Correlation Analysis of Quantitative Microscopic Characteristics and Polysaccharide Content of Dendrobium officinale from Guangxi

Liuyuan FAN, Hua ZHU, Jianbei TENG, Zhonghua DAI*, Piaoling HUANG

1. Liuzhou Traditional Chinese Medical Hospital (Liujcouh Si Ywcuengh Yihyen), Liuzhou 545001, China; 2. Guangxi University of Chinese Medicine, Nanning 530200, China

Abstract [Objectives] The paper was to investigate the correlation between the microscopic characteristic constants and the index component polysaccharide content of Dendrobium officinale from Guangxi. [Methods] The paraffin sections of D. officinale were made, and the microscopic characteristic constants of D. officinale stem, including cross section area, vascular bundle area and phloem area, were determined by NIS-Elements D microscopic image processing software. The contents of polysaccharides in D. officinale were determined by UV-Vis spectrophotometry, and the quantitative values of microscopic characteristics of D. officinale with different growth years were analyzed by SPSS 23.0 (IBM SPSS Statistics 23.0) data statistical software. The correlation between quantitative microscopic characteristics of D. officinale and its polysaccharide content was analyzed. [Results] According to the correlation between the index component of D. officinale and its quantitative microscopic characteristics, the measurement indexes, the percentage of vascular bundle in the cross section area and the percentage of hard structure in the cross section area, were strongly negatively correlated with polysaccharide content. [Conclusions] The microscopic characteristic constant values of vascular bundles of D. officinale can be used as a method to verify polysaccharide content.

Key words Dendrobium officinale, Microscopic characteristics, Polysaccharides, Correlation analysis

1 Introduction

Since modern times, the identification of traditional Chinese medicine has developed from simple identification of microscopic characteristics to quantification of microscopic characteristics and determination of microscopic tissues, and its quality is controlled by combining with chemical methods. This method has become a practical technology for the identification of crude drugs, the judgment of authenticity and amerits and the determination of their content. The identification and quantitative analysis of traditional Chinese medicine can be the microscopic quantification of the special structure of traditional Chinese medicine powder, or the microscopic measurement of the specific tissue structure of the horizontal and vertical sections of crude drugs. There are many kinds of microscopic quantitative methods for medicinal materials powder, while reference and non-reference microscopic quantitative methods are mainly used[1]. Microscopic measurement is a method to measure the specimen of special tissue structure of crude drug sections observed under a microscope by using a slide with graduated scale and a graduated eyepiece[2].

Dendrobiumofficinaleis the stem of the perennial epiphytic herbD.officinaleKimura et Migo. It is sweet in flavor and mild cold in property, with channel tropism of stomach, lung and kidney.D.officinalehas unique effect of invigorating the stomach and generating fluid, nourishing yin and clearing heat, and is mainly used for the treatment of flaccidity disease with syndrome of fluid consumption, dry mouth and polydipsia, insufficient stomach yin, reduced appetite and retching, persistent deficiency heat after illness, yin deficiency with effulgent fire, osteopyrexia and fever, blurred vision, and limp wilting sinews and bones[3]. The medicinal part ofD.officinalisis the stem, and polysaccharide in the stem is one of its main effective components, which has pharmacological effects such as tumor inhibition, immune regulation and hypoglycemic action, and meets the needs of modern society for drugs with cancer suppression, immune function improvement, and hypoglycemic effect[4-6], with a good development prospect. The importance ofD.officinalehas increased since it was separated into separate item from multiple sources ofDendrobiummedicinal materials in theChinesePharmacopoeia(2010 edition). In addition to magistral medicine,D.officinaleis also used as crude drug, and is often used as medicine or health care products.

In recent years, there are many reports on the microscopic identification ofD.officinale, most of which focus on "with or without" and "yes or no" of the microscopic characteristic molecules of its medicinal powder and the characteristic structure of its section tissue, while there are few reports on the correlation analysis of its quantitative microscopic characteristics and composition[7-10]. According to the traditional identification experience,D.officinalewith thickening flesh and less slag is better, that is, the less the hard structure (such as catheter), the better the material. Therefore, the fiber and hard structure affecting firmness and texture ofD.officinalecan be selected as the microscopic quantitative characteristics. The fiber and hard structure of powderedD.officinalehave been crushed and broken to different degrees, and the microscopic quantitative method is not suitable for their determination. Therefore, the tissue structure of the sections was selected for microscopic measurement.

