DESC:FIELD OF THE INVENTION
The present invention relates generally to a natural and effective composition for the treatment of oral sub-mucosal fibrosis (OSMF), more particularly a topical composition that promotes apoptosis of fibroblast in OSMF.
BACKGROUND OF THE INVENTION
Oral submucous fibrosis (OSMF) is a chronic disease that causes tissue fibrosis, scars, and precancerous lesions. It often occurs in the buccal mucosa and is involves Inflammation, Progressive fibrosis, Tissue fibrosis, Scars and Precancerous lesions. Although occasionally preceded by, or associated with, the formation of vesicles, it is always associated with a juxtaepithelial inflammatory reaction followed by fibroelastic change of the lamina propria and epithelial atrophy that leads to stiffness of the oral mucosa and causes trismus and an inability to eat”. Rao, N.R., Villa, A., More, C.B. et al. Oral submucous fibrosis: a contemporary narrative review with a proposed inter-professional approach for an early diagnosis and clinical management. J of Otolaryngol - Head & Neck Surg 49, 3 (2020).
OSMF is also known by other names including idiopathic scleroderma of the mouth, juxtaepithelial fibrosis, idiopathic palatal fibrosis, diffuse oral submucous fibrosis, and sclerosing stomatitis. Id.
Worldwide, the number of cases of OSMF was estimated to be 2.5 million in 1996. The prevalence of OSMF in India has been estimated to range from 0.2–2.3% in males and 1.2–4.6% in females, with a broad age range from 11 to 60?years. Id. Currently A marked increase in incidence has been observed after the widespread marketing of commercial tobacco and areca nut products, generally known as Gutkha, which is sold in single-use packets. Further patients with OSMF have been reported with higher risk of developing oral squamous cell carcinoma (OSCC).
As with other lifestyle related diseases, primary prevention at population and individual levels needs to be improved by a legislation that bans the sale of gutkha and similar products. It is to be noted that several Indian states have had success in this regard. Since May 2013, gutkha is banned in 24 states and 5 union territories of India, under the provision of centrally enacted Food Safety and Regulation (Prohibition) Act 2011.
One of the most important factors in management of OSMF is the early diagnosis. Dependent on their dominant symptoms, patients may seek consultation from either primary care physicians (PCP) or dentists. When examined by a dentist, the diagnostic and treatment approach is likely to be focused on the oral signs and symptoms. Conversely, when patients present to a PCP, the focus of management is likely to be general, with the oral condition under-investigated and under-managed. In most cases, the OSMF is not managed as a multidisciplinary team.
The treatment of OSMF is difficult, and the present treatment methods are mainly aimed at relieving the signs and symptoms of the disease. Usually, OSMF is treated with drugs in the early stages, followed by physical therapy in severe cases and surgery in the late or advanced stages. Many studies have shown that a combination of drugs, including steroids, enzymes, antioxidants, multivitamins, and minerals, can alleviate the signs and symptoms of OSMF, however a related drug therapy trials have been disappointing.
In terms of immunosuppression, steroids can activate sensitized lymphocytes against specific antigens to release soluble factors, however they do not improve the abnormal accumulation of fibrotic tissue. Enzyme hyaluronidase can target MMP-1 or MMP-2 to destroy abnormal fibrotic tissue compensating for the lack of effect of steroids in improving the abnormal accumulation of fibrotic tissue.
A potential cause of OSMF is the progressive loss of diseased mucosal blood vessels, which can lead to epithelial atrophy. Therefore, vasodilators Pentoxifylline (PTX), a trisubstituted methylxanthine derivative, owing to its ability to relax and dilate blood vessels, ensuring increased blood supply to the ischemic tissue and enabling nutritional and therapeutic drugs to reach the affected tissue.
Several studies have tested various natural or synthetic antioxidants and reported that using them can improve the condition of betel nut damage to the mouth. Natural antioxidants include lycopene, aloe vera, curcumin, and spirulina. In addition to the abovementioned drugs, allicin, colchicine, omega 3, and oxitard have a certain effect on the treatment of OSMF. There is evidence showing that allicin has important anti-inflammatory effects.
Many studies have been conducted to understand the treatment modalities in case of OSMF. Studies also indicate that the naturally occurring herbal medicines have an immense potential in the management of precancer OSMF.
There is a constant demand for new therapies to treat & prevent OSMF. Scientific & research interest is drawing its attention towards naturally derived compounds as they are considered to have less toxic side effects compare to current treatments.
Though it is known the role of herbs to possess effective anti OSMF activities. However, till date they have not been combined together to prepare effective formulation for the treatment of OSMF. However, difficulty occurs in formulating a topical composition that provides effective treatment in patients with OSMF. The present invention provides a natural formulation that provides for the treatment of OSMF & malignant transformation of OSMF.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide a safe dental care composition that providing effective treatment for oral sub-mucosal fibrosis.
