Granularity and Laxative Effect of Ultrafine Powder of Dendrobium officinale

Abstract

Constipation is a common disorder that is a significant source of morbidity among people around the world ranging from 2% to 28%. Dendrobium officinale Kimura et Migo is a traditional herbal medicine and health food used for tonicity of the stomach and promotion of body fluid production in China. This study aimed to prepare the ultrafine powder of Dendrobium officinale (UDO) and investigate its laxative effect and potential mechanism in mice with diphenoxylate-induced constipation. Results indicated that the mean diameter (d₅₀) of UDO obtained by ball milling was 6.56 μm. UDO (62.5, 125, and 250 mg/kg, p.o.) could significantly enhance the gastrointestinal transit ratio and promote fecal output. Moreover, UDO treatment resulted in significant increases in the serum levels of acetylcholinesterase (AChE), gastrin (Gas), motilin (MTL), and substance P (SP), and obviously decreased serum contents of somatostatin (SS). Taken together, UDO, which can be easily obtained through milling to a satisfactory particle size, exhibited obvious laxative effect in diphenoxylate-induced constipated mice, and the mechanism might be associated with elevated levels of AChE, Gas, MTL, SP, and reduced production of SS. UDO has the potential for further development into an alternative effective diet therapy for constipation.

Introduction

Constipation is one of the most common chronic functional gastrointestinal (GI) ailments affecting the health-related quality of life of the worldwide population ranging from 2% to 28%. Constipation symptoms are usually characterized by difficulty during defecation and incomplete evacuation of stool with symptoms such as hard stools, infrequent stools, and unsuccessful defecation.¹ Occurrence of constipation is not only discomforting but also may lead to abdominal distension, restlessness, gut obstruction and perforation, and could be a risk factor for colorectal cancer.² There have been some agents developed to relieve constipation such as osmotic and stimulant laxatives, 5-HT4 agonists, and prosecretory agents.³ However, these drugs are usually associated with potentially adverse side effects such as tolerance induction, melanosis coli, or cathartic colon, which compromises their applications to some extent.⁴ In recent years, herbal plants and medicinal foods have been receiving increasing attention as promising alternative therapeutic choices for the treatment of constipation.⁵

Dendrobium officinale Kimura et Migo (Dendrobium officinale), a well-known and widely used traditional Chinese medicinal herb (called “Tie-Pi-Shi-Hu” in Chinese), has been used for thousands of years for maintaining tonicity of the stomach and promoting the production of body fluids.⁶ It is classified as one of the most precious species among the Dendrobium genus and specified in the Chinese Pharmacopeia (2015 version) with an individual category among more than 70 Dendrobium species in China.⁷ During the past decades, tremendous amounts of efforts have been devoted to investigating the biological activities of D. officinale, including beneficial effects on colon health, promotion of salivary secretion,⁸ immunomodulatory activities,⁹ and ameliorating pulmonary functions.^10,11 According to traditional Chinese medicine theory, D. officinale was commonly used in GI tract diseases. Besides, many studies have proved the enhanced effect of D. officinale on constipation in mice with spleen deficiency, including improved spleen-deficiency constipation symptoms and body weight, regulating the balance of molecular diversity of intestinal Lactobacillus and intestinal bacteria, and enhanced the intestinal enzyme activity through different pathways.^12
–14 Due to these properties, it is presumed that D. officinale has beneficial effects on the attenuation of constipation.

Nowadays, D. officinale, which has a relatively wide margin of safety, is widely used in daily life as a healthy food approved by the China Food and Drug Administration (CFDA).¹⁵ The stem of D. officinale can be made into soup and dishes as a high-quality agricultural vegetable in diets, drink products, functional liquid, as well as functional capsules and powder for health promotion effect.^16,17 As a functional food source, D. officinale has been the focus of considerable interest in diet and disease research, especially in the form of ultrafine powder, which has been widely applied in the food industry.¹⁸ Indeed, the ultrafine powder has become an important form of various functional and medicinal herbs especially for those precious herbal species such as Radix Ginseng, Radix Panacis Quinquefolii, and D. officinale,¹⁹ for the sake of higher solubility and absorption, enhanced bioavailability, and thus improved biological effect.

