Efficacy of Lycium barbarum (Goji berry) mouthwash for managing periodontitis: a randomized clinical trial

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, affecting approximately 20–50% of the adult population worldwide.² It is more prevalent in males compared to females. It is primarily caused by the interaction of the gingival and periodontal tissues with the microorganisms in the oral biofilm. This host-microbial interaction triggers a massive influx of various pro-inflammatory mediators, microbial by-products, proteolytic enzymes, and free reactive oxygen species (ROS) leading to periodontal tissue destruction and inflammation.^3–6 Due to increased ROS during periodontal disease, it is also referred to as free radical-mediated tissue injury.⁷ Some of the common putative periodontal microbes associated with periodontitis are Porphyromonas gingivalis, Tanerella forsythia, Treponema denticola, Campylobacter rectus, and Fusobacterium nucleatum.⁷ Apart from the microbial etiology, other risk factors such as smoking, diabetes mellitus, HIV, nutritional deficiency, medications, poor oral hygiene, and genetics influence the severity of periodontal inflammation.⁸

To control periodontal inflammation the primary and most vital step is the removal of oral biofilm formed around the teeth by performing effective mechanical debridement with 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, studies have shown that complete debridement of calculus is technically demanding, as many times removal of hard and soft tissue deposits from deep pockets (>5 mm) and interdental regions becomes challenging.^11,12 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 for the management of biofilm from deep and tortuous pockets additional periodontal therapy and use of adjuncts are needed.^12–15

It is also noted that the efficacy of SRP is dependent on the patient’s 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. Additionally, studies have shown that even after a good plaque control regime; posterior, palatal, and lingual surfaces of the teeth retain some amount of plaque. Thus for many patients, adjuncts such as mouthwash and gels along with regular toothbrushing are indicated.^11,17–24 Various chemical plaque control agents with 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 periodontitis. Among these agents, chlorhexidine gluconate is the most popular and routinely used agent for managing periodontitis. Additionally, recent reports have shown no additional benefits of using chlorhexidine and the development of antimicrobial resistance among many oral bacteria to chlorhexidine molecules.^25–29 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 is also known to have cytotoxic effects on the gingival fibroblasts,²⁶ periodontal ligament,²⁷ and osteoblastic cells.²⁸

Thus, there is an emerging trend to use natural and herbal extracts with antioxidant and antimicrobial properties to treat periodontal diseases. Herbal extracts from neem, Tulsi, guava, green tea, turmeric, curcumin, pomegranate, and many more plants have been tried to effectively treat gingivitis and periodontitis.^30–34 Since periodontal disease causes a massive release of ROS and increases oxidative stress, adjuncts with antioxidant potential are often used with SRP to control the oxidative stress present locally in the gingival and periodontal tissues. 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.^35–46

Goji berry, scientifically known as Lycium barbarum (L. barbarum), is a fruit native to southeast Europe, China, and Asia.³⁵ The fruit belongs to the family of Solanaceae and is consumed either in both fresh and dried form. It has shown powerful antioxidant, antimicrobial, immuno-modulating, and anticancer properties.^36–46 Soesanto et al. (2021) showed that ethanolic extract of L. barbarum is effective against oral bacteria (Streptococcus mutans and P. gingivalis) at 100 μg/mL.⁴⁷ Previous in-vitro studies have also reported that the minimal inhibitory concentration (MIC) of L. barbarum was comparable to chlorhexidine, however, its efficacy was less as compared to the antibiotic doxycycline. 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 periodontitis. Therefore, this clinical study aims to evaluate the efficacy of L. barbarum mouthwash along with SRP for patients with periodontitis compared to chlorhexidine for the first time.

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 steps in the clinical trials are explained as follows:

Figure 1. Schematic representation of the study design (Created in Biorender.com).

Figure 2. Participant 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 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 15% glycerol. To this around 15% propylene glycol and 1% Tween solution was added to formulate the mouthwash. 0.1% menthol was added to adjust 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 a 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 color, odor, 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)
		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

Ninety subjects aged 20 to 50 years (both male/females) visiting the outpatient department were screened for the presence of localized/generalized periodontitis according to the 2017 classification of periodontal disease and selected based on the following exclusion and inclusion criteria:

Inclusion criteria:

• 1. Participants in the age group of 20-50 years diagnosed with stage I to stage II localized/generalized periodontitis (Grades A to B).

• 2. Participants with a minimum of 24 functional teeth.

Exclusion criteria:

• 1. Individuals with severe periodontitis (localized or generalized stage III and stage IV) were excluded.

• 2. Participants with any allergic reactions to chlorhexidine.

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

• 4. Participants who were on any anti-inflammatory, antibiotics, or analgesics in the last six months were excluded.

• 5. Pregnant or lactating mothers.

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

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

• 8. Participants undergoing orthodontic treatment.

• 9. 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.

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

Results

Of the 90 participants assessed for eligibility, 30 participants were excluded as they did not meet the eligibility criteria. The rest 60 participants were allocated into test (n=30) and control (n=30) groups. The mean age of 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; females: 14 and the control group was males: 14; females: 16 (Table 2). Out of the 30 patients in each group, 14 patients in the test group and 16 patients in the control did not come for the follow-up visit due to the sudden lockdown imposed by COVID-19. Additionally, one patient in test group reported bitter taste of the goji berry mouthwash and discontinued the mouthwash. Thus, for analysis 15 patients in the test and 14 patients in the control were included in the analysis. The comparison between goji berry mouthwash and chlorhexidine mouthwash showed 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 showed a significant reduction in the mean PPD, Pi, and Gi from baseline to one month in both the control and test groups. The CAL reduced significantly only in the chlorhexidine group compared to the goji berry group (Table 3, Table 4). 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 the baseline (2.76 ± 1.06).

