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The accumulation and dynamic trends of triterpenoid saponin in

vegetative organs of Achyranthus bidentata Blume.

Li Jinting1,2 and Hu Zhenghai1*

�1 Key Laboratory of Resource Biology and Biotechnology in Western China (Northwest

University)�Ministry of Education, Xi'an, 710069, China 2 College of Life Science, Henan Normal University�Xinxiang, 453007, China�

Abstract

The relationship between structural features of various vegetative organs and triterpenoid saponin

accumulation in Achyranthus bidentata Blume was investigated using anatomy, histochemistry and

phytochemistry. The results showed that the primary and secondary structures of roots, and the

structures of stems and leaves of A. bidentata, were similar to those of ordinary dicotyledonous

plants. The enlargement of its roots, however, was primarily associated with growth and

differentiation of tertiary structures. There were collateral medullary vascular bundles in addition to

the normal vascular bundles in the stem. The tertiary structure was not only main parts in the roots

of A.bidentata, but also important storage region of triterpenoid saponin in its growth and

development. The stem may be the essential transport organ of triterpenoid saponin, while palisade

tissue cells may be the primary synthesis location. In November, the total quantity of triterpenoid

saponin and overall biomass in the roots reach a maximum level. This was the best time, therefore,

to harvest the roots and corresponded to the traditional harvest period. Despite the withered

appearance of leaves, stems also contained substantial amounts of triterpenoid saponin, and it was

recommended that the stems of A. bidentata should be utilized.

Keywords: A. bidentata; anatomy; oleanolic acid; triterpenoid saponin.

Supported by the National Natural Science Foundation of China (30470105), the Special Fund of the Education

Department of Shanxi Province of China�2006, JK177�and the Natural Science Foundation of Education

Department of Henan Province of China (2007180031).

*Author for correspondence.

Tel: +86-029-88302684; Fax: +86-029-88303572; E-mail: Zhenghaihu@sina.com.cn.

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Introduction

A. bidentata, a herb of the Amaranthaceae family, has become one of the important Chinese

traditional medicinal plants. In China this specie is mainly distributed in the Guhuaiqingfu area of

Henan Province. Its dried roots contain several secondary metabolites such as saponin, ecdysterone,

polysaccharide, and betaine. Several saponins have been separated and identified from A.bidentata,

and include triterpenoid saponin with oleanolic acid aglycon, which is a traditional indicator of

quality according to Chinese Pharmacopoeia(China Pharmacopoeia Committee 2005). Triterpenoid

saponin is widely distributed in higher plants and is an important secondary metabolite. Not only

does it possesses antibiosis and pest resistance characteristics (Haralampidis et al. 2002;

Papadopoulou et al. 1999), but also may reduce cholesterol levels and have anti-cancer,

anti-inflammatory and analgesic properties (Huang et al. 2006; Gao et al. 2003; Hu et al. 2005).

Several studies have reported on the separation, identification, chemical structure and biological

activities of saponins (Khamidullina et al. 2006; Sparg et al. 2004; Meng et al. 2002; Wang et al.

1996). The few reports that are available mainly focus on root development(Li and Hu 2006; Zhang

and Hu 1988; Wei et al. 1997). However, there are no studies on stem and leaf structures. The

relationship between plant structure and the accumulation of active substances has been explored in

other species, including Aloe arborescens mill. and Lamium album (Liao et al. 2006; Tamars et al.

2001). This dissertation investigated the relationship between organ structure and accumulation of

triterpenoid saponin in A. bidentata. Data obtained in this research can be used to determine the

most appropriate harvesting stage and parts and contribute to standardization of cultivation

techniques, thus improving output and quality of the medicinal plant.

Results

Structures and histochemical observation of roots

The developmental anatomy of A. bidentata roots has been reported (Li and Hu 2006). In addition

to the primary and secondary structures of normal dicotyledonous plants, its roots also have tertiary

structures formed by the differentiation of extra cambium (Fig I, 1, 2, 3). The tertiary vascular

bundles were neatly arranged in a concentric ring, separated by parenchyma cells.

