Ait., Euphorbia taihsiensis (Chaw et Koutnik) Oudejians, Pharmacognostic identification

1 Introduction

EuphorbiamaculataL. is a plant of the genusEuphorbia. It is included in theChinesePharmacopoeia(2012 edition) with the same group of plant,EuphorbiahumifusaWilld.ex Schlecht. It has the effects of clearing heat and detoxifying, cooling blood and arresting bleeding, removing dampness and relieving jaundice. Clinically, it is used for the treatment of dysentery, hemoptysis, hematuria, metrorrhagia, boils, carbuncle, damp heat and jaundice[1]. In recent years, studies have shown that it has pharmacological activities such as protecting liver injury[2-3], anti-cervical cancer[4-5], anti-tumor and promoting angiogenesis[6-8], thus it has gradually attracted people’s attention. In Guangxi, six kinds ofSect.Chamaesyceplants, in whichEuphorbiamaculataL.,EuphorbiathymifoliaL.,EuphorbiaprostrataAit.,Euphorbiataihsiensis(Chaw et Koutnik) Oudejians are common. They are similar in appearance, often mixed together, and the properties of the medicinal materials are very similar, which is easy to cause confusion. In view of this, to ensure the safety and effectiveness of clinical medication, we used the pharmacognostic identification method to distinguish them.

2 Materials and methods

2.1 InstrumentsLeica DFC500 microsystems (Leica Geosystems Technologies GmbH, Germany); Leica DM2500 optical microscope (Leica Geosystems Technologies GmbH, Germany); Leica RM 2145 rotary microtome (Leica Geosystems Technologies GmbH, Germany).

2.2 ReagentsThin layer chromatography silica gel G (chemically pure, Qingdao Haiyang Chemical Co., Ltd., China; 95% ethanol, absolute ethanol, petroleum ether (60-90 ℃), chloroform, ethyl acetate, methanol; reagents are all analytical pure, water is pure water. The four kinds of medicinal materials were collected from areas of Guangxi Zhuang Autonomous Region and identified by professor Cai Yi from Guangxi University of Chinese Medicine asSect.Chamaesyceplants:E.maculataL.,E.thymifoliaL.,E.prostrataAit.,E.taihsiensis(Chaw et Koutnik) Oudejians, respectively. And the sample information of each batch of medicinal materials was listed in Table 1.

Table 1 Sample information of four species of Sect. Chamaesyce Plants

2.3 MethodsCut the dry medicinal materials produced in different areas into pieces and ground to powder, and screened with No.4 sieve (65 mesh) for later use, and then conducted identification using the classical pharmacognostic identification method.

3 Results and analysis

3.1 Common characteristics of the four kinds of medicinal materialsSmall and thin, shrunken and curled. The roots are slender and tawny. The stem is thin with forked branches, the surface is tawny or reddish brown, smooth and glabrous or sparsely white with fine fluff. It is brittle and easy to break. The section is yellow-white and hollow. The leaf is single opposite, the stalk is short or almost sessile. The leaves are mostly shrunken or fallen off, and are elliptical or oblong after being flattened, 5-10 mm long and 4-6 mm wide; light green or gray-green, slightly purplish red; the apex is blunt, slightly concave, the base is oblique, and the leaf margins are entire or with small serrations. Capsules are triangular in shape, with a smooth surface or densely covered with white fluff. The seeds are small, ovoid, brown. These medicinal materials are odorless, slightly astringent (Fig.1 and Table 2).

Table 2 Identification characteristics of 4 common species of Sect. Chamaesyce in Guangxi

3.2 Microscopic identification featuresThe four medicinal materials are of the same genus, same group, but different species, they have similar organization structures. The following is the tissue structure ofE.thymifoliaas a representative.

3.2.1Cross section of root. There are 2-3 rows of suberin lamella cells, oblong in shape. The cortical parenchyma cells are larger, similar to long oval or square-like shape. The phloem is narrow, with small cells and tightly arranged, with scattered laticifers. The cambium is not obvious. Xylem accounts for about 2/3, and most of the vessels are single and arranged radially (Fig.2).

Note: A. E. thymifolia, B. E. maculata, C. E. prostrate, D. E. taihsiensis.Fig.1 Pictures of four medicinal materials

3.2.2Cross section of stem. There is one row of epidermal cells, in square-like or oval shape. The cortex is thick, composed of 5-8 rows of cells. The phloem is narrow and laticifers are visible. Secretory cells can be seen in the cortex and phloem and contain secretions. The cambium is not obvious. The xylem is connected in a ring shape, and most of the vessels are single and arranged radially. Sclereids can be seen intermittently in a ring on the outside of the vascular bundle. The pith is broad, with large cells, round-like, and loosely arranged (Fig.3).

3.2.3Cross section of leaf. The upper and lower epidermis are one row of epidermal cells, round-like or oval shape, with different sizes. The palisade tissue is in short cylindrical shape and passes through the main vein. The outer side of the vascular bundle is a semi-circular vascular bundle sheath, which is composed of several large closely arranged cells, containing secretions, on the same side as the xylem. The phloem is narrow with scattered laticifers (Fig.4).

