Identification of Tibetan Medical Material Dracocephalum tanguticum Maxim.

Ling TAN, Pei QUN, Hongna SU, Xuelian ZHAO, Yisong LI, Yanfei HUANG, 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; 4.Institute of Tibetan Plateau, Southwest Minzu University, Chengdu 610041, China; 5.Ethnic Medicine Institute, Southwest Minzu University, Chengdu 610041, China

Abstract [Objectives]To explore morphological identification, macroscopical identification, microscopic identification, thin layer chromatography(TLC)identification of Tibetan medical material Dracocephalum tanguticum Maxim., and provide experimental data for its identification and application.[Methods]The Tibetan medical material was identified by means of original plant, characters, powder, paraffin section and thin layer chromatography(TLC).[Results]Tibetan medical material D. tanguticum Maxim.was obviously distinguished in character identification and microscopic identification, and the TLC method was simple and feasible.[Conclusions]The results will provide the source work foundation for the formulation of the quality standard of Sichuan Province(draft)for Tibetan medicinal material "D. tanguticum Maxim." and the development of pharmaceutical preparations for medical institutions.

Key words Tibetan medical material, Dracocephalum tanguticum Maxim, Original plant, Character identification, Microscopic identification, TLC identification

1 Introduction

DracocephalumtanguticumMaxim.refers to the dry herb ofD.tanguticumMaxim.in Labiaceae, also known as Tangut dragonhead[1], Longsai dragonhead[2].D.tanguticumMaxim.first recorded inYuewangyaozhenis a common clinical medicine in Tibetan medicine featured by sweet flavour, bitter taste and cold nature, which is often used for the treatment of gastric ulcer, bacon disease, liver heat, hemostasis, grasserie, irregular menstruation and other diseases, and the seedlings are used for the treatment of ascites and edema[3-8].The traditional Chinese medicine is composed of 25 famous Tibetan prescription preparations such as Sanwei Ganlu pill, Qishiwei Songshi pills, which was included in 1995 edition standards issued by the Ministry of Health.Currently, there are many studies on the active components of the Tibetan medicinal materialD.tanguticumMaxim.[9-13].Pharmacological experiments such as liver protection and anti-hypoxia effect are mainly studied in terms of efficacy[14-15], while few efforts have been dedicated to its identification[16].This paper intends to study morphological identification, macroscopical identification, microscopic identification and TLC identification of the Tibetan medicinal materialD.tanguticumMaxim., in order to provide the source work foundation for the formulation of the quality standard of Sichuan Province(draft)for Tibetan medicinal material "D.tanguticumMaxim." and the development of pharmaceutical preparations for medical institutions.

2 Instruments and materials

2.1 InstrumentsThe instruments used in the test included Olympus BX41 optical microscope(Olympus, Japan), ZF3 UV transmission reflection analyzer(Shanghai Jiapeng Technology Co., Ltd.), Mettler AE240 electronic analytical balance[Mettler Toledo Instruments(Shanghai)Co., Ltd.], DHG-9240A electric constant temperature drying oven(Shanghai Precision Test Equipment Co., Ltd.), and silica gel G plate(Yantai Xinnuo Chemical Co., Ltd.).

2.2 MaterialsThe agents used in the test included oleanolic acid(batch number BWB50565, Beijing Century Oko Biotechnology Co., Ltd., purity ≥98%); ursolic acid(batch number BWB50564, Beijing Century Oko Biotechnology Co., Ltd., purity≥98%); chloral hydrate test solution, dilute glycerol, 0.5% sarranine, 0.25% fast green, FAA stationary liquid.Besides, concentrated sulfuric acid, methanol, ethyl acetate, petroleum ether(69-90 ℃), ethanol, dichloromethane and iodine were analytically pure.

The herb was collected from the field and purchased from local Tibetan hospitals and medicinal materials markets in 2017-2018, which was identified asD.tanguticumMaxim.by Professor Liu Yuan at Southwest Minzu University.The sources of samples are shown in Table 1.