In the primary structures of roots, cells of the epidermis, cortex and primary xylem did not show

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any color reaction, while light pink color appeared in the pericycle, primary phloem, and

parenchyma cells between primary phloem and primary xylem (Fig II, 9). In the secondary root

structures, the secondary phloem and the parenchyma cells of the phelloderm appeared a claret

color (Fig II, 10, 12). With the further development of roots, claret appeared in extra cambium cells

formed by the dedifferentiation of parenchyma cells outside of secondary phloem and close to the

pericycle (Fig II, 10); the cells of tertiary phloem formed by the differentiation of extra cambium

cells also showed claret (Fig II, 11).

Structures and histochemical observation of stems

The stems of A.bidentata were tetragonal and consisted of, from outside to inside, epidermis, cortex

and a vascular cylinder (Fig I, 4). There was one layer of epidermal cells, long or somewhat

rectangular, neatly arranged in a dense pattern, with some protuberance in the external walls, and

nonglandular hair. There were three to five rows of cortex parenchyma cells with chloroplasts;

some cells contained brownish-yellow substances. Fully developed collenchyma cells were present

in edges and corners. The vascular cylinder consisted of vascular bundles, pith and pith ray.

Vascular bundles were collateral, with the primary phloem fibers being relatively developed in the

corners of the stems. The phloem was narrow and consisted of sieve tubes, companion cells and

phloem parenchyma cells; xylem consisted of vessels, parenchyma cells and wood fibers (Fig I, 5).

Vessels in the xylem congregated as a group with wood fiber around the vessel cluster. There were

several layers of vascular cambium cells between xylem and phloem. In the center of the stems

were fully developed pith, which consisted of a large number of parenchyma cells, and there were

two collateral medullary vascular bundles close to the center (Fig I, 4, 6). Phloem cells of the

normal vascular bundles and medullary vascular bundles appeared a claret coloration, while no

color reaction was observed in other tissues (Fig II, 13).

Structures and histochemical observation of leaves

The leaves of A. bidentata were typically bifacial, with blades consisting of epidermis, mesophyll

and veins. The epidermis consisted of a layer of irregular flat cells, with nonglandular hair in both

the adaxial and abaxial epidermal layers, while stomata were distributed only in the abaxial

epidermis. There were three to four layers of palisade tissue cells in mesophyll, with several

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chloroplasts. Some cells contained calcium oxalate cluster crystals. There were a few spongy tissue

cells with an irregular cell shape, containing few chloroplasts, and intercellular space (Fig I, 7).

Both the abaxial surfaces of the main leaf vein protruded, and there were collenchyma cells inside

the adaxial and abaxial epidermis. Collenchyma cells beneath the epidermis were connected to

palisade tissues on both sides of the leaf. The collateral vascular bundles in the median vein were

arranged in a discontinuous ring (Fig I, 8). The histochemical test of mature leaf slices showed

claret in palisade tissue and phloem cells of main vein vascular bundles, while no color appeared in

other tissues (Fig II, 14, 15). In senile leaves, the chloroplast envelope membrane disappeared,

chloroplasts disintegrated and were concentrated to form vesicles. There was no red color in the

mesophylls, indicating there was no triterpenoid saponin accumulation in the cells, or no

triterpenoid saponin that was there had been transported to other locations before aged leaves fall

off (Fig II, 16).

There was no detectable saponin in control samples of root, stem and leaf.

HPLC determination results of oleanolic acid in A. bidentata structures

The histochemical results indicated that the roots, stems and leaves of A. bidentata contained

triterpenoid saponin. For further authentication, we used HPLC to determine the contents of

triterpenoid saponin in various vegetative organs at different developmental stages, using oleanolic

acid as evaluation index. The results showed that various vegetative organs contained different

levels of oleanolic acid; chromatograms are shown in Fig. 1.

