Effects of Zingiber officinalis (WILLD.) ROSC. Membranes on minor recurrent aphthous stomatitis: a randomized pragmatic trial

Journal of Traditional Chinese Medical Sciences (2018) 5, 58e63

Available online at www.sciencedirect.com

ScienceDirect journal homepage: http://www.elsevier.com/locate/jtcms

Effects of Zingiber officinalis (WILLD.) ROSC. Membranes on minor recurrent aphthous stomatitis: a randomized pragmatic trial Qian Du a, Shenglou Ni a, Lin Guo a, Wenjie Song a, Kun Zhao a, Ni Liu a, Xinrong Wang a, Xun Ma a, Yanling Fu a,*, Quanming Tan b a b

Beijing University of Chinese Medicine, Beijing 100029, China Singapore Thong Chai Medical Institution, Thong Chai Building 169874, Singapore

Received 8 January 2018; received in revised form 8 February 2018; accepted 18 February 2018

Available online 17 March 2018

KEYWORDS Dried ginger rhizome membrane; Recurrent aphthous stomatitis; Epidermal growth factor; Tumor necrosis factor-a

Abstract Objective: To evaluate the effects of dried ginger rhizome (DGR; Zingiber officinalis (WILLD.) ROSC.), prepared as a membrane, in minor recurrent aphthous stomatitis (miRAS) treatment and explore its mechanism of action by detecting changes in levels of epidermal growth factor (EGF) and tumor necrosis factor (TNF)-a in saliva. Methods: Fifty-nine miRAS patients were enrolled in this study. The number of participants in the dried ginger rhizome membrane (DGRM) group was 30, and 29 were in the placebo membrane (PM) group. Sixty sealed envelopes containing either type of membrane were coded randomly. Investigators and participants were blinded to group assignments. A visual analog scale (VAS) was used for pain, follow-up information for healing time, and enzyme-linked immunosorbent assays to measure the concentrations of EGF and TNF-a. Results: In terms of VAS, there was a significant difference between pre- and post-DGRM treatment (P < .001), but not so for the PM group (P > .05). A significant difference was observed in the healing time between the two groups (6.08 (2.712) vs. 8.04 (2.142) days). The mean healing time in the DGRM group was shorter than that in the PM group (P < .05). In both groups, the salivary EGF concentration decreased significantly after treatment (P < .05), but the mean level in the DGRM group was significantly lower than that in the PM group (P < .05). The mean TNF-a level in both groups was increased significantly after treatment (P < .05), but patients who used DGRMs had a significantly lower level than that in the PM group (P < .05).

* Corresponding author. E-mail address: [email protected] (Y. Fu). Peer review under responsibility of Beijing University of Chinese Medicine. https://doi.org/10.1016/j.jtcms.2018.02.004 2095-7548/ª 2018 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Effects of Zingiber officinalis membranes on miRAS

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Conclusion: The present study provides evidence that DGRMs are effective treatment for RAS. Dried ginger rhizome has obvious effects on pain relief, shortening of healing time, reducing the EGF level in saliva, and has an inhibitory effect on TNF-a release. ª 2018 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).