Table 1 Information on the source of Dracocephalum tanguticum Maxim.

3 Methods and results

3.1 Plant morphologyD.tanguticumMaxim.herbs, aromatic.Rhizome short, fibrous roots numerous, stem 25-45 cm, square or 4-angled, retrorse pubescent toward apex, subglabrous basally, internodes 3-6 cm, with short branches in leaf axils.Leaves shortly petiolate, petiole 2.5-7.0 mm; leaf blade elliptic, base broadly cuneate, 2.5-8.0 cm long, 2-5 cm wide, pinnatisect, lobes in 2-3 pairs, lanceolate; blade adaxially green, abaxially gray-green, densely gray-white pubescent, margin entire, involute.Verticillasters in 5-9 upper stem nodes, usually with 4-6-flowered discontinuous spikes; bracts leaflike, very small, with 1 pair of segments, both surface pubescent, ciliate, ca.1/2-1/3 as long as calyx.Calyx tubular, ca.1.6 cm, densely spreading pubescent, golden glandular, purplish, apex 5-lobed, 2-lipped; upper lip 3-lobed, lower lip 2-lobed.Corolla purple-blue to dark purple, 2-lipped, upper lip arched, shorter than lower lip.Filaments pubescent.Nutlets smooth(Fig.1).Fl.Jun-Aug, fr.Aug-Sep.

3.2 Macroscopical identificationStem 4-angled, 1.5-4.0 mm in diam., grayish green or purplish red, nodular, brittle, easily broken, central section with yellow wood pith or hollow.Leaves opposite, shortly petiolate, petiole 2-10 mm, pinnatipartite to pinnatisect, 2-5 cm if entire, obliquely spreading at an obtuse angle with midrib, needle-like or linear, 1.5-3.5 cm wide, both surfaces pubescent.Flowers blue-purple or yellow-green(Fig.1).Taste fragrant, acrid, sweet, slightly bitter, cold in nature.

Note: a.Original plant; b.Herbarium; c.Fresh whole grass; d.Medical materials.

3.3 Microscopic identification

3.3.1Leaf cross-sections.The upper epidermis is composed of 1 row of cells, rectangular, tangentially elongated, covered by cuticle.There are more conical non-glandular hairs with small verrucous protuberations and fewer stomata.Glandular hairs are flattened at the tip, few and almost inconspicuous.The palisade tissue is composed of two rows of palisade cells, neatly arranged, dense, containing chlorophyll.The spongy tissue is composed of several layers of irregular cells, including chloroplasts.Several vascular bundles are surrounded by spongy tissue layer in a row.The lower epidermis is composed of 1 row of small quasi-circular or polygonal cells, with dense conical non-glandular hairs composed of 1-3 cells, occasionally curled, erect or inverted.The lower epidermis has 2-4 rows of collenchyma, with parenchyma cells inside; the vessels are inverted and surrounded by vascular bundle sheath composed of parenchymal cells.The xylem is broad, neatly arranged; the phloem is narrow, and cells are semicircular, densely arranged.The main vein tissue is adaxially V-shaped concave, abaxially protruded conspicuously(Fig.2).

Note: 1.Glandular hairs; 2.Upper epidermis; 3.Palisade tissue; 4.Vascular bundle; 5.Spongy tissue; 6.Non-glandular hair; 7.Xylem; 8.Phloem; 9.Collenchyma cell; 10.Lower epidermis.a.Contour plot; b, c.Detailed plot.

3.3.2Cross section of stem.The section is square; the epithelial cells are in 1 row, tangentially elongated, elliptic; the outer wall is slightly thickened, covered by cuticle; the cortical cells have several rows of parenchymal cells, loosen; four corners are distinctly ridged, with more than 10 rows of collenchyma cells inward; the vascular bundles are more developed at four corners, with several small vascular bundles between two adjacent corners; the phloem is narrow; the xylem is developed at four corners, composed of vessels, xylem parenchyma and a large number of wood fiber bundles; the medulla is broad, composed of large parenchyma cells(Fig.3).

