a

Original Article ISSN 0101-2061 (Print)

Food Science and Technology ISSN 1678-457X (Online)

DOI: [Link]

Effect of environmental factors on the polysaccharide content of Dendrobium officinale

Daopeng TAN1 , Yao ZENG1, Jiangmei WANG1, Yanliu LU1,2, Shudong SUN3, Lin QIN1,2* , Yuqi HE1,2* 

Abstract
Dendrobium officinale is a traditional Chinese medicine and nourishing food in China. Polysaccharide content was used as a
criterion for evaluating the quality of D. officinale. At present, epiphytic cultivation is the main cultivation method of D. officinale
in China. In the present study, D. officinale was collected under various growing conditions to compare its polysaccharide
content in an attempt to discover the most favorable growing conditions for D. officinale, which can be used to guide the artificial
cultivation production of D. officinale.
Keywords: Dendrobium officinale; polysaccharide; quality control.
Practical Application: This investigation provides valuable guidance for the cultivation production of D. officinale.

1 Introduction
In recent years, the R&D of natural functional products polysaccharides have been selected as the only quality markers of
from herbs has attracted more and more attention in the world D. officinale, and polysaccharide content was used as a criterion
(Ruiz-Cisneros et al., 2022; Wang et al., 2022a, b; Yin et al., 2022). for evaluating the quality of D. officinale.
The genus Dendrobium is one of the largest genera in the orchid
To obtain a large amount of green and organic D. officinale
family, containing 1500-2000 species (Hou et al., 2017). Among
them, Dendrobium officinale Kimura et Migo is the main source herbs, it is now common to grow D. officinale in China by attaching
of the traditional Chinese medicine Dendrobii caulis, which is trees (Figure 1) (Tan & He, 2020). There have been several reports
widely distributed throughout the world, such as the United States, in the literature that the cultivation sites and cultivation techniques
Australia, and Japan (Tang et al., 2017). In particular, D. officinale of D. officinale affect its polysaccharide content (Tan et al., 2020;
is widely grown in various regions of China, including Zhejiang, Zeng et al., 2020). However, these reported samples are limited
Anhui, Hunan, Guizhou, Fujian, Guangxi, and Yunnan provinces in number and do not accurately reflect the main growing factors
(Liu et al., 2020; Yan et al., 2015; Yang et al., 2020). of polysaccharide content of D. officinale. Therefore, in this study,
D. officinale was collected under various growing conditions to
D. officinale is firstly recorded in the “Shen Nong’s Herbal compare its polysaccharide content in an attempt to discover the
Classic” (Dong Han Dynasty, A.D.25–220) and used as traditional most favorable growing conditions for D. officinale, which can be
Chinese medicine or functional food in China (Yin et al., 2021). used to guide the artificial cultivation production of D. officinale.
In traditional medicine, D. officinale was as a tonic to nourish
Yin, clear heat, nourish stomach, and replenish body fluid
(Cakova et al., 2017; Shin et al., 2017) and used for various 2 Materials and methods
diseases or as beverages (Cakova et al., 2017; Tan et al., 2023). 2.1 Chemicals
In term of modern pharmacological effects, D. officinale exhibits
The reagents required for the polysaccharide assay including
various pharmacological effects such as enhancing immunity,
phenol, anhydrous ethanol, concentrated sulfuric acid, and
anti-fatigue, antioxidant, hypoglycemia, hypotension, and others
(He et al., 2022; Huang et al., 2019; Lv et al., 2020). glucose standards were purchased from Merck (Darmstadt,
Germany), Sinopharm Chemical Reagent Co., Ltd (Shanghai,
According to current phytochemical investigations, more China), and Kelong Chemical Reagent Factory (Chengdu,
than 190 compounds have been isolated from D. officinale, China), respectively.
including polysaccharides, alkaloids, amino acids, flavonoids
and other nutritional components (He et al., 2022). Among
2.2 Plant materials
them, polysaccharides have been shown to regulate intestinal
homeostasis and protect against carbon tetrachloride-induced Under the premise of ensuring sustainable utilization and
liver injury in mice, and was considered to be its most important representativeness, the D. officinale samples were collected
active components. In the Chinese Pharmacopoeia (Ch.P., from the Good Agricultural Practices (GAP) bases located in
2020 edition) (Chinese Pharmacopoeia Commission, 2020), Guizhou and Zhejiang Province of China in 2019. All samples

Received 12 Nov., 2022

Accepted 26 Dec., 2022
1
Guizhou Engineering Research Center of Industrial Key-technology for Dendrobium Nobile, School of Pharmacy, Zunyi Medical University, Zunyi, China
2
Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou, China
3
Anlong County Xichengxiushu Agriculture and Forestry Co., Ltd., Anlong, Guizhou, China
*Corresponding author: 505181163@[Link]; [Link]@[Link]

