Pharmacognostic Identification of Hedyotis auricularia and Mitracarpus villosus

Piaoling HUANG, Feipeng HUANG, Xinxin LU, Zhonghua DAI, Hailin LU

Guangxi University of Chinese Medicine, Nanning 530001, China

Abstract [Objectives] To conduct the pharmacognostic identification of Hedyotis auricularia and Mitracarpus villosus in Guangxi and provide a scientific basis for their identification. [Methods] The characteristics of original plants were studied by origin identification method; the properties and characteristics were studied by character identification method; and the microscopic features of the roots, stems, leaves and medicinal powder of H. auricularia and M. villosus in Guangxi were studied by paraffin method and powder slicing method. [Results] (i) Origin identification. H. auricularia: Leaves leathery, apex acuminate, base cuneate; petiole shorter; cyme axillary; corolla hairy at throat; fruit indehiscent at maturity; testa black after drying. M. villosus: Leaf apex short pointed, base attenuate, blade sessile; flowers small, clustered in axillary; fruits dehiscent by lid at or below middle at maturity, seeds dark brown. (ii) Character identification. Fracture surface of H. auricularia uneven, white in outer layer and sepia in inner layer. Fracture surface of M. villosus hollow, uneven and white. (iii) Microscopic identification. H. auricularia: Root phloem thick, cambium visible, duct cells quasi-polygonal, large; rays obvious. Stem transection quasi-circular square, often with non-glandular hairs on epidermis; calcium oxalate raphides present in leaf parenchymal cells. Power grayish brown, starch granules single-grained; calcium oxalate raphides frequent, calcium oxalate clustered crystals occasional; catheter spiral, rarely annular, stomata infinitive. M. villosus: Root parenchyma cells with scattered calcium oxalate raphides, calcium oxalate clustered crystals and brownish red substances visible. Stem transection quasi-square, edge angle with 4 short narrow wings. Powder brown, simple starch granules numerous, compound starch granules also present; calcium oxalate raphides numerous, calcium oxalate clustered crystals and calcium oxalate square cubic crystals also present; catheter spiral, stomata paracytic. [Conclusions] The above transaction microscopic characteristics of the roots, stems and leaves and powder characteristics can be used as the identification features of H. auricularia and M. villosus.

Key words Hedyotis auricularia, Mitracarpus villosus, Pharmacognostic, Identification

1 Introduction

Guangxi is located in low latitude, belonging to subtropical monsoon climate, with warm climate, plenty of heat and a wide variety of plants, and there are often many similar points of identification between similar species.HedyotisauriculariaL. belongs toHedyotis, Rubiaceae[1]. The whole plant ofH.auriculariais often used medically, and it has the effects of clearing heat and removing toxicity, and cooling blood and dispersing swelling[2]. It has a good therapeutic effect on cold, sore throat, cough, skin eczema and venomous snake bite[3-4]. In theNationalCompilationofChineseHerbalMedicine, it has many alias such as auricled hedyotis herb, featured by bitter taste and cold nature[5]. It was first recorded in theRawHerbPropertiesAssembly(ShengcaoYaoxingBeiyao) and then listed in theLingnanCollectionRecordofMedicinalHerbs(LingnanCaiyaoLu): "Herbs, leaves opposite, pointed and slender, margin entire, arising from axils." According to this description, it is consistent withH.auriculariain Rubiaceae[6-8]. Modern studies have found that the chemical components ofH.auriculariaare mainly alkaloids, flavonoid glycosides, amino acids and β-sitosterol, while β-sitosterol is one of the phytosterol components in plants and has anti-inflammatory, hylipid-lowering and anti-tumor effects[5,9-10].Mitracarpusvillosus(Sw.) DC., belonging toMitracarpus, Rubiaceae, is born on highways and wastelands, mainly distributed in India, tropical South America, tropical East and West Africa, and in Hainan, Guangdong, Guangxi and other places of China[3].M.villosusis an invasive alien species[11-15].H.auriculariaandM.villosushave similar characters. Therefore, this paper conducted pharmacognostic identification and comparison ofH.auriculariaandM.villosusin Guangxi from origin, character, microscopic feature, and leaf morphology-venation graph, in order to provide a reference for further development and utilization ofH.auriculariaandM.villosus.