The quantitative microscopic characteristics and polysaccharide content ofD.officinalewith different growth years were measured. The correlation between quantitative microscopic characteristics and polysaccharide content ofD.officinalewith different growth years was analyzed by SPSS 23.0 (IBM SPSS Statistics 23.0) statistical software. Using the quantitative technique and method of microscopic characteristics, the data related to the quality of medicinal materials can be measured, so as to provide the experimental data for identification and quality control ofD.officinale.

2 Materials and methods

2.1 Source ofD.officinalesamplesD.officinalefrom Guangxi was cultivated in Wangfan Village of Yantian Village Committee, Zhenlong Township, Hengxian County (Hengxian XiancaotangDendrobiumofficinaleprofessional cooperative). Annual, biennial and triennialD.officinalefrom Guangxi adopted organic cultivation and potted mode; light: black shading net, shading degree 70%; substrate: decomposed pine bark, organic fertilizer; water: mountain spring water. All the experimental samples were identified as authentic medicinal materials by Meng Ping, the associate researcher of Guangxi Academy of Agricultural Sciences. The medicinal materials were annual, biennial and triennial branches collected from triennial pot.

The materials used in the paraffin section test were all freshly picked stems ofD.officinale, which were divided into 3 groups with annual, biennial and triennial samples respectively, and 10 branches were randomly selected from each group. Three samples were selected from the upper, middle and lower part of the middle part of each branch, and then 5 samples were randomly selected from different parts of different sample groups to make microsections for observation and measurement. There were 45 samples in total, as shown in Table 1.

Table 1 Paraffin section number of Dendrobium officinale sample

2.2 Instruments and reagentsUV-1780 UV-Vis spectrophotometry [Shimadzu Instruments (Suzhou) Co., Ltd.]; Sorvall ST 16R high speed refrigerated centrifuge [Thermo Fisher Scientific (China) Co., Ltd.]; BSA224S electronic balance [Sartorius Scientific Instruments (Beijing) Co., Ltd.]; H22-X3 infrared furnace (Hangzhou Joyoung Household Electric Appliances Co., Ltd.); RM2235 rotary microtome, HistoCore Arcadia Cparaffin embedding machine, metal embedding mold [Leica Microsystems (Shanghai) Co., Ltd.]; HI120 water bath-slide drier (Shanghai Leica Instruments Co., Ltd.); DHG-9240A electrothermal blowing dry box (Shanghai Yiheng Scientific Instruments Co., Ltd.); Gilson Pipetman P1000L Pipette (Gilson, USA); Merck Millipore Direct-Q3, 5, 8 pure water/ultra-pure water purification system (Merck KGaA); NIKON Eclipse Ni Nikon positive research grade microscope, Nikon DS-Ri2 camera, NIS-Elements D microscopic image processing software (Nikon, Japan); tissue embedding box (Jiangsu Shitai Experimental Equipment Co., Ltd.); micrometer (Motic Industrial Group Co., Ltd.).

The reagents used in the test included formaldehyde, glacial acetic acid, glycerin, ethanol, xylene, paraffin, sarranine, brilliant green, gelatin, phenol, sulfuric acid, Canada balsam, TO-type biological film transparent agent, D-anhydrous glucose.

2.3 Preparation of cross-sectional paraffin sectionAccording to the investigation results of methodology for paraffin section preparation, each sample was sectioned, and the production process was as follows: sampling→fixing→rinsing→dehydrating→transparent→wax immersing→ embedding→slicing→adhering→dewaxing→staining→sealing[11].

2.3.1Sampling. Due to the large amount of annual group, it was used as the material for exploring conditions of paraffin section. The stem of neatD.officinalewith straight stalk was selected, and the outer surface of the stem was washed with water and wiped dry. After rinsing and disinfection with 70% ethanol, the stem was cut into segments of about 0.5 cm with a scalpel, put into small reagent bottles with lid, and fixed with fixing solution.

2.3.2Fixation and softening. Fixation: The fixed solution (70% ethanol: formaldehyde: acetic acid: glycerol) was put into a 50 mL small reagent bottle, and more than 10 stem segments ofD.officinalewere put into each fixed solution. The small reagent bottles were put into PC-3 plastic vacuum suction dryer for air extraction. After air extraction for 30 min, the two suction valves of the vacuum suction dryer were tightened. At this time, the pressure gauge of the vacuum suction dryer was kept at the negative pressure level and maintained overnight. The samples were kept in the fixing solution for more than 48 h before proceeding to the next step.

