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10.12688/f1000research.129891.1

Research Article

Articles

Lycium barbarum (Goji berry) mouthwash is a viable alternative to 0.2% chlorhexidine gluconate for managing chronic periodontitis: a randomized clinical trial

[version 1; peer review: 1 approved with reservations, 1 not approved]

Sanghavi

Amee

Conceptualization Data Curation Investigation Methodology Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing 1 Shettigar

Laasya

Data Curation Formal Analysis Methodology Resources Visualization Writing – Original Draft Preparation Writing – Review & Editing 1 Chopra

Aditi

Conceptualization Data Curation Investigation Methodology Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0003-4805-5943 a 2 Shah

Ashmeet

Conceptualization Data Curation Formal Analysis Methodology Visualization Writing – Original Draft Preparation Writing – Review & Editing 1 Lobo

Richard

Conceptualization Formal Analysis Investigation Methodology Resources Supervision Validation Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0003-3202-1108 3 Shenoy

Padmaja A

Conceptualization Data Curation Formal Analysis Investigation Methodology Resources Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0001-6781-5579 b 4 Gadag

ShivaPrasada

Investigation Methodology Resources Validation Writing – Original Draft Preparation Writing – Review & Editing 5 Nayak

Usha Y

Formal Analysis Investigation Methodology Project Administration Resources Supervision Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-1995-3114 5 Shravya S

Mangalore

Formal Analysis Investigation Methodology Resources Writing – Original Draft Preparation Writing – Review & Editing 6 Kamath

Shobha Ullas

Formal Analysis Investigation Methodology Resources Supervision Validation Writing – Original Draft Preparation Writing – Review & Editing 6 Nayak

Prajna P

Data Curation Investigation Software Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing 7 1Manipal College of Dental Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India 2Periodontology, Manipal College of Dental Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India 3Department of Pharmacognosy, Manipal College of Pharamcuetical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India 4Microbiology, Kasturba Medical College and Hospital, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India 5Pharmacuetics, Manipal College of Pharmacuetical Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India 6Biochemistry, Kasturba Medical College and Hospital, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India 7Public Health Dentistry, Manipal College of Dental Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India

a aditi.chopra@manipal.edu b padmaja.shenoy@manipal.edu

No competing interests were disclosed.

20 3 2023

2023

302

10 1 2023

2023

This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Removal of the microbial deposits (plaque and calculus) by performing effective scaling and root planing (SRP) is the primary and fundamental requirement for managing periodontal disease. Various adjuncts with antimicrobial, antioxidant, and anti-inflammatory properties are used as adjuncts to SRP for managing chronic periodontitis. However, with a rapid rise in antimicrobial resistance to several antimicrobial agents along with the increased risk of adverse effects, the use the conventional chemotherapeutic agents for managing periodontal disease is slowly declining. Hence, there is a urgent need to explore new plant-based products for treating periodontal disease. Lycium barbarum (L. barbarum), or goji berry, has recently gained popularity for managing chronic inflammatory and infectious diseases. However, its efficacy in managing periodontal diseases has never been explored. Hence the present study aims to evaluate the efficacy of L. barbarum mouthwash along with SRP compared to chlorhexidine for managing chronic periodontitis.

Methods: The study is designed as a randomized clinical trial with 57 adult participants (Males:29; Females:28) with chronic periodontitis. The participants were divided randomly into two groups: One group used L. barbarum mouthwash and the other group used ‘0.2% chlorhexidine gluconate’ mouthwash. The changes in the gingival index (Gi), plaque index (Pi), bleeding on probing (BOP), clinical attachment loss (CAL), probing pocket depth (PPD), microbial load, and antioxidant levels (protein thiol ) in saliva were noted at the baseline, at 15 days and one month.

Results: A statistically significant difference was noted in the Pi (P-value = 0.791), Gi (P-value= 0.594), PPD (with P-value= 0.134), and microbial levels (P-value = 0.188) in both groups from the baseline. The protein thiol levels in saliva were increased only in the goji berry group.

Conclusion: L. barbarum mouthwash along with SRP was found to be effective in managing periodontal disease.

Periodontitis Periodontal disease Oral health Dental Hygiene Lycium Barbarum Goji berry Mouthwash Chlorhexidine Herbal Antioxidants

The author(s) declared that no grants were involved in supporting this work.

Introduction

Periodontitis is defined as a chronic immuno-inflammatory multifactorial disease that affects the soft tissue around the teeth. ¹ Periodontitis is considered the 6th most common disease that affects approximately 20–50% of the adult population worldwide. ² It is more prevalent in males as compared to females. It is primarily caused by the interaction of the gingival tissues with the microorganisms in the oral biofilm. This host-microbial interaction triggers a massive influx of various pro-inflammatory mediators, microbial byproducts, proteolytic enzymes, and free reactive oxygen species, in the gingival and periodontal tissues leading to periodontal tissue destruction. ³ ^– ⁶ Due to increased oxidative stress during periodontal disease, it is also referred to as free radical-mediated tissue injury. ⁷ Some of the common putative periodontal microbes associated with chronic periodontitis are Porphyromonas gingivalis, Tanerella forsythia, Treponema denticola, Campylobacter rectus, and Fusobacterium nucleatum. ⁷ Apart from the microbial etiology, other risk factors that exaggerate periodontal inflammation include smoking, diabetes mellitus, HIV, nutritional deficiency, medications, poor oral hygiene, and genetics. ⁸

The primary and most vital step to control periodontal inflammation is to control the biofilm formed around the teeth by effective mechanical debridement by either hand or machine-driven instruments. ⁹ ^, ¹⁰ Effective scaling and root planning (SRP) can reduce gingival inflammation, thereby preventing disease progression and restoring gingival health. ¹¹ However, mechanical debridement is technically demanding, as many times removal of hard and soft tissue deposits from pockets greater than five mm becomes challenging. Additionally, it is difficult to completely remove the smooth or burnished calculus from deep and circuitous periodontal pockets, furcation areas, root concavities, and irregular roots owing to a lack of good visibility and accessibility to such areas. ¹² ^, ¹³ Hence SRP in deep and tortuous pockets often exhibits residual subgingival biofilm and calculus, thereby warranting a need for additional periodontal therapy. ¹² ^– ¹⁵

The efficacy of professional prophylaxis is also dependent on the individuals’ compliance and motivation to maintain a meticulous oral care regime and effective plaque control at home. ¹⁶ SRP alone may not be sufficient to maintain the required plaque control if patient compliance is poor and the patient does not effectively follow oral hygiene instructions at home. Studies have shown that even after a good plaque control regime, posterior, palatal, and lingual surfaces of the teeth will retain some amount of plaque, and this warrants the need for adjuncts to regular toothbrushes at home. ¹¹ ^, ¹⁷ ^– ²³

