Lycium barbarum (Goji berry) mouthwash is a viable alternative to 0.2% chlorhexidine gluconate for managing chronic 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 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

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)
		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).

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

  Exclusion criteria:

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

• 2. Participants with any allergic reactions to chlorhexidineg

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

• 4. Participants on any concurrent medications

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

• 6. Pregnant or lactating mothers

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

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

• 9. 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.

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.

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.