Anti-protease activity of Lavender on Chronic Periodontitis patients – An Ex-vivo study

 

Dr. Janhvi Manohar1, Dr Asha2

1Student, Saveetha Dental College, Chennai

2Department of Periodontics, Saveetha Dental College, Chennai

*Corresponding Author E-mail: janhvin7@gmail.com

 

ABSTRACT:

Aim: The aim of this ex-vivo study was to compare the anti-collagenase activity of lavender extract to doxycycline on gingival tissue biopsies obtained from five chronic periodontitis patients who underwent periodontal flap surgeries. Background: Periodontitis is a chronic inflammatory disease characterized by periodontal tissue damage due to an interaction between bacteria and the host immune system. The most important event in chronic periodontitis is the destruction of periodontal tissue, most importantly, collagen. Bacteria releases toxins which cause an increase in matrix metalloproteinases (MMPs) as a host modulatory response. Doxycycline is the FDA approved medication for periodontitis. The use of herbal extracts for the treatment of periodontal diseases as opposed to anti-biotics seem to have minimal side-effects and are inexpensive. This study aimed to evaluate the in-vitro inhibitory effect of lavender on collagenase enzyme on gingival biopsies. Materials and Methods: Gingival tissue samples were extracted from 5 patients with chronic periodontitis. Tissue extracts were treated with the drug solution and lavender extract, the inhibition was analysed by gelatin zymography, and the percentage of inhibition was determined by sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE) and other gel documentation systems. Results: The activity of collagenase enzyme was markedly decreased with the use of doxycycline. Lavender showed a 40% inhibition of the enzyme collagenase activity at the concentration, 1500µg/ml and the control, doxycycline showed. Conclusion: This present study concluded that lavender extract showed significantly potent anti-collagenase activity when compared to doxycycline on gingival tissue biopsies obtained from chronic patients. Thus, it can be used as a substitute to doxycycline.

 

KEYWORDS: Periodontitis, lavender extract, doxycycline, anti-protease activity, gelatin zymography.

 

 

 

INTRODUCTION:

Chronic periodontitis is an inflammatory disease caused by an interaction between periodontal immune response and invasion of bio-film in a susceptible host and is characterised by destruction of periodontal tissue, most importantly, collagen. [1], [2]

 

The 1999 World Workshop on the Classification of Periodontal Diseases proposed a classification based on the differences in the clinical presentation of periodontal disease also including differences in the genetic constitution of the host, microbiota involved, associated systemic diseases and environmental factors. These criteria led to a classification of periodontitis into four major types, aggressive periodontitis, chronic periodontitis, necrotizing periodontal disease and periodontitis due to systemic conditions. [3] The host immune response identifies invading pathogens and tries to neutralize or kill the microorganisms. In the process, it elaborates variety of pro-inflammatory mediators, cytokines, and proteolytic enzymes such as matrix metalloproteinases (MMPs) which cause soft and hard tissue destruction seen in periodontitis. [4] Collagenases are protein enzymes which belong to a group of cathepsins. These cathepsins are precise in their action on substrates like gelatin and collagen. Collagenases belong to the group of MMPs which are highly homozygous Zn2+, they consist of endopeptidases that cleave all the constituents of the extra cellular matrix [5] Periodontal pocket formation and clinical loss of attachment are the clinical manifestations of periodontitis and reduction in probing pocket depth is one of the fundamentals for a successful therapy of periodontitis. [6] Thus, this host response which is essentially protective by intent paradoxically can also result in tissue damage. The progression of the disease is as follows, an acute exudative vasculitis occurs following 2 to 4 days of plaque accumulation, this is termed as the initial lesion wherein loss of perivascular collagen occurs due to MMPs and cytokines. Within a week, there is development of early lesion characterized by infiltration of PMNs which causes continued loss of connective tissue. Next stage is the established lesion following which the lesion progresses to become a destructive lesion where there is loss of alveolar bone and the periodontal ligament. [7]

 