Softening: The fixed materials were respectively placed in softener (glycerin: nitric acid: water) to soften for a certain time, and set aside.

2.3.3Dehydration. The fixed and softened materials were taken out and dehydrated by ethanol gradient. First, the materials were rinsed twice with 50% ethanol for 30 and 20 min, respectively; the materials were then dehydrated step by step in ethanol. The steps were as follows: 50% ethanol for 30 min→50% ethanol for 20 min→70% ethanol for 30 min→80% ethanol for 1 h→90% ethanol for 1 h→95% ethanol for 1h→absolute ethanol for 30 min→absolute ethanol for 30 min.

2.3.4Transparency. The dehydrated and non-shrinking materials were transferred to xylene 1, xylene 2 and xylene 3 in turn, and the steps were as follows: xylene 1 for 1 h → xylene 2 for 1 h → xylene 3 for 1.5 h.

2.3.5Wax immersing. Pre-treatment of wax immersion: Approximately 20 mL of xylene solution was poured into a 50 mL reagent bottle, and then added with a small spoon of broken wax, capped, and dissolved in 45 ℃ water bath. The bottle was taken out, and the experimental operation of "adding a small spoon of broken wax, capping and dissolving in 45 ℃ water bath" was repeated until the added wax reached saturation state. Finally, it was capped and placed in an incubator at 50 ℃ for 2-3 d. The wax immersing procedure was as follows: pure xylene + pure paraffin (50 ℃) overnight → pure paraffin I 1 h→ pure paraffin II 1 h → pure paraffin III 2 h.

2.3.6Embedding. Temperature setting of HistoCore Arcadia H paraffin embedding machine: embedded wax tank 60 ℃, left and right thermal insulation tank 70 ℃, operating table 60 ℃. After the metal embedding mold was removed from the thermal insulation tank, it was placed on the corresponding position of the embedding machine, and then appropriate amount of wax liquid was dropped into the groove of the metal embedding mold at a quick speed. Afterwards, the metal embedding mold was level-shifted to a small ice table, making the wax on the bottom solidify slightly. Finally, the tissue was removed from wax III and quickly inserted vertically into the bottom of the mold (the cross section of the stem was parallel to the bottom of the mold), and the embedding box was covered. When the wax was gradually congealed, a label was put on it. The embedding mold with tissue was frozen on the freezing table until the wax block was separated from the mold naturally. The wax block was taken out and stored in a refrigerator.

2.3.7Slicing, adhering and baking. Slicing: The embedded wax block was trimmed around the embedded box with a blade, and the embedded box was clamped in the incision groove of the rotary microtome after trimming, so that the cut surface of the wax block was parallel to the blade of the slice and clamped tightly. The sharpness of the cutter and the hardness of the wax block would directly affect the quality of the slice. After the slice thickness was appropriately adjusted, pieces of continuous wax tapes were cut out, lightly supported with a brush, gently picked up with tweezers and placed in the developing slot.

Adhering: The wax tape (serial slice) of a certain length or single wax piece broken with a blade was flattened in warm water (about 45 ℃), and then spread straight on the glass slide boiled with adhesive.

Baking: The successfully made slices were put into an oven at 60 ℃ for 4 h.

2.3.8Dewaxing and rehydrating. According to the references[12-14], the baked slices were successively placed into TO (35 min)→absolute ethanol (20 min)→water (15 min).

2.3.9Staining, color fixing and sealing. According to the reference[12], sarranine-bright green staining method was adopted. After rehydration, the slices were stained in sarranine solution (30 min)→bright green solution (10 s)→TO color fixing (5 min)→dropping a drop of neutral gum→sealing the slice→drying in oven at 35 ℃.

2.4 Observation and measurement of microscopic characteristics of cross section of stem in different treatment groups

2.4.1Verification of the reliability of measurement data. (i) Verification of accuracy. In order to verify the accuracy of the NIS-Elements D microscopic image processing software of the microcamera system in measuring size, the micrometer was used as the standard for measurement. The micrometer was measured 6 times at 40X magnification and 400X magnification, and the 6 times measurements were operated by 6 people. The measurement data were recorded and theRSDvalues were calculated. (i) Verification of repeatability. In order to verify the repeatability of the microsystem, the system was used to randomly measure the cross section of 5 samples ofD.officinalestem (magnification 40X) and 5 vascular bundles in single section (magnification 400X) for 6 times, and the 6 measurements were performed by 6 people. TheRSDvalue was calculated.