Introduction Recurrent aphthous stomatitis (RAS) is a relatively common disease affecting the oral mucosa. According to epidemiologic data, 2%e66% of the worldwide population is affected by RAS.1‒2 It is characterized by painful, recurrent ulcerations of the oral mucosa and can be classified clinically into three categories based on the diameter of mouth ulcers: minor (<1 cm), major (>1 cm), and herpetiform. Minor recurrent aphthous stomatitis (miRAS) is the most common manifestation, and occurs in 75%e80% of patients. Ulcers due to miRAS are <1 cm in diameter (usually 2e5 mm) and heal spontaneously in 7e14 days. Although common, RAS has an unpredictable disease course.3‒4 The exact pathogenesis of RAS is not known,5e9 but inflammation plays an important part in it. Considerable evidence suggests that focal immune dysfunction exerts a significant influence. Thus, anti-inflammatory and immunosuppressant drugs have been administered widely to miRAS patients. However, efficacious therapies that can prevent repeated bouts of ulceration are lacking. Mainstream treatments aim at alleviating pain, reducing functional disability, and facilitating healing.10,11 Traditional Chinese medicine (TCM) could be used to treat RAS. Dried ginger rhizome (DGR; Zingiber officinalis (WILLD.) ROSC.) is a common dietary adjunct contributing to the taste and flavor of foods, and is also an important Chinese herb. Dried ginger rhizome is a frequently used ingredient in TCM-based treatment of RAS.12 Some studies have shown that DGR has anti-inflammatory, antioxidant, analgesic and antiseptic effects.13e15 Yang et al reported that DGR is a transient receptor potential cation channel subfamily V member 1 (TRPV1) agonist. Initially, DGR can sensitize sensory endings within inflamed tissue and produce a “warm” feeling, then desensitize the tissue and relieve pain.16,17 Some researchers have shown that water extracts of DGR have good effects against pain caused by different stimuli, and can reduce the pain caused by inflammation.18 Luo et al used a component of DGR, 6-gingerol,19 to treat oral stomatitis in mice, and showed a shortened healing time and improvement of the cure rate.20 We evaluated the effects of a water extract of DGR, in the form of an oral membrane, on miRAS.

Methods Ethical approval This was a double-blind, placebo-controlled clinical pragmatic trial undertaken at the Beijing University of Chinese

Medicine (BUCM; Beijing, China). The study was approved by the Ethics Committee of BUCM (2017BZHYLL0308). All participants were informed of the purpose, general contents, and data use of our study. The trial was registered in the Chinese Clinical Trial Registry (ChiCTR-OPR-17013127).

Patient selection This was an exploratory study so the patient cohort was small. Participants were selected from the general population of Beijing using flyers posted at various locations as well as e-mails to universities. People were enrolled after a diagnosis had been made by Professor Yanling Fu in the Guo Yi Tang Outpatient Department of BUCM in 2017. Inclusion criteria were: (i) age > 18 years; (ii) a clear history of RAS occurring no less than four times a year; (iii) presentation with one or two ulcers measuring
10 mm in diameter for
48 hours and yet to receive treatment; (iv) ulcers that took > 5 days to resolve without treatment. Individuals were excluded if they: (i) had underlying systemic disease(s) or a history of immunologic disorder(s); (ii) were taking immunomodulatory agents or systemic nonsteroidal anti-inflammatory drugs < 1 month before study commencement;4 (iii) were smokers; (iv) were pregnant; (v) were (or had a history of) abusing drugs or alcohol; (vi) could not provide written informed consent.

Preparation membranes Dried ginger rhizome (DGR; Z. officinalis (WILLD.) ROSC.) was purchased from a Tong Ren Tang outlet in Beijing. Using a ratio of DGR: water of 1:10, medicinal components were extracted by boiling in water for 6 hours. Then, extracts were filtered, concentrated, dried, ground and stored. The resulting powder was mixed with polyvinyl alcohol (PVA)1788 and prepared into a dried ginger rhizome membrane (DGRM). A placebo membrane (PM) containing the resulting powder at one-tenth the concentration of DGR and PVA1788 was prepared after we failed to create a DGRM at one-twentieth concentration of DGR.21,22 There were no other ingredients in either type of membrane. In dry storage, both types of membrane are nonadherent and can be applied readily as required. Upon contact with saliva at a lesion site, a sticky hydrogel is formed which adheres to the mucosa. The prepared membranes were cut into squares of size 1.5 cm, and sealed in small plastic bags. The latter were encoded by a research pharmacist (Lin Guo) so that investigators and patients were both blinded to the type of membrane contained within.

60 We selected three pieces of membranes randomly to test for 6-gingerol, which is the main component of DGR as listed in the Pharmacopoeia of People’s Republic of China.23 A 6-gingerol measurement standard (Y12A8H41703, Shanghai, China) was used, and the mean content was 0.0535 mg.