Recent therapeutic efforts are focusing on altering or modulating this host response, leading to emergence of a new comprehensive treatment strategy combining traditional periodontal therapies with host modulatory therapy. [8] Gurumoorthy K et al., explained in this study the role of viruses in the etiology of periodontitis namely Epstein barr virus, Herpes simplex, cytomegalovirus etcetera. [9] Moore et al., reported the role of Wolinella recta, Actinomyces israelii I, Wolinella HS, Lactobacillus minutus and Fusobacterium D 2 in contributing to periodontal disease. [10] Newer bacterial species had a role to play in periodontal diseases. These were two uncultivated phlotypes namely, clone BU063 from the Bacteroidetes and clone BU063 from the Bacteroidetes and a group of named species Atopbium parvulum and Atopobium rimae. [11] However, the microbial profiles of samples obtained from chronic periodontitis patients varied based on geographical area, mean pocket depth, gender, age and smoking status as reported by Haffajee AD et al. [12] High levels of IL-1β, TNF-α, IL-2 and IFN-γ and their ratios to IL 4 and IL 10 were also found to be high in the cases of periodontal disease as reported by Gorska R et al. [13]

 

MMPs can cleave other molecules found within the cell such as collagenase-2 (MMP-8) can cleave elastin, laminin, fibronectin, collagenase, gelatin, etcetera. [14] These substrates are synthesized by tissue cells and inflammatory cells such as neutrophils, keratinocytes, fibroblasts, epithelial cells, microorganisms and macrophages [5] Progress in elucidating the role of MMPs in ECM degradation has led to a new concept involving the chemotherapeutic inhibition of these enzymes. [5] Collagenase activity is inhibited by a number of endogenous physiologic inhibitors like α2-macroglobulin, tissue inhibitors of matrix metalloproteinases (TIMPs) [5], phenytoin [15], eriochrome black T [16], phosphonamides [17], retinoic acid [18], etcetera, To date, subantimicrobial dose doxycycline is the only Food and Drug Administration approved systemic therapy, prescribed as a host response modifier in treatment of periodontal disease which downregulates the activity of MMPs. [19] However, tetracyclines when used for an extended period of time has several side-effects such as staining of teeth, resistance to anti-biotics by microbes, photosensitivity, discoloration of the nails, and onycholysis etcetera. [20] For long, herbal extracts have been used for treating various diseases including periodontitis as they have almost no side-effects and are economically available. However, there aren’t enough studies that state that herbal extracts such as lavender, amla, etcetera, have therapeutic effects when employed in the case of chronic periodontitis. Herbs that may help treat gingivitis include chamomile, echinacea, green tea, peppermint, sage, clove, and myrrh. A mouthwash combination that includes sage oil, peppermint oil, mint oil, menthol, chamomile tincture, expressed juice from Echinacea purpurea, myrrh tincture, clove oil, and caraway oil has been used successfully to treat gingivitis. [21]

 

Lavender (Lavundula angustifolia L.) is a species of flowering plants with its active constituents being essential oil monoterpenoids (including linaloyl-acetate, linalool, 1-terpinen-4-ol), leaves contain rosmarinic acid, tannins, coumarins, triterpenes and phenolic acids. [22]. According to the study done by Danielle Cristina Machado Costa et al., linalool-rich L. alba essential oils has antimicrobial, antipeptidase and antikeratinase activities. [23] In other studies, lavender has been known to decrease salivary cortisol levels, which is usually a stress marker [24] and co-incidentally, salivary cortisol levels have been found to be high in chronic periodontitis patients. [25] Taking into account the same, this study aimed to evaluate the in-vitro inhibitory effect of lavender on collagenase enzyme on gingival biopsies.

 

MATERIALS AND METHODS:

Collection of gingival tissues:

Gingival tissue biopsies were obtained under aseptic conditions following administration of local anaesthesia from 5 patients who were diagnosed clinically with chronic periodontitis and therefore underwent periodontal flap surgery. Patients were chosen based on their periodontal diagnosis of probing depths ranging from five to eight millimetres and clinical loss of attachment. Immediately following excision, the tissue samples were preserved in a protease inhibitor in Eppendorf tubes and stored at 20ºC until it was transported to the lab for processing.

 

This study followed the methodology of Abraham. S., et al. [5]

 

Extraction of plant extract:

The Lavendula plant material were acquired from Herbal care and Cure Centre. Dried herbs were ground in a pestle and mortar, extracted in boiling water and cooled prior to sonication for 15 mins to extract minimum components from within cells as shown in Figure 1. The following day the debris was removed via filtration with Whatman no.1 filter paper and filtrate were passed through a 0.2µm membrane into clean, pre-weighed glass vials. The resulting filtrates were fan dried and weighed. The dried material was stored at -20ºC and resuspended in water at 10mg/ml for use in the assay.