Note: 1.Epidermal cells; 2.Collenchyma; 3.Cortex; 4.Fiber bundle sheath; 5.Phloem; 6.Xylem ray; 7.Xylem; 8.Vessel; 9.Medulla.

3.3.3Cross section of roots.The cross section of roots is quasi-circular, and the phellem layer is composed of multiple layers of cells, with small amount of mauve brown materials; the epidermis is composed of 1 layer of closely arranged irregular cells, and the outer wall of partial cells is protruded outward, forming lateral roots or gyrate fibrous roots.The outer cortex is composed of several layers of irregular parenchyma cells, with a few intercellular spaces.The cortex is broad and composed of rectangular parenchyma cells.The phloem cells are tightly arranged, the vessels are transverse and relatively dense, arranged in a ring.The xylem rays are fine and orderly(Fig.4).

Note: 1.Cork layer; 2.Phloem; 3.Cambium; 4.Wood ray; 5.Xylem(primary); 6.Hollow; 7.Fibrous root; 8.Wood fiber bundle; 9.Xylem(secondary).

3.3.4Powder identification.The power of the product is light green or gray green, and appears yellow fluorescence under ultraviolet light.Unicellular non-glandular hairs are more, obtusely tipped, tapered or curved, partially slender.Multicellular non-glandular hairs are folded, generally composed of complete 1-4 cells; the basal diameter is about 45-50 μm, and the wall thickness is about 2-7 μm.Keroid texture or verrucous protuberances are visible, or sometimes branched.Vessels are mostly spiral vessels and bordered pit vessels, while double spiral vessels and reticulate vessels are visible, and the diameter is about 5-42 μm.The pit of bordered pit vessel is dense, and that of reticulate vessel is broad.The cells in the lower epidermis are pentagonal or irregularly polygonal, with a diameter of 14-25 μm.There are more subsidiary cells, causing stomatal inequality.The wood fibers are long prismatic, slightly curved, mostly in clusters, colorless, with a diameter of 8-45 μm.The cell wall is slightly thickened, and twill or circular holes can be seen on the surface.The corky cells are yellowish brown, with a slightly thick cell wall and a hexagonal or polygonal surface.The glandular hairs are very few, with flat and round head, and the diameter is about 12-30 μm; the stalks are short, mostly single-celled; sclereids are common(Fig.5).

Note: 1.Non-glandular hair; 2.Vessel; 3.Wood fiber; 4.Cork cells; 5.Branched non-glandular hairs; 6.Lower epidermal cells; 7.Xylem parenchyma; 8.Sclereid.

3.4 TLC identification

3.4.1Preparation of test solution.Approximately 1 g ofD.tanguticumMaxim.powder(screened by No.3 sieve)was added with 10 mL of methanol and treated by ultrasound for 30 min.After filtration, the solution obtained was severed as the test solution.

3.4.2Preparation of reference solution.Accurately 1 mg of oleanolic acid and ursolic acid reference was weighed, and 1mL of methanol was added to prepare 1 mg/mL reference solution for later use.

3.4.3Optimization of TLC condition.The extraction method was investigated and optimized.(i)The solution was first added with diethyl ether and treated by ultrasound for 30 min.After filtration, the filtrate was recovered under reduced pressure(30 ℃), and added with 5 mL of methanol to sever as the test solution.(ii)The solution was first added with methanol and treated by ultrasound for 30 min.After filtration, the filtrate was directly used as the test solution.It was found that the color of samples after methanol extraction was clear.