2.4.2Definition of sample parameter. (i) Cross section area. Parameter definition: According to the literature[10], in the microscopic structure of the cross section ofD.officinalestem, the outer layer of epidermis is covered by thick cuticle, that is, the smooth waxy layer observed by the naked eyes when the leaf sheath falls off. When the stem is used medically, the outermost leaf sheath is usually removed. Therefore, the measured area is the area within the cuticle (including cuticle) when the NIS-Elements D microscopic image software is used for processing.

Measurement method: The panorama of sample sections was taken with a microscope at low magnification (4X), and the jigsaw was processed with Photoshop software. The area surrounded by thick cuticle was sketched on the cross section panorama with the mouse in NIS-Elements D microscopic image processing software, which was the area index required for experimental measurement. When using NIS-Elements D microscopic image software, the measuring eyepiece should be in 4X mode.

(ii) Vascular bundle area. Parameter definition: Vascular bundle refers to the structure formed by bundle arrangement of vascular bundle sheath, xylem and phloem in plant tissue[15-16]. After being made into paraffin sections and stained with sarranine-brilliant green, vascular bundle sheath and xylem in vascular bundle will be dyed red, while phloem will be dyed blue.

Measurement method: 30 vascular bundles were selected from each section and shot in a single photo at high magnification (40X) (one vascular bundle was a picture), and the 30 vascular bundles should be evenly distributed on each vascular bundle layer. In NIS-Elements D microscopic image processing software, the area of vascular bundle was sketched by the mouse in the single vascular bundle map taken successfully. When using NIS-Elements D microscopic image processing software, the measuring eyepiece should be in 40X mode.

(iii) Hard structure area. Parameter definition: ①Mechanical tissue refers to the tissue that provides primary support and protection to plants, mainly including mechanical collenchyma and sclerenchyma, which are vascular bundle sheath and fiber in the microstructure ofD.officinalestem; ②Other lignified cells mainly refer to ducts and xylem parenchyma, while other microscopic parts ofD.officinalestem, such as epidermis, basic tissues and sieve tubes of phloem, are all thin-walled[15]. Therefore, the medical trait ofD.officinaleis closely related to ① and ② structures mentioned above, so the calculation formula of hard structure is as follows: Hard structure area=Vascular bundle area-Phloem area.

Measurement method: 30 vascular bundles were selected from each section and shot in a single photo at high magnification (40X) (one vascular bundle was a picture), and the 30 vascular bundles should be evenly distributed on each vascular bundle layer. In NIS-Elements D microscopic image processing software, the area of hard structure was circled by the mouse in the single vascular bundle map taken successfully (Fig.1). When using NIS-Elements D microscopic image processing software, the measuring eyepiece should be in 40X mode.

Note: A. Vascular bundle area; B. Phloem area.

(iv) Number of vascular bundles in a single section. Measurement methods: The vascular bundles in the cross section panorama ofD.officinalewere counted using NIS-Elements D microscopic image processing software.

2.5 Measurement of polysaccharide contentAccording to the polysaccharide detection method ofD.officinaleinChinesePharmacopoeia(Part I, 2020 edition)[17], the polysaccharide content of annual, biennial, triennialD.officinalewas determined by phenol-sulfuric acid method.

2.6 Statistic analysisThe mean and variance of quantitative microscopic characteristics of annual, biennial and triennialD.officinalewith different growth years were calculated by SPSS 23.0 (IBM SPSS Statistics 23.0) statistical software, and the correlation between quantitative microscopic characteristics and polysaccharide content ofD.officinalewith different growth years was analyzed.

3 Results and analysis

3.1 Reliability evaluation results of measurement data of microscopic system

3.1.1Verification of the accuracy of microscopic image processing software. TheRSDvalues measured at 40X magnification and 400X magnification were 0.05% and 0.12%, respectively, which showed a small error range with the theoretical value, indicating that the microscopic system was accurate and reliable for measuring the microscopic area. The results are shown in Table 2.