Chemical plaque control measures are often advocated as adjuncts to professional oral prophylaxis and daily home care. ²⁴ Various chemical plaque control agents that have antimicrobial and anti-inflammatory are being used in the form of mouthwash, gels, gum paints, fibers, varnishes, microspheres, chips, tablets, powder, and capsules for managing gingivitis and chronic periodontitis. Among all these agents, chlorhexidine gluconate is considered the most popular and routinely used agent for managing periodontal disease. However, prolonged use of chlorhexidine is contraindicated owing to various side effects like altered taste sensation, staining of the teeth and soft tissues such as tongue and mucosa, increased calculus formation, and parotid gland swellings. ²⁵ Chlorhexidine has also been shown to have cytotoxic effects on the gingival fibroblasts, ²⁶ periodontal ligament, ²⁷ and osteoblastic cells. ²⁸ Recent studies have even reported the emergence of antimicrobial resistance among oral bacteria to chlorhexidine molecules. ²⁹ Thus, there is an emerging trend to use natural and herbal extracts for treating periodontal disease. Herbal extracts from neem, Tulsi, guava, green tea, turmeric, curcumin, pomegranate, and many more plants have been tried to effectively treat gingival and periodontal diseases. ³⁰ ^– ³⁴ Recently, goji berry, commonly known as Wolfberry, Himalayan goji, or Tibetan goji, has gained a lot of popularity due to its strong antioxidant and anti-inflammatory properties.

Goji berry is scientifically known as Lycium barbarum (L. barbarum). L. barbarum is native to southeast Europe, China, and Asia. It comes under the family of Solanaceae. ³⁵ The fruit in the form of berries is consumed in both fresh and dried form. L. barbarum contains abundant antioxidants such as scopoletin, vitamin C analogs, carotenoids (zeaxanthin and β-carotene), flavonoids, quercetin, etc. These constituents have powerful antioxidant, immuno-modulating and anticancer properties. ³⁶ ^– ⁴⁶ The stems and berries of goji berry plant was effective against many various Gram-negative bacteria and Gram-positive bacteria. Soesanto et al (2021) also showed that ethanolic of L. barbarum was effective against caries-causing bacteria ( Streptococcus. Mutans) and P. gingivalis at 100 μg/mL. ⁴⁷ Previous in-vitro studies have also found that the minimal inhibitory concentration (MIC) of L. barbarum was comparable to chlorhexidine, however, its efficacy was less as compared to the antibiotic doxycycline. ⁴⁸ ^, ⁴⁹ At 50 μg/mL, ethanolic extract of goji berry could inhibit most of the periodontal pathogens. ⁴⁸ ^, ⁴⁹

However, no clinical study has yet assessed the effectiveness of goji berry mouthwash as an adjunct to SRP for the management of periodontal diseases. Therefore, this clinical study aims to evaluate the efficacy of L. barbarum mouthwash along with SRP for patients with chronic periodontitis compared to chlorhexidine for the first time. ⁴⁷

The objectives of the study include

To evaluate the effect of L. barbarum mouthwash on the Pi, Gi, BOP, PPD, and CAL at baseline, 15 days and one month compared to 0.2% chlorhexidine gluconate mouthwash.

To evaluate and compare the change in the protein thiol levels in saliva at 15 days, and one month compared to baseline.

To evaluate and compare the reduction in the microbial count at end of one month compared to baseline in participants using L. barbarum mouthwash compared to chlorhexidine mouthwash

Methods Trial design

The study was designed as a randomized, double-blind single-centered parallel arm study with an allocation ratio of 1:1. The study was conducted at Manipal’ from 2019 to 2020 following the “Helsinki Declaration of 1975 (as revised in 2000)”. The trial was initiated after receiving ethical clearance from the Kasturba Medical Hospital Institutional Ethics Committee with IEC no 117/2019. The trial has been registered at the ‘Clinical trial registry (CTRI/2019/05/019042)’ and was done following the CONSORT and SAGER guidelines ( Figures 1 and 2). ⁴⁸ The step in the clinical trials are explained as follows:

Figure 1. Schematic representation of the study design (Created in Biorender). Figure 2. CONSORT flow diagram.

Formulation of mouthwash: The mouthwash was prepared at the “Manipal College of Pharmaceutical Sciences, Manipal”. L. barbarum mouthwash was prepared by preparing an ethanolic extract of dried goji berry using Soxhlet’s method and dissolving the accurately 50 ug/ ml of weighed goji berry extract for formulating the mouthwash as described previously. ⁴⁹ A concentration of 50 ug/ml was mixed in 1.15 of 100% alcohol and then homogenized using of 15% glycerol. To this around 15% propylene glycol, 1% Tween solution was added to formulate the mouthwash. 0.1% menthol was added to the adjust to the taste of the mouthwash. The volume was adjusted to 100% by using distilled water. 100% alcohol was added to serve both as a preservative and a dissolvent.

Assessment of stability of mouthwash

The shelf life of the mouthwash was measured as per the ICH guidelines and the samples were tested for three months under three different conditions: “35°±2% C with relative humidity of 60% ± 5%; 25°C± 2°C and relative humidity: 60% ± 5%; and 40°C ± 2°C with a relative humidity of 75% ± 5%”. The mouthwash was kept in opaque plastic bottles and one of the bottles was kept in the stability compartment (Thermo lab, India). The samples were analysed at baseline, one, two, and three months by visual observation and UV spectrum analysis (Shimadzu UV-1601PC, Japan) and analysed for its physical parameters and stability. The mouthwash showed no change in the colour, odour, consistency, or no phase separation at three months. The sample showed no change when observed under the ultraviolet analysis at 274 nm. This indicated that the mouthwash was stable till the end of three months ( Table 1).

Table 1. Physical properties and evaluation of the stability of the mouthwash.

Temperature	Evaluation parameters	Observation (in months)
Temperature	Evaluation parameters	Baseline	1	2	3
Room temperature (3 – 5 ± 2%°C)	Visual appearance	Slight yellowish-brown	Slight yellowish-brown	Slight yellowish-brown	Slight yellowish-brown
	Precipitation/Phase separation	Nil	Nil	Nil	Nil
	Homogeneity	Good	Good	Good	Good
Room temperature (25°C ± 2°C) Relative humidity (60% ± 5%)	Visual appearance	Slight yellowish-brown	Slight yellowish-brown	Slight yellowish-brown	Slight yellowish-brown
	Precipitation/Phase separation	Nil	Nil	Nil	Nil
	Homogeneity	Good	Good	Good	Good
Room temperature 40°C ± 2°C Relative humidity (75% ± 5%)	Visual appearance	Slight yellowish-brown	Slight yellowish-brown	Slight yellowish-brown	Slight yellowish-brown
	Precipitation/Phase separation	Nil	Nil	Nil	Nil
	Homogeneity	Good	Good	Good	Good

Clinical study design

Around ninety subjects aged 20 to 50 years (both male/females) visiting the outpatient department were screened for the presence of chronic periodontitis according to the following exclusion and inclusion criteria:

Inclusion criteria: 1.

Participants in age group of 20-50 years diagnosed with Stage 1 to stage II generalized periodontitis (Grades A to B).

Participants with a minimum of 24 functional teeth.

Exclusion criteria:

Individuals with severe periodontitis (localized and generalized or Stage III and Stage IV) were excluded.