Food Sci. Technol, Campinas, 43, e127422, 2023 1

 Polysaccharide differences in Dendrobium officinale

Figure 1. Growing environment of D. officinale.

were authenticated by Associate Professor Daopeng Tan Preparation of glucose standard curve
(pharmacognosy, Zunyi medical university).
The glucose standard was weighed precisely and prepared
into 180 μg/mL of glucose solution, i.e. the reserve solution.
2.3 Polysaccharides content determination The reserve solution was diluted and prepared as 30.47, 60.93,
Sample preparation 91.40, 121.87, 152.33 and 182.80 μg/mL, and the phenol-sulfuric
acid color development reaction was performed according to
Samples of D. officinale was dried at 60 °C, crushed, and sieved the method of polysaccharide assay, and the absorbance was
through No.3. Take about 0.06 g of sample, weighed precisely, and determined together with the samples.
refluxed with 40 mL of water, heated for 2 h. Cooled, transferred
into a 50 mL volumetric flask, and fixed with water to the scale Method validation
line. 4000 rpm, centrifuged for 15 min. 2 mL of supernatant is
taken, 10 mL of anhydrous ethanol is added, mixed well, and left The linearity, precision, repeatability, stability, and recovery
for 1 h at 4 °C. Centrifuge at 4000 rpm for 20 min and discard were checked for method validation.
the supernatant. Add 8 mL of 80% ethanol solution, centrifuge
at 4000 rpm for 20 min, discard the supernatant, and repeat 2.4 Statistical methods
once. Dissolve the precipitate with heated water, allow to cool,
transfer to a 10 mL volumetric flask, add water to the scale to SPSS18.0 was used for statistical analysis, and t-test was
obtain the sample solution to be tested. used to calculate the comparison between two groups, One-way
ANOVA was used to calculate the comparison between more
than two groups, and the measurement data were expressed as
Polysaccharide assay Mean ± SD, and P < 0.05 was statistically different.
Take 1.0 mL of the sample to be measured into a 10 mL stoppered
test tube, add 1.0 mL of 5% phenol solution (ready to use) and 5 mL 3 Results and discussion
of concentrated sulfuric acid, shake well rapidly, heat in boiling water
3.1 Sample collection
for 20 min, then ice bath for 5 min. 200 μL of the reaction solution
was taken into a 96-well plate, and the absorbance was measured The samples were collected at five Dendrobium cultivation
at 488 nm with an enzyme marker, and the polysaccharide content sites in Dushan county, Danzhai county, and Xingyi city of
was calculated by substituting into the standard curve. Guizhou province, and Taizhou and Yueqing city of Zhejiang

2 Food Sci. Technol, Campinas, 43, e127422, 2023

 Tan et al.

province (Figure S1). Dendrobium samples were collected equation of the standard curve was y=215.18x-0.9978, r=0.9999.
according to different altitudes, different epiphytic species and The results indicated that there was a good linear relationship
different illuminance. Eight to ten samples of D. officinale were between the absorbance and concentration of glucose in the
collected for each factor, 380 samples were collected in total. range of 30.47~182.80 μg/mL.
The information in detail is listed in Table 1. The fresh stems
were dried, ground into powder, passed through a sieve with Precision was evaluated by using variability assessed with
300 mesh, and stored at -80 °C for subsequent analysis. six replicates within one day. The variation (RSD%) for precision
was shown in Table 2. The repeatability was conducted using six
replicates of the same sample, and the variations of repeatability
3.2 Validation of the method was 0.63% (Table 3). The stability of the sample solution was
Linear regression equations (e.g., y = ax + b) were constructed investigated at 0, 0.5, 1, 2, 4, 6, and 12 h. The RSD% of absobances
by absorbance (x) of each analyte against analyte concentrations of the analyzed sample was 2.39%(Table 4), indicating that the
(y; μg/mL). The results are shown as Figure 2, and the linear samples were stable at least 12 h.

Table 1. Sample information of D. officinale.

