Oral leukoplakia (OL) is the most common potentially malignant disorder of the oral mucosa.  Leukoplakia may occur anywhere on the oral mucosa and its prevalence varies from 0.2% to 5.2% of the Indian population.  The tobacco chewing habit both in smoking and smokeless form is the most common cause for oral leukoplakia. The tobacco consumption has progressively increased in younger generation. The other factors predisposing to leukoplakia include infections like candida albicans, HPV (16 and 18), atrophic epithelial lesions, persistent trauma and electrogalvanic current. [2,3]
Many oral squamous cell carcinomas develop from potentially malignant disorders.  It has been reported that between 16% and 62% of oral squamous cell carcinomas are associated with oral leukoplakia.  Early diagnosis and treatment of these potentially malignant disorders is required to prevent their progression to malignancy.  Hence, it is important for the clinician to diagnose leukoplakia in its initial stages and manage effectively.
Leukoplakia can be treated by medical and surgical therapies. The first line of treatment is cessation of any habit or removal of irritational agent. Medical line of treatment uses chemopreventive agents in the form of topical application or systemic administration such as local bleomycin, retinoids, systemic beta carotene, lycopene, and a mixture of green tea used both topically and systemically based on its low cost and ease of application. [1,6]
Current treatment modalities include cessation of tobacco chewing habit and lycopene as a powerful antioxidant, a member of carotenoid family of phytochemicals obtained from tomatoes, other red fruits and vegetables (red carrots, watermelons, papayas).  Lycopene exhibits the highest physical quenching rate constant with singlet oxygen, thereby known for its antioxidizing property.  The conjugated polyene structure of lycopene provides an electron-rich system which neutralizes harmful free radicals and prevent cell death.  Tomato products and lycopene consumption reduces the risk of upper aero-digestive tract cancers of oral cavities, pharynx, larynx and oesophagus.  The ability of Lycopene is supposed to modify intercellular exchange junctions, thereby effective in potentially malignant disorders. 
Many herbal medicines have been tried in management of oral diseases including premalignancies. Calendula officinalis is one among the herbal medicines which has the potential antioxidant property. Calendula officinalis also known as pot marigold is an annual herb belonging to family Asteraceae.  Pharmacological studies have confirmed that Calendula officinalis has a broad range of biological activity such as antimicrobial, anti-inflammatory, immunomodulatory, wound healing, antiviral and anti-tumoral property. 
Calendula officinalis extracts can be applied externally for the treatment of skin ulcers, conjunctivitis and ulcers. [14,15] The internal use of calendula officinalis also provides relief for pain arising from stomach ulcers and inflammation [14,15] Its cytotoxic effect on tumor cells taken as an in vitro study was outlined 20 years ago.  Calendula is effective in preventing acute dermatitis in patients of cancer undergoing postoperative irradiation. 
The properties of Calendula officinalis are shown to control the bacterial growth into biofilm, against periodontopathogenic bacteria, and oral inflammatory processes that require healing intervention.  Plant is reported to have hepatoprotective and hypoglycemic activity due to its antioxidant property.  Research has shown that Calendula officinalis contains flavonoids, polyphenols, lutein, flavoxanthin, lycopene, auroxanthin, beta carotene which possesses antioxidant property  and hence can be utilized in treatment of leukoplakia.
In the light of above properties of Calendula officinalis, this study was proposed to assess the effectiveness in management of homogenous leukoplakia and also as a cost effective treatment modality in comparison with lycopene gel for treatment of homogenous leukoplakia.
Review of Literature
The term “leuko” means white and “plakia” means plaque/patch in Greek. Hence, the literal meaning of the term leukoplakia is a “white plaque”. The first definitive terminology associated with leukoplakia to describe a white lesion was given by Schwimmer in 1877. [21,22]
- Pindborg (1972) defined leukoplakia as a white patch or plaque on the mucous membrane that cannot be removed by rubbing and cannot be classified as any other diagnosable disease. 
- WHO (1978) defined leukoplakia as “a white patch or plaque that cannot be characterized clinically or pathologically as any other disease”. 
- Axell et al. (1984) defined leukoplakia as whitish patch or plaque that cannot be characterized clinically or pathologically as any other disease and is not associated with any physical or chemical causative agent except for the use of tobacco. 