Although the laxative effect of D. officinale has been investigated in some animal models, the underlying mechanism remains to be elucidated. The present study was designed to investigate the laxative effect and potential mechanism of the ultrafine powder of Dendrobium officinale (UDO) in diphenoxylate-induced constipation mice. The D. officinale was milled by ball milling to obtain UDO. As a well-known effective antidiarrheal agent, diphenoxylate was used to induce constipation in the present study through the inhibited intestinal motility and propulsion.^20,21 The GI transit ratio, the time for the first defecation of a black stool, and the changes in number, weight, and water content of the feces were evaluated.²² Bedsides, to further probe the possible mechanism of the observed laxative effects of UDO, the levels of acetylcholinesterase (AChE), gastrin (Gas), motilin (MTL), substance P (SP), and somatostatin (SS) were also determined.²³

Materials and Methods

Materials and drugs

D. officinale was acquired from Guangdong Academy of Forestry (Guangzhou, China). The Indian ink was purchased from Shanghai Yuanmu Biotechnology Technology Co., Ltd. (Shanghai, China). Pyridostigmine bromide (PB) and diphenoxylate were purchased from Guangzhou Feibo Biological Technology Co., Ltd. (Guangzhou, China). All enzyme-linked immunosorbent assay (ELISA) kits were purchased from Beijing Cheng Lin Biotechnology Technology Co., Ltd. (Beijing, China).

Animals

One hundred twenty ICR mice (60 males and 60 females, 20–22 g) were purchased from Guangdong Medical Laboratory Animal Center (Foshan, China) and allowed to acclimate for 7 days before the constipation experiment. All the mice were kept at standard condition (20–23°C, relative humidity 50% ± 5% with 12-h light/12-h dark cycles) and given free access to food and water. Animal experiments were carried out in accordance with procedures approved by the Animal Experimental Ethics Committee of Guangzhou University of Chinese Medicine (dSPF 2014 021), and the experimental protocols followed the “Guide for the Care and Use of Laboratory Animals.”

Preparation and characterization of UDO

D. officinale was dried at 50°C for 24 h, and then precrushed for 60 min using a ball mill. After that, the powders were screened through mesh size of 80 and then milled in the high-frequency oscillatory type ball mill. After that, the powder was screened through mesh size of 500 to obtain the UDO. The particle size of UDO was analyzed using a Dry Dispersion Laser Particle Size Analyzer (3003A; Jinan Winner Particle Instruments Joint Stock Co., Ltd., Jinan, China).

Induction of constipation by diphenoxylate

All the mice were fasted for 16 h before the constipation experiment. Ten male and 10 female mice were used as the intact group, orally administrated with distilled water (0.1 mL/10 g). Subsequently, 50 male and 50 female mice were orally administrated with diphenoxylate (50 mg/kg) to induce constipation. After administration with diphenoxylate for 30 min, the constipated mice were randomly divided into the following five groups: the intact and vehicle group with distilled water (0.1 mL/10 g); three treatment groups with UDO (62.5, 125, 250 mg/kg); and the positive control group with PB (40 mg/kg). All the drugs were previously prepared in 0.5% India ink before use.

Measurement of GI transit ratio on constipated mice

After administration with different treatments for 20 min, 10 mice (5 males and 5 females) in each group were scarified and dissected to obtain the small intestine. The length of the entire small intestine and translated distance by India ink were measured to obtain the GI transit ratio. The GI transit ratio was calculated as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{{ \rm{GI \ transit \ ratio = }}{{{ \rm{Translated \ distance \ by \ India \ ink}}} \over {{ \rm{Length \ of \ entire \ small \ intestine}}}}{ \rm{ \times 100 \% }}} \end{align*} \end{document}

In addition, serum was collected from the heart following surgery before cervical dislocation and then kept at −80°C for the subsequent analysis.