Insitutreatment method: since oleanolic acid and ursolic acid are isomers, the difference inRfvalues is too small to be distinguished on thin layer plate.According to the reference[17], in situ treatment was performed with 1% iodine-dichloromethane solution(thin layer plate was dropped with samples and then immersed in 1% iodine-dichloromethane solution until just above the starting line, quickly removed and then covered with glass plate for 30 min; as the residual liquid was removed, the samples were spread on thin layer plate, and colored), theRfvalue changed.TheRfvalue was 0.51 withoutinsitutreatment, and theRfvalues of oleanolic acid and ursolic acid were 0.71 and 0.42 afterinsitutreatment, showing obvious resolution(Fig.6).

Note: a.TLC picture without in situ treatment; b.TLC picture of in situ treatment.

TLC development system was investigated with benzene: ethyl acetate(4∶1), toluene∶ethyl acetate∶glacial acetic acid(12∶4∶0.5), cyclohexane∶ethyl acetate∶glacial acetic acid(10∶3∶0.1, 10∶3∶0.5, 10∶5∶0.5), cyclohexane∶ethyl acetate∶formic acid(10∶3∶0.5), petroleum ether(60-90 ℃)∶ethyl acetate∶methanol(8∶2∶1, 8∶1∶1), respectively.Afterinsitutreatment with petroleum ether(60-90 ℃)∶ethyl acetate∶methanol(8∶2∶1), cyclohexane∶ethyl acetate∶glacial acetic acid(10∶5∶0.5), the resolution of oleanolic acid and ursolic acid was good, and the spots were clear.Among them, petroleum ether(60-90 ℃)∶ethyl acetate∶methanol(8∶2∶1)led to more spots, so it was selected as the final method.There were more tailing phenomenon and the color development was fuzzy when benzene∶ethyl acetate(4∶1)was used as the developing solvent, and the resolution of other developing solvents was not ideal with fuzzy color development.Meantime, the color developing effects of different color developing agents were investigated.It was found that the color of the sample was obvious when 10% sulfuric acid-ethanol solution was used as the color developing agent, and reddish purple spots were clear under natural light and 365 nm UV light.

3.4.4TLC conditions and sample color inspection.According to the TLC method inChinesePharmacopoeia(2015 edition)[18], 2 μL of solutions obtained in Section3.4.1and3.4.2were absorbed and dotted on the same silica gel G thin layer plate.After in situ treatment with 1% iodine-dichloromethane solution, the plate was dried, and developed with petroleum ether(60-90 ℃)∶ethyl acetate∶methanol(8∶2∶1)as the developing agent.Afterwards, the plate was taken out, dried, and sprayed with 10% sulfuric acid-ethanol and heated at 105 ℃ until the spots were colored.The results showed that the purple-red spots were clear under natural light and 365 nm UV light, and the 16 samples showed spots of the same color on the corresponding positions with the reference samples(Fig.7).

Note: 1-16.16 batches of D. tanguticum Maxim.samples; 17.Oleanolic acid; 18.Ursolic acid.The image on the left is in natural light and the image on the right is in UV light.

4 Conclusions and Discussion

The morphological characteristics, microscopic identification of root, stem and leaf tissue structure characters of Tibetan medicineD.tanguticumMaxim.are in line with the general structural characteristics of dicotyledon belonging to Labiatae.The main characteristics of powder can be identified from non-glandular hairs, vessels, corks cells, branched non-glandular hairs and sclereids, and the characteristics and microscopic identification methods are intuitive.A TLC method was established for the identification of oleanolic acid and ursolic acid in Tibetan medicineD.tanguticumMaxim.by different extraction solvents, development systems, color developing agents and whether performinginsitutreatment.The results showed that afterinsitutreatment, the Rf values of oleanolic acid and ursolic acid, which were mutual isomers, were significantly different and the spots were clear.The study will provide the source work foundation for the formulation of the quality standard of Sichuan Province(draft)for Tibetan medicinal material "D.tanguticumMaxim." and the development of pharmaceutical preparations for medical institutions.