Participants with any allergic reactions to chlorhexidineg

Participants with any systemic diseases such as hypertension, diabetes mellitus, cardiovascular, renal, and neurological diseases.

Participants on any concurrent medications

Participants currently using anti-inflammatory, antibiotics, or analgesics in the last six months

Pregnant or lactating mothers

Participants with any oral abusive habits such as smoking, alcohol, betel nut chewing, gutka, paan, supari, areca nut

Participants who had undergone any periodontal treatment/surgery in the last six months

Participants undergoing orthodontic treatment.

10.

Participants using any other oral hygiene agents (mouthwash or gels) will be excluded to remove confounding bias arising due to the difference in plaque control measures.

All participants who satisfied the above-mentioned criteria were recruited after obtaining an oral and written signed informed consent.

Randomization, allocation concealment and blinding

After screening the participants, around 60 participants were recruited after obtaining oral and written informed consent. This sample size was based on p-value <0.05, alpha value = 0.05 two-tailed, power = 0.8, and the effect size = 0.7. Of 60 participants, three participants refused the to take part in the study after scaling, and the remaining fifty-seven participants were randomly allocated using the computer-generated random sequence to either the test group ( L. barbarum, n = 30) or the control group (0.2% chlorhexidine gluconate mouthwash, n = 27). The allocation of participants into two groups was done by an investigator who was not the part of the either samples collection, periodontal therapy or periodontal examination. Following recruitment, all participants were assigned to another investigator who was blinded about the randomization and grouping. The participants were also blinded about their group. Following grouping, the following biologic samples were collected:

a. Microbiology plaque sample collection

The plaque from the subgingival region was collected from the pocket with the maximum probing depth in each quadrant using a sterile site-specific Gracey curette. After collection, the curette was gently submerged in a reduced transport medium (Thioglycolate bath) for testing the total colony Forming Units (CFU). The number of colonies were calculated on the plated blood agar and then converted to CFU/mL using the following formula: CFU/mL = (no. of colonies × dilution factor)/volume of the culture plate.

b. Saliva Collection

Following plaque collection, a stringent method for saliva collection was followed. All participants were requested to sit comfortably in an upright position. Following this, around 2ml of saliva was collected by the ‘spitting method’ and without any stimulation. ⁵⁰ All participants were requested toward spiting the saliva into an Eppendorf vial. The collected saliva was then stored immediately in a refrigerator at -80 degree Celsius. The Eppendorf tubes used to store saliva samples were numerically marked according to the participant number. The saliva collected was sent for biochemical analysis for evaluation of the protein thiol levels using Ellman's Reagent. The absorbance of the agent was measured after incubation at room temperature at 412 nm for around five minutes and the concentration of protein thiol was determined with the standard curve of glutathione.

c. Examination of the clinical periodontal parameters

Following saliva sample collection, a periodontal examination was done for all the sextants. The following periodontal parameters were recorded: Gi by Loe & Silness, 1963; percentage of sites with BOP; PI by Silness & Loe 1964; PD and CAL. All the clinical evaluation was done by the investigator who was blinded about the patient’s grouping. The BOP, PPD, and CAL were recorded by Williams periodontal probe (Hu-Friedy, USA). The sites with BOP were checked by noting the presence and absence of bleeding on all four surfaces (buccal, lingual, mesial, and distal) for all the teeth. The percentage of sites with BOP was considered by the percentage of the teeth with BOP to the total teeth present. The PPD and CAL were calculated at buccal, mesial, distal, and lingual. The deepest pocket depth at each surface was recorded. The average of each surface was considered as the reading for that tooth. The mean PPD and CAL were assessed by adding the reading from each tooth and dividing it with the total number of teeth.

Following periodontal examination and sample collection, a thorough SRP was initiated for all the participants. The scaling was assessed by the supervisor to ensure the complete removal of plaque and calculus has been achieved. All patients were educated to brush their/teeth in modified bass technique for two minutes twice daily, to nullify any confounding effects arising due to differences in the oral hygiene measures. All participants were given the opaque amber color bottle which were coded (AX or BX). This was done to blind the patients and investigators regarding the type of mouthwash given to the participants. All patients were instructed to use 10 ml mouthwash diluted with 10 ml of water twice a day for a month. Patients were recalled after 15 days and one month for revaluation. At each recall visit Pi, Gi, % of sites with BOP, CAL, and PPD were noted. The plaque from same site and saliva samples were collected at each recall visit. The investigation for each participant, at each recall visit, was done by the same investigator.

Statistical analysis

Data obtained was analysed by the ‘SPSS version 26.0, IBM’. The descriptive data such as the frequency and for categorical data; mean and standard deviation for all the numerical data was analysed using ‘Kolmogorov–Smirnov test’. The normality of the distribution was checked for all variables. The inter-group comparisons of all the assessed outcomes were done using an ‘independent sample t-test’. The comparisons between the goji berry mouthwash and chlorhexidine mouthwash was done to measure any significant increase/reduction from baseline to 15 days and one month using Repeated measures ANOVA. Intergroup comparison of reduction of all the variables at follow-up was done using ANCOVA after adjusting the respective baseline scores. The P-value of less than 0.05 was considered to be significant.

Results

The study results showed that the mean age of study participants in the test group was 35.42 ± 11.79 years and in the controls was 32.12 ± 12.85. The gender wise distribution in the test group was Males: 16; Female: 14 and Control group was Males: 13; Females: 14 ( Table 2). The comparison between goji berry mouth group and chlorhexidine group for of all the study variables was found to have no significant differences in the mean values for Pi (P = 0.470), Gi (P = 0.239), BOP (P = 0.450), PPD (P = 0.216), CAL (P = 0.220), and Microbial level (P = 0.251) ( Table 3). The intra-group comparison using the ‘repeated measures of ANOVA with Greenhouse Geisser correction’ followed by a ‘post-hoc analysis with Bonferroni adjustment’. This showed that the mean PPD, Pi, and Gi, was changed from baseline to one month in both the control and test groups. The CAL was reduced significantly only in the chlorhexidine group compared to the goji berry group ( Table 3, Table 4 and Figure 3). A significant difference was noted in the antioxidant levels (protein thiol) in saliva in the goji berry group at the end of one month. No change in the salivary antioxidant level was noted in the chlorhexidine group. No significant differences were reported in the log-transformed microbial CFU counts in both groups at any given time. In the case of the test group, the mean Pi reduced from 1.6 ± 0.38 at baseline and was 0.89 ± 0.17 at one month. However, in the case of the goji berry mouthwash, the mean PPD was reduced during the 15-day follow-up (1.73 ± 0.45) compared to baseline (2.76 ± 1.06). The intergroup comparison at one-month follow-up (15 days and one month) after adjusting the respective baseline scores was done using ANCOVA. The control group had a significant reduction in CAL (P = 0.001) as compared to the test group. There were no differences seen in the mean values for Pi (P = 0.791), Gi (P = 0.594), PPD (P = 0.134), protein thiol levels (P = 0.211), and microbial levels (P = 0.188) between the two groups ( Table 4). No harms were reported by any patient. One patient reported bitter taste of the mouthwash and discontinued the mouthwash.

