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ORIGINAL ARTICLE
Year : 2023 | Volume
: 9
| Issue : 4 | Page : 430-437
Study on the resolving phlegm effect of D-limonene in mice with spleen deficiency and phlegm-dampness syndrome
Jie Luo, Long-Jing Wang, Yang-Song Li, Jin Zhang, Gui-Rong Zhang, Fei Long, Teng Peng, Jie Yan, Bao-Jie Zhu
School of Pharmacy/School of Modern Chinese Medicine Industry, Chengdu University of Traditional Chinese Medicine; State Key Laboratory of Characteristic Chinese Drug Resources in Southwest China, Chengdu, China
Correspondence Address:
Prof. Fei Long
School of Pharmacy, Chengdu University of Traditional Chinese Medicine, State Key Laboratory of Characteristic Chinese Drug Resources in Southwest China, Chengdu 611137
China
Prof. Teng Peng
School of Pharmacy, Chengdu University of Traditional Chinese Medicine, State Key Laboratory of Characteristic Chinese Drug Resources in Southwest China, Chengdu 611137
China
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/2311-8571.391114
Objective: According to Traditional Chinese Medicine theory, spleen deficiency and phlegm-dampness syndrome (SDPDS) are caused by abnormal water metabolism in the body because of spleen dysfunction. The purpose of this article was to evaluate the efficacy of D-limonene (DL) in resolving phlegm in mice with SDPDS from the perspective of regulating the level of aquaporin 3 (AQP3). Methods: The model of SDPDS was induced in mice using the multifactor modeling method, which combines internal and external dampness. An artificial climate box was used to create a humid environment, whereas the irregular diet was caused by different feeding methods on odd-even days. The mice were divided into blank control, model group, DL low-dose, DL high-dose, and positive groups. The mice were modeled and treated for 7 day. Levels of gastrin and amylase (AMS) in the serum, mucus secretion in the trachea, and AQP3 in the tissue near the gastric cardia. Results: DL significantly reduced mucus secretion in the trachea (P < 0.001). It also increased the level of AMS in the serum (P < 0.01) and decreased the level of AQP3 in the gastric tissue (P < 0.01). Conclusions: Mice with SDPDS exhibited disturbed water metabolism and significantly increased AQP3 levels. DL can restore the levels of AQP3 and plays an important role in resolving phlegm. This study may also help expand the efficacy of natural drugs containing DL and improve the utilization of natural drug resources.
Keywords: Aquaporins, D-limonene, phlegm, spleen deficiency and phlegm-dampness syndrome, stomach
In the clinical research of Traditional Chinese Medicine (TCM), spleen deficiency and phlegm-dampness syndrome (SDPDS) is a common syndrome in various chronic diseases. Excessive intake of high-fat foods attenuates spleen and stomach functions. According to TCM theory, the main function of the spleen is to transport and transform food and fluid into nutrients to nourish the body. Water absorption depends on this function.[1] Huang Di Nei Jing states that all edema and bloating caused by dampness were inseparable from the function of the spleen. When the transporting and transforming functions of the spleen are decreased, it leads to abnormal water metabolism. The normal circulation of water is affected and produces pathological products, such as phlegm.[2] Aquaporin 3 (AQP3) is one of the AQPs that control the water balance in the digestive system.[3] Abnormal expression of AQP3 can cause an imbalance in the metabolism of water and fluids, and phlegm and other pathological products may form in the body. The theory of TCM states that dampness can accumulate into phlegm, which is the pathogenesis of SDPDS.[4] Clinical studies in TCM have shown that SDPDS are important causes of hyperlipidemia,[5] type 2 diabetes mellitus,[6] gout,[7] coronary heart disease,[8] and essential hypertension.[9]
D-limonene (DL) is a natural monocyclic monoterpene with a lemon-like fragrance.[10] Its aromatic properties and low toxicity have made it widely used in various fields such as agriculture, cosmetics, the food industry, and medicine in recent years.[11],[12],[13] DL also possesses various pharmacological properties such as insecticidal, antibacterial, antioxidant, anticancer, and gallstone dissolution effects.[11] Studies have shown that DL can act on the respiratory mucosa and relieve bronchospasms, facilitating the elimination of phlegm.[14] It is also an active ingredient in eucalyptol, limonene, and pinene enteric soft capsules (
), which is a domestically listed drug.[15] Furthermore, it is worth noting that it exists in many Chinese Medicines that resolve phlegm. Pingwei San (
), a classic dispelling dampness prescription, is composed of Cangzhu (
), Houpo (
), Chenpi (
), and Gancao (
). The first three Chinese medicines have phlegm-reducing effects and contain DL.[16],[17],[18]
TCM has been practiced for 1000 of years and has unique effects on treating complex chronic diseases.[19] With fewer side effects, TCM can be an alternative therapy for treating chronic diseases.[20] However, the modernization of TCM has been hindered by its complex composition and unclear mechanism of action.[21] Active ingredients are the material basis for the efficacy of TCM, which works through drug targets in the body, such as receptors, enzymes, and ion channels.[22] Clarifying the interactions between each active component and the related drug targets could contribute to research on the mechanisms of Chinese medicine and the utilization of natural products.