Note:1.suberinlamella;2.laticifer;3.cortex;4.secretorycell;5.phloem;6.xylem.Fig.2 CrosssectionofrootofEuphorbiathymifo-liaL.plantNote:1.sclereid;2.epidermis;3.cortex;4.laticifer;5.secretorycell;6.phloem;7.pith;8.xylem.Fig.3 CrosssectionofstemofEuphorbiathy-mifoliaL.plantNote:1.upperlowerepidermis;2.palisadetissue;3.vascularbundlesheath;4.xy-lem;5.phloem;6.laticifer;7.spongytissue;8.lowerepidermis.Fig.4 CrosssectionofleafofEuphorbiathymifoliaL.plant

3.3 Leaf surface characteristicsThe cells on the upper surface are polygonal, the vertical walls are straight, and anisocytic pores can be seen. The cells on the lower surface are polygonal, the vertical wall is deeply wavy and curved, with irregular pores, multicellular non-glandular hairs can be seen (Fig.5).

3.4 Whole herb powderYellow-green. Secretory cells are 29.4-42.1 μm in diameter. Laticifers have a diameter of 3.8-8.1 μm, single root, containing yellow, granular secretions. The pollen grains are spherical and 17.3-23.5 μm in diameter, golden yellow, and have smooth outer walls. Sclereids (stone cells) are 15.1-30.2 μm in diameter, multicellular non-glandular hairs, and 111.8-250.1 μm in length. Calcium oxalate square crystal has a diameter of 7.5-23.5 μm. The seed coat fragment vessels are in threaded or reticulated shape, with a diameter of 8.4-18.6 μm. Fiber has a diameter of 17.8-28.1 μm (Fig.6).

The difference in microscopic structure:E.thymifolia,E.maculata, andE.prostratahave obvious non-glandular hairs, whileE.taihsiensisdoes not.

Note:A.uppersurface;B.lowersurface;1.epidermalcell;2.pore;3.non-glandularhairsFig.5 LeafsurfacepictureofEuphorbiathymifoliaplantFig.6 WholeherbpowderpictureofEuphorbiathymifolia

4 Thin-layer identification of medicinal materials

Took 1 g of each of 4 kinds of medicinal powders from different production areas, added 50 mL of 80% ethanol, ultrasonically extracted for 60 min, filtered, evaporated the filtrate, added 60 mL of water-isobutanol (1∶1) mixture to dissolve it, and allowed to stand for a certain time to separate into two layers, discarded the isobutanol solution. Added isobutanol to the aqueous solution and extracted 20 mL twice each time, and discarded the isobutanol solution. Poured the water into a conical flask, added 2.5 mL of hydrochloric acid, hydrolyzed it in a water bath at 60 ℃ for 1 h, and took it out to cool down. Extracted twice with isobutanol, discarded the aqueous solution, combined the isobutanol solution, washed with 30 mL of water, discarded the aqueous solution, placed the isobutanol solution in an evaporating dish and evaporated to dryness, and added 1 mL of ethanol to dissolve the residue, then obtained the test solution. Performed the test using the thin-layer chromatography (General Rules 0502 of theChinesePharmacopoeia2015 Edition Volume IV). Pipetted 2 μL of each of quercetin, kaempferol reference substance and test substance solution, separately dripped on the same silica gel G thin layer plate, used chloroform-ethyl acetate-formic acid (5∶3∶0.3) as the developing agent, and expanded, sprayed with 3% ethanol solution of aluminum trichloride, took it out to dry, and viewed under ultraviolet light (365 nm). In the chromatogram of the test substancesE.maculata,E.prostrata,E.taihsiensis, andE.thymifolia, the fluorescent spots of the same color appeared at the position corresponding to the chromatogram of the reference substance (Fig.7).

Note: 1. kaempferol (reference substance); 2. quercetin (reference substance); 3. PFDJ-1; 4. PFDJ-2; 5. QGC-1; 6. QGC-2; 7.TXDJ-1; 8. TXDJ-2; 9BDJ-1; 10. BDJ-2.Fig.7 Thin layer chromatography of Sect. Chamaesyce

5 Conclusions

The above experiment showed that the four main medicinal materials are very similar, the fluff can be seen inE.thymifoliaandE.maculata, whileE.prostrataandE.taihsiensishave not. Besides, their tissue structure is basically the same. ExceptE.taihsiensishas no non-glandular hairs, the powder of all other three medicinal materials has secretory cells, laticifers, cells, calcium oxalate square crystals, fibers, vessels, and seed coat fragments. Through ultrasonic extraction with 80% ethanol, extraction with isobutanol, extending with chloroform∶ethyl acetate∶formic acid (5∶3∶0.3), developing color with 3% aluminum trichloride ethanol solution, under ultraviolet light (365 nm), the fluorescent spots of the same color appeared on the corresponding position of the chromatogram of the reference substance (quercetin, kaempferol). In conclusion, it is not easy to distinguish the four medicinal materials by character identification and microscopic identification, while using the thin-layer chromatography, it is able to effectively distinguish them.