Measurement of fecal output on constipated mice

After administration with different treatments, 10 mice (5 males and 5 females) in each group were moved into the metabolic cage individually and observed for 6 h. The time for the first black feces, the number, weight, and character of the feces collected during the experiment were recorded. The feces were weighed to obtain the fecal wet weight, then dried at 105°C for 48 h after the experiment, and weighed again to obtain the fecal dry weight. The fecal water content was calculated as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}& { \rm{Fecal \ water \ content \ }}\\ & \quad= {{{ \rm{ ( Fecal \ wet \ weight \ - \ Fecal \ dry \ weight ) }}} \over {{ \rm{Fecal \ wet \ weight}}}}{ \rm{ \times 100 \% }} \end{align*} \end{document}

Determination of levels of AChE, Gas, MTL, SP, and SS in the serum

The levels of AChE, Gas, MTL, SP, and SS in the serum from different groups were determined by using an ELISA kit according to the manufacturer's instructions.

Statistical analysis

All data are expressed as the mean ± standard deviation (SD). The difference between different treatments was assessed by one-way ANOVA, followed by Duncan's multiple range test using SPSS. P < .05 means a statistically significant difference between different groups.

Results

Particle size of UDO

The UDO was produced in the high-frequency oscillatory type ball mill and its particle size distribution was determined by a Dry Dispersion Laser Particle Size Analyzer. The results are shown in Figure 1. The diameter of ultrafine powder D. officinale ranged from 1 to 31 μm, and the mean diameter (d₅₀) was 6.56 μm, which fell within the satisfactory level and therefore applied in the subsequent study.²⁴

FIG. 1.

The particle size distribution of UDO (μm). UDO was produced in the high-frequency oscillatory type ball mill and its particle size was measured using a Dry Dispersion Laser Particle Size Analyzer. UDO, ultrafine powder of Dendrobium officinale.

Measurement of GI transit ratio in constipated mice

To investigate the GI peristalsis function, we measured the GI motility in mice. The representative character of the small intestine and GI transit ratio are exhibited in Figure 2 and Table 1, respectively. Diphenoxylate significantly reduced the GI transit ratio to 27.08% ± 5.79% (P < .01) compared to 54.76% ± 6.88% in the normal group. In contrast, UDO oral administrations (62.5, 125, and 250 mg/kg) dose dependently increased the India ink propelling ratio to 43.07% ± 7.88%, 51.38% ± 6.39%, and 62.46% ± 5.91%, respectively (all P < .01), when compared with the vehicle group. Besides, PB (40 mg/kg) remarkably accelerated the India ink pass through the small intestine to obtain a higher GI transit ratio at 75.33% ± 6.12% (P < .01).

FIG. 2.

Representative photographs of small intestine in constipated mice after administration of different treatments. The symbol “a” indicates the translated distance by India ink and “b” represents the length of entire small intestine.

Table 1.

Effect of Ultrafine Powder of Dendrobium officinale on Gastrointestinal Transit Ratio in Diphenoxylate-Induced Constipated Mice

Groups	Intestinal transit length (cm)	Intestinal length (cm)	Intestinal transit ratio (%)
Intact	21.86 ± 3.06	39.95 ± 3.03	54.76 ± 6.88
Vehicle	10.99 ± 2.13^##	40.89 ± 2.74	27.08 ± 5.79^##
PB (40 mg/kg)	31.66 ± 2.69^**	42.13 ± 3.29	75.33 ± 6.12^**
UDO (62.5 mg/kg)	17.96 ± 2.96^**	41.83 ± 1.36	43.07 ± 7.88^**
UDO (125 mg/kg)	21.52 ± 2.19^**	41.81 ± 3.76	51.38 ± 6.39^**
UDO (250 mg/kg)	24.48 ± 3.62^**	39.25 ± 3.46	62.46 ± 5.91^**

Data represent the mean ± SD (n = 10). Asterisks designate statistically significant differences: ^## P < .01 (the vehicle vs. intact group); ^** P < .01 (the vehicle vs. UDO groups).