Randomization and masking The research pharmacist numbered 60 opaque and sealed envelopes. She assigned the DGRM group or PM group (1:1 distribution) randomly based on a random number table (excluding repeating numbers). Researchers distributed envelopes containing the drugs in sequence and maintained records of the number assigned to each patient. The research pharmacist did not participate in other activities during the study.

Interventions Patients were required to undergo intervention within 48 hours of aphthous stomatitis onset. Baseline parameters were recorded during the first visit. The application of a membrane was demonstrated to participants. They were instructed to use one membrane piece each time twice dailydno less than 30 minutes apart from eating and before sleepduntil it dissolved. Usually, the membrane dissolved in 20e30 minutes. Drinking, eating or talking during membrane application was prohibited. The total duration of treatment for each patient was the healing time of aphthous stomatitis.

Therapeutic evaluation Patients were required to provide two saliva samples. One sample was collected at the first visit (i.e., before treatment) and the other was just after healing. We measured the levels of epidermal growth factor (EGF) and tumor necrosis factor (TNF)-a in saliva. The pain level was recorded before application of the first membrane and 1 day after treatment using a visual analog scale (VAS), which is a tool can be used to indicate the degree of pain. It consisted of a 10-cm horizontal line, and the end of the line is (0) indicating “no pain” and the other end is (10) denoting “unbearable pain”. The investigators recorded the patient’s tolerance to the membranes including the DGRM and any adverse reactions that may be associated with the membranes.

Saliva collection and measurement of EGF and TNF-a Saliva was collected from fasting patients under standard conditions between 9 AM and 11 AM Patients were prohibited from drinking water during the entire sampling process. Unstimulated whole saliva (3 mL) was collected using sterile plastic centrifuge tubes. Samples were centrifuged for 10 minutes at 4 C (428  g), and stored at 80 C for further analyses.

Q. Du et al. For determination of salivary levels of EGF and TNF-a, we used a Human EGF enzyme-linked immunosorbent assay (ELISA) kit (HU9916; BioTSZ, San Francisco, CA) and Human TNF-a ELISA kit (HU9890; BioTSZ).

Statistical analyses Data were analyzed using SPSS v20.0 (IBM, Armonk, NY) and expressed by mean (standard deviation). Statistical analyses were used Wilcoxon ranKolmogorov-Smirovum test. P < .05 was considered significant.

Results Fifty-nine individuals were recruited for our study. They were assigned randomly to the DGRM group (n Z 30) and PM group (n Z 29). Their mean age was 29.47 (8.93) years. Ten people withdrew due to 3 cases suffering from common cold and 7 people failing in contact. Finally, 49 patients were evaluated at the last visit: 26 and 23 in the DGRM and PM groups respectively.

VAS and healing time The VAS for ulcers before treatment between the DGRM group and PM group was well-matched upon study entry. However, there was a significant difference between before treatment and after 1 day of treatment in the DGRM group (P Z .000), but there was no significant difference in the PM group (P Z .337) (Table 1). A difference was also observed in the healing time between the two groups (6.08 (2.712) vs. 8.04 (2.142) days, respectively) (Fig. 1). The healing time in the RZM group was shorter compared with that in the PM group (P Z .002).

Epidermal growth factor level There was no significant difference in the salivary level of EGF at the first visit between the two groups (P > .05). However, the EGF concentration of the two groups after healing decreased significantly (Table 2). The EGF level after healing in DGRM group was significantly lower than that in the PM group (P Z .024), suggesting that the DGR could reduce the EGF level in saliva significantly.

Tumor necrosis factor-a level No significant difference in the salivary level of TNF-a before treatment between the two groups was displayed (P > .05). In the two groups of patients after healing, the

Table 1

Change in the VAS between the two groups.

