Effect of topical Zingiber cassumunar on painful diabetic neuropathy: a double-blind randomized-controlled trial

Background Plai or Zingiber cassumunar Roxb. was registered into the Thai Traditional Medicine list since 2011. However, there is limited evidence regarding Plai as a treatment in painful diabetic neuropathy (PDN). Therefore, this study aimed to evaluate the efficacy of topical Zingiber cassumunar. Methods A RCT was conducted in patients with PDN during February to March 2019. All participants received oral gabapentin 300 mg before bed as a standard regimen. The intervention group (n=16) received Plai balm 15%w/w 0.5 gram to apply on their feet three times a day and the control group (n=15) received placebo balm to similarly apply. Pain score at baseline, 2 nd and 4 th weeks were assessed and compared. Patients’ quality of life, and adverse events, were collected. Mean pain scores before and after treatment in each group and between groups were also analyzed. Results At the end of week two and week four, the Plai group showed statistically significant lesser mean pain scores than the placebo group by -1.47 (95%CI: -1.96 to -1.30, p-value < 0.001), and by -1.51 (95%CI: -1.92 to -0.13, p-value = 0.027), respectively. Moreover, the Plai group had more cases number/ percentages with at least 50% pain score reduction than the placebo group [12/16 (75%) vs 3/15 (20%), p-value = 0.004]. However, there was no statistically significant difference in quality of life between the two groups (overall p-value = 0.366). Adverse event was not found in any groups. Conclusions Zingiber cassumunar balm (Plai) was efficacious for pain reduction in painful diabetic neuropathy. Registration Registered with the Thai Clinical Trials Registry; TCTR20200221001.


Introduction
Diabetic polyneuropathy is one of the complications of diabetes mellitus.There are many signs and symptoms, such as painful diabetic neuropathy (PDN), orthostatic hypotension, cardiac autonomic neuropathy, foot injuries, and wounds. 1 The incidence of diabetic patients who have developed neuropathy, is approximately 20%.In addition, 50-70% of them must undergo surgery for non-traumatic amputation. 2 PDN is the damage of nerves, that causes pain in the limbs.Patients will suffer from aching and feeling like shooting and stabbing pain.These symptoms will occur at the limbs during nighttime, and usually affect daily life activities, sleeping patterns and work, as a result, patient quality of life will be decreased. 3,4esently, non-steroidal analgesic medicines or opioids have been widely used for symptoms relieving.However, opioids are not only ineffective but have many side effects when treating PDN.6][7][8] Capsaicin cream is effectively used for pain relief in PDN, but after a week, it might cause severe burn effect and finally require opioids for pain relief. 9ai or Zingiber cassumunar Roxb. is registered on the Thai Traditional Medicine list since 2011, 10 and categorized as a topical anti-inflammatory agent.The important extracted components from Zingiber cassumunar are (E)-4-(3, 4dimethoxyphenyl) but-3-en-1-ol (compound D), (E)-1-(3, 4-dimethoxyphenyl) but-1, 3-diene (DMPBD) and zerumbone. 11[22][23] Although there were two successful studies of mixed multi-herbal preparation using Zingiber cassumunar Roxb.as its main component in treating diabetic foot ulcers, 24,25 there still has been no study regarding the use of Plai as a treatment in PDN.Therefore, this study aimed to evaluate the efficacy of a topical Zingiber cassumunar preparation by comparing with placebo on PDN.

Patients
This study was conducted at Selaphum Hospital, Selaphum District, Roi Et, Thailand.Participants were patients diagnosed with PDN.Neuropathic Pain Diagnostic Questionnaire (Thai version of DN4) was used for pain assessment, 26 and monofilament test was used as a sensory screening tool in these patients. 8Written informed consent was obtained from all patients.

Recruitment
Patients aged 20-60 years who had been diagnosed with diabetic mellitus for at least 1 year, tested for HbA1c within six months, suffering from pain intensity on a scale as moderate or more [Numerical Rating Scale (NRS), pain score ≥ 4], and with defined positive monofilament test result, were included.Patients who had been diagnosed with diabetic mellitus less than one year, with other caused neuropathy, clinically significant cardiovascular, foot ulcer and/or infection, pregnancy and lactation, and allergy to Plai, were excluded.