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Fig 1. Chromatograms of oleanolic acid reference substance and different organs samples of A. bidentata

1-Oleanolic acid; A- Oleanolic acid reference substance; B-Root; C-Stem; D-Leaf

There were dynamic variation of oleanolic acid contents in various vegetative organs at different

developmental stages (Table 1; Fig. 2). During the whole growth period, oleanolic acid in the roots

revealed a “high-low-high” pattern. In early August, when A. bidentata was in a vegetative growth

phase, the aerial parts grew vigorously, and the amount of oleanolic acid in the roots was the

highest (7.76%). On 15-20 August was a period of reproductive growth when inflorescence and

flowers occurred. At that time, oleanolic acid levels decreased quickly in the roots and reached the

lowest annual level (1.03%) in September. With fruits maturation, however, the amount of

oleanolic acid in roots increased gradually again and tended to stabilize after reaching 2.95% in

October. The amount of oleanolic acid in both stems and leaves was highest in August, and then

began to decrease gradually. In October, oleanolic acid levels in stems tended to stabilize, but levels

in leaves continued to decline. After November, when the roots of A. bidentata were fully

developed, the aerial parts had withered and oleanolic acid levels in the leaves were extremely low

(0.06%). At that time, the relative levels of oleanolic acid were roots>stems>leaves.

Table 1 Oleanolic acid (OA) contents in different vegetative organs of A. bidentata

OA contents (%) Sampling time

Root Stem Leaf

Early August 7.76 4.09 4.13

Early September 1.03 2.76 3.37

Early October 2.95 1.17 0.25

Early November 2.90 1.09 0.06

Early December 2.59 1.47

Fig 2. Accumulation trends of oleanolic acid in different organs of A. bidentata

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Discussion

Triterpenoid saponin is the important medicinal ingredient of A. bidentata, which is a traditional

medicinal plant used to treat a variety of conditions. Saponins are widely distributed in higher

plants, yet their specific form and distribution in different plant organs are highly diverse (Cao et al.

2004; Liu et al. 2005). Histochemical analysis of A. bidentata vegetative organs indicated that, in

the root primary structures, saponins were mainly distributed in the pericycle, primary phloem and

parenchyma cells between primary phloem and primary xylem. In the secondary root structures,

saponins were mainly distributed in secondary phloem and parenchyma cells of the phelloderm. As

roots developed, extra cambium was formed by the dedifferentiation of parenchyma cells outside of

the secondary phloem and close to the pericycle, and the tertiary vascular bundles were formed by

the differentiation of the extra cambium. Saponins accumulated in the extra cambium cells, as well

as in the phloem cells of tertiary vascular bundles.

Saponins were mainly found in the phloem cells of normal vascular bundles and medullary vascular

bundles of stems, and in palisade tissue and phloem cells of the main vein vascular bundles in

leaves. Because the enlargement of roots in the growth process was primarily related to the

occurrence and differentiation of tertiary structures, and tertiary structures were dominate in

mature root of A. bidentata, and so tertiary structures were the main storage site of triterpenoid

saponin. The red substances in stems were present mainly in the phloem of vascular bundles. In

leaves, however, they were found mainly in the palisade tissue cells and the phloem of vein

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vascular bundles. It was speculated, therefore, that saponins in A. bidentata were first synthesized

in the leaves and then transported to roots for storage via the phloem of veins and stem vascular

bundles. The disappearance of saponins from leaves when they withered also supports the

speculation that the leaves acted primarily as synthesis, and not storage locations. Additional

further investigation will be needed to clarify the roles of these various structures in the synthesis

and transfer process.

The accumulation patterns of triterpenoid saponins in various vegetative organs were quantified by

using HPLC. The amount of oleanolic acid changes with developmental stage of vegetative organs.

The observed variability was similar to the oleanolic acid distribution patterns observed in such

medicinal plants as Abrus mollis Hance and Herba sambuci chinensis (Huang et al. 2006; Zou et al.

2005). In early August, the proportions of oleanolic acid in the roots, stems and leaves of A.

bidentata were 7.76%, 4.09% and 4.13%, respectively, which were the maximum measured levels.