PB, pyridostigmine bromide; SD, standard deviation; UDO, ultrafine powder of Dendrobium officinale.

Measurement of fecal output in constipated mice

The character of feces and fecal output are shown in Figure 3. Diphenoxylate can obviously prolong the time for the first black feces and reduce the number, weight, and fecal water content (Fig. 3B, all P < .01). Besides, the feces in the vehicle group were dry and hard, compared with the intact mice (Fig. 3A). Administration of UDO also dose dependently exhibited a significant ameliorating effect in constipated mice, with shortened time for the first black feces and increased number and weight (Fig. 3B, all P < .01). Also, the water content was normalized from 30.61% ± 6.75% to 34.59% ± 7.08%, 45.23% ± 7.05%, and 60.41% ± 5.75%, respectively (125 and 250 mg/kg, all P < .01), and the feces seemed wet and soft compared with the vehicle group (Fig. 3A). PB also showed potent therapeutic effect against diphenoxylate-induced constipation with obviously restored fecal output and softened fecal character (all P < .01).

FIG. 3.

Effect of UDO on the character of feces and fecal output in diphenoxylate-induced constipated mice from different groups. (A) Representative character of feces; (B) fecal output (a: time for the first black feces; b: number of feces; c: weight of feces; d: fecal water content). Each bar represents the mean ± SD (n = 10). Asterisk signs designate statistically significant differences: ^## P < .01 (the vehicle vs. intact group); **P < .01 (the vehicle vs. UDO groups); \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$^{\Delta \Delta}$$ \end{document} P < .05 (PB vs. UDO groups). SD, standard deviation.

Levels of AChE, Gas, MTL, SP, and SS in the serum

The changes of AChE, Gas, MTL, SP, and SS in the different groups are shown in Figures 4 and 5. In the vehicle group, constipated mice induced by diphenoxylate showed lower serum levels of AChE, Gas, MTL, and SP with a higher concentration of SS compared with the intact mice (all P < .01). On the contrary, UDO of three test doses could significantly increase the serum levels of Gas and MTL and decrease the levels of SS (all P < .01). Furthermore, UDO at doses of 125 and 250 mg/kg was shown to obviously increase the serum levels of AChE (all P < .01) and SP (125 mg/kg, P < .05; 250 mg/kg, P < .01) when compared with the vehicle. PB was also observed to significantly attenuate the decline of serum AChE, Gas, MTL, and SP levels, and counteract the increase of SS induced by diphenoxylate (all P < .01).

FIG. 4.

Levels of AChE, Gas, MTL, and SP in the serum: (A) AChE; (B) Gas; (C) MTL; and (D) SP. Each bar represents the mean ± SD (n = 10). Asterisk signs designate statistically significant differences: ^## P < .01 (the vehicle vs. intact group); *P < .05, **P < .01 (the vehicle vs. UDO groups); ^△△ P < .05 (PB vs. UDO groups). AChE, acetylcholinesterase; Gas, gastrin; MTL, motilin; PB, pyridostigmine bromide; SP, substance P.

FIG. 5.

Levels of SS in the serum from different groups. Each bar represents the mean ± SD (n = 10). Asterisk signs designate statistically significant differences: ^## P < 0.05 (the vehicle vs. intact group); **P < .05 (the vehicle vs. UDO group); ^△ P < .05, ^△△ P < .01 (PB vs. UDO group). SS, somatostatin.

Discussion

Constipation is a symptom-based chronic functional GI disorder with high prevalence. Due to the unsatisfactory outcome of current agents, investigations of functional medicinal herbs and foods have kindled intensive interest as a potential efficacious alternative in recent years. Indeed, the use of herbal remedies in the treatment of constipation is a common practice in many countries, including China, for many years. D. officinale is a famous Chinese functional and medicinal herb frequently used to tonify the stomach and promote body fluid production, which might be closely associated with the laxative effect in the GI system.