Table 2. Demographic data of the groups.

Groups	Goji berry group (case group)	Chlorhexidine group (Control group)	p-value
Age (in Years)	35.42 ± 11.79	32.12 ± 12.85	0.29 ^a
	Male-Female	Male-Female
Gender	16-14	13-14	0.11 ^b

Based on: One-way ANOVA/Chi Square test.

Table 3. Inter-group and intra-group comparison of baseline, 15 days, and 1-month follow-up scores.

	Group	Baseline	15 days	1 month	P-value ¥
	Group	Mean ± SD	Mean ± SD	Mean ± SD	Baseline vs 15 days	Baseline vs 1 month	15 days vs 1 month
Plaque index	Control	1.45 ± 0.42	1.03 ± 0.50	0.92 ± 0.43	0.104	0.027	0.334
	Test	1.6 ± 0.38	1.04 ± 0.36	0.89 ± 0.17	0.000	0.000	0.076
	P-value #	0.470
Gingival index	Control	1.4 ± 0.64	0.87 ± 0.47	0.81 ± 0.43	0.001	0.036	0.214
	Test	1.35 ± 0.38	0.85 ± 0.31	0.75 ± 0.24	0.000	0.000	0.092
	P-value #	0.239
Percentage of sites with bleeding on probing	Control	80.7 ± 0.42	42.7 ± 0.49	10.7 ± 0.42	0.001	0.034	0.210
	Test	84.8± 0.32	40.8± 0.29	09.0± 0.32	0.000	0.000	0.090
	P-value #	0.450
Probing pocket depth	Control	2.35 ± 0.56	1.62 ± 0.56	1.78 ± 0.35	0.004	0.006	0.186
	Test	2.76 ± 1.06	1.73 ± 0.45	2.15 ± 0.68	0.002	0.035	0.327
	P-value #	0.216
Clinical attachment level	Control	2.03 ±0.90	1.52 ± 0.65	1.33 ± 0.43	0.020	0.005	0.174
	Test	2.62 ± 1.16	2.29 ± 0.99	2.02 ± 0.61	0.103	0.248	0.260
	P-value #	0.220
Biochemical analysis	Control	233.06 ± 144.49	235.48 ± 103.39	225.06 ± 72.11	0.874	0.899	0.760
	Test	132.68 ± 56.17	227.05 ± 72.21	248.30 ± 68.31	0.000	0.000	0.272
	P-value #	0.021
Microbial level	Control	7.44 ± 0.43	7.35 ± 0.584	7.33 ± 0.567	0.183	0.130	0.756
	Test	7.57 ± 0.77	7.19 ± 0.74	7.04 ± 0.85	0.222	0.564	0.264
	P-value #	0.251

Test: L. barbarum mouthwash; Control: chlorhexidine gluconate mouthwash.

N for Case group = 15; Control group = 14. Values in bold indicate significant difference.

P-value for inter-group comparisons (independent sample t-test);

^¥

P-value for intra-group comparisons (Repeated measures ANOVA).

Table 4. Inter-group comparisons at 1-month follow-up after adjusting for baseline values.

Outcomes	Adjusted baseline	I month		P-value
Outcomes	Adjusted baseline	Control Mean ± SE	Test Mean ± SE	P-value
Plaque index	1.61	0.92±.092	0.88±0.09	0.791
Gingival index	1.46	0.82±0.100	0.75±0.10	0.594
Percentage of sites with BOP	78.4	12.9 ±0.56	13.1 ±0.42	0.494
Probing pocket depth	2.06	1.77±0.254	2.15±0.26	0.134
Clinical attachment level	2.33	1.33±1.22	2.02±0.11	0.001
Biochemical analysis	182.77	225.06 ±16.83	248.30 ±16.83	0.211
Microbial level	7.45	7.33 ±0.17	7.05 ±0.19	0.188

Values in bold indicate significant difference.

Figure 3. Changes in gingival index, plaque index, bleeding on probing, pocket depth and clinical attachment, microbiologic colony-forming units, and antioxidant levels of protein thiol variables at each recall visit. Discussion

The present study is the first clinical trial aimed to evaluate the role of goji berry in treating any dental diseases. The study evaluated the effectiveness of goji berry mouthwash compared to chlorhexidine for managing chronic periodontitis in both males and female patients. Based on the results, it was noted that goji berry mouthwash can control the gingival inflammation, BOP, and plaque formation at 15 days following SRP with statistically significant difference compared to chlorhexidine group. A significant in the Gi, Pi, BOP, PPD was noted in participants using goji berry mouthwash. To our knowledge, this is first clinical trial comparing the potential of L. barbarum and chlorhexidine (gold standard antimicrobial agent) as an adjunct to SRP for the management of chronic periodontitis. The most significant outcome was the statistically significant increase in the protein thiol levels in saliva in the goji berry group alone, suggesting the advantage of using the goji berry mouthwash over chlorhexidine . This could be attributed to powerful antioxidants in goji berry mouthwash compared to chlorhexidine.

Goji berry is considered a ‘superfood’ with good antimicrobial, anti-inflammatory, and antioxidant properties. The constituents of L. barbarum have been tried for various inflammatory and infectious disease, however its role in treating periodontal inflammation has not been explored. The use of L. barbarum could improve the local antioxidant levels and this in turn helps in controlling the clinical symptoms of inflammation in chronic periodontitis. The study is crucial, especially with the emergence of resistance to chlorhexidine molecules among oral bacteria and numerous side effects of using chlorhexidine for long time. ⁵¹ Recent systematic reviews have shown that there are limited benefits of using chlorhexidine gluconate in patients with chronic periodontitis owing to the development of resistance among oral bacteria to chlorhexidine molecule and cytotoxicity to soft tissues by chlorhexidine. ⁵² Hence the use of plant-based products with antioxidant and anti-inflammatory properties is advocated to control the local oxidative stress in the gingival tissues and preclude the harmful effects of chlorhexidine. ⁵³