Here, A. bidentata was in the vegetative growth stage, and aerial parts grew vigorously. Large

amount of anabolites were produced and accumulated and then transported to roots, which also

grew quickly. Tertiary structures in the roots differentiated at a rapid rate. Levels of triterpenoid

saponin in roots and aerial parts increased rapidly. This pattern of growth and saponin accumulation

suggests that fertilizer application in early August may be appropriate in order to improve the

biomass and quality of roots.

On15-20 August, A. bidentata entered into a reproductive phase. At that time, the amount of

oleanolic acid quickly dropped, reaching the annual lowest level (1.03%) in September (full fruits

period). When that occurred, the proportion of oleanolic acid in the stems decreased dramatically

(from 4.09% to 2.76%) but levels in the leaves were unchangeable. This difference may be due to

rapid consumption of nutrients by the developing reproductive organs, thus causing of rapid

decrease of triterpenoid saponin content in roots. We determined that the oleanolic acid level in

mature A. bidentata fruits was 7.73%. It indicated that the decrease of triterpenoid saponin content

in roots and stem is closely related to reproductive growth of A. bidentata which was not beneficial

to the accumulation of saponins in roots. The result is in agreement with standpoint of others (Cao

et al. 2004). Hence, flowers should be removed or other measures taken to inhibit the growth and

development of reproductive organs, and consumption of nutrients, in order to maximize the

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quality and quantity of A. bidentata roots.

With the development and the ripening of fruits, the consumption of nutrients decreased relatively

and the oleanolic acid contents in roots began to increase again, reaching a relatively stable 2.95%

in October. At the same time, the amount of oleanolic acid in stems decreased to 1.17% and also

began to stabilize. Whereas the contents of oleanolic acid in leaves decreased rapidly with the

increase in the number of withered leaves. After November, the roots of A. bidentata were fully

developed and mature, the aerial parts of plants began to die back and wither, resulting in an

extremely low quantity (0.06%) of oleanolic acid. The proportions of oleanolic acid in various

vegetative organs of A. bidentata, from highest to lowest, were roots > stems > leaves.

The accumulation pattern of active ingredients, and plant growth and development, are two

important indicators that can help determine the most suitable harvest periods for (root) medicinal

plants (Han et al. 2003). The determination of the best harvest period should be based not solely on

the maximum level of triterpenoid saponin in the roots in a phenological phase, but on multiple

factors such as total biomass of roots and extraction ratio of triterpenoid saponin, etc. Although

maximum triterpenoid saponin levels in A. bidentata roots were reached in August, the biomass

was low. In October, saponin levels tended to stabilize, while root biomass was increasing. By

November, the aerial parts of plants were beginning to die back and wither. Roots, on the other

hand, achieved maximum biomass and high triterpenoid saponin levels following a rapid growth

period. This time in November, therefore, would be the ideal period to harvest A. bidentata roots in

order to obtain the highest biomasses and concentration of saponin.

Materials and Methods

Plant Materials

Seeds of A. bidentata were sown in the Botanical Garden of Northwest University in China on 1

July in 2006 and emerged on 7 July. All plant material used in this study was harvested at each

developmental stage in August to December 2006.

Anatomical experiment

The root, stem and leaf at each developmental stage were fixed in a formalin-acetic acid-alcohol

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(FAA) solution immediately. Samples were embedded in paraffin and sectioned into the 8-10 µm

slices by Leica microtome. The sections were stained with safranino and pastgreen as previously

described (Li and Hu 2006), observed and imaged under Leica-DMLB microscope.

Histochemistry experiment

Histochemical analysis was performed according to the Liu Shibiao’ method (Liu and Hu 2005)

with modification. Frozen sections (30-40µm) were cut with Leica-CM1850 cryostat, mounted on

poly-L-lysine–coated slides, and soaked into a 5% lead acetate solution for 10min. This process

resulted in the precipitation of saponons in tissues. The sections were then placed in a mixture of

5% vanillic aldehyde-glacial acetic acid and perchloric acid for 5 to 10min for colour development.