2 Materials and methods

2.1 Materials

2.1.1Sample source. The experimental samples were collected from Baqiao Village, Shuangqiao Town, Wuming District, Nanning City, Guangxi Province, and identified as the whole grass ofM.villosus(Sw.) DC. andH.auriculariaL. by associate professor Guo Min at Guangxi University of Chinese Medicine.

2.1.2Reagents. 95% Ethanol (Guangxi Beilunhe Medical Industry Group Co., Ltd.); formalin, glacial acetic acid and chloral hydrate solution (Sinopharm Chemical Reagent Co., Ltd.).

2.1.3Instruments. Electrothermal blowing dry box (DHG9340A, Shanghai Yiheng Scientific Instrument Co., Ltd.); paraffin slicer (RM2235, Leica Microsystems Wetzlar GmbH); high speed grinder (DFY-300C, Wenling Big Machinery Co., Ltd.); fluorescence biological microscope (Ni-U, Nanjing Hengqiao Instrument Co., Ltd.).

2.2 Methods

2.2.1Origin identification. Origin identification was performed in the following steps: observing plant morphology → consulting literature → checking specimens.

2.2.2Character identification. The appearance and morphology of medicinal materials were observed through visual observation, hand touch, smelling and sipping. The identification content generally included shape, size, color, surface characteristics, texture, fracture surface, flavor, taste and so on.

2.2.3Paraffin section. Paraffin sectioning method included sampling, fixation, washing and dehydration, transparency, wax impregnation, embedding, slicing and patching, dewaxing, dyeing, dehydration, transparent, sealing and other steps. Sections can be stored for a long time.

2.2.4Powder slice making. (i) Powder preparation.H.auriculariaandM.villosuswere dried in an oven at 60 ℃, then crushed with a grinder, passed through No.4 sieve, and stored in the dryer for later use. (ii) Temporary slice making of power. A small amount of powder was taken by a small medicine spoon and loaded to a clean slide, then added with 1 or 2 drops of dilute glycerin. The cover glass was covered over the glass slide slantingly at 45°, and excess liquid was wiped from another side of the cover glass. It can be used to observe the characteristics of starch granules and other powders. A slice was made by chloral hydrate solution, and permeabilized to make the characteristics of fiber and catheter more obvious. After cooling, half a drop of dilute glycerin was added, and the cover glass was covered slantingly at 45°. The power slice was placed under an optical microscope to observe the microscopic characteristics of tissues, which were photographed and preserved with an optical microscope.

3 Results and analysis

3.1 Origin identification

3.1.1H.auricularia. Herbs, perennial, hairchested, suberect or recumbent; stems flattened, 4-angled, terete when old, with adventitious root on nodes, branchlets densely puberulent. Leaves opposite, leathery, lanceolate to elliptic, (3.5-8.0) cm×(1.0-3.5) cm, apex acuminate, base cuniform, margin entire, surface green; petiole short, 2-7 mm, brown-yellow tomentose; stipule membranous, hairy, united to short sheath. Cymes axillary, densely capitate, peduncle absent; corolla white, lobes 4, outside glabrous, inside only hairy at throat. Fruits globose, sparsely tomentose or subglabrous, indehiscent at maturity. Seeds 2-6 per chamber, testa black when drying (Fig.1A).

Fig.1 Original plants of Hedyotis auricularia (A) and Mitracarpus villosus (B)

3.1.2M.villosus. Herbs, erect, multibranched, 35-80 cm tall. Stem quadrilateral when young, terete when old, sparsely hirtellous. Leaves opposite, grassy, oblong to lanceolate, (1.7-3.5) cm × (0.3-1.6) cm, apex acute, base attenuate, margin entire, sessile. Flowers small, fascicled in axils, corolla funnel-shaped, lobes 4, glabrous. Fruits subglobose, epidermis coarse or sparsely pubescent, dehiscent by lid at or below middle at maturity. Seeds dark brown, suboblong (Fig.1B).