It is one another object of the present invention to provide a natural composition that provides effects of promoting apoptosis and inhibition of TNF- a & TGF-ß and collagen synthesis.
It is yet another object of the present invention to provide a natural composition that exhibits synergistic effects in mitigating cell cycle & inducing apoptosis of cancer cells in OSMF.
It is yet another object of the present invention to provide gel formulation for topical oral application.
SUMMARY OF THE INVENTION
The following presents a simplified summary of one or more embodiments in order to provide a basic understanding of such embodiments. This summary is not an extensive overview of all contemplated embodiments and is intended to neither identify key or critical elements of all embodiments nor delineate the scope of any or all embodiments. Its sole purpose is to present some concepts of one or more embodiments in a simplified form as a prelude to the more detailed description that is presented later.
In accordance with one aspect of the present invention provides an oral dental composition comprising of
a) Picrasma quassioides or Indian quassia,
b) Carum carvi or fennel,
c) Piper nigrum or black pepper,
d) Caesalpinia bonduc or bonduc nut,
e) Withania somnifera or ashwagandha,
f) Myristica fragrans or nutmeg,
g) Nigella sativa or black cumin,
h) Ocimum Sanctum or Tulsi,
i) Mimosops elengi or Bakula,
j) Acacia catechu or Khadira,
k) Cuminum cyminium or Shvetajiraka,
l) Boerhavia diffusa or Punarnava
The combination of the present composition promotes apoptosis of fibroblasts & inhibit TNF- a, TGF-ß & activation of collagen as well as targeting glycolytic pathway for the treatment of cancer cells. The composition of the present invention exhibits synergistic effect in both mitigating cell cycle & apoptosis of cancer cells.
Said composition has application in treating oral sub-mucosal fibrosis. Said composition is a gel formulation for topical oral application.
The foregoing has outlined rather broadly the features and technical advantages of the present invention so that those skilled in the art may better understand the detailed description of the invention that follows. Additional features and advantages of the invention will be described hereinafter that form the subject of the claims of the invention. Those skilled in the art should appreciate that they may readily use the conception and the specific embodiment disclosed as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the invention in its broadest form.
BRIEF DESCRIPTION OF THE FIGURES
FIG.1 is a graphical representation of the CTC50 (%) values of 8% Picrasma quassiodes, 0.8% Caesalpinia bonduc, 1.6% Withania somnitera, 10% Myristica fragrans, 10% Mimusops elengi, and the dental composition.
FIG. 2 is a graphical representation of the relative gene expression fold of TNF-a in HaCaT cells which are – untreated (control), treated with LPS, treated with 20µM/mL Dexamethasone and LPS, treated with 250 µg/ml and 500 µg/ml dental composition (SQUA CARE GEL) and LPS.
FIG. 3 is a graphical representation of the relative gene expression fold of TGF-ß in HaCaT cells which are – untreated (control), treated with LPS, treated with 20µM/mL Dexamethasone and LPS, treated with 250 µg/ml and 500 µg/ml dental composition (SQUA CARE GEL) and LPS.
FIG. 4 is a graphical representation of the relative gene expression fold of TNF-a in KB31 cells which are – untreated (control), treated with LPS, treated with 20µM/mL Dexamethasone and LPS, treated with 250 µg/ml and 500 µg/ml dental composition (SQUA CARE GEL) and LPS.
FIG. 5 is a graphical representation of the relative gene expression fold of TGF-ß in KB31 cells which are – untreated (control), treated with LPS, treated with 20µM/mL Dexamethasone and LPS, treated with 250 µg/ml and 500 µg/ml dental composition (SQUA CARE GEL) and LPS.
FIG. 6A is an image of the liver of female Sprague Dawley rat on 15th day after treatment with dental composition (SQUA CARE GEL) at a dose of 2000 mg/kg weight.
FIG. 6B is an image of the kidney of female Sprague Dawley rat on 15th day after treatment with dental composition (SQUA CARE GEL) at a dose of 2000 mg/kg weight.
FIG. 6C is an image of the spleen of female Sprague Dawley rat on 15th day after treatment with dental composition (SQUA CARE GEL) at a dose of 2000 mg/kg weight.
FIG. 6D is an image of the heart of female Sprague Dawley rat on 15th day after treatment with dental composition (SQUA CARE GEL) at a dose of 2000 mg/kg weight.