 

Figure 1: Preparation of lavendula extract

 

Concentration of drug:

To determine the optimal inhibition of collagenase enzyme by Lavendula extract, different concentrations of extract (500, 1,000, 1,500µg/ml) and doxycycline (300 µg/ml) were added to 50µl of tissue homogenate and incubated for 60min at room temperature in a series of vials.

 

Extraction of Tissue Collagenase:

Frozen tissue sample was allowed to thaw to room temperature and 100mg (wet weight) of tissue from each patient was homogenized with Tris buffer (saline, 0.9%; Tris, 0.05 M; Triton X-100, 0.25%; and CaCl2, 0.02 M) and centrifuged at 6000 rpm for 30 minutes at 4°C. The resultant supernatant was separated and used for the analysis as shown in Figure 2.

 

Figure 2: Extraction of homogenized tissue collagenase

 

Collagenase assay by Gelatin Zymography:

Pre-treated tissue extracts were subjected to sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide containing 0.1% SDS and 1g/l gelatin under nonreducing conditions without prior boiling as shown in Figure 3. After electrophoresis, gels were washed in 2.5% Triton X-100 for 1 hour to remove SDS and allow protein to denature, and subsequently immersed in a mixture containing Tris-HCl 50 mM/l (pH 7.5) and CaCl2 20 mM/L for 16 hours at 37°C. The gels were then stained with 0.25% Coomassie Brilliant Blue R250/40% ethanol/10% acetic acid, and destained in 25% ethanol/8% acetic acid. Enzymatic activities were detected as clear bands of gelatin lysis against the blue background. To measure the relative collagenase levels, clear zones were scanned and the percentage of inhibition was analyzed by multi-image gel documentation systems.

 

Figure 3: Pretreated tissue samples subjected to sodium dodecyl sulphate-poly acrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide.

 

RESULTS:

The enzymatic activities were detected as bands on gelatin gel by zymography technique. Figure 1 shows the activity of collagenase enzyme in doxycycline and increasing concentrations of lavender extract. The collagenase activity decreases with increasing concentrations of lavender. Assessing the collagenase activity demonstrated that the concentration of lavender extract required to inhibit upto 40% of the enzyme was 1500µg/ml which is almost similar to the results exhibited by triphala. [5] Figure 4 illustrates the reduction in collagenase activity incubated with the drugs expressed as percentage of reduction from control. Doxycycline shows 40% inhibition at 300µg/ml which is the same amount of inhibition showed by 1500µg/ml of lavender extract. The control, as expected, showed the maximum collagenase activity, 100%.

 

Figure 4: The clear zones obtained by gelatin zymography scanned by multi gel documentation systems

 

Figure 5: Bar graph depicting the collagenase activity of the tissue samples in Control, varying amounts of Lavender extract and in doxycycline.

 

In Chart 1, the bar graph depicts the comparison of collagenase inhibition activities of the drugs. Results are expressed in percentage. Asterisk (*) denotes significance at P <0.001, Duncan’s post-hoc test one-way ANOVA.

DISCUSSION:

Research in the field of pathogenesis of periodontal disease has shown that various enzymatic activities which are directed toward the destruction of the pathogen result in destruction of periodontal connective tissue and MMPs and collagenase are groups of enzymes which are responsible to a large extent. [1] Collagenase, upto a large extent, is one of the major causes for loss of attachment and gingival recession in chronic periodontitis. According to recent studies, it has been suggested that PMNLs (Polymorphonuclear neutrophils) are a major source of these collagenases and gelatinases that cause breakdown of connective tissue. [26] Thus, its apparent that the level of the PMN type of MMPs increase with the severity of the periodontal disease and simultaneously, fibroblast type MMPs are not expressed in adequate amounts. [27] The level of MMPs in the body is usually tightly regulated. When there is a disruption in this balance, thus increasing the levels of MMPs, the production of the enzyme collagenase increases beginning the cascade of events leading to periodontitis. The complex process of ECM degradation comprises of extra cellular pathways such as plasminogen dependent pathway (serine proteases), osteoplastic pathway, acid cathepsins mediated intra cellular phagocytic pathway and MMP pathway. [27] In several other studies, activity of MMP was assessed in both periodontitis and gingivitis patients. Increased levels of collagenases (MMP 8 and MMP 1) and gelatinases, MMP 2 and MMP 9, were present in inflamed gingival tissue samples obtained from chronic periodontitis patients wherein the activity of these proteinases completely relate to the pocket depth at the lesion site of the donor, to the severity of the inflammation and the level of enzymes obtained in an active form rather than its latent form increased with the severity of the periodontal disease, as stated by Sorsa T et al. [28]