Goji berry has numerous antioxidants, which could be utilized to combat the proinflammatory mediators and oxidative stress in the periodontal tissues. ⁵⁴ ^– ⁵⁶ Some of the important antioxidants in L. barbarum are the Lycium barbarum polysaccharides (LBPs), caffeic acid, coumaric acid, scopoletin, linoleic acid, zeaxanthin dipalmitate, kaempferol, caffeoylquinic and coumaric acid. ⁵⁵ ^– ⁵⁷ Studies have also identified different types of flavonoids such as catechin, epicatechin, and quercetin in goji berries. Flavonoids have a good antimicrobial effect on the various periodontal pathogen and this could justify the reduction in the PI and control the gingival inflammation in participants using goji berry mouthwash. The alcoholic group (-OH) in the goji berry is also another antimicrobial agent that can control the microbial growth in oral biofilm. ⁵⁸ Goji berry also contains monoterpenes (like sabinene, phellandrene, γ-terpinene), phenolic acid (chlorogenic acid), citric acid, tartaric acid, oxalic acid, malic acid, and vitamin C. These compounds have shown superior anti-inflammatory and antibacterial properties. ⁵⁹ LBP in goji berry is also a potent inhibitor of proinflammatory cytokines. It can inhibit matrix metalloproteinase (MMP-1), Tumor necrosis factor, Interleukin-2, and PGE2. This can helps to control connective tissue destruction, which is a crucial aspect in the progression of periodontal disease. Goji berries can help to improve fibroblast healing and increase the production of Type I collagen. ⁴¹ ^, ⁶⁰ This can be attributed to the ability to reduce gingival inflammation and release of various antioxidants. Chlorogenic acid in the goji berry can even inhibit nitric oxide and inducible nitric oxide synthase which are some of the vital pathways activated during periodontal inflammation. ⁶¹ ^– ⁶³ At the molecular level, the polysaccharides in the goji berries (LBP) influence the ‘antigen-presenting cells’ such as dendritic cell. This improves the adaptive immune response at the site of inflammation and control tissue destruction. ⁶¹ Direct effect of chlorogenic acid, yet another important constituent in goji berry, on the oxidative stress-induced secretion of proinflammatory cytokines and mRNA expression has also been proven. ⁶² ^, ⁶³

Based on our results and exitsing evidence, it can be concluded that goji berry can be an effective adjunct to oral prophylaxis compared to chlorhexidine in patients with chronic periodontitis. ⁶⁴ ^, ⁶⁵ Although our study has proven that goji berry mouthwash is an effective adjunct to SRP for managing periodontitis, future studies should compare the efficacy of goji berry with other herbal or plant-based adjuncts with long term follow up. One should also note that the study tested the role of goji berry for only a short recall time, as prolonged exposure to chlorhexidine mouthwash is associated with many side effects. Future studies can explore the effect of goji berry on longer recall visits. Future studies can also evaluate the effect of L. barbarum on specific periodontal pathogens such as red complex bacteria or P. gingivalis or specific biomarkers for periodontitis.

Conclusion

L. barbarum is a promising ‘superfood’ with many potential health benefits. It has antioxidant and antimicrobial properties that can be utilized for developing novel formulations for treating patients with chronic periodontitis. L. barbarum mouthwash effectively reduced plaque scores, gingival inflammation, and bleeding on probing. Goji berry can be tried instead of chlorhexidine gluconate for managing periodontal disease.

Ethical statement

The study was conducted after receiving ethical approval from Kasturba Medical College and Kasturba Hospital Ethic committee with IEC no: IEC no 117/2019. The trial has been registered at the ‘Clinical trial registry (CTRI/2019/05/019042)’.

Data availability

Figshare. Data on study titled: Lycium barbarum (Goji berry) mouthwash is a viable alternative to 0.2% chlorhexidine gluconate for managing chronic periodontitis: a randomized clinical trial. DOI: https://doi.org/10.6084/m9.figshare.21834939.v1. ⁶⁵

This project contains the following data: -

CONSORT checklist

Study protocol

Figshare. Goji berry mouthwash as alternative to chlorhexidine for managing chronic periodontitis. DOI: https://doi.org/10.6084/m9.figshare.21780164.v1. ⁶⁴

This project contains the following data: -

Study data

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

The datasets related to our study is also available with the corresponding author and can be shared on reasonable request via email to aditi.chopra@manipal.edu.

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Chopra

: Data on study titled: Lycium barbarum (Goji berry) mouthwash is a viable alternative to 0.2% chlorhexidine gluconate for managing chronic periodontitis: a randomized clinical trial.Dataset. figshare. 2023. 10.6084/m9.figshare.21834939.v1

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Reviewer response for version 1

Muniz

Francisco Wilker Mustafa Gomes

1 Referee https://orcid.org/0000-0002-3945-1752 1Graduate Program in Dentistry, Department of Periodontology, Universidade Federal de Pelotas, Pelotas, State of Rio Grande do Sul, Brazil

Competing interests: No competing interests were disclosed.

9 10 2023

2023

This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

recommendation

reject

This is the first round of peer review of this referee. Several queries were raised as stated below:

Overall

A very short follow-up period was followed for the chosen outcomes, such as BOP, CAL and PPD. A longer follow-up period, to detect any important change, is necessary. The antiplaque and antigingivitis efficacy may be important to the literature, though.

Throughout the manuscript the term “periodontal disease” is used, maybe, referring to periodontitis. The term periodontal disease is very broad, and do not mean only periodontitis. Authors must revise it.

Title

Moreover, the term “chronic periodontitis” should not be used since 2018. Please see Caton, J. G., Armitage, G., Berglundh, T., Chapple, I. L., Jepsen, S., Kornman, K. S., ... & Tonetti, M. S. (2018). A new classification scheme for periodontal and peri‐implant diseases and conditions–Introduction and key changes from the 1999 classification. Journal of periodontology, 89, S1-S8. Prefer the term “periodontitis” whenever appropriate, and this must be applied to all manuscript.

Additionally, lower gain of CAL was detected in the test group. Title must not state that similar results were detected between groups.

Abstract

Authors wrote: “A statistically significant difference was noted in the Pi (P-value = 0.791), Gi (P-value= 0.594), PPD (with P-value= 0.134), and microbial levels (P-value = 0.188) in both groups from the baseline.” However, none of the showed p-values mean statistical significance. The sentence is not understandable. Does the comparison mean within or between comparisons?

Conclusion may also be misleading. L. barbarum mouthwash presented similar clinical efficacy, when compared to chlorhexidine. The SRP performed may be the main reason to the so-called “effective in managing periodontal disease”. Please provide a interpretation aligned with your aims.

Introduction

The term “exaggerate” in the first paragraph should be revised, as may be not be the most appropriate term in an epidemiological point of view. In addition, authors have cited both risk factors and indicator factors associated with periodontitis.

Reference is necessary for the following sentence: “However, mechanical debridement is technically demanding, as many times removal of hard and soft tissue deposits from pockets greater than five mm becomes challenging.” In well-trained clinicians, this may not be a problem.

Provide citation for the following sentences:

“Recently, goji berry, commonly known as Wolfberry, Himalayan goji, or Tibetan goji, has gained a lot of popularity due to its strong antioxidant and anti-inflammatory properties.”

“The stems and berries of goji berry plant was effective against many various Gram-negative bacteria and Gram-positive bacteria.”

Italicize the word “in vitro.”

Reference #47 may not be necessary in the following sentence: “(…) efficacy of L. barbarum mouthwash along with SRP for patients with chronic periodontitis compared to chlorhexidine for the first time. ⁴⁷”

Why salivary levels of protein thiol were assessed in the present study? What is the relevance for the present study. This is not properly explained in this section.

Objective

Pi, Gi, BOP, PPD, and CAL must be explained, as this is the first time their appear in the text.

Methods

Figure 1 do not represent the flowchart of the study, and do not follow what CONSORT recommends. Figure 1 is illustrative and important, but the sentence must be revised.