Location Epiphytic trees Longitude and Latitude Altitude(m) Illuminance Sample size
Dushan Pinus E107°31’2”N5°56’5” 852 sunny 10
Dushan Pinus E107°31’2”N5°56’5” 852 shady 10
Dushan Pinus E107°31’2”N25°55’47” 918 sunny 10
Dushan Pinus E107°31’2”N25°55’47” 918 shady 9
Dushan Cunninghamia lanceolata E107°31’2”N5°56’5” 852 sunny 8
Dushan Cunninghamia lanceolata E107°31’2”N5°56’5” 852 shady 9
Dushan Cunninghamia lanceolata E107°31’2”N25°55’47” 918 sunny 9
Dushan Cunninghamia lanceolata E107°31’2”N25°55’47” 918 shady 10
Dushan Cyclobalanopsis glauca E107°31’2”N5°56’5” 852 sunny 8
Dushan Cyclobalanopsis glauca E107°31’2”N5°56’5” 852 shady 9
Dushan Quercus fabri E107°31’2”N25°55’47” 918 sunny 9
Dushan Quercus fabri E107°31’2”N25°55’47” 918 shady 9
Dushan Quercus fabri E107°31’2”N5°56’5” 852 sunny 9
Dushan Quercus fabri E107°31’2”N5°56’5” 852 shady 9
Danzhai Camellia sinensis E107°79’90”N26°25’23” 891 shady 28
Xingyi Cyclobalanopsis glauca. E105°24’54”N24°58’37” 835 sunny 9
Xingyi Cyclobalanopsis glauca. E105°24’54”N24°58’37” 835 shady 8
Xingyi Cyclobalanopsis glauca. E105°27’8”N24°59’12” 978.5 sunny 10
Xingyi Cyclobalanopsis glauca. E105°27’8”N24°59’12” 978.5 shady 9
Xingyi Cyclobalanopsis glauca. E105°27’57” N24°54’3” 1122 sunny 8
Xingyi Cyclobalanopsis glauca. E105°27’57” N24°54’3” 1122 shady 9
Xingyi Albizia julibrissin E105°24’54”N24°58’37” 835 sunny 9
Xingyi Albizia julibrissin E105°24’54”N24°58’37” 835 shady 10
Xingyi Albizia julibrissin E105°27’8”N24°59’12” 978.5 sunny 9
Xingyi Albizia julibrissin E105°27’8”N24°59’12” 978.5 shady 9
Xingyi Albizia julibrissin E105°27’57” N24°54’3” 1122 sunny 9
Xingyi Albizia julibrissin E105°27’57” N24°54’3” 1122 shady 9
Xingyi Cyclobalanopsis glauca E105°24’54”N24°58’37” 835 sunny 8
Xingyi Cyclobalanopsis glauca E105°24’54”N24°58’37” 835 shady 9
Xingyi Cyclobalanopsis glauca E105°27’8”N24°59’12” 978.5 sunny 9
Xingyi Cyclobalanopsis glauca E105°27’8”N24°59’12” 978.5 shady 9
Xingyi Cyclobalanopsis glauca E105°27’57” N24°54’3” 1122 sunny 9
Xingyi Cyclobalanopsis glauca E105°27’57” N24°54’3” 1122 shady 9
Taizhou Amygdalus persica E121°6′57″N28°26′26″ 34 sunny 9
Taizhou Cinnamomum camphora E121°6′57″N28°26′26″ 34 shady 10
Yueqing Myrica rubra E121°9′53″N28°22′7″ 166 sunny 9
Yueqing Myrica rubra E121°9′53″N28°22′7″ 166 shady 8
Yueqing Ziziphus jujuba E121°7′14″N28°26′16″ 28 sunny 9
Yueqing Ziziphus jujuba E121°7′14″N28°26′16″ 28 shady 8
Yueqing wooden pile E121°7′14″N28°26′16″ 28 sunny 10

Food Sci. Technol, Campinas, 43, e127422, 2023 3

 Polysaccharide differences in Dendrobium officinale

Table 2. Precision of polysaccharide content in D. officinale. Table 5. Accuracy of polysaccharide content in D. officinale.
No. Absorbance Mean RSD No. Recovery RSD
1 0.5422 0.5372 0.63% 1 98.62%
2 0.5397 2 100.30%
3 0.5377 3 100.73%
2.00%
4 0.5361 4 98.05%
5 0.5344 5 95.27%
6 0.5331 6 99.52%

Table 3. Repeatability of polysaccharide content of D. officinale.

No. Content Mean RSD
1 39.50%
2 38.18%
3 37.53%
38.35% 2.05%
4 38.73%
5 38.68%
6 37.45%

Table 4. Stability of polysaccharide content in D. officinale.

Time (h) Absorbance Mean RSD
0 0.5353 0.5266 2.39%
0.5 0.5241 Figure 2. Standard curve of glucose solution.
1 0.5359
2 0.5089
4 0.5371
6 0.5097
12 0.5353

The recovery test was used to evaluate the accuracy of the

method. Precisely weighed six powders of D. officinale in parallel,
and about 23 mg of dextran was added to each of them. The test
samples were prepared according to the method of polysaccharide
assay, and the absorbance was determined according to the method
of preparation of glucose standard curve. The recovery of each spiked
reference standards was calculated by the formula recovery%=
[(found amount−original amount)/spiked amount] × 100%.
The results are shown in Table 5. The recoveries of polysaccharides Figure 3. Overall profile of polysaccharide content in D. officinale of
ranged from 95.27% to 100.73% with the RSD value of 2.00%, 380 batch.
indicating that the method is accurate.