The purpose of this study was to evaluate the efficacy of DL in resolving phlegm in SDPDS in mice from the perspective of regulating the abnormal expression of AQP3 [Figure 1].
Figure 1: Mechanism of resolving phlegm effect of D-limonene. SDPDS: Spleen deficiency and phlegm-dampness syndrome Methods Animals and groups
Forty adult KM mice of both sexes (half male and half female) weighing 20 ± 2 g were obtained from Chengdu Dashuo Experimental Animal Co., Ltd. (License No. [SCXK (Chuan) 2020–2030]). Experimental mice acclimatized for 1 week were randomly distributed into five groups (eight mice per group): blank control (BC), model group (MG), DL low-dose (DL-L), DL high-dose (DL-H), and positive groups (PG). Mice had ad libitum access to food and water. All animal experiments followed the relevant regulations of the animal room of the laboratory at Chengdu University of TCM (No. SYXK [Chuan] 2020-124). All animal procedures were carried out in compliance with the guidelines for scientific animal procedures approved by the Ethics Committee of the Experimental Animal Ethics Committee of Chengdu University of TCM.
Main reagents and instruments
The gastrin (GAS) ELISA kits (Quanzhou Ruixin Biotechnology Co., Ltd.), amylase (AMS) (Quanzhou Ruixin Biotechnology Co., Ltd.), and AQP3 (Quanzhou Ruixin Biotechnology Co., Ltd.) were usedPingwei Wan (Beijing Tongrentang Natural Drugs [Tangshan] Co., Ltd.), DL (No. PS020126; Chengdu Push Bio-Technology Co. Ltd.)An A580 Double-beam Ultraviolet-visible Spectrophotometer (Aoyi Instrument [Shanghai] Co., Ltd.) and Spectra Max i3 Microplate Reader (Meigu Molecular Instrument [Shanghai] Co., Ltd.) were used.Model establishment
After 7 day of adaptive feeding, the SDPDS model was established according to the literature,[23] including an improper diet and dampened environment. To create humid conditions for establishing the SDPDS model, the bottom of the mouse cage was covered with moist sawdust. Then, the cages were placed in an artificial climate box (RH = 90% ±4%, T = 23°C ± 2°C) for 8 h daily, which could optimize the modeling method of SDPDS mice by accurately controlling the temperature and humidity and maintaining a stable value.[24],[25] To simulate an irregular diet, on odd days, the mice were administered lard (4.2 mL/kg) by gavage and provided sufficient high-fat feed.[26] On even days, the mice were only gavaged with water at 4°C. The mice were modeled for 7 days, during which BC was fed normally. Throughout the experiment, the general condition of the mice was observed, including their mental, urinary, and defecation status and color gloss.
Administration
The BC and MG were administered purified water 2 mL/100 g by gavage. PG was treated with Pingwei Wan 4.5 mg/kg and was administered intragastrically. The intragastric administration of DL was 140 and 560 mg/kg, respectively.[27],[28],[29] Each group was treated for 7 consecutive days.