DETAIL DESCRIPTION OF THE INVENTION
Before undertaking the detailed description of the invention below it may be advantageous to set forth definitions of certain words and phrases used throughout this patent document: the terms “include” and “comprise ” as well as derivatives thereof mean inclusion without limitation; the term “or ” is inclusive meaning and/or; the phrases “associated with” and “associated therewith ” as well as derivatives thereof may mean to include be included within interconnect with contain be contained within connect to or with couple to or with be communicable with cooperate with interleave juxtapose be proximate to be bound to or with have a property of or the like. Definitions for certain words and phrases are provided throughout this patent document those of ordinary skill in the art should understand that in many if not most instances such definitions apply to prior as well as future uses of such defined words and phrases.
The term "comprising", which is synonymous with "including", "containing", or "characterized by" here is defined as being inclusive or open-ended, and does not exclude additional, unrecited elements or method steps, unless the context clearly requires otherwise. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
The term “CTC50” means Critical Threshold Concentration of a drug or any composition. It is the concentration at which 50% of its maximum effect is observed. The lower the CTC50 value the more potent is the drug or composition.
The term “cytotoxicity” means the ability of a substance or process to damage or kill cells.
The term “MTT assay” means the widely used and well-known colorimetric method to assess cell metabolic activity and viability, based on the reduction of the yellow dye MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)) to a purple formazan product by active mitochondria in living cells, which is then measured spectrophotometrically.
The term “OSMF” refers to oral sub-mucosal fibrosis, which is a chronic, potentially malignant condition of the oral cavity characterized by fibrosis and stiffness, often associated with areca nut chewing.
The term “SQUA CARE GEL” refers to the dental composition comprising of 8% Picrasma quassiodes, 0.8% Caesalpinia bonduc, 1.6% Withania somnitera, 10% Myristica fragrans, 10% Mimusops elengi, and the dental composition comprising of 8% Picrasma quassiodes, 3.2 % (w/w) Carum carvi, 1.6 % (w/w) Piper nigrum, 0.8 % (w/w) Caesalpinia bonduc, 1.6 % (w/w) Withania somnifera or Ashwagandha, 2 % (w/w) Myristica fragrans or Nutmeg, 3 % (w/w) Nigella sativa, 4 % (w/w) Ocimum Sanctum or Tulsi, 10 % (w/w) Mimosops elengi or Bakula, 5 % (w/w) Acacia catechu or Khadira, 5 % (w/w) Cuminum cyminium or Shvetajiraka, and 2 % (w/w) Boerhavia diffusa or Punarnava. The composition is in a gel form.
Various terms as used herein should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
Applicants have recognized that there is a need for an oral care composition that provides safe and effective dental care that does not have any toxic substances and that is stable and effective to the perborate based available oral care composition.
In the main embodiment, the present invention provides an oral composition comprising of,
a) Picrasma quassioides or Indian quassia,
b) Carum carvi or fennel,
c) Piper nigrum or black pepper,
d) Caesalpinia bonduc or bonduc nut,
e) Withania somnifera or ashwagandha,
f) Myristica fragrans or nutmeg,
g) Nigella sativa or black cumin,
h) Ocimum Sanctum or Tulsi,
i) Mimosops elengi or Bakula,
j) Acacia catechu or Khadira,
k) Cuminum cyminium or Shvetajiraka,
l) Boerhavia diffusa or Punarnava
Said composition has application in treating oral sub-mucosal fibrosis (OSMF).
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Picrasma quassioides or Indian quassia is 8 % (w/w). Picrasma quassioides is a small tree or shrub, commonly referred to as “nigaki” bitterwood, that belongs to the Simaroubaceae family. The extracts and isolated compounds from P. quassioides exhibit notable anti-inflammatory, anti-oxidant, antibacterial, antitumor, and neuroprotective properties. Alkaloids are the main anti-inflammatory components in Picrasma, among which indole alkaloids such as ß-carboline alkaloids and canthinone alkaloids are the main ones.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Carum carvi is 3.2 % (w/w). Carum carvi is generally known as caraway which is typically a biennial. Caraway is rich in several major compounds including carvacrol, carvone, a-pinene, limonene, ?-terpinene, linalool, carvenone, and p-cymene. It contains essential oils rich in nutraceutical compounds used as food supplements and plant-based medicine. carvone possesses antioxidant and antimicrobial properties. Its medicinal and health potentials are mainly attributed to its antioxidant, antibacterial, antifungal, anti-inflammatory, antidiabetic, insecticide, and immunomodulatory properties.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Piper nigrum is 1.6 % (w/w). It is generally known as pepper. It contains a plethora of alkaloids and related compounds, with piperine being the most abundant, followed by methyl piperine and pipernonaline. Notably, this fruit is rich in alkaloids like piperine and piplaritine, which are known to freshen breath and reduce gum swelling. Piperine, classified as an amide alkaloid, exhibits diverse properties