Figure 2, in fact, must be placed in the Results section, as recommended by CONSORT.

Do not use Stage 1, use “stage I.” Provide reference for this inclusion criterion

Do not mistake exclusion criteria as the opposite of inclusion criteria. For instance, if only generalized periodontitis was included. It is not necessary to exclude those with localized periodontitis.

What is “chlorhexidineg”?

Revise the following sentence: “Participants currently using anti-inflammatory, antibiotics, or analgesics in the last six months.” It seems that patients that used antibiotics in the last six months were included.

Revise exclusion criteria #10, as the sentence is in the future.

Sample size calculation is not properly described. Readers to not know the necessary number of patients to detect important measures in the primary outcome. For instance, the effect size of 0.7 is referring to what outcome?

Why allocation stated uneven (30:27) at baseline (Figure 2), as a 1:1 allocation ratio was established in the Methods?

In fact, allocation concealment is not properly explained.

Both mouthwashes may be very distinct in the general characteristics, such as taste, color and visual aspects. Therefore, this referee does not understand the following sentence: “The participants were also blinded about their group.”

It is well-established that the best periodontal examination must be performed in six sites per tooth. It is not clear why authors have performed it only in four.

Further information regarding chlorhexidine is necessary. Was it manufactured by the authors? What percentage was used? Provide details.

Chlorhexidine, such as those commercially available, must not be diluted in water. The lower efficacy of chlorhexidine must not be ruled out in the present study.

It is not clear for how long (who many seconds) volunteers should rinse.

Calibration, if any, is not reported.

Results

In Table 3, why p-values for the comparison between groups are reported only for baseline?

The following sentence should be better placed in the Methods: “(…) repeated measures of ANOVA with Greenhouse Geisser correction’ followed by a ‘post-hoc analysis with Bonferroni adjustment’.”

Figure 3 and Table 3 are repetitive results. Both of them are not necessary.

Authors wrote: “One patient reported bitter taste of the mouthwash and discontinued the mouthwash.” What mouthwash?

Discussion

Effectiveness was not tested. Do not mistake it with efficacy.

Only mild periodontitis patients were included in the present study. Do not generalize it. This type of patient is not discussed.

The following sentences are not correct: “Based on the results, it was noted that goji berry mouthwash can control the gingival inflammation, BOP, and plaque formation at 15 days following SRP with statistically significant difference compared to chlorhexidine group. A significant in the Gi, Pi, BOP, PPD was noted in participants using goji berry mouthwash.” No differences between groups were detected.

Again, chlorhexidine is not largely used for the management of periodontitis. In fact, its use as adjuvant to periodontitis treatment is not well-established, and the previously mentioned clinical practice guideline do not recommend it.

A broader discussion regarding the L. barbarum on periodontal tissues is warranted.

There is no discussion regarding the very short follow-up period.

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Are sufficient details of methods and analysis provided to allow replication by others?

Reviewer Expertise:

Periodontology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

References 1

: Treatment of stage I-III periodontitis-The EFP S3 level clinical practice guideline. J Clin Periodontol .2020;47 Suppl 22(Suppl 22) : 10.1111/jcpe.13290 4-60

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: A new classification scheme for periodontal and peri-implant diseases and conditions - Introduction and key changes from the 1999 classification. J Clin Periodontol .2018;45 Suppl 20: 10.1111/jcpe.12935 S1-S8

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Chopra

Aditi

Periodontology, Manipal College of Dental Sciences, Manipal, Manipal, India

Competing interests: No potential conflict of interest to be reported

4 11 2023

Firstly, we would like to thank all the reviewers and the editor for spending their valuable time and providing us with their valuable comments and suggestions. We truly appreciate your guidance in improving our work. We have tried to address the comments and revised the manuscript accordingly. The changes in the manuscript have been highlighted in track change mode in the revised manuscript.

Reviewer 2

A very short follow-up period was followed for the chosen outcomes, such as BOP, CAL, and PPD. A longer follow-up period, to detect any important change is necessary. The antiplaque and antigingivitis efficacy may be important to the literature, though. Throughout the manuscript the term “periodontal disease” is used, maybe, referring to periodontitis. The term periodontal disease is very broad, and do not mean only periodontitis. The authors must revise it.

Response: We thank the reviewers for their valuable insight. We agree with the reviewer that a long-term follow-up is necessary, however, due to various side effects of chlorhexidine it is not advised to use for a long time. Additionally, there are studies that have found the development of antimicrobial resistance in periodontal pathogens against Chlorhexidine, hence the use of herbal mouthwashes is constantly being researched. We have also revised the term periodontal disease to periodontitis as suggested. The changes in the manuscript have been highlighted in track change mode in the revised manuscript.

Chlorhexidine is an excellent mouthwash to control biofilm and gingival inflammation. However, literature does not state that this mouthwash is useful for the treatment of periodontitis. Other treatments are more efficacious for this periodontal disease. Therefore, title must be revised, as it may be misleading in its current format. Please see Sanz, M., Herrera, D., Kebschull, M., Chapple, I., Jepsen, S., Berglundh, T., ... & Wennström, J. (2020). Treatment of stage I–III periodontitis—The EFP S3 level clinical practice guideline. Journal of Clinical Periodontology, 47, 4-60. Moreover, the term “chronic periodontitis” should not be used since 2018. Please see Caton, J. G., Armitage, G., Berglundh, T., Chapple, I. L., Jepsen, S., Kornman, K. S., ... & Tonetti, M. S. (2018). A new classification scheme for periodontal and peri‐implant diseases and conditions–Introduction and key changes from the 1999 classification. Journal of periodontology, 89, S1-S8. Prefer the term “periodontitis” whenever appropriate, and this must be applied to all manuscript.

Response: we thank the reviewers for sharing the article. Based on your suggestion we have now revised the title as follows: Efficacy of Lycium barbarum (Goji berry) mouthwash for managing periodontitis: a randomized clinical trial. We have now revised the terminology chronic periodontitis to periodontitis as requested. The changes in the manuscript have been highlighted in track change mode in the revised manuscript.

Additionally, lower gain of CAL was detected in the test group. Title must not state that similar results were detected between groups.

Response: we have now revised the interpretation as requested both in abstract and result/ discussion section.

Abstract

Response: we have now revised the result section and removed the following as they were baseline comparisons: that “statistically significant difference was noted in the Pi (P-value = 0.791), Gi (P-value= 0.594), PPD (with P-value= 0.134), and microbial levels (P-value = 0.188) in both groups.” Instead, we have now added the follow-up comparisons. The p-values are for intergroup comparison. The changes in the manuscript have been highlighted in track change mode in the revised manuscript.

Response: We thank the reviewers for their valuable suggestions. We have now revised the conclusion as follows: Goji berry mouthwash along with SRP was effective in reducing the Gi, Pi, BOP, and PPD. Goji berry mouth also increased the antioxidant levels in saliva in patients with periodontitis. Thus, goji berry mouthwash is a viable adjunct to SRP for patients with periodontitis. The changes in the manuscript have been highlighted in track change mode in the revised manuscript.