Group

BT

AOT

P

DGRM group (n Z 30) PM group (n Z 29)

2.73 (0.944) 2.52 (1.122)

1.33 (1.155) 2.35 (1.143)

.000 .337

DGRM: dried ginger rhizome membrane; PM: placebo membrane; BT: before treatment; AOT: after 1 day of treatment; VAS: visual analogue scale.

Effects of Zingiber officinalis membranes on miRAS

61

Discussion DGR and pain relief

Figure 1 Healing time of patients in the two groups. DGRM: dried ginger rhizome membrane; PM: placebo membrane.

Table 2 Differences in salivary levels of EGF before treatment and after healing in the two groups. Group

BT (pg/mL)

AH (pg/mL)

P

DGRM group (BT: n Z 30; AH: n Z 26) PM group (BT: n Z 29; AH: n Z 23)

49.64 (12.070)

26.44 (10.870)

.000

45.64 (13.260)

33.82 (11.370)

.001

EGF: epidermal growth factor; DGRM: dried ginger rhizome membrane; PM: placebo membrane; BT: before treatment; AH: after healing.

Table 3 Differences in salivary levels of TNF-a before treatment and after healing in the two groups. Group

BT (pg/mL)

AH (pg/mL)

P

DGRM group (BT: n Z 30; AH: n Z 26) PM group (BT: n Z 29; AH: n Z 23)

32.21 (6.837)

38.18 (7.840)

.002

29.33 (6.245)

44.64 (5.960)

.000

TNF: tumor necrosis factor; DGRM: dried ginger rhizome membrane; PM: placebo membrane; BT: before treatment; AH: after healing.

saliva concentration of TNF-a was significantly higher than that before treatment (Table 3). However, at the last collection, for patients who used a DGRM, the TNF-a level was significantly lower compared with that of the PM group after treatment (P Z .002), suggesting that DGRMs may have inhibitory effects on TNF-a expression.

Adverse drug effects Adverse drug effects (e.g., hypersensitivity, infection, taste-bud malfunction) were not observed in any participant (Fig. 2).

Our study showed that the change in ulcer-based pain in the DGRM group was significantly different before treatment and after 1 day of treatment (Table 1), suggesting that DGRMs had an obvious analgesic effect. Pain affects the quality of life of RAS patients. Severe pain can lead to eating problems and, subsequently, inadequate nutrition. The mechanism of action of RAS is not known, but pain relief is one of the main principles for treating RAS.24 Dried ginger rhizome is an “acrid” and “warm” herb, which may form part of its effect on pain relief. Yang et al showed that DGR is a strong agonist of TRPV1 channels that can produce a warm feeling through sensitization of the sensory endings in inflamed tissue before desensitizing the tissue towards pain.16 Hitomi et al proposed that 6-gingerol and 6-shogaol, extracted from DGR, could inhibit the stimulant-induced release of substance P and generation of action potentials in cultured rat sensory neurons to elicit analgesic effects.25 Li et al compared the concentrations of five components (zingerone, 6-gingerol, 6-shogaol, 8gingerol, and 10-gingerol) and antioxidant activity of fresh, dried, stir-fried and carbonized ginger extracts, and found DGR to be maximal in both parameters.26 Ma et al designed rat experiments that showed water extracts of DGR to have good antipyretic analgesic effects, possibly by inhibiting the activity of ring oxidase and lipoxygenase and reducing the amount of prostaglandins and leukotrienes generated. The identity of the exact antipyretic component within water extracts of DGR, the pathways through which it modulates the temperature-regulating center and pain response, and other pharmacologic effects have yet to be elucidated.18