3.3 Analysis of polysaccharide content of D. officinale samples are qualified. As shown in Figure 3, the polysaccharide
Overall profile of polysaccharide content of D. officinale content in 380 D. officinale samples was significantly different,
and the highest polysaccharide content was 61.69%. 42% of the
A total of 380 D. officinale samples were collected according 380 samples were qualified. Among them, the polysaccharide
to different regions, altitudes and illuminance (Figure S1). content of D. officinale was mainly concentrated between 20%
The overall profile of polysaccharide content in 380 D. officinale and 35%.
samples was analyzed to determine the polysaccharide content.
The horizontal coordinate in the figure is the sample number, the
Effect of different origins on polysaccharide content of
vertical coordinate is the percentage content of polysaccharide
D. officinale
in D. officinale, and the dotted line is the standard limit of
25.00% of polysaccharide content in D. officinale as stipulated For the polysaccharide content of D. officinale from five
in the Chinese Pharmacopoeia 2020 edition, a part of herbs and regions, including Dushan county, Danzhai county, and Xingyi city
beverages, and higher than this line means that the D. officinale of Guizhou province, and Taizhou and Yueqing city of Zhejiang

4 Food Sci. Technol, Campinas, 43, e127422, 2023

 Tan et al.

province were analyzed as shown in Figure 4. The polysaccharide polysaccharide content of D. officinale was found at 28 m above
content of D. officinale in Yueqing city was the highest among sea level. Altitude was found to be the main factor affecting the
the five regions, and the polysaccharide content of D. officinale polysaccharide content of D. officinale.
in Xingyi city was the lowest. The polysaccharide contents of D.
officinale in Yueqing city were compared with those of Dushan, Effect of different tree species on polysaccharide content of
Denzhai, Xingyi and Taizhou respectively, and P<0.05, which D. officinale
was statistically different, and the polysaccharide contents of D.
officinale in Yueqing city were significantly different from those The areas with more epiphytic tree species were selected to
of Dushan, Denzhai, Xingyi and Taizhou. The polysaccharide analyze the effects of different tree species on the polysaccharide
contents of Xingyi city was statistically different from those contents of D. officinale in Dushan county of Guizhou province.
of Dushan, Denzhai, Taizhou and Yueqing by two-by-two As shown in Figure 6, there was no significant difference in the
comparison with P<0.05, and the polysaccharide contents of polysaccharide contents of D. officinale attached to different
D. officinale in Xingyi city was significantly lower than those tree species such as Cyclobalanopsis glauca, Quercus fabri,
of the other four origins. Cunninghamia lanceolata, and Pinus, however, the polysaccharide
content of D. officinale at an altitude of 918 m was significantly
higher than that of the sample at an altitude of 852 m. Those
Effect of different altitudes on polysaccharide content of
results indicated that the main influencing factor is related to
D. officinale
the altitude, but not to the epiphytic tree species.
Analysis was conducted to compare the polysaccharide content
of D. officinale at different altitudes (Figure 5.). The samples Effect of illuminance on polysaccharide content of D. officinale
collected in Guizhou province ranged from 835 m to 1122 m in
altitude, and the highest polysaccharide content of D. officinale For the analysis of the effect of illuminance on the
was found at 918 m in altitude. The samples collected in Zhejiang polysaccharide content of D. officinale, the polysaccharide
Province ranged from 28 to 166 m above sea level, and the highest content of D. officinale with shade and sunny growth was
compared. It was found that the polysaccharide content
of sunny-grown D. officinale was generally higher than
that of the shady-grown. The passing rate of sunny-grown
D. officinale was generally high, and all samples of sunny-
grown D. officinale at an altitude of 1122 m passed in this
sampling. The mean values of the polysaccharide content
of D. officinale at different elevations of sunny and shady
grown D. officinale were subjected to paired t-test, and the
results showed that the polysaccharide content of sunny-
grown D. officinale was significantly higher than that of the
shady-grown (Figure 7).

Figure 4. The effect of origin on polysaccharide content of D. officinale

(‾x ±SD, n=19-162, a compared to Xingyi, *P < 0.05, b compared to
Yueqing, *P < 0.05).

Figure 5. The effect of altitudes on polysaccharide content of D. officinale

(‾x ±SD, n=17-71). Figure 6. The effect of tree on polysaccharide content of D. officinale.

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 Polysaccharide differences in Dendrobium officinale

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Supplementary Material
Supplementary material accompanies this paper.
Figure S1. Sample collection of D. officinale
This material is available as part of the online article from [Link]

8 Food Sci. Technol, Campinas, 43, e127422, 2023