- Bouquot JE (1994) defined leukoplakia as a chronic white mucosal macule which cannot be scraped off, cannot be given another specific diagnostic name and does not typically disappear with removal of known etiologic factors. 
- Axell et al. (1996) defined leukoplakia as predominantly white lesion of the oral mucosa that cannot be characterized as any other definable disease. 
- WHO (1997) defined as predominantly white lesion of the oral mucosa that cannot be characterized as any other definable lesion. 
- Warnakulasuriya S et al. (2007) – defined this lesion as “a white plaque with an increasing questionable oral cancer risk after excluding other known diseases and disorders that do not increase the risk. 
Incidence and Prevalence
Leukoplakia is a potentially malignant disorder with a prevalence varying from 0.2% to 5.2% in Indian subcontinent. The overall rate of conversion to malignancy for leukoplakia ranges from 0.1% to 17%.  In India, malignant transformation ranges between 0.13% and 10%. .
Warnakulasuriya et al. listed the following factors which are associated with increased risk for malignant transformation in potentially malignant disorders. 
- Female gender
- Long duration of leukoplakia
- Leukoplakia in non-smokers (idiopathic leukoplakia)
- Location on the tongue and/or floor of the mouth
- Size >200 mm2
- Non-homogeneous type
- Presence of C. albicans
- Presence of epithelial dysplasia
Age and Gender
Oral leukoplakia is most commonly found in men, and its prevalence increases with age. It mainly affects men over 40 years. 
The etiopathogenesis of tobacco has been related to the following factors which are classified as exogenous and endogenous agents.  The lesion develops due to the influence of exogenous factors such as smoking, local irritants, occlusal disharmony, bacterial products, galvanic irritations etc. and also endogenous factors which include heredity, hormonal factors, nutritional factors etc.
- Tobacco – Leukoplakia occurs more frequently in tobacco chewers as compared to tobacco smokers.  A dose response relationship has been reported between tobacco usage and the prevalence of oral leukoplakia. The most common form of tobacco chewing in India is pan with betel quid (smokeless tobacco).
- Alcohol – Drinking alcohol is an independent risk factor for oral leukoplakia, regardless of beverage type or drinking pattern.  Its effect may be synergistic to other well-known etiological factors (physical irritants).
- Candidiasis – About 10% of oral leukoplakia satisfy the clinical and histological criteria for chronic hyperplastic candidiasis (candidal leukoplakia). Epithelial dysplasia is reported to occur four to five times more frequently in candidal leukoplakia.  This change is more common in the speckled variant than in homogeneous leukoplakia. In patients of speckled leukoplakia with superimposed candida infection carcinomatous changes are more commonly seen.
- Viral agents – The contributory role of viral agents (human papilloma virus strains 16,18) in the pathogenesis of oral leukoplakia is seen with regard to exophytic verrucous leukoplakia. 
- Deficiencies – Deficiency of serum levels of vitamin A, B12, C, beta-carotene, and folic acid has also been suggested as predisposing factors in development of oral leukoplakia.
Classification of Leukoplakia
Oral Leukoplakia has been classified in two main types by Warnakulasuriya S et al (2007) :
- Homogeneous leukoplakia (also termed “thick leukoplakia”) is a well-defined white patch of uniform, flat appearance and texture. (Fig. 1) It is slightly elevated compared to surrounding mucosa, and often has a fissured, wrinkled or corrugated surface texture, with the texture generally consistent throughout the whole lesion. 
- Non homogenous leukoplakia is a lesion of non-uniform appearance. They are classified into speckled, nodular and verrucous type.
- Verrucous type is a thick, keratinized non-homogenous lesion with an elevated, proliferative or corrugated surface appearance. (Fig. 2)
- The nodular type is a variant of non-homogenous leukoplakia characterized by pinhead-sized white nodules on an erythematous base. (Fig. 3)
- The speckled type is a non-homogenous lesion of mixed white (keratotic) and red (atrophic) color. (Fig. 4)
Banoczy , distinguished three types of oral leukoplakia:
- Leukoplakia simplex comprises of 50% of cases of leukoplakia and is a white, homogenous, keratinized lesion, slightly elevated from the surface of oral mucosa.