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Caesalpinia bonduc is 0.8 % (w/w). Caesalpinia bonduc (L.) Roxb., a pantropical leguminous scandent shrub belonging to the family Caesalpiniaceae, has been extensively studied for its diverse uses by various researchers. Commonly known as Fever Nut, this plant has yielded isolated flavonoids from its young twigs and leaves, which have demonstrated anticancer activity and high antioxidant capacity. Phytochemical studies have revealed the presence of flavonoids, alkaloids, tannins, saponins, anthracenics, quinoid derivatives, phenolics, coumarins, anthocyans, leucoanthocyans, triterpenoids, steroids, and mucilages in the root of C. bonduc. Additionally, Latakaranja, a variety of C. bonduc, has been explored phytochemically, uncovering chemical constituents such as bonducin, heptocasane, phytosterol alkaloids, flavonoids, triterpenoids, proteins, saponins, steroids, tannins, and glycosides.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Withania somnifera is 1.6 % (w/w). Ashwagandha (Withania Somnifera, WS), belonging to family Solanaceae, is an Ayurvedic herb also known as Indian winter cherry and Indian ginseng. It contains biologically active chemical constituents such as alkaloids (including isopelletierine, anaferine, cuseohygrine, and anahygrine), steroidal lactones (such as withanolides and withaferins), and saponins. Notably, Withaferin A and 3-b-hydroxy-2,3-dihydrowithanolide F isolated from Withania somnifera exhibit promising antibacterial, antitumoral, immunomodulating, and anti-inflammatory properties.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Myristica fragrans is 2 % (w/w). Commenly known as nutmeg, derived from the seed kernel of Myristica fragrans, is extensively utilized in the food industry as a culinary product. Its inflammatory properties attributed to the presence of anti-inflammatory compounds like mono terpenes, including terpene, pinene, and sabinene. The main constituents of Myristica fragrans include alkyl benzene derivatives such as myristicin, elemicin, and safrole, along with terpenes like alpha-pinene and beta-pinene, myristic acid, trimyristin, neolignan (myrislignan), and macelignan.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Nigella sativa is 3 % (w/w). Commonly known as Black seed or Kalonji, is a natural herb belonging to the family Ranunculaceae, primarily cultivated in India. The volatile oil obtained from the seeds contains various constituents such as thymoquinone, carvacrol, thymol, a- and ß-pinene, 4-terpineol, and nigellone. Nigella sativa has been found effective in attenuating the severity of chemotherapy-induced oral mucositis and improving pain and swallowing function in acute myeloid leukemia patients receiving chemotherapy.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Ocimum Sanctum is 4 % (w/w). Commonly known as holy basil, tulasi or tulsi (from Sanskrit), is an aromatic perennial plant in the family Lamiaceae. The plant and its oil contain diverse phytochemicals, including tannins, flavonoids, eugenol, caryophyllenes, carvacrol, linalool, camphor, and cinnamyl acetate, among others.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Mimosops elengi is 10 % (w/w). Commonly known as Bakula, contains active components with diverse biological and pharmacological activities. These include antibacterial, antifungal, anticarcinogenic, and cytotoxic activities. Additionally, it has been reported as having antiulcer and anti-inflammatory properties.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Acacia catechu is 5 % (w/w). It belongs to the Mimosoideae family, is renowned for its therapeutic properties in treating oral ailments such as dental caries, gingivitis, and pyorrhea. Rich in flavonoids, tannins, and phenolic compounds including catechin, epicatechin, epigallocatechin, and quercetin, it has been extensively studied for its phytochemical composition.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Cuminum cyminium is 5 % (w/w). commonly known as cumin, is a slender annual herb extensively cultivated in many regions of India, where its seeds are utilized as a significant condiment. a valuable source of iron and minerals, cumin exhibits antioxidant, antibacterial, antifungal, anti-inflammatory, antidiabetic, insecticidal, and immunomodulatory effects. Its composition includes various bioactive compounds such as monoterpene hydrocarbons, oxygenated monoterpenes, oxygenated sesquiterpenes, saturated and unsaturated fatty acids, aldehydes, ketones, and esters.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the concentration of Boerhavia diffusa is 2 % (w/w). It is commonly known as punarnava in Indian traditional medicine, and is a perennial weed categorized under the Nyctacinaceae family. The roots of this medicinal herb serve as a rich source of various bioactive compounds, including rotenoids, flavonoids, flavonoid glycosides, xanthones, purine nucleosides, lignans, ecdysteroids, and steroids. Punarnava has been recognized for its protective properties against inflammation, prostatic hyperplasia, and cancer, among other conditions.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the composition is a gel formulation. Said gel formulation can be directly applied to the affected areas in the mouth of a human suffering from OSMF.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the composition shows anti-inflammatory properties. Said composition showed down regulation of inflammation markers TNF-a and TGF–ß gene expression.
In another embodiment, the invention provides dental composition for treating OSMF, wherein, the composition shows anti-cancer properties by showing cytotoxic properties against cancerous cells.