Study design and oversight
This double-blind randomized-controlled trial was conducted within four weeks, from the 1 st of February to the 1 st of March 2019.All the authors were involved in the design and performance of the study, which was conducted according to the Declaration of Helsinki.The Khon Kaen Hospital Institute Review Board (KKHIRB) in Human Research (the oversighting IRB for Selaphum Hospital) approved the study protocol on 19 th December 2018 (KE61099).This trial was retrospectively registered with the Thai Clinical Trial Registry (TCTR20200221001) on 19 th February 2020, because "registration before recruitment" was missed, but authors finally registered it within one year after completion.Registration before recruitment is not a prerequisite for KKHIRB or Selaphum Hospital's study.The study protocol did not differ from the registered one.

REVISED Amendments from Version 1
This revised version has more details about producing and components of Plai balm and placebo balm in Method.Adverse events assessment and data collection were added to the Study procedures.Details about the 2 patients that were excluded were also added to the Results.In addition, the limitations were added to the Discussion.
Any further responses from the reviewers can be found at the end of the article Study treatment and procedures Pain assessment was commenced at week 0 and indicated by using The World Health Organization Quality of Life (WHOQOL-BREF-THAI) 27 with NRS, pain characteristic, and pain mapping to follow-up side effect of Plai and its adverse events as baseline.Consequently, patients returned to clinic at the following week one, two, and four, to monitor and assess the accuracy of practice on frequency, and dosage of balms' use.The patients' quality of life in week 4 were observed and recorded.
Data about adverse events such as burning sensation and pruritus were collected by history taking and examination at week 1, 2, and 4, similar to the primary outcomes, 8,26 but edema/erythema/papules were collected by visual examination.
Block-of-four randomization was applied to this study, concealment was done by telephone calls to research assistants.Participants were divided into two groups.All participants received oral gabapentin of 300 mg, one tablet a day before bedtime as a standard regimen.The intervention group (n=16) received Plai balm 15% w/w 0.5 gram to apply on their feet three times a day and the control group (n=15) received the placebo balm to apply similarly.

Preparation of the Plai balm and the placebo balm
To obtain the 100 grams of Plai balm, we mixed 15 grams of Plai oil with a balm base (which contained paraffin 10 g, Vaseline 50 g, camphor 10 g, Borneo camphor 5 g, menthol 5 g, and wintergreen oil 5 g).The placebo balm was made by increasing the first two components to paraffin 15 g and Vaseline 60 g, without adding Plai oil.Other active plants' ingredients were still similar to the Plai balm.Both kinds of balm were loaded into a 60 g-sized case: 18 Plai balm and 17 placebo balm were labeled by numbers according to their randomization scheme (1-35).The contents inside the cases were not known to the test subjects or to the treating physicians.

Endpoints
The mean differences of pain scores in NRS at week two and four between the two groups were the first endpoint.The other endpoint was the comparison of patients' numbers/percentages who have at least 50% pain reduction at week two and four between the groups.Additionally, The World Health Organization Quality of Life (WHOQOL -BREF-THAI) scores changes at week four were compared between the groups.

Statistical analysis
The preliminary study suggests that the number of participants should be 24-36 people.Consequently, this study recruited 35 participants, which were divided into two groups.Generalized estimating equation (GEE) was used to analyze the mean differences of pain scores at week two and four.Fisher's exact test was used to analyze the differences in patients' numbers/percentages who had at least 50% pain reduction at week two and four between the groups.Independent t-test was used to analyze the differences of The World Health Organization Quality of Life (WHOQOL-BREF-THAI) scores changes at week four between the groups.Mann-Whitney U-test was used to compare skew continuous data.

Study patients
There were 826 diabetic mellitus type 2 patients treated at Selaphum Hospital during the study period.Although 88 of them had developed PDN, only 46 patients were able to participate according to the age criteria.Eleven of them were later excluded by other exclusion criteria (2 had foot ulcers, and 9 declined to participate).As a result, 35 of them were randomly assigned to each group.The intervention group had 18 participants receiving Plai balm, and the control group had 17 participants receiving placebo balm.Two participants in each group were later excluded due to follow-up loss (Figure 1).The patients' characteristics, pain characteristics, and pain mapping of PDN at baseline, were similar among the two groups (Tables 1, 1.1, and 1.2).
Using GEE analyses by controlling confounding factors (age, sex, quality of life score), showed that the Plai group had statistically significant less pain at week two by -1.47 (95%CI: -1.96 to -1.30, p-value <0.001) when compared to  the placebo group.At week four, the Plai group's pain score was still decreasing and statistically significant less than the placebo group by -1.51 (95%CI: -1.92 to -0.13, p-value <0.027) (Table 2.1).