DGR and levels of EGF and TNF-a in saliva The protective properties of saliva are not limited to cleaning, flushing, cushioning, lubrication and mineral supplementation. Saliva can deliver various anti-viral, antibacterial, anti-fungal and multiple skin-growth factors of the human body itself. Therefore, considerable attention should be paid to the qualitative and quantitative analyses of saliva.27 We measured levels of EGF and TNF-a in saliva. The stability of the epithelial layer of the oral mucosa is dependent upon the relative rates of proliferation of the basal cell layer and sloughing of the keratinocyte layer. If the former decreases or the latter increases, the mucosal epithelium thins and becomes prone to ulceration. Under normal physiologic conditions, the oral epithelium is protected by the mucosal defense systems, including salivary secretions. The basic function of saliva is to protect and maintain the integrity of the mucous membranes of the oral and upper gastrointestinal tracts.28 Considerable evidence suggests that salivary EGF has a preventive effect against oral mucosal diseases and can prevent oral mucosal injury, thus maintaining the integrity of oral mucosa. In addition, EGF is beneficial to mucosal repair and healing, and is involved in repair mechanisms.29e31

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Q. Du et al.

Figure 2

Photographs of an ulcer before (A) and six days after treatment (B).

The EGF concentration was higher in both groups pretreatment (Table 2), which was likely due to the body’s anti-ulcer response. However, the time needed for the EGF level to decrease was shorter in the DGRM group, which paralleled the faster healing of wounds using DGR (Fig. 1, Table 2). This finding suggests that EGF is beneficial to ulcer healing. The TNF-a level was higher in both groups posttreatment, suggesting that oral ulcers could activate the body’s immune system. However, the level of TNF-a after treatment in the DGRM group was lower than that in the PM group, suggesting that DGRMs may have had an inhibitory effect on TNF-a expression (Table 3). Recurrent aphthous stomatitis seems to be regulated mainly by the immune system. However, the part of the immune system involved in its pathogenesis as a reaction to a still-unknown antigen is not clear.32 TNF-a participates in inflammation and the immune response. Several studies have highlighted a possible role for TNF-a, and its increased production could predispose individuals to RAS. Song et al found that mean levels of TNF-a in patients with major, minor, and herpetiform types of RAS were significantly higher than those in healthy controls. TNF-a level may also be associated with the severity and stage of RAS.33 It has been assumed that the therapeutic effect of thalidomide and corticosteroids in RAS is regulated through their ability to inhibit TNF-a production. Taken together, these observations suggest that increased production of TNF-a could predispose individuals to RAS. Some researchers have suggested that TNF-a, a pro-inflammatory cytokine, gets involved in the inflammatory process, and is closely related to the clinical manifestations and recurrent nature of oral ulceration. Patients with active RAS display high levels of TNF-a and its receptor in serum, so the positive response of complex aphthous stomatitis to TNF-a inhibitors is not surprising.34,35

Limitations This is a small size clinical trial with a high rate of withdrawal. We measured levels of EGF and TNF-a in saliva but other components of saliva need to be evaluated too. We focused only on 6-gingerol, but other components in water decoctions need to be investigated in future studies.

Conclusion The present study provides evidence that DGRMs are effective treatment for RAS. Dried ginger rhizome has

obvious effects on pain relief, shortening of healing time, reducing the EGF level in saliva, and has an inhibitory effect on TNF-a release. Recurrent aphthous stomatitis treatment could be achieved through analgesia, promotion of wound healing, and immune suppression.

Funding The clinical trial was supported by Beijing University of Chinese Medicine Research Project (2010072220010).

Conflicts of interest None declared.

Author contributions Qian Du designed the study, took part in the whole process including participants enrollment, data analyses, etc., and wrote the manuscript. Yanling Fu designed the study, revised the manuscript and provided fund support. Shenglou Ni offered valuable advice on the study design, guided the statistical analyses partly, and revised the manuscript. Lin Guo assisted in creating the DGR membranes, was in charge of blinding and grouping, and partly measured drug concentrations. Xun Ma measured drug concentrations. Kun Zhao, Ni Liu and Xinrong Wang were responsible for the distribution of drugs to patients and collected samples. Wenjie Song recorded and analyzed data. Quanming Tan carefully polished the article.

Acknowledgements We are grateful to Professor Jian Ni and Associate Professor Xingbin Yin (Beijing University of Chinese Medicine; BUCM) for their advice on DGRM preparation, and to Professor Jianxin Chen (BUCM) for his expertise in statistical analyses.

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