The control materials were soaked for 20 days in FAA fixing solution (prepared using 50% ethanol)

to remove saponins. The prepared sections were observed under Leica-DMLB microscope and

imaged.

Phytochemical analysis

Sample preparation

The root, stem and leaf were harvested in August, September, October, November and December,

followed by baking dry at 60°C, crushing with a minitype grinder (Model FW80 , Beijing Kewei

Yongxing Apparatus Co., Beijing , China) , and sieving through a 40-mesh screen. Because of the

low temperature in December, all leaves were sear and fell out, and the oleanolic acid content of the

leaves at this stage was not measured. A 0.5g sample was placed in 10ml of methanol, followed by

ultrasonic extraction for 40min and concentration using a rotary vacuum evaporator (Model

SENCO R204B, Shanghai Shenke Technique Co., Shanghai, China). To the concentrated samples

was added 10 ml of 4 mol/L hydrochloric acid for hydrolysing at 85°C for 1h. After being allowed

to cool, 10 ml of chloroform were added to the sample, followed by two countercurrent extractions

at 60°C, each extraction being 15min. The bottom liquid was harvested and evaporated under

reduced pressure. The residue was dissolved in methanol (3ml), filtrated through a Millipore filter

(0.22µm) and subjected to High-performance liquid chromatography (HPLC) analysis.

High-performance liquid chromatography analyses

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High-performance liquid chromatography (HPLC) was used to determine oleanolic acid in A.

bidentata as described previously (Flores-Sánchez et al. 2002). The HPLC system consisted of a

Shimadzu LC-10Atvp HPLC apparatus (Kyoto, Japan), diode array detector (SPD-M10Avp), Class

VP-LC workstation and a Shim-pack VP-ODS column (150 mm×4.6mm). The assay conditions

were mobile phase: methanol-water-glacial acetic acid (90:10:0.05); flow rate: 0.9 ml/min; column

temperature: 30�; detection wavelength: 210 nm. An oleanolic acid standard was purchased from

the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China).

Quantification was based on calibration curves obtained from the samples to which standards of

different concentrations had been added over the linear range of 0.04-8µg. The reproducibility of

the method was controlled by analysing a standard five times (20µl). The relative standard

deviation (RSD) of peak area was 1.156%. Recovery efficiency was determined under the same

operating conditions. The mean recovery rate was 97.83%. For a stability test, the samples were

analyzed by HPLC after 0, 2, 4, 6, 8, 10h under room temperature. Oleanolic acid was stable under

room temperature for at least 10h in methanol, with the RSD being 2.644%.

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Figure I:

Structur of root, stem and leaf.

1. Primary structure of root; 2. Secondary structure of root; 3. Tertiary vascular in portion of root�

4. Secondary structure of stem; 5. Secondary structure of stem; 6. Medullary bundle in portion

of stem; 7. Structure of leaf; 8. Structure of the medial vein in blade

Abbreviation:

E, Epidermis; C, Cortex; PP, Primary phloem; Pe, Pericycle; PX, Primary xylem; SX, Secondary

xylem; CC, cork cambium; AB, tertiary vascular bundles; VB, Vascular bundle; MB, Medullary

bundle.

Figure �:

Histochemical analysis of root, stem and leaf.

9. Cells of the primary and the pericycle of root (light red); 10.Secondary phloem, supernumerary

cambium and phelloderm of root (purple); 11. Secondary phloem and phloem of tertiary vascular

bundles of root, (purple); 12. Cells of phelloderm in root (purple); 13 Phloem cells of the vascular

bundle and medullary bundle in stem (purple); 14-15. Cells of palisade and phloem of the medial

vein in leaf(purple); 16. Mesophyll cells in old leaf without red staining.

1

Figure I

7

AB

3

1 2

MB

VB

4

5 6

7

VB

8

2

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11 12

13 14

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