Secondary outcomes
Numbers/percentages of patients who had at least 50% pain reduction of pain by NRS scores at week four were found significantly more in the Plai group when compared to the placebo group [12/16 (75%) vs 3/15 (20%), p-value = 0.004] (Table 3).The overall integration of WHOQOL-BREF-THAI 4 components was used.Physical domain, psychological domain, social relationships, and environmental domain analyzed patients' quality of life scores at week four.Overall and each domain's patients' quality of life scores were no different between the two groups (Table 4).

Adverse events
No adverse events such as edema, erythema, papules, pruritus, and burning sensation, were found in this study.

Discussion
This double-blind randomized-controlled trial was conducted in PDN patients who had pain by NRS scores of four or more within four weeks, from the 1 st February to the 1 st March 2019 at Selaphum Hospital, Selaphum District, Roi Et, Thailand.Majority of participants were elderly female patients who had been diagnosed with poor controlled type 2 diabetic mellitus for years.Those patients met the criteria of having PDN, which obviously presented similar pain characteristics and pain mapping between the groups.
Based on this study's method, all participants received oral gabapentin 300 mg, 1 tablet daily before bed as a standard regimen.The use of Plai balm 15% w/w 0.5 gram significantly showed pain reduction on PDN.The pain scores by NRS  were decreased more in the Plai group compared to the placebo group [by mean differences of -1.47 (95%CI: -1.96 to -1.30, p-value <0.001) at week two, and by mean difference of -1.51 (95%CI: -1.92 to -0.13, p-value = 0.027) at week four].These results derived from the effects of Plai balm on PDN.
Until now (December 2022), there has been no other trial that studied the efficacy of Plai on reducing pain in PDN.However, previous studies had shown that Plai being main component in mixed herbal preparations, is safe 24 and effective 25 in treating diabetic foot ulcers.7][18][19] These mechanisms in combination, might be the cause of pain reduction.Nevertheless, this study has been the first one to show the effect on PDN.Whether a neuroprotective effect also occurs in the peripheral nerves, is needed to be investigated.
In this study, not only Plai was found to be effective in pain reduction, but also safe with no adverse event reported, neither redness, swelling, burning or rash.These were similar to a systematic review done in 2017 by Chongmelaxme et al. 15 Even in two more recent studies, with very similar application but more mixed herbs, adverse events were still not found. 24,25However, quality of life scores were not significantly different between groups at week 4, even though the quality-of-life scores were improved in each groups.This could be explained by the standard treatment both groups had received: 300 mg gabapentin, once a day.This might be also the main reason for similar changes in the quality-of-life scores.
Strengths of this study were 1) This has been the first RCT comparing treatment efficacy of Plai to placebo, 2) Pain reduction was found significantly different between the groups, and 3) Pain and quality of life scores were measured, reported, and analyzed using their standard methods.Weaknesses of this study might be that; 1) It has low sample size, 2) Some patients were lost from each group, and 3) Cost-effectiveness was not collected and analyzed.Future research with similar objectives may be conducted with larger sample sizes and more diverse settings.
Limitations of this study included 1) Authors could not know the reasons of losing following-up in four patients, and this might be from serious adverse events, and 2) Authors did not added any color or smell to the placebo balm to make it more similar to the Plai balm like Klaphajone et al. 18 However, both of them were only slightly different in their color and smell, and the color or smell added may cause allergy.If the treating physicians and patients did not open the case to campare them directly, it was very hard to differentiate.

Conclusion
In conclusion, Zingiber cassumunar balm (Plai) was efficacious and safe for reducing pain in patients with painful diabetic neuropathy (PDN).However, quality of life scores changes in the Plai group were not significantly different from the placebo group.
Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Yes
Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Fifteen Aprila Fajrin
Clinical and Community Department, Faculty of Pharmacy, Universitas Jember, Jember, East Java, Indonesia I think the author has conducted this research well.But it needs some explanation for the following area: There is no detail about the preparation of topical Zingiber cassumunar.And what is the composition of Plai balm, just Zingiber cassumunar or there are some active plants? 1.
The author needs to explain why patients were excluded from this research and give detailed explanations.

2.
In the result, I found the adverse events, but in the methods, there was no description of how this test was performed and also the assessment.

3.
Almost half of the references are too old, the author can choose another reference that is more up-to-date.

4.
The author needs to explain the limitation of this study.5.

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate?Yes Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?

Table 1 .
Baseline characteristics of the patients.

Table 2 .
Mean changes of NRS scores in each groups.

Table 2 .
1. Mean of NRS scores differences between groups at week 2 and 4.

Table 3 .
Numbers/percentages of patients who had at least 50% pain reduction in each groups at week 4.