Table 2 Verification results of the accuracy of microscopic image processing software

3.1.2Repeatability results. The area of vascular bundles in cross section ofD.officinalestem showed good repeatability at 40X and 400X magnification. The results are shown in Table 3.

Table 3 Investigation of repeatability in measuring the area of vascular bundle of Dendrobium officinale stem by image processing software μm2

3.2 Microscopic measurement results and analysis of cross section of stem

3.2.1Cross section area and number of vascular bundles. In the cross section, a column of epidermal cells were stained, and the cells were flat and slightly oval. The basic parenchyma cells were unstained, polygonal and uniform in size, with a thick yellow cuticle outside, resembling a square. The area surrounded by the cuticle was clearly visible, and the vascular bundles were scattered (Fig.2). The cross section area and number of vascular bundles in annual, biennial, triennialD.officinalestem in different treatment groups are shown in Table 4.

Note: A. Annual (upper part-section 1); B. Biennial (upper part-section 3); C. Triennial (lower part-section 4).

3.2.2Vascular bundle area and hard structure area in cross section. For collateral vascular bundle, the phloem accounted for less proportion and the degree of lignification was higher, and the surrounding sclerenchyma cells were stained red by saffranine reagent; the sclerenchyma cells near the lateral side of phloem were obviously thickened. The thicknesses of vascular bundle sheath cells of annual, biennial, triennialD.officinalewere significantly different. The thicknesses of sclerenchyma cells of vascular bundle sheath near the lateral side of phloem successively were annual < biennial < triennial; and the thickening degrees of xylem cells in the whole vascular bundle were triennial > biennial > annual (Fig.3). The area of single vascular bundle and the measured area of hard structure are shown in Tables 2-5. The total vascular bundle area and the total hard structure area of the cross section ofD.officinalestem were estimated, and the calculation results are shown in Table 4.

Note: A, B and C represent the vascular bundles of annual, biennial, triennial D.officinale, respectively; 1. Xylem; 2. Phloem; 3. Vascular bundle sheath.

3.3 Polysaccharide contentAccording to the measurement method of polysaccharide content ofD.officinaleinChinesePharmacopoeia(Part I, 2020 edition), the regression equation of the standard curve wasA=63.344C+0.001, and the correlation coefficient was 0.999 7 (n=6). The polysaccharide contents of annual, biennial, triennialD.officinaleare shown in Table 6.

Table 4 Statistics of measurement indexes in cross section of Dendrobium officinale stem

Table 5 Quantitative difference analysis of microscopic characteristics of Dendrobium officinale n=15)

Table 6 Determination of polysaccharide content of Dendrobium officinale (n=3)

3.4 Correlation analysisThe polysaccharide content ofD.officinalewas negatively correlated with the percentage of vascular bundle in cross section area and the percentage of hard structure in cross section area, and the correlation coefficient was greater than 0.8.

4 Conclusions

The correlation between polysaccharide content and quantitative microscopic characteristics ofD.officinalewas obtained, which linked tissue morphology with quality evaluation. According to the correlation between the index component ofD.officinaleand its quantitative microscopic characteristics, the percentage of vascular bundle in the cross section area and the percentage of hard structure in the cross section area were strongly negatively correlated with polysaccharide content, indicating the less the vascular bundle and hard structure, the higher the saccharides content.

5 Discussion

(i) The sampling action should be clean and neat, and whether the edge of the material is complete should be concerned. The section obtained with incomplete edges may be damaged, so those with incomplete edges should not be used.

(ii) In this experiment, when the number of vascular bundles ofD.officinalestem was counted by NIS-Elements D microscopic image processing software, it was observed in the shape of zigzag under the microscope and could not be counted repeatedly[18]. The area of cross section should be delineated according to the periphery of the cuticle, and the area of vascular bundle and phloem should be delineated along the cell wall.

(iii) When measuring polysaccharide content by phenol-sulfuric acid method, the moisture absorption and the degree of moisture absorption of sulfuric acid have great influence on polysaccharide content, so the well-preserved sulfuric acid should be used in the experiment.

(iv) In the later research, further correlation between the two indexes of the percentage of vascular bundle in the cross section area and the percentage of hard structure in the cross section area and the results of chemical composition can be analyzed, and the correlation between quantitative results of microscopic characteristics and quality of medicinal materials from different producing areas can be studied by statistical regression analysis[19].