Introduction

Response: We thank the reviewers for their valuable suggestions. We have now revised the term as follows: Apart from the microbial etiology, other risk factors that influence the severity of periodontal inflammation include smoking, diabetes mellitus, HIV, nutritional deficiency, medications, poor oral hygiene, and genetics. The changes in the manuscript have been highlighted in track change mode in the revised manuscript.

Response: We have now added two references for this sentence as requested. The changes in the manuscript have been highlighted in track change mode in the revised manuscript.

Heitz-Mayfield L.J., Trombelli L., Heitz F., Needleman I., Moles D. A systematic review of the effect of surgical debridement vs. non-surgical debridement for the treatment of chronic periodontitis. J. Clin. Periodontol. 2002;29((Suppl. S3)):92–102. doi: 10.1034/j.1600-051X.29.s3.5.x.

Sherman P.R., Hutchens L.H., Jr., Jewson L.G., Moriarty J.M., Greco G.W., McFall W.T., Jr. The effectiveness of subgingival scaling and root planing. I. Clinical detection of residual calculus. J. Periodontal. 1990;61:3–8. doi: 10.1902/jop.1990.61.1.3.

Provide citation for the following sentences: “Recently, goji berry, commonly known as Wolfberry, Himalayan goji, or Tibetan goji, has gained a lot of popularity due to its strong antioxidant and anti-inflammatory properties.” And “The stems and berries of goji berry plant was effective against many various Gram-negative bacteria and Gram-positive bacteria.”

Response: We thank the reviewer for their suggestion, We have now added reference no 38-48 , which discusses the antimicrobial and antioxidant properties of the goji berry as suggested. The changes in the manuscript have been highlighted in track change mode in the revised manuscript.

Italicize the word “in vitro.”

Response: We thank you for your suggestion, We have now Italicized the word as requested.

Response: We have removed reference no 47 from the sentences mentioned above.-

Why salivary levels of protein thiol were assessed in the present study? What is the relevance of the present study? This is not properly explained in this section.

Response: we thank the reviewers for their valuable insight and key appraisal. we have now added the importance of assessment of local antioxidant levels in oral cavity as immune defense mechanisms in the discussion and introduction section. Since protein thiol is an important antioxidant during periodontal disease and the ability of goji berry to increase it levels in saliva as additional advantage over chlorhexidine. The changes in the manuscript have been highlighted in track change mode in the revised manuscript.

Objective: Pi, Gi, BOP, PPD, and CAL must be explained, as this is the first time they appear in the text.

Response: We have now added the full forms for each of the abbreviations in the objective section as suggested.

Methods: Figure 1 does not represent the flowchart of the study, and does not follow what CONSORT recommends. Figure 1 is illustrative and important, but the sentence must be revised.

Response: We thank the reviewers for the recommendation, we have now revised the figure 1.

Figure 2, in fact, must be placed in the Results section, as recommended by CONSORT.

Response: We thank the reviewer for their suggestion, we have now placed figure 2 in the result section.

Do not use Stage 1, use “stage I.” Provide reference for this inclusion criterion

Response: We have now revised the term Stage 1 to Stage I as suggested.

Response: We thank the reviewer for their valuable insight. We have now revised the criteria as suggested : Participants in age group of 20-50 years diagnosed with stage I to stage II localized/generalized periodontitis (Grades A to B).

What is “chlorhexidineg”?

Response: We apologise for the Typo error, we have now corrected it as Chlorhexidine

Response: We have now revised the sentence as follows: Participants who were on any anti-inflammatory, antibiotics, or analgesics in last six months were excluded

Revise exclusion criteria #10, as the sentence is in the future.

Response: We have now revised the sentence as follows: Participants using any other oral hygiene agents (mouthwash or gels) were excluded to remove confounding bias arising due to the difference in plaque control measures

Response: We thank the reviewer for pointing our mistake and not mentioning the effect size linked outcome. The effect size was calculated based on clinical attachment loss.

Why allocation stated uneven (30:27) at baseline (Figure 2), as a 1:1 allocation ratio was established in the Methods?

Response: We thank the reviewer for their insight. We apologize for the mistake in numbers mentioned in the concealment. Allocation was done at ratio of 1:1 as mentioned in the method. We have now corrected and uploaded the revised figure as suggested.

In fact, allocation concealment is not properly explained.

Response: We thank the reviewer for their insight. We have now added an explanation on how the allocation concealment was done using opaque amber-colored bottles to mask the color of the mouthwash.

Both types of mouthwash may be very distinct in general characteristics, such as taste, color, and visual aspects. Therefore, this referee does not understand the following sentence: “The participants were also blinded about their group.”

Response: We agree with the reviewer that both goji berry and chlorhexidine mouthwash, mouthwash were stored in similar amber-colored bottles to mask the color of the mouthwash. However, differences in the taste will remain as a major factor in patient blinding/ concealment

It is well-established that the best periodontal examination must be performed in six sites per tooth. It is not clear why authors have performed it only in four.

Response: We agree with the reviewers and apologize for the ambiguity in the sentence we have performed periodontal examination on buccal, lingual, mesial, and distal (in the interdental area the probing was done both from the buccal and lingual side of the interdental area. We have now revised the sentence in the manuscript.

Further information regarding chlorhexidine is necessary. Was it manufactured by the authors? What percentage was used? Provide details.

Response: We have added the percentage of chlorhexidine (0.2%)

Chlorhexidine, such as those commercially available, must not be diluted in water. The lower efficacy of chlorhexidine must not be ruled out in the present study.

Response: We agree with the reviewers. however, the chlorhexidine used our study 0.2% was prepared and was diluted in 10 ml of water.

It is not clear for how long (who many seconds) volunteers should rinse.

Response: We have now added the time the mouthwash should be used in manuscript.

Calibration, if any, is not reported.

Response: Since single trained investigator examined the baseline and follow-up visits, calibration was not done.

Results

In Table 3, why p-values for the comparison between groups are reported only for baseline?

Response: The p-values for comparison between groups at follow-ups and mentioned in the last columns of the same table.

Response: We have now revised the statement and mentioned it in Methods section.

Figure 3 and Table 3 are repetitive results. Both of them are not necessary.

Response: We agree with reviewer suggestions. We have now removed the figure 3.

Authors wrote: “One patient reported bitter taste of the mouthwash and discontinued the mouthwash.” What mouthwash?

Response: we have now added which group (Test group) patient reported bitterness.

Discussion: Effectiveness was not tested. Do not mistake it with efficacy.

Response: We have modified the title according to the suggestion and instead of effectiveness mentioned it as efficacy

Only mild periodontitis patients were included in the present study. Do not generalize it. This type of patient is not discussed.

Response: We have added this in the limitations this points our results

Response: We agree with the reviewers, we apologize for the error and interpreatation. we have now corrected the results section accordingly.

Response: we thank the reviewers for their recommendation, we have now added these points to our introduction and discussion sections

A broader discussion regarding the L. barbarum on periodontal tissues is warranted.