Intergroup comparison at one-month follow-up (15 days and one month) was done using ANCOVA after adjusting the respective baseline scores. 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.

Discussion

The present study is the first clinical trial that aimed to evaluate the role of goji berry mouthwash for the treatment of periodontitis. The study assessed the efficacy of goji berry mouthwash compared to 0.2% chlorhexidine mouthwash in controlling the gingival inflammation, plaque formation, BOP, PPD, CAL, microbial profile, and antioxidant levels (protein thiols) in the saliva. We found that the mean reduction in Gi, Pi, BOP, and PPD with goji berry mouthwash was comparable to chlorhexidine mouthwash. The reduction in CAL was better for chlorhexidine mouthwash compared to goji berry mouthwash. However, we did not find any statistically significant difference between the goji berry mouth and chlorhexidine mouthwash for Gi, Pi, BOP, and PPD at the end of one month. Since goji berry mouthwash showed comparable results to chlorhexidine, it can be used along with SRP for managing periodontitis, especially in the current times when oral bacteria are becoming resistant to chlorhexidine.^50–53 Additionally, since prolonged use of chlorhexidine is not advised to prevent the onset of various side effects such as alterations in taste, staining, and increased calculus, alternatives to chlorhexidine becomes important. Costa et al. (2017) in a systematic review and meta-analysis also concluded that adjunctive use of chlorhexidine with SRP provides only a minor PPD reduction compared to SRP alone. Therefore, clinicians should consider the small additional gain in PPD reduction, negligible effect on CAL, and potential for adverse effects when prescribing chlorhexidine to their patients.⁵²

In such situations, the use of herbal mouthwash like goji berry would be a viable alternative.⁵¹ The present study also found that goji berry mouthwash increased the protein thiol levels in saliva, unlike chlorhexidine. This is a positive finding that goji berry would be advantageous compared to chlorhexidine in increasing the antioxidant levels in periodontal disease. Since periodontitis is linked with increased oxidative stress and free radical-mediated tissue injury, use of compounds with antioxidant properties are often used. The increase in antioxidant levels in saliva upon use of goji berry mouthwash would help to reduce and control the inflammation. The antioxidant properties of goji berry are attributed to the presence of constituents like Lycium barbarum polysaccharides (LBPs), catechin, epicatechin, quercetin, chlorogenic acid, citric acid, coumaric acid, scopoletin, linoleic acid, kaempferol, and coumaric acid.^50–57 The flavonoids in goji berries are proven to have good antimicrobial effects and this justifies the reduction in the PI and control of gingival inflammation with goji berry mouthwash.⁵⁸ The reduction in gingival inflammation can also be attributed to the ability of goji berry to inhibit proinflammatory cytokines, radical scavenging activities, and interactions with other antioxidants. Studies have shown that goji berries can improve fibroblast healing, increase collagen formation, and reduce oxidative stress.^41,59–62

Sanghavi et al. (2022) evaluated the antimicrobial, anti-adhesion, anti-biofilm, and cytotoxic properties of L. barbarum against five potential periodontal pathogens (P. gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Prevotella intermedia, Tanerella forsythia) and found that goji berry extract could inhibit the growth of periodontal pathogens, however, the zone of inhibition of was less when compared to doxycycline and chlorhexidine. The authors also reported that goji berry was compatible with gingival fibroblast and oral keratinocytes at a concentration of 1 mg/mL. The study also found that goji berry has good anti-adhesion property against P. gingivalis. The anti-adhesion properties of LBE (96%) were comparable compared to chlorhexidine (96.3%). However, the anti-biofilm activity of chlorhexidine (96%) was found to be slightly better than that of goji berry extract (91.6%).⁴⁹ The ability of goji berry mouthwash to reduce the PI and GI can also be explaind by it ability to modulate the immune cells. Studies by Du et al. (2014) and Ren et al. (2012) also found that goji berry supplementation enhances the maturation and increases the recruitment of neutrophils and monocytes to sites of infection.^63,64 These properties of LBP would be beneficial in lowering the microbial load and controlling periodontal inflammation. Lai et al. (2022) conducted an in-vitro and animal study to assess the efficacy of alkaline phosphatase activity and osteogenic potential of human periodontal ligament stem cells upon treatment with goji berry and found that goji berry can enhance the proliferation and migration of stem cells. This could facilitate superior healing and control of gingival inflammation. The study also showed a reduction in alveolar bone resorption and confirmed the positive effect of L. barbarum for managing periodontitis.⁶⁵

Hence based on our results and existing evidence, it can be stated that goji berry has a promising role inmanaging periodontitis. However, one should also note that the present study evaluated the role of goji berry for only a short recall time for one month and prolonged exposure to chlorhexidine mouthwash was avoided to prevent side effects. One should also note that although the patients were blinded by using coded amber-coded colored bottles to mask the color of the mouthwash, the taste of each mouthwash was different. The study was conducted on patients with mild-moderateperiodontitis, therefore future studies should assess the efficacy of goji berry mouthwash on severe cases of periodontitis with longer follow-up. Future studies should also evaluate the effect of goji berry on specific periodontal pathogens or local and systemic inflammatory markers for periodontitis.

Conclusion

Goji berry mouthwash along with SRP was effective in reducing the Gi Pi, BOP, and PPD in patients with periodontitis. No statistically significant difference was noted between the use of goji berry mouthwash and chlorhexidine mouthwash. However, goji berry mouthwash was more effective than chlorhexidine in increasing the antioxidant levels in saliva.

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: 10.6084/m9.figshare.21834939.⁶⁶

This project contains the following 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.