In the present work, the laxative effect and potential mechanism of D. officinale were investigated in diphenoxylate-induced constipated mice. Results indicated that the classical biomarkers of fecal output, such as fecal pellet number, weight, and moisture content, were greatly increased and the defecation time was obviously shortened in UDO-treated mice, compared to the nontreated counterparts, indicating that the defecation was significantly facilitated by administration of D. officinale. The feces were also observed to be wet and soft in the UDO-treated mice. It was reported that the enterosystemic circulation of body fluid plays an important role in the occurrence of constipation, which involves moisture intake, saliva secretion, gastric acid secretion and intestine secretion through the GI tract, and so on. It is therefore important to maintain internal euhydration of the body as a prevention of constipation.²⁵ D. officinale is a herbal medicine that has been clinically used to promote body fluid production.²⁶ Previous investigations demonstrated that D. officinale polysaccharides could ameliorate symptoms of salivary secretion of patients with Sjögren's syndrome and in the SS mouse model through increasing the expression of aquaporin-5 in labial glands.⁸ Some studies have indicated that D. officinale could reduce the inhibitory effect on salivary gland secretion and enhanced saliva secretion in normal rabbits.²⁷ Another study showed that D. officinale recipes improved gastric fluid excretion in 30 clinical cases with an efficiency rate over 83.40%.²⁸ Based on these findings and its traditional application, it was hypothesized that the laxative effect of UDO observed in the present work might be associated with the promotion of the release of body fluid, thereby increasing the fecal moisture content and intestinal secretion. However, further in-depth investigation on its exact mechanism is warranted.

Regulation of the motility of the GI tract has been an important strategy for the treatment of constipation. The transit process of the entire GI tract reflected the overall GI motor activity. Therefore, measurement of GI transit ratio is a reliable indicator of constipation.²⁹ In the present work, the laxative effect of UDO was observed in the constipated mice induced by diphenoxylate, as evidenced by significantly enhanced GI transit length and ratio and obviously recovered defecation in the diphenoxylate-induced constipated mice. Hence, the laxative effect of UDO might be intimately related to alternations in the intestinal motility, which produced an increase in the intestinal transit and colonic movement.

Stools are formed from the nondigestible components of food, after water is either absorbed or secreted in the large intestine. When the GI peristalsis is active, the stool is propelled forward, and the mucus can provide lubrication when the stool is moving.³⁰ AChE, Gas, MTL, SP, and SS all play an important role in regulating GI motility and constipation,^31

–37 and were found to be changed after administration with diphenoxylate. As one of the major neurotransmitters of the enteric nervous system, acetylcholine (ACh) can regulate the contraction and relaxation of muscle, and the secretion of mucus. As peptides from the CCK-gastrin family, Gas was structured as a pair of heptadecapeptide amides. Besides, Gas was present at a lower level in patients with slow transit constipation.³⁸ MTL is a hormone released from the endocrine M cells from the duodenal mucosa, after duodenal acidification, stimulating GI smooth muscle contraction.^39,40 SP, one of the three classic members of the tachykinin family, is expressed by intrinsic neurons from cell bodies within the intestinal wall and plays an important role in intestinal motility, secretion, and vascular functions.^39,40 Furthermore, the levels of SP and SP nerve fiber density were found to be reduced in the constipated patients.^41,42

SS is a hypothalamic cyclic polypeptide consisting of 14 amino acids, which are produced by D cells in the gastric antrum and pancreas, mucosal δ-cells in the intestinal epithelium, and intrinsic neurons in the myenteric and submucosal plexuses along the digestive tract. Besides, SS can inhibit GI motility and the release of a variety of hormones such as MTL and Gas in the GI tract.^35,43 SS was also found to be present at higher levels in patients with severe idiopathic constipation.³¹