Response: We thank the reviewers for their recommendation. We have now added the role of the L. barbarum on periodontal pathogens, periodontal ligament fibroblast, neutrophils and wound healing

References:

Gan L, Zhang SH, Liu Q, et al.: A polysaccharide-protein complex from Lycium barbarum upregulates cytokine expression in human peripheral blood mononuclear cells. Eur. J. Pharmacol. 2003;471(3):217–222. 12826241 10.1016/s0014-2999(03)01827-2

Bo R, Liu Z, Zhang J, et al.: Mechanism of Lycium barbarum polysaccharides liposomes on activating murine dendritic cells. Carbohydr. Polym. 2019;205:540–549. 30446138 10.1016/j.carbpol.2018.10.057

Du X, Wang J, Niu X, Smith D, Wu D, Meydani SN. Dietary wolfberry supplementation enhances the protective effect of flu vaccine against influenza challenges in aged mice. J Nutr 2014;144: 224–9.35.

Ren Z, Na L, Xu Y, Rozati M, Wang J, Xu J, et al. Dietary supplementation with lacto-wolfberry enhances the immune response and reduces pathogenesis to influenza infection in mice. J Nutr 2012;142:1596–602.

There is no discussion regarding the very short follow-up period.

Response: We agree with the reviewer and we have added these points as the limitations of the study

10.5256/f1000research.142608.r173857

Reviewer response for version 1

Sharma

Rajinder K.

1 Referee https://orcid.org/0000-0001-7839-1097 Anand

Deepti

1 Co-referee 1Department of Periodontics and Oral Implantology, Postgraduate Institute of Dental Sciences, Rohtak, Haryana, India

Competing interests: No competing interests were disclosed.

6 6 2023

2023

recommendation

approve-with-reservations

Abstract:

In abstract, results have been mentioned as significant in both the groups, however the p values mentioned don't corroborate the significant results. Please do the necessary corrections.

Introduction:

It needs to be brief & relevant. The objectives of the study may be omitted in the introduction.

Methodology

Please describe in detail the method of randomization of 57 participants in test group(30) and control group(27).

Were the participants recruited on consecutive order or in pooled manner?

Out of 30 participants in test group, 15 were dropped out, only 15 were included in statistical analysis. Sample size calculation was 27 patients in each group. Rationale of sample size calculation is not served in this study. 'Around' 60 participants were recruited; Please avoid vague terms like 'around'. Please mention 57.

Out of 27 allocated to control group, 16 were lost to follow up hence only 11 are left for follow up completion, however in the flow chart it is mentioned, 14 participants were analyzed. Please look into it.

Results

In the result section, demographic data is with reference to patients recruited whereas all other data is with reference to patients who completed the study. Similar data may be included in the result section, either all of the patients recruited or only the patients who have completed the follow up.

All the results have been presented in overall average values. PPD is an important parameter influencing the outcomes of periodontal intervention. Data needs to be segregated as per PPD.

Discussion:

Please focus the discussion on the results of the overall study period of one month, unless there is some interesting finding otherwise.

It takes 6-8 weeks for healing to complete after SRP, Results of SRP are required to be estimated after this period. However the follow up of study period was one month; Please comment on it.

A significant part of discussion is only review of literature with reference to Guji berry; Please avoid it as much as possible. Comment on the findings of your study may be the focus of discussion .

Conclusion:

Conclusions are not based on the results of the study. For example "L.barbarum is a promising superfood with many potential health benefits", is not the conclusion of the study.

L. barbarum has been investigated as an adjunct to SRP. Negative control is missing in the study. Hence the results can't be attributed exclusively to L.barbarum.

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Periodontitis, periodontal medicine

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

Chopra

Aditi

Periodontology, Manipal College of Dental Sciences, Manipal, Manipal, India

Competing interests: nil

6 11 2023

Reviewer 1

Abstract: In the abstract, results have been mentioned as significant in both the groups, however the p values mentioned don't corroborate the significant results. Please do the necessary corrections.

Response: We thank the reviewers for their valuable insight. We have now revised the abstract and interpretation of the results.

Introduction: It needs to be brief & relevant. The objectives of the study may be omitted in the introduction.

Response: We thank the reviewer for their suggestion. We have now revised and shortened the introduction/background section in the abstract.

Methodology: Please describe in detail the method of randomization of 57 participants in test group(30) and control group(27). Were the participants recruited on consecutive order or in pooled manner?

Response: We have now added the method of randomization (computer-generated random sequence) in the abstract. The participants were recruited in as when they come to the OPD and randomized once the consequent order.

Out of 30 participants in test group, 15 were dropped out, only 15 were included in the statistical analysis. The sample size calculation was 27 patients in each group. The rationale of sample size calculation is not served in this study. 'Around' 60 participants were recruited; Please avoid vague terms like 'around'. Please mention 57.

Response: We thank the reviewer for their valuable suggestion. We have now revised paper accordingly and removed the word around along number of participants

Response: We thank the reviewer for their valuable suggestion. We have now revised paper accordingly and provided the corrected CONSORT flow diagram

Results

Response: We thank the reviewer for their valuable suggestion. We have now revised paper accordingly and included the data related to the patients who were recruited

All the results have been presented in overall average values. PPD is an important parameter influencing the outcomes of periodontal intervention. Data needs to be segregated as per PPD.

Response: We thank the reviewers for their valuable insight. However, since it study was conducted on a small number of participant where segregation of PPD was not possible due to similarity in the amount of PPD, we have mentioned the data as combine reduction in PPD from baseline, moreover with segregation we found that all the patient fall under 1-3 mm of PPD and CAL loss due to recession of 1-2 mm .

Discussion:

Please focus the discussion on the results of the overall study period of one month, unless there is some interesting finding otherwise. It takes 6-8 weeks for healing to complete after SRP, Results of SRP are required to be estimated after this period. However, the follow-up of study period was one month; Please comment on it.

Response: We thank the reviewer for the critical appraisal of these key findings. We have now added these points to the limitations of the study. We have kept the follow-up at 1 month as long-term use of chlorhexidine mouthwash is not recommended owing to the development of various side effects

A significant part of discussion is only review of literature with reference to Goji berry; Please avoid it as much as possible. Comment on the findings of your study may be the focus of discussion.

Response: We thank the reviewers for their valuable insight. We have now revised the manuscript accordingly. We have shortened the evidence on goji berry and discussed the clinical implications of our results and limitations of the study

Conclusion:

Conclusions are not based on the results of the study. For example "L.barbarum is a promising superfood with many potential health benefits", is not the conclusion of the study.

Response: We thank the reviewers for their valuable insight. We have revised the conlucsion and removed the line suggested above.

L. barbarum has been investigated as an adjunct to SRP. Negative control is missing in the study. Hence the results can't be attributed exclusively to L.barbarum.

Response: We thank the reviewers for their valuable insight and agree with fact that the results are due to the combined effects of SRP with goji berry mouthwash. Since SRP is primary and the fundamental step that should be performed and matched in both the group we aimed to compared goji berry mouthwash with chlorhexidine. We have added these points to our discussion as you have suggested.