- Leukoplakia verrucosa comprises of 27% of cases of leukoplakia which include white, verrucous proliferations with wrinkled surface, and
- Leukoplakia erosive that comprise 17% of leukoplakia and is identical with erythro leukoplakia type of leukoplakia. It presents as white lesions with erythematous areas, erosions, fissures.
Types of Oral leukoplakia
- Fig 1 : Homogenous leukoplakia
- Fig 2: Verrucous leukoplakia
- Fig. 3 : Nodular Leukoplakia
- Fig. 4 : Speckled Leukoplakia
Oral leukoplakia has been classified by Pindborg et al  as:
Proliferative verrucous leukoplakia (PVL) is a distinct clinical form of oral leukoplakia. PVL has a high rate of malignant transformation which has been described by the WHO. It is multifocal progressive lesions, found most commonly in women. The sites most commonly involved are lower gingiva, tongue, buccal mucosa, and alveolar ridge. 
Oral erythroleukoplakia (OEL) is a non-homogenous lesion of mixed white and red components. It a fiery red patch that cannot be characterized clinically or pathologically as any other definable disease. OEL demonstrates a higher rate of malignant transformation s compared to homogeneous leukoplakia. 
Sublingual keratosis is a soft white plaque in the sublingual region with a wrinkled surface, an irregular but well-defined outline and sometimes a butterfly shape. 
Candida leukoplakia (CL) is a chronic, discrete elevated lesion. The clinical presentation of candida leukoplakia ranges from translucent, whitish area to large, dense, opaque plaque which is hard and rough to touch. 
Oral hairy leukoplakia (OHL) is a white patch with a corrugated or hairy surface and most commonly present on the lateral borders of the tongue. It is caused by the reactivation of a previous Epstein-Barr virus infection. 
Management of Leukoplakia
- Elimination of risk factors – In managing Leukoplakia, the risk factors must be eliminated such as tobacco abuse, alcohol, superimposed candida infection over the lesion.
- Habit counseling – Counseling alone or counseling with medication is used for tobacco cessation and should be started on the day of diagnosis for leukoplakia.
- Nicotine substitutes – Nicotine Patch (Nicotinell and Novartis – Transdermal patches), Nicotine chewing gums (Nicorette), Nicotine spray and Nicotine inhaler are used to stop the habit of tobacco usage. 
- Biopsy – Biopsy is recommended in cases of lesion which dont regress in size even after the cessation of tobacco habit. Most of the cases of leukoplakia with less than 2cms of size, excisional biopsy is indicated. Conservative treatment is advisable in patients with leukoplakia with no signs of dysplasia irrespective of clinical form of the lesion.  In cases of mild moderate or severe dysplasia, both conservative and surgical methods have shown improvement in treating leukoplakia.
- Surgical treatment – Conventional surgery or laser ablation, electrocauterization or cryosurgery and photodynamic therapy are effective in eradicating OL or preventing eventual malignancy.
- Conservative treatment – The use of chemopreventive agents such as vitamins, green tea, lycopene, protease inhibitor and anti-inflammatory drugs are helpful in resolving the lesion.
Chemoprevention can be defined as the use of specific natural or synthetic chemical agents to reverse, suppress, or prevent carcinogenesis before the development of invasive malignancy. 
Various chemopreventive agents have a protective role against cancer development and its progression. They include antioxidants, carotenoids – retinoids, Beta – carotene, lycopene, vitamin C and vitamin E, protease inhibitors, green tea and NSAIDS. 
Wattenberg (1985)  has classified chemopreventive agents based on mechanism of action into three broad categories:
- Inhibitors of carcinogen formation includes the endogenous formation of N-nitroso carcinogens which are inhibited by ascorbic acid and tocopherol.
- Blocking agents prevent the reaction of carcinogens with cellular targets such as aryl alkyl isothiocyanates.
- Suppressing Agents inhibit the promotion and progression stages of carcinogenesis. The use of retinoids and carotenoids(Lycopene), anti-inflammatory drugs, protease inhibitors have shown reduced incidence of cancer.
Tomatoes, watermelon, guava, rosehips, pink grapefruit and apricots are major source of lycopene compounds. (Table 1) Lycopene is considered as major source of carotenoids in diet and also provides red color to tomato products.
