Pharmacognostic study of Clematidis armandii the determination of index components

3.1. Microscopic identification

1) The structure of the stem.

Clematis armandii Franch: a list of pericytes, round, the outermost layer of the cortex is almost square, arranged closely, and have no cell gap. The inner parenchyma cells the cortex are irregular round, and have cell gap. There are two sclerenchyma rings in the phloem, and more from the outer layer of thick-walled cells 6-9, was not continuous cap structure; continuous arcuate inner mostly composed of 1-2 layers of thick-walled cells box. Xylem wide, large ducts, wood fibers, wood parenchyma cells were wood of rings is not obvious. Myeloid round, wood technology, there is the cell gap, sometimes by a thin wall of the central marrow of non-wood cells or hollow (Fig. 1).

Clematis montana Buch.-Ham.: Hydrangea vine biennial stem visible by decadent tissue rhytidome. The cap structure of outer ring by the discontinuous 8-17 layers of thick wall cells, the inner ring is not obvious. Xylem large, scattered holes arrangement, ray cells lignified, rings clear. With the wooden through stems of distinction (Fig. 2).

Fig. 1Clematis armandii Franch Stems

Fig. 2Clematis montana Buch.-Ham. stems

2) Leaf structure of the organization.

Clematis armandii Franch Stems’s leaf epidermal cells are square or rectangular category, closely arranged neatly outside the cuticle. Palisade tissue is typically 2 column cells, the spongy tissue of 5-6 rows of cells. Main vein vascular bundle surrounded by a thick fibrous oval, semicircular phloem, xylem and more from the catheter components.

Clematis montana Buch.-Ham. has mainly 4-5 rows of cells in the spongy tissue, cyclic structure with wooden main veins Vascular outside without a thick fibers pass to be different.

3) The organizational structure of the petiole.

Irregular cross-section through the petiole wooden round. Epidermal cells an outer cuticle. Outermost cortical cell types square, arranged very close, no cell gap; cortical parenchyma cells in the inner irregular round, there is the cell gap. Vascular bundles are mostly six, evenly distributed, thick-walled cells in the outer layer of 2-6, was cap. Phloem tissue mostly irregular round or square class. Xylem from the catheter, wood parenchyma cells and wood fibers, rays broad, often small vascular bundles visible secondary cell wall thicker, slightly wood. Pith cells round, the cell gap (Fig. 3).

Clematis montana Buch.-Ham. adaxial petiole pale heart-shaped cross-section, of vascular 6, but both sides of the vascular bundles near the center of the shaft surface often see two small secondary vascular bundle, vascular bundle outside layer of thick-walled cells often 3-10 can pass the difference between wooden (Fig. 4).

Fig. 3Clematis armandii Franch Stems petiole

Fig. 4Clematis montana Buch.-Ham. petiole

3.2. TLC identification of the chemical composition

1) Preparation of the test solution and reference solution.

a) Preparation of the test solution;

2 g medicinal powder (over 4 screens), 100 ml alcohol, reflux for 2 hours, filtration, the filtrate evaporated, the residue was dissolved with 5 ml ethanol, static, the supernatant as the standard medicine solution.

b) Preparation of reference solution;

Beta-sitosterol, add anhydrous ethanol, as the reference solution (1 mg/ml).

2) TLC conditions and results.

According to Thin-layer chromatography (Chinese pharmacopoeia 2010 edition), draw the test solution of 8 μ L, 4 μ l reference solution, respectively, on the same silica gel G thin layer plate. Developer: 1) chloroform:acetone (25:1); 2) cyclohexane:ether:acetate (20:5.5:2.5); 3) toluene:chloroform:acetone:methanol (8:5:1:1); 4) chloroform:ethyl acetate (10:1); 5) petroleum ether:ethyl formate:formic acid (6:2:0.1), spray with 10 % sulfuric acid ethanol solution, the color clear heating at 105 ℃ to spots, respectively the sunlight and UV lamp (365 nm).