Serum index detection
After fasting for 12 h, blood samples from the mouse eyeballs were centrifuged at 4000 rpm for 10 min, and the supernatant was collected. The GAS and AMS were measured according to the manufacturer's instructions.
Mucous secretion in trachea detection
Mucus secretion was detected by an intraperitoneal injection of 10 mL/kg phenol red (5%) dissolved in saline. Mice were sacrificed by cervical dislocation 30 min after dosing. The soft tissue overlying the trachea was removed and placed in an EP tube containing 3 mL of saline. NaHCO3 (5%, 1 mL) was added to the EP tube, and the samples were centrifuged at 4000 rpm for 10 min. The optical density (OD) of the supernatant was measured at 546 nm using a microplate reader.
Gastric tissue aquaporin 3 detection
The gastric tissue homogenate was prepared and centrifuged at 3000 rpm for 20 min, and the supernatant obtained was kept at −20°C. Then, AQP3 was determined according to the manufacturer's instructions.
Data analysis
All statistical analyses used Prism Version 9.0 (GraphPad Software Crop., San Diego, CA, USA) and presented as the mean ± standard deviation. ANOVA with Tukey's multiple comparison tests was used to compare group differences. Differences were considered statistically significant at P < 0.5 was considered a significant difference.
ResultsD-limonene could improve the general condition of spleen deficiency and phlegm-dampness syndrome mice
During the experiment, there were no obvious changes in urinary and defecation status, color gloss, or BC weight. Compared with BC, mice with SDPDS were obese with a greasy coat, sluggish activity, ease of getting tired and lying down, and loose stools, which accorded with the general manifestation of spleen deficiency and phlegm dampness.[23],[26] After treatment, the consistency of the stools and the weight of the mice in the administration groups improved.
D-limonene could improve the function of the spleen in spleen deficiency and phlegm-dampness syndrome mice
As shown in [Figure 2], the serum levels of GAS and AMS in mice with MG decreased significantly (P < 0.05), indicating that the model was successful.
Figure 2: (a) Comparison of serum gastrin level in each group after the experiment. (b) Comparison of serum amylase level in each group after experiment.*P < 0.05 and **P < 0.01 compared to model group, #P < 0.05 compared to blank control. GAS: Gastrin, AMS: Amylase, BC: Blank control, MG: Model group, DL-L: DL Low dose, DL-H: DL High dose, PG: Positive groupAfter treatment, the GAS level in the PG increased, whereas the GAS level in the two DL administration groups (DL-L and DL-H) was lower than in the MG. Compared to the MG, the levels of AMS in DL-H (P < 0.01) and PG (P < 0.05) were higher, and the level of AMS in DL-H was higher than that in DL-L.
D-limonene could decrease the tracheal mucous secretion of spleen deficiency and phlegm-dampness syndrome mice
As shown in [Figure 3], the OD value of each MG was higher than that of the BC group. This indicated that mucous secretion in the trachea of the modeling groups increased, resulting in a large amount of phlegm.
Figure 3: Comparison of mucous secretion in each group after the experiment. **P < 0.01 and ***P < 0.001 compared to MG, ###P < 0.001 compared to BC. OD: Optical density, BC: Blank control, MG: Model group, DL-L: DL Low dose, DL-H: DL High dose, PG: Positive groupCompared with the MG, mucous secretion in DL-L (P < 0.01), DL-H (P < 0.001), and PG (P < 0.001) decreased, suggesting that DL can inhibit the hypersecretion of airway mucus and reduce phlegm generation. Mucus secretion in the DL-H group was comparable to that in the PG, indicating that DL-H had a better inhibitory effect on airway mucus hypersecretion than DL-L.
D-limonene could decrease gastric tissue aquaporin 3 in spleen deficiency and phlegm-dampness syndrome mice
As shown in [Figure 4], the level of AQP 3 in the gastric tissue of MG was significantly higher than that in BC (P < 0.01). Compared with the MG group, the level of AQP 3 in the gastric tissue of mice in the DL-L (P < 0.01), DL-H (P < 0.01), and PG (P < 0.05) groups decreased after treatment. The restoration of AQP 3 activity differed in the DL-L group compared with the DL-H and PG groups.