EXAMPLES
EXAMPLE 1
CYTOTOXICITY EFFECT OF DENTAL COMPOSITION ON CANCER CELL LINES
The dental composition used for all the studies comprised of,
a) 8 % (w/w) Picrasma quassioides
b) 3.2 % (w/w) Carum carvi,
c) 1.6 % (w/w) Piper nigrum,
d) 0.8 % (w/w) Caesalpinia bonduc,
e) 1.6 % (w/w) Withania somnifera or Ashwagandha,
f) 2 % (w/w) Myristica fragrans or Nutmeg,
g) 3 % (w/w) Nigella sativa,
h) 4 % (w/w) Ocimum Sanctum or Tulsi,
i) 10 % (w/w) Mimosops elengi or Bakula,
j) 5 % (w/w) Acacia catechu or Khadira,
k) 5 % (w/w) Cuminum cyminium or Shvetajiraka,
l) 2 % (w/w) Boerhavia diffusa or Punarnava
Human oral carcinoma (KB31) cell line and human keratinocyte (HaCaT) cell line were used to test the cytotoxic effects of said dental composition. To determine the cytotoxic effects, the general procedure of MTT assay was carried using both the cell lines. MTT assay a colorimetric method used to assess cell metabolic activity and viability, based on the reduction of the yellow dye MTT to a purple formazan product by active mitochondria in living cells, which is then measured spectrophotometrically.
Determination of cell cytotoxicity by MTT Assay
The monolayer cell culture was trypsinized and the cell count was adjusted to 100,000 cells/mL using DMEM-MG (Dulbecco's Modified Eagle's Medium with high glucose (4500 mg/L) and L-glutamine) containing 10% FBS. To each well of the 96 well microtitre plate, 0.1 mL of the diluted cell suspension was added. After 24 hours, when a partial monolayer was formed, the supernatant was flicked off. The monolayer was washed once with Dulbecco's phosphate buffered saline (DPBS) and 100 µL of different test concentrations of test samples was added on to the partial monolayer in microtitre plates. The untreated cells were maintained as cell control for comparison. The plates were then incubated at 37°C with 5% CO2 for 24 h. After 24 h microscopic examination was carried out and observations was noted. Then, the test solutions in the wells were discarded and 100 µl of MTT with PBS was added to each well. The plates were gently shaken and incubated for 3 hours at 37°C in 5% CO2 atmosphere. The supernatant was removed and 100 µl of DMSO was added. Then the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 570 nm.
Table 1: In vitro cytotoxicity test of dental composition in terms of percentage cell viability against Human Keratinocyte (HaCaT) cell line by MTT assay.
Concentration (µg/mL) Percentage Cell viability after treatment (Mean ± SD) CTC50
(µg/mL)
1000 60.74 ± 1.94

>1000
500 75.34 ± 2.52
250 80.54 ± 3.38
125 87.92 ± 2.05
62.5 92.70 ± 2.26
31.25 95.12 ± 2.27
15.625 97.23 ± 0.84
7.8 98.04 ± 0.80
The cytotoxicity said dental composition was determined in terms of percentage cell viability and it was found to be 60.74 ± 1.94 % at higher concentration (1000µg/mL) with CTC50 of >1000 µg/mL on HaCaT cell line. The dental composition was found to be toxic in HaCaT cell line at higher concentrations.
Initially, the test sample was estimated for cytotoxicity at different concentrations, and two concentrations with above 80% viability at 24 hours (250 µg/ml and 125µg/ml) were identified for further analysis.
Table 2: In vitro cell - proliferative activity for the non-toxic concentration of the of dental composition in Human Keratinocytes (HaCaT) cell line.
Concentration (µg/ml) Cell proliferative activity over control (%)
250 17.42 ± 1.10
125 7.94 ± 0.98
The dental composition showed ‘17.42 and 7.94 %’ of cell proliferative activity in terms of percentage over the cell control at higher and lower non-toxic concentrations of 250 and 125µg/mL respectively, after being examined for 72 hours by cell-proliferation assay.
Table 3: In vitro cytotoxicity of dental composition in terms of percentage cell viability against Human Oral Carcinoma (KB31) cell line by MTT assay.
Concentration (µg/mL) Percentage Cell viability after treatment (Mean ± SD) CTC50
(µg/mL)
1000 43.31 ± 0.68

926.3
500 73.72 ± 1.32
250 83.98 ± 0.83
125 88.16 ± 1.02
62.5 91.65 ± 1.28
31.25 94.51 ± 0.30
15.625 97.86 ± 0.94
7.8 99.40 ± 0.30
The cytotoxicity of dental composition was determined in terms of percentage cell viability and it was found to be 43.31 ± 0.68 % at higher concentration (1000µg/mL) with CTC50 of 926.3µg/mL (0.09263%) on KB31 cell line. The dental composition was found to be toxic in Human Oral Carcinoma (KB31) cells at higher concentrations.