Chloroform-ethyl acetate (10:1) used as a Developer will have good separation effect (Fig. 5).

3.3. Determination of beta-sitosterol

1) Preparation of the solution.

a) Preparation of the test solution;

1 g medicine powder (over 3 screens), accurately weighed, were set stoppered Erlenmeyer flask, precision chloroform 50 ml, sonication (power 120 W , frequency 40 kHz) 2 hours, cooled, shake, filtration, the filtrate evaporated and the residue dissolved in methanol, set 5 ml flask, microporous membrane (0.45 μm) filter.

b) Preparation of standard solution;

Beta-sitosterol accurately weighed, add methanol, as the standard solution (65 μg/ml).

Fig. 5Clematidis armandii Herbs TLC: 1-3: Clematis armandii Franch; 4-6: Clematis armandii Franch; 7: Liangshan prefecture Sichuan; 8: Anba prefecture Sichuan; 9: Beichuan County Sichuan; 10: Zhaotong Yunnan; 11: Slices; 12: Beta-sitosterol

Fig. 6High performance liquid chromatography (HPLC) figure (1 – Beta-sitosterol)

a) Reference substance

b) Blank solvent

c) Clematis armandii Franch

d) Clematis armandii Franch

2) Chromatographic conditions.

TC-C18 column (Agilent, 4.6 mm×250 mm, 5 μm); mobile phase:methanol-water (97:3); detection wavelength: 204 nm; column temperature: 30°C; flow rate: 1.0 ml/ min; injection volume: 10 μL. Number of theoretical plates beta-sitosterol of not less than 3000.

3) Methodological study.

a) Methods specificity study;

Respectively pipette beta-sitosterol standard solution, blank solution, test solution (Clematis armandii Franch, Clematis montana Buch.-Ham.) each 10 μl, injection, according to 2.3.2 test, both beta-sitosterol standard solution and test solution appear beta-sitosterol absorption peaks at about 23 min (Fig. 6).

b) Linear relation;

Take beta-sitosterol standard solution (65 μg /ml), respectively, sample 2 μl, 4 μl, 6 μl, 10 μl, 12 μl, 16 μl, with the sample volume ($X$, μg) for horizontal , peak area values ($Y$) for the vertical axis, the establishment of a standard curve, the linear regression equation $Y=$ 210328$X+$6688.5, ($r=$ 0.9997), the linear range of 0.13-1.04 μg (Fig. 7).

Fig. 7The standard curve of Beta-sitosterol

c) Precision test;

At a concentration of 65 μg/ml of the standard solution, continuous injection six times, measured the peak area, RSD is 1.67 %, indicating that equipment’s precision is good.

d) Repeatability;

Parallel to take No. 1 and No. 4 Sample 6 copies, accurately weighed, according to the sample preparation method, measured the peak area by sample analysis, the No. 1 sample’s peak area RSD is 2.31 % and the No. 4 sample’s peak area RSD is 2.11 %, indicating that the reproducibility of the test method is well.

e) Stability test;

Take the same test solution, according to the above chromatographic conditions measured peak area after the preparation of 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, Clematis armandii Franch and Clematis montana Buch.-Ham. peak area RSD values were 2.19 %, 2.94 %, indicating that the test solution was stable within 24 h.

f) Recovery test;

Accurately weigh sample No. 1 and No. 4 each 0.5 g (1 g Clematis armandii Franch contains beta-sitosterol 170.266 μg, 1 g Clematis montana Buch.-Ham contains beta-sitosterol 304.505 μg), each 6 parts, precision adding beta-sitosterol standard solution, according to the sample preparation method, and measuring beta-sitosterol content, calculate recoveries, the results in Table 2 and Table 3.

4) The content determination of the sample.

Take samples collected from six different regions and buy medicinal materials from five different places, according to the sample preparation method, preparation of sample solution, determine the content of beta-sitosterol, the results are shown in Table 4.