Figure 4: Comparison of gastric tissue aquaporin 3 in each group after the experiment. *P < 0.05 and **P < 0.01 compared to model group; ##P < 0.01 compared to blank control. AQP 3: Aquaporin 3, BC: Blank control, MG: Model group, DL-L: DL Low dose, DL-H: DL High dose, PG: Positive group Discussion Relationship between spleen deficiency and phlegm-dampness syndrome, D-limonene, and Pingwei San
According to the TCM theory, spleen deficiency is a source of phlegm production. The main physiological function of the spleen is to govern the transportation and transformation of cereal essence. A spleen malfunction could cause water and fluid to stagnate in the body and accumulate in the phlegm, which is the pathogenesis of SDPDS. In this study, the SDPDS model was established using a multifactor modeling method that combines internal and external dampness. The artificial climate box created a highly humid environment that led to spleen deficiency syndrome. Furthermore, an improper diet causes gastrointestinal dysfunction and abnormal water metabolism and produces pathological products, such as phlegm.
DL is used clinically as an expectorant to resolve phlegm by acting on the respiratory mucosa and relieving bronchospasm. According to the theory, phlegm is a pathological product of abnormal water metabolism. Furthermore, in [Table 1], DL is found in many Chinese medicines with the effect of resolving phlegm, including Chenpi (
), Juhong (
), Foshou (
), Houpo (
), and Cangzhu (
). [Table 2] shows many prescriptions that contain Chinese medicines with DL, such as Pingwei San (
), Erchen Tang (
), Banxia Houpo Tang (
), and Sanzi Yangqin Tang (
). It suggests that DL may be one of the material bases of Chinese medicine with resolving phlegm effect.
Pingwei San possesses drying dampness, resolves the phlegm effect, and strengthens the spleen and stomach effects, first recorded in Jian Yao Ji Zhong Fang.[51] The first three Chinese medicines possess phlegm-reducing effects and contain DL. In clinical research, Pingwei San has a positive effect on treating patients with SDPDS.[52] It is also used as an intervention drug in animal models of SDPDS and has an obvious effect on regulating abnormal water metabolism.[53] The results of this study show that Pingwei San has drying dampness and resolving phlegm. This suggests that DL may be the material basis for the efficacy of Pingwei San.
Chinese medicine generally comprises a mixture of multiple components. Its curative effect is achieved through multitarget interventions. Its chemical composition and mechanism of action are complex, and its current technical means are still difficult to detect. This is why it is difficult for Chinese medicines to pass the examination and approval of foreign drugs or obtain approval from the international registered drug market. This paper studies the mechanism of action of a single effective component co-existing in most aromatic and dampness-removing Chinese medicines, which provides ideas for the further study of the basis of the effectiveness of Chinese medicine. In addition, DL is the main active ingredient in Eucalyptol, Limonene, and Pinene Enteric Soft Capsules (
) and Compound Limonene Capsules (
), which are domestic listed drugs. The former has antitussive and expectorant effects, whereas the latter is used clinically to treat gallbladder diseases. As the common active ingredient in two listed drugs, elucidating further the mechanism of the expectorant effect of DL is helpful for the study of the new clinical application of compound limonene capsules and re-evaluation of eucalyptol, limonene, and pinene enteric soft capsules.
The connection between spleen deficiency and phlegm-dampness syndrome, gastrin, and amylase
GAS, a hormone that regulates gastrointestinal function, is important in digestive tract movement, food absorption, and metabolism.[54] Maintaining gastrointestinal digestive function is closely related to the level of GAS secretion. It is generally believed that a change in GAS results in gastrointestinal disorders, which are important etiologies of spleen deficiency syndrome.[55] GAS is a recognized index used to measure spleen deficiency syndrome. Many studies have revealed decreased GAS levels in rats with spleen qi deficiency.[56] In this study, the level of GAS in the MG was significantly decreased, suggesting that SDPDS mice had gastrointestinal disorders caused by spleen deficiency. However, the GAS level in the DL group was lower than that in the MG, indicating that DL did not reflect the regulatory effect of GAS in this study.