EXAMPLE 2
SYNERGISTIC EFFECT OF THE DENTAL COMPOSITION
To determine the synergistic effect of dental composition, the most important ingredients of the composition were tested for their cytotoxicity efficiency w.r.t the CTC-50 values and compared to the dental composition.
The Critical Threshold Concentration (CTC 50) of a drug refers to the concentration at which 50% of its maximum effect is observed. Whether a lower or higher CTC 50 is better depends on the drug’s intended use and pharmacokinetics:
• Lower CTC 50 ? Higher potency: The drug is effective at a lower concentration, which may reduce the required dosage and minimize side effects. This is generally desirable for efficiency.
50% stock solutions were prepared for Picrasma quassiodes, Caesalpinia bonduc, Withania somnitera, Myristica fragrans, Mimusops eleng. Each of the solution at different concentrations was tested for cytotoxicity properties by MTT assay in KB31 cell line following the same procedure as explained in Example 1.
Table 4: In vitro cytotoxicity of Picrasma quassiodes in terms of percentage cell cytotoxicity against Oral Cancer (KB31) cell line by MTT assay
Concentration (%) Percentage of cell toxicity (Mean ± SD) CTC 50 (%)
10 10.17 ± 3.46

49.03
8 7.34 ± 3.63
6 5.79 ± 1.06
4 3.55 ± 0.38
2 1.88 ± 0.48
Table 5: In vitro cytotoxicity of Caesalpinia bonduc in terms of percentage cell cytotoxicity against Oral Cancer (KB31) cell line by MTT assay.
Concentration (%) Percentage of cell toxicity (Mean ± SD) CTC 50 (%)
1 9.22 ± 0.35

4.99
0.8 7.39 ± 0.35
0.6 5.93 ± 0.27
0.4 3.56 ±0.38
0.2 1.10 ± 0.14
Table 6: In vitro cytotoxicity of Withania somnitera in terms of percentage cell cytotoxicity against Oral Cancer (KB31) cell line by MTT assay.
Concentration (%) Percentage of cell toxicity (Mean ± SD) CTC 50 (%)
1.8 3.57 ± 0.75

17.20
1.6 2.32 ± 0.54
1.4 1.99 ± 0.81
1.2 1.45 ± 0.34
1.0 1.31 ± 0.28

Table 7: In vitro cytotoxicity of Myristica fragrans in terms of percentage cell cytotoxicity against Oral Cancer (KB-3-1) cell line by MTT assay
Concentration
(%) Percentage of cell toxicity (Mean ± SD) CTC 50 (%)
12 14.97 ± 6.15

16.62
10 9.60 ± 3.14
8 6.09 ± 1.07
6 5.42 ± 0.24
4 4.86 ± 0.14
Table 8: In vitro cytotoxicity of Mimusops elengi in terms of percentage cell cytotoxicity against Oral Cancer (KB-3-1) cell line by MTT assay
Concentration (%) Percentage of cell toxicity (Mean ± SD) CTC 50 (%)
12 12.01 ± 1.78

53.14
10 10.17 ± 1.38
8 8.19 ± 1.89
6 6.57 ± 1.14
4 4.58 ± 1.95
FIG.1 compares the CTC50 (%) values of 8% Picrasma quassiodes, 0.8% Caesalpinia bonduc, 1.6% Withania somnitera, 10% Myristica fragrans, 10% Mimusops elengi, and the dental composition comprising of 8% Picrasma quassiodes, 3.2 % (w/w) Carum carvi, 1.6 % (w/w) Piper nigrum, 0.8 % (w/w) Caesalpinia bonduc, 1.6 % (w/w) Withania somnifera or Ashwagandha, 2 % (w/w) Myristica fragrans or Nutmeg, 3 % (w/w) Nigella sativa, 4 % (w/w) Ocimum Sanctum or Tulsi, 10 % (w/w) Mimosops elengi or Bakula, 5 % (w/w) Acacia catechu or Khadira, 5 % (w/w) Cuminum cyminium or Shvetajiraka, and 2 % (w/w) Boerhavia diffusa or Punarnava. The data clearly shows that the CTC50 (%) of the combination of the ingredients in the dental composition is significantly less compared to the CTC50 (%) of the important ingredients individually.