3.2 Character identification

3.2.1H.auricularia. Plants light and tough. Roots longitudinally striate, tough, hard to break off; fracture surface not flattened, white in outer layer, sepia in inner layer. Stems white tomentose; old stems yellowish brown, terete; young stems green, tabular, tough, hard to break off; fracture surface not flattened, old stems white brown, young stems white. Leaves green, opposite, leathery, adaxial vein slightly concave, abaxial vein slightly convex; blade slightly curled, broadly oval when spreading. Flavor mild, taste astringent (Fig.2A).

3.2.2M.villosus. Plants light and tough. Roots longitudinally striate, lilac, hard to break off; fracture surface hollow, not flattened, white. Stems pale green, white tomentose, quadrilateral when young, terete when old, brittle, easy to break off; fracture surface flattened, hollow. Leaves opposite, apical one fascicled, yellow-green or pale green, densely white tomentose, papery, adaxial vein slightly concave, abaxial vein slightly convex; blade slightly curled, broadly oval when spreading. Flavor mild, taste astringent (Fig.2B).

3.3 Microscopic identification

3.3.1Root transaction. (i)H.auricularia. Root transection quasi-circular. Cork layer composed of several columns of flat cork cells, outer cells fragile. Cortex thin, with only several columns of parenchyma cells, sparsely arranged, gapped. Vascular bundle ectophloic, phloem thick, cambium visible; xylem broad, duct cells quasi-polygonal, large; rays obvious (Fig.3A).

(ii)M.villosus. Root transection quasi-circular. Cork layer composed of 1-2 columns of cork cells, outer cells fragile. Cortex thin, with only several columns of parenchyma cells, obround or suboblong, with scattered calcium oxalate raphides, calcium oxalate clustered crystals and brownish red substances visible. Vascular bundle ectophloic, phloem narrow, cambium invisible; xylem broad, occupying most area of transection (Fig. 3B).

3.3.2Stem transaction. (i)H.auricularia. Stem transection quasi-circular square. Epidermal cells single-columned, quasi-circular, often with non-glandular hairs. Cortex broad, consisting of 7 layers of parenchyma cells. Epidermal cells short columnar, large, tightly arranged in palisade shape; endothelial cells quasi-rectangular, densely arranged. Vascular bundle ectophloic, phloem narrow, cambium unconspicuous, xylem broad. Pith part broad, located in center, occupying most area of transection. Parenchyma cells quasi-polygonal, sometimes broken and hollow in middle part (Fig.4A).

Note: A1, B1. Full micrographs of stem transaction of H. auricularia and M. villosus; A2, B2. Local magnification of stem transaction of H. auricularia (1. Non-glandular hairs; 2. Epidermis; 3. Cortex; 4. Phloem; 5. Xylem; 6. Pith) and M. villosus (1. Collenchyma; 2. Epidermis; 3. Cortex; 4. Phloem; 5. Xylem; 6. Pith).

(ii)M.villosus. Stem transection quasi-square; edge angle with 4 short narrow wings composing of several columns of collenchyma. Epidermal cells single-columned, quasi-circular, small. Cortex narrow, consisting of several columns of parenchyma cells, suboblong; endothelial cells large, densely arranged. Vascular bundle ectophloic, phloem narrow, with brownish red substances; cambium unconspicuous, xylem broader. Pith part broad, located in center, occupying most area of transaction (Fig.4B).

3.3.3Leaf transaction. (i)H.auricularia. Epidermal cells single-columned, often non-glandular hairy in lower epidermis; upper epidermal cells quasi-circular to quasi-square, large; lower epidermal cells quasi-circular, small. Palisade cells single-columned, cylindrical, not reaching midrib, brownish red substances visible in cells; spongy tissue quasi-circular, loosely arranged, calcium oxalate raphides and brownish red substances present in parenchymal cells. Midrib with several columns of collenchyma in inner side of upper and lower epidermis; vascular bundle of midrib ectophloic, half-moon-shaped (Fig.5A).

Note: 1. Upper epidermis; 2. Palisade tissue; 3. Spongy tissue; 4. Lower epidermis; 5. Collenchyma; 6. Xylem; 7. Phloem; 8. Non-glandular hairs.

(ii)M.villosus. Epidermal cells single-columned, upper epidermal cells quasi- rectangular, relatively large; lower epidermal cells quasi-circular, often with non-glandular hairs in epidermis, non-glandular hairs in lower epidermis longer. Palisade cells single-columned, cylindrical, not reaching midrib; spongy tissue loosely arranged, with brownish red substances. Midrib with several columns of collenchyma in inner side of upper and lower epidermis; vascular bundle of midrib ectophloic, quasi-half-moon-shaped (Fig.5B).