In the present study, lower levels of MTL, Gas, SP, and AChE and higher levels of SS were found in the vehicle group, which coincided with previous studies.^23,44,45 However, after administration with UDO, significantly higher levels of MTL, Gas, SP, and AChE and a lower level of SS were observed compared with the constipated mice. Based on these observations and their physiological functions, the modulatory effect exerted by UDO on the serum levels of MTL, Gas, SP, AChE, and SS might be intimately associated with the laxative effect of UDO in diphenoxylate-induced mice, which was similar to that observed in D. officinale aqueous extract in carbon-induced constipation in ICR mice.²³

D. officinale is widely used in daily life as a healthy food, and increasing attention has been paid to the development of UDO in both its food and medicinal applications. As a health food, the UDO was made into a granule, capsule, and tablets, which were convenient and portable for use in daily life.⁴⁶ Besides, UDO can be made into Chinese herbal medicine powder, which exhibited higher utilization of effective component and improved medicinal quality.⁴⁷ To date, ultrafine powder has found various applications in ceramics, electric materials, biotechnology, and food material as well as in the pharmaceutical field.⁴⁸ Besides, ultrafine powder has some notable characteristics such as improved surface absorbability, promoted dispersion uniformity, elevated solvency and fragrance preserving ability that conventional particle materials do not possess. The absorption of ultrafine powder can also be boosted, which improves the utilization of effective component, enhances bioavailability, and reduces dosage, as well as maximizes the therapeutic efficacy,^49,50 compared to the traditional solvent extraction, which may be labor-intensive, requires long extraction time, and has low extraction yield. Hence, promotion of the use of ultrafine powder can allow rational use of herbal medicine resources, the realization of clinical efficacy with minimal doses, and maximal utilization of drug materials, which would be more suitable for the development of functional foods than native materials.

The mechanical method is a commonly used traditional processing method for ultrafine powder preparation, of which ball mills have been successfully applied for grinding raw materials to ultrafine powder due to its convenient operation, higher grinding rate, and lower energy consumption compared with other fine grinding technique.⁵¹ In the present work, a volume-based median particle size (d₅₀) of around 6.56 μm was achieved via the ball milling technique, which provided a convenient and effective way for UDO preparation. When compared with the aqueous extract of D. officinale, the preparation and preservation method for ultrafine powder was relatively easier and simpler. Also, the ultrafine powder was found to achieve a significant therapeutic effect at much lower doses and fewer administration times. Besides, UDO might contain the insoluble and sparingly soluble substances, which the water extract of D. officinale failed to possess. The absence of these substances might lead to the distinction of therapeutic effects between UDO and water extract of D. officinale.¹⁹

However, further definitive studies are merited to clarify in greater detail its underlying mechanism. Also of interest and importance are further identification and illumination of its active components responsible for the observed laxative effect, and some of these endeavors are already initiated in our laboratory. These findings in the future might provide further insight into the potential application of UDO and the active constituents contained therein in the treatment of constipation.

In the present study, the UDO was prepared through ball milling to obtain an appropriate particle size. Besides, the UDO obtained proved to effectively alleviate constipation in diphenoxylate-induced constipation mice with enhanced GI transit ratio and favorably regulated fecal output such as pellet number, weight, moisture content, and the mechanism might be associated with its modulatory effect on the levels of MTL, Gas, SP, AChE, and SS. The results obtained might provide further experimental evidence for the traditional use of D. officinale. UDO has the potential for further development into an alternative effective diet therapy for constipation.

Footnotes

Acknowledgments

This work was supported by grants from the Forestry Science and Technology Innovational Specific Project of Guangdong Province (No. 2014KJCX019-02 and 2016KJCX006), the HongKong, Macao, and Taiwan Science & Technology Cooperation Program of China (No. 2014DFH30010), Science and Technology Planning Project of Guangdong Province, China (No. 2013B090600007 and 2013B090600026 and 2013B090800052), the Science and Technology Major Project of Guangdong Province (No. 2013A022100001), and the Guangdong International Cooperation Project (No. 2013508102016).

Author Disclosure Statement

No competing financial interests exist.

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