 

With this understanding, possibility of host modulation so as to reduce the destructive aspects of the host response and hence reduce damage to the periodontium has been investigated. The first line of drugs, only FDA approved systemic therapy, showing anti-collagenase activity are tetracyclines. Studies have demonstrated that tetracycline could significantly inhibit collagenase activity in gingival crevicular fluid and gingival tissue, even at lower dosage than traditional antimicrobial dosages, i.e. sub antimicrobial dosage. [26] Thus, 300µg/ml was used in this study to compare its activity with lavender extract.

 

Tetracyclines cause the inhibition of collagenases by binding to Ca2+ or Zn2+. These two cations are necessary for the activation of MMPs such as gelatinases and collagenases. [29] Another theory states the inhibition of the synthesis of neutrophil derived oxygen radicals thus suppressing migration of neutrophils and its degranulation. [30] However, long term treatment has adverse side-effects such as nausea, vomiting, anorexia, fatty liver, etcetera. This instigated a growing need for drugs with reduced side-effects and are cost-effective. Ayurvedic herbs and drugs have been used therapeutically since ancient times to treat diseases including periodontal diseases. [1] Due to the rising side-effects in the newer drugs, a natural alternative was used in this study, Lavendula. Lavender extract has around 31.06% anti-collagenase activity according to the study conducted by Thring et al. [31]

 

In this study, collagenase inhibition activity was assessed by gelatin zymography and the enzymatic activities detected as clear bands of gelatin lysis further scanned by multi-image gel documentation systems. The amount of collagenase activity decreased with increasing levels of lavendula extract. 1500µg/ml of the lavendula extract showed almost similar amount of anti-collagenase activity as was seen with 300µg/ml of doxycycline. Even though the dosage has to be increased to obtain optimum results, the side-effects are reduced when herbal drugs are used. The inhibitory effect of lavender was also statistically significant giving P <0.001. The anti-collagenase activity increased by 10% with every 500µg/ml increase in the extract. Thus, it can be laid out that 2000µg/ml has 50% inhibitory effect against the collagenase enzyme and it can completely be inhibited by concentrations as low as 4500µg/ml can provide 100% inhibition of collagenase. As stated by Page et al., periodontitis is the most common type of destructive periodontal disease which majorly affects people over the age of 35 years. [32] This statement correlated with our average value of subjects who were chosen for this study as the mean age was calculated to be around 40 years of age shown in Table 1. The average probing depth of the site chosen for the collection of all subjects was 5.8mm and the average clinical loss of attachment for the chosen site was 8.49mm.

 

TABLE 1: Mean values of the parameters such as the probing depth, site specific P.D, clinical attachment loss, site specific CAL and gingival index five generalised chronic periodontitis patients

SUBJECTS

VARIABLES

MEAN VALUE

GENERALIZED CHRONIC PERIODONTITIS PATIENTS

AGE

40.4

FULL MOUTH – PROBING DEPTH

2.706

FULL MOUTH – CLINICAL LOSS OF ATTACHMENT

2.458

SITE SPECIFIC – PROBING DEPTH

5.864

SITE SPECIFIC – CLINICAL LOSS OF ATTACHMENT

8.49

GINGIVAL INDEX

1.94

 

According to A. Prashar et al, lavender oil is cytotoxic to human skin cells in vitro (endothelial cells and fibroblasts) at a concentration of 0.25% (v/v). [33] Thus our study states that around 2000µg/ml is an optimum dosage and safe dosage for chronic periodontitis. However, further studies are required to assess side-effects with regular usage of lavender extract and its method of administration. 

 

CONCLUSION:

In the light of observations from the current study, it can be concluded that doxycycline and lavendula possess anti-collagenase activity in vitro.  Lavender has good anti-collagenase activity in chronic periodontitis, is more cost-effective when compared to doxycycline and can be used as a safe substitute for tetracyclines. Thus, lavender may have therapeutic potential as a host modulation agent in periodontal diseases.

 

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Received on 03.02.2020            Modified on 28.02.2020

Accepted on 25.03.2020      ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2020; 10(2):95-100.

DOI: 10.5958/2231-5691.2020.00018.0