EXAMPLE 3
ANTI-INFLAMMATORY ACTIVITY OF DENTAL COMPOSITION
Tumor Necrosis Factor – alpha (TNF-a) is a key player in the inflammatory process it is a type of cytokine which is a signalling protein produced by immune cells like macrophages. TNF-a helps regulate the immune response by promoting inflammation. Tumor Growth Factor – beta (TGF–ß) is a critical regulator of immune system by balancing inflammation and immune tolerance. TGF–ß Promotes inflammation in the presence of interleukin-6 (IL-6) and further induces the differentiation of T-helper 17 (Th17) cells, which are associated with auto immune and inflammatory diseases.
Anti-inflammatory effect of the dental composition also referred to as SQUA CARE GEL was determined by quantitative RT-PCR technique using gene specific primers. The qRT-PCR analysis at 500 µg/mL and 250 µg/mL revealed the modulatory effect of SQUA CARE GEL on the mRNA expression of TNF-a and TGF–ß genes over lipopolysaccharide (LPS) induced cells. LPS induces inflammation, primarily through the activation of TLR4, leading to the release of pro-inflammatory cytokines like TNF-a, while TGF-ß, a cytokine with anti-inflammatory and anti-fibrotic properties, can be upregulated in response to LPS, potentially modulating the inflammatory response
The HaCaT cells, and the KB31 cells were treated with dental composition (SQUA CARE GEL) and subjected to cell lysis followed by RNA isolation and cDNA synthesis by general protocol known in prior art.
The mRNA expression levels of TNF-a and TGF–ß genes were measured by qRT-PCR using CFX Opus 96 Real time PCR system (BIO RAD) which directly detects the RT-PCR products without downstream processing. This is achieved by monitoring the increase in fluorescence of a dye labelled DNA probe, in which one is specific for the gene of interests and another is specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, it is used as an endogenous assay control. 20µL of the reaction mixture was subjected to PCR for amplification of genes by using cDNA and specifically designed primers. The endogenous control probe, specific for the GAPDH gene, served to standardize the amount of RNA samples. The 40 cycles of two step PCR reactions consist of a 5 min period at 95°C followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing Tm for 30 seconds and extension at 72°C for 45 seconds. This was followed by final extension at 72°C for 10 min.
As depicted in FIG. 2 and FIG. 3, when HaCaT cells are treated with LPS, the cells show high expression of mRNA of TNF-a and TGF–ß. Whereas, when the cells are treated with positive control drug, Dexamethasone, the inflammatory genes are less expressed. Dexamethasone is a synthetic glucocorticoid known to reduce the release and expression of both TNF-alpha (a pro-inflammatory cytokine) and TGF-beta (a cytokine with both pro and anti-inflammatory roles), modulating innate immunity and potentially inhibiting fibrosis. The dental composition (SQUA CRE GEL) significantly reduces the expression of TNF-a, comparable to Dexamethasone, and reduces the expression of TGF–ß more efficiently than Dexamethasone.
Similarly, as depicted in FIG. 4 and FIG. 5, when KB31 cells are treated with LPS, the cells show high expression of mRNA of TNF-a and TGF–ß. Whereas, when the cells are treated with positive control drug, Dexamethasone, the inflammatory genes are less expressed. Dexamethasone is a synthetic glucocorticoid known to reduce the release and expression of both TNF-alpha (a pro-inflammatory cytokine) and TGF-beta (a cytokine with both pro and anti-inflammatory roles), modulating innate immunity and potentially inhibiting fibrosis. The dental composition (SQUA CRE GEL) significantly reduces the expression of TNF-a, comparable to Dexamethasone, and reduces the expression of TGF–ß more efficiently than Dexamethasone.
These results clearly show that the dental composition has the anti-inflammatory properties.
EXAMPLE 4
Toxicity/Safety Test of the Dental Composition by acute oral administration in adult healthy female rats
The present study was conducted in rats (Female Sprague Dawley) by using OECD 423 guidelines. In the acute toxicity study, rats were fasted prior to dosing (without food but not water over-night). Followed the period of fasting, the animals were weighed and the dental composition was administered at a starting dose level of 2000 mg/kg bodyweight of animal in a single dose orally. After administration of the dental composition, food was withheld for further 3-4 hours in rats. After administration, animals were observed first 4 hours, then periodically for 24 hours and daily thereafter, for a total of 14 days.
Animals at a starting dose of 2000 mg/kg weight didn’t show any mortality.
All the animals were observed to be normal throughout the experimental period.
As shown in Table 9, during the study period the dental composition treated animals body weights were significantly increased when compared to day 0 bodyweight of animals. Increased body weight in animals during the study is a normal pattern with healthy animals.
This showed that the dental composition did not have any adverse effect on the health of the animals and was safe to use.