3.3.4Microscopic features of powder. (i)H.auricularia. Powder grayish brown. Starch granules numerous, granule elliptic, small, multi-aggregated, 2-5 μm in diam. Calcium oxalate raphides many, fascicled or scattered in parenchyma cells, 6-110 μm in diam.; calcium oxalate clustered crystals occasional, small, 10-15 μm in diam. Non-glandular hairs common, single-celled or composed of several cells, 68-210 μm long, often with warty prominences on surface. Catheter spiral, or annular, rarely pit bordered, 9-40 μm in diam. Fibers usually bundled, cell cavity large, pits often visible. Stomata infinitive, subsidiary cells 3-5. Pollen grains common, yellow or pale yellow, spherical, 28-40 μm in diam. (Fig.6A).

Note: A. H. auricularia (1. Stomata; 2. Catheter; 3. Starch granules; 4. Pollen grains; 5. Fibers; 6. Non-glandular hairs; 7. Calcium oxalate raphides; 8. Calcium oxalate clustered crystals; 9. Brown substances); B. M. villosus (1. Stomata; 2. Calcium oxalate raphides; 3. Calcium oxalate clustered crystals, calcium oxalate cubic crystals; 4. Non-glandular hairs; 5. Fibers; 6. Starch granules; 7. Catheter; 8. Pollen grains; 9. Brown substances).

(ii)M.villosus. Powder brown. Starch granules numerous, granule circular, 3-6 μm in diam., composite grains often composed of 2-3 single starch granules. Calcium oxalate raphides common, 50-124 μm long, fascicled or scattered in cells; calcium oxalate clustered crystals present in parenchymal cells, 6-12 μm in diam.; calcium oxalate cubic crystals scattered, small, 3-13 μm in diam.; non-glandular hairs common, single-celled or composed of several cells, 100-236 μm long, occasionally with warty prominences on surface. Catheter spiral, 6-24 μm in diam.; fibers usually bundled, cell wall thick, pit visible. Stomata paracytic. Pollen grains common, pale yellow, spherical to oval, with particles on surface, 22-25 μm in diam.(Fig.6B).

4 Conclusions

From the above studies, it can be concluded thatH.auriculariaandM.villosuscan be distinguished by the following features.

(i) Origin identification.H.auricularia: Leaves leathery, apex acuminate, base cuneate; petiole shorter; cyme axillary; corolla hairy at throat; fruit indehiscent at maturity; seeds 2-6 per chamber, testa black when drying.M.villosus: Leaf apex short pointed, leaf base attenuate, leaves sessile; flowers small, clustered in axils; fruits dehiscent by lid at or below the middle at maturity, seeds dark brown, suboblong.

(ii) Character identification. Fracture surface ofH.auriculariauneven, white in outer layer and sepia in inner layer. Fracture surface ofM.villosushollow, uneven and white.

(iii) Microscopic identification.H.auricularia: Root phloem thick, cambium visible, duct cells quasi-polygonal, large; rays obvious. Stem transection quasi-circular square, often with non-glandular hairs on epidermis. Calcium oxalate raphides present in leaf parenchymal cells. Power grayish brown, starch granules single-grained; calcium oxalate raphides frequent, calcium oxalate clustered crystals occasional; catheter spiral, rarely annular, occasionally pit bordered; stomata infinitive, pollen grains common, yellow or pale yellow.M.villosus: Root transection parenchyma cells with scattered calcium oxalate raphides, calcium oxalate clustered crystals and brownish red substances visible. Stem transection quasi-square, edge angle with 4 short narrow wings, consisting of several columns of collenchyma. Powder brown, simple starch granules numerous, compound starch granules also present; calcium oxalate raphides numerous, calcium oxalate clustered crystals and calcium oxalate square cubic crystals also present; catheter spiral, stomata paracytic, pollen grains common.

The above results can offer basic research data for the identification and quality evaluation ofH.auriculariaandM.villosusin Guangxi, provide a scientific basis for their identification, and ensure the safety of drug use.