AMS is an important human digestive enzyme. Insufficient AMS secretion affects food digestion and nutrient absorption.[57] Furthermore, water metabolism is closely related to the transport function of the spleen. Many studies have shown that AMS levels in spleen deficiency syndrome decline significantly and can increase after treatment.[58],[59] The recovery of AMS levels refers to the recovery of the transport and transformation functions of the spleen, which is helpful for the normal distribution of water and grain in the body. Therefore, AMS level is a recognized indicator of spleen deficiency syndrome.[60] In this study, the experimental results show that the level of AMS was decreased significantly in spleen deficiency syndrome. The AMS levels in the two DL administration groups were higher than in the MG group. This suggests that DL may improve splenic function by regulating water metabolism and normalizing the absorption and distribution of fluid and food.
Resolving phlegm and aquaporin 3
Phlegm is a pathological product of metabolic disorders of body fluids. The spleen is the source of phlegm in TCM. Autopsy material showed that thick liquid filled the lung tissue of COVID-19 patients, which impaired breathing function and may lead to respiratory and circulatory failure as the disease progressed.[61] Because phenol red can be partially secreted from the trachea, phenol red secretion in the trachea is an expedient and efficient experimental approach for detecting the effects of expectorants.[62] The results of this study indicated that mucus secretion in the trachea of SDPDS mice increased, resulting in a large amount of phlegm. After treatment, phlegm secretion in the treatment groups decreased, suggesting that DL can inhibit airway mucus hypersecretion and reduce phlegm generation.
AQPs, closely associated with water metabolism, are transmembrane proteins that allow water to cross biological membranes. It mainly participates in the transmembrane transport of water molecules and water metabolism in the body and plays an important role in regulating the balance of water metabolism. AQPs are widely distributed in organs and tissues, such as the kidneys, lungs, and digestive system.[63] The TCM theory also states that body fluid metabolism is mainly related to the lungs, spleen, kidneys, and triple energizer.[64] Normal expression of AQPs may be the basis of molecular biology for transporting water and body fluids, whereas abnormal expression of AQPs is an important factor in developing phlegm syndrome.[65] Nei Jing states that the spleen transports body fluid to the stomach. Once the function of the spleen declines, water cannot be transported normally, resulting in the stagnation of the body, which may form an excessive fluid and even phlegm dampness. In rats with damp retention syndrome in middle Jiao, many studies have shown that the level of AQP3 in gastric cardia tissues increased significantly and recovered significantly after treatment.[66],[67] AQP3 is expressed in the gastrointestinal tract and other tissues. High AQP3 expression can lead to increased water permeability and promote water transport to gastrointestinal cells.[67] This may be the molecular basis of spleen deficiency syndrome, which leads to abnormal spleen and stomach water infusions.[68] In this study, after the intervention of Pingwei Wan, the level of AQP3 distributed in the gastric cardia mucosa decreased, indicating that the transport of water to gastric cells was inhibited, which might be one of the molecular mechanisms of its efficacy in drying dampness and strengthening the spleen for the syndrome of damp retention in middle-jiao. The expression of AQP3 in the DL group also declined; therefore, it is speculated that DL may be the material basis for the efficacy of this formula.
ConclusionsIn this study, the experimental results showed that DL could resolve phlegm in mice with SDPDS by regulating AQP3 expression. Thus, DL helps restore the normal function of the spleen by improving water metabolism in the body. The mechanism underlying the clinical expectorant effect of DL was further clarified, providing a scientific basis for TCM clinical treatment of diseases caused by SDPDS. Moreover, this study may help expand the efficacy of natural DL drugs. It may be useful for exploring the new clinical application of Compound Limonene Capsules and re-evaluating Eucalyptol, Limonene, and Pinene Enteric Soft Capsules.
Financial support and sponsorship
This research was sponsored by the Program of Scientific Research Innovation for Colleges and Universities in Sichuan Province (18TD0017), the Research Project of Sichuan TCM Administration (2021MS448), and the Technological Innovation R and D Project of the Chengdu Bureau of Science and Technology (2021-YF05-02298-SN).
Conflicts of interest
There are no conflicts of interest.
References
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