Table 9: Body weight of rats during the study

Animal ID

Dose
Treatment
Before After
Day 0 Day 3 Day 7 Day 14
RA 01 RR421000
2000 mg/kg
(Limit test) 188.4 192.7 195.8 200.1
RA 02 186.3 190.1 194.7 199.4
RA 03 185.5 189.5 193.6 198.8
Mean 186.7 190.8 194.7 199.4
SD 1.5 1.7 1.1 0.65
RA 04 RR241000
(2000 mg/kg)
(Main Test) 185.4 189.6 193.6 199.7
RA 05 186.1 190.1 194.2 200.1
RA 06 185.4 189.7 193.7 198.2
Mean 185.6 189.8 193.8 199.3
SD 0.4 0.26 0.32 1
At the end of the treatment period (Day15), all the animals were sacrificed by using Isoflurane and subjected to gross pathological examination. FIG. 6 depicts the photographs of liver, kidney, spleen, and heart of one of the tested animals which are clearly normal and do not show shrinkage or other abnormalities. The median lethal dose of dental composition is more than 2000 mg/kg body weight. The lethal dose value of dental composition in female rats after single oral treatment of above 2000 mg/kg body weight was found to be non-toxic and is classified as Category 5.
EXAMPLE 5
Treatment of OSMF
The effect of the dental composition in treating oral sub-mucosal fibrosis (OSMF) was studied by oral application of the dental composition in the affected mouth areas of patients.
A 48-year-old male suffering from Stage III OSMF with reduced mouth opening, 25 mm mouth opening, was treated with the dental composition on both right and left buccal mucosa thrice a day and recalled after 2 weeks. After 2 weeks the mouth opening increased to 35 mm.
Another 69-year-old male suffering from Stage III OSMF with reduced mouth opening, 23 mm mouth opening, was treated with the dental composition on on both right and left buccal mucosa thrice a day and recalled after 2 weeks. After 2 weeks the mouth opening increased to 36 mm.
Table 10: Effect of treatment with the dental composition for two weeks on mouth opening ability of Stage III OSMF patients
S. No. Pre-treatment mouth opening capacity Post-treatment mouth opening capacity % increase in mouth opening capacity
1. 25 mm 35 mm 40 %
2. 23 mm 36 mm 56 %
This shows that the dental composition is effective in enabling a patient suffering with oral sub-mucosal fibrosis to increase his/her mouth opening capacity.
,CLAIMS:We claim,
1. A dental care composition comprising of:
a) Picrasma quassioides,
b) Carum carvi,
c) Piper nigrum,
d) Caesalpinia bonduc,
e) Withania somnifera,
f) Myristica fragrans,
g) Nigella sativa,
h) Ocimum Sanctum,
i) Mimosops elengi,
j) Acacia catechu,
k) Cuminum cyminium,
l) Boerhavia diffusa.
2. The dental care composition as claimed in claim 1, wherein, the concentration of Picrasma quassioides is 8 % (w/w).
3. The dental care composition as claimed in claim 1, wherein, the concentration of Carum carvi is 3.2 % (w/w).
4. The dental care composition as claimed in claim 1, wherein, the concentration of Piper nigrum is 1.6 % (w/w).
5. The dental care composition as claimed in claim 1, wherein, the concentration of Caesalpinia bonduc is 0.8 % (w/w).
6. The dental care composition as claimed in claim 1, wherein, the concentration of Withania somnifera is 1.6 % (w/w).
7. The dental care composition as claimed in claim 1, wherein, the concentration of Myristica fragrans is 2 % (w/w).
8. The dental care composition as claimed in claim 1, wherein, the concentration of Nigella sativa is 3 % (w/w).
9. The dental care composition as claimed in claim 1, wherein, the concentration of Ocimum Sanctum is 4 % (w/w).
10. The dental care composition as claimed in claim 1, wherein, the concentration of Mimosops elengi is 10 % (w/w).
11. The dental care composition as claimed in claim 1, wherein, the concentration of Acacia catechu is 5 % (w/w).
12. The dental care composition as claimed in claim 1, wherein, the concentration of Cuminum cyminium is 5 % (w/w).
13. The dental care composition as claimed in claim 1, wherein, the concentration of Boerhavia diffusa is 2 % (w/w).
14. The dental care composition as claimed in claim 1, wherein, the composition has a pH of 5.5-6.5.
15. The dental care composition as claimed in claim 1, wherein, the composition has anti-inflammatory properties by down regulation of tumour necrosis factor-alpha (TNF-a) and transforming growth factor-beta (TGF–ß) gene expression.
16. The dental care composition as claimed in claim 1, wherein, the composition in a gel formulation induces cytotoxic effects against cancerous cells.
17. The dental care composition as claimed in claim 1, wherein, the composition is a gel formulation for topical oral application.
18. The dental care composition as claimed in claim 1, wherein, the composition in a gel formulation has application in treating oral sub-mucosal fibrosis.
19. The dental care composition as claimed in claim 1, wherein, the composition in a gel formulation is non-toxic and safe for oral administration.