Lycopene content of common fruits and vegetables (Table 1) 
Lycopene content [mg/100 g wet basis] / Source
- 0.7-20 Fresh Tomatoes
- 3.7 Cooked Tomatoes
- 2.3-7.2 Fresh Watermelon
- 2-5.3 Fresh Papaya
- 5,2-5.5 Pink Guava
- 0.4-3.4 Pink Grapefruit
- 0.01-0.05 Apricot
Chemical properties of lycopene  – Lycopene is a representative of the hydrocarbon carotenoids with the molecular formula of C40H56, has an acyclic open-chain structure consisting of 13 double-bonds. The conjugated polyene structure is responsible for the antioxidant properties of lycopene. The most common isomer of lycopene found in foods is all-trans-isomer, which is also the thermodynamically most stable configuration.  The carotenoids present in lycopene are divided into two main groups –
- Highly unsaturated hydrocarbons which consists of lycopene, α-, β-, and γ-carotene. They contain carbon- and hydrogen-atoms, but lack oxygen.
- Xanthophylls consists of at least one oxygenated group on their terminal rings of β-cryptoxanthin, lutein, and zeaxanthin.
Bioavailability and Metabolism of Lycopene
The absorption of lycopene from dietary sources occurs in the range of 10 to 30% in humans.  The dietary sources of lycopene are ingested and is taken up by dietary lipid micelles and incorporated into the mucosa of the small intestine. (Fig. 5) The micelles are packaged into chylomicrons, which are then transported to the liver using the lymph system. Lycopene molecules are distributed into lipoprotein fractions and are carried to the plasma, from where they are distributed to the target organs. 
Fig. 5 – Absorption and metabolism of lycopene
Studies have shown that absorption from processed tomato products is better than from raw tomatoes. The bond between lycopene and macromolecules within the food-matrix is comparatively strong, so the bioavailability from dietary sources is remote. Processing of the food like cooking or chopping by which the bioavailability can be enhanced. 
Two major hypotheses have been proposed to explain the anticarcinogenic and anti atherogenic activities of lycopene  :
Non oxidative mechanisms
Non – oxidative mechanism
- Antiproliferative and prodifferentiation activities  – Lycopene inhibits the proliferation of cancer cells including those in cells of breast, lung and endometrium. Lycopene suppresses the carcinogen-induced phosphorylation of regulatory proteins such as p53 and Rbanti oncogenes and stops the cell division at Go-G 1 phase of the cell cycle.
- Hypocholesterolemic effects  – Lycopene acts as a hypocholesterolemic agent by inhibiting macrophage 3- hydroxy-3-methyl glutaryl co-enzyme A (HMGCoA) reductase, the rate-limiting enzyme in cholesterol synthesis. Mohitpal et al concluded that it can be used for the prevention of heart disease and supplementation of carotenoids like lycopene may help to prevent common forms of cancer. Many of the epidemiological studies are concerned with the use of lycopene in prevention of cardiovascular disease and cancer. (Fig. 6)
Fig. 6 – Use of lycopene in prevention of cardiovascular disease and cancer
Recently, lycopene has been found to inhibit proliferation of cancer cells those present in areas of breast, prostate, lung, and endometrium. It is also helpful in neurodegenerative disease, and bone disorders.
- Antioxidative activity  – Oxidative stress is one of the major contributors of increased risk of cancer and lycopene being the most potent antioxidant among various carotenoids can be helpful in treatment of oxidative stress. Lycopene basically traps the singlet oxygen and reduce mutagenesis.
- Mechanism of quenching of reactive oxygen by lycopene  – The quenching of oxygen by lycopene occurs through physical or chemical means. Physical quenching of lycopene predominates leaving the carotenoid part intact, whereas chemical quenching is responsible for the decomposition of the carotenoid (bleaching). Physical quenching involves the transfer of excitation energy from oxygen to the carotenoid, thereby producing ground state oxygen and a carotenoid in the excited triplet state. The excess energy is dissipated as heat through rotational and vibrational interactions with the surrounding solvent and structures. The regeneration of the ground state carotenoids allows it to again function as a catalyst and undergo additional cycles of oxygen quenching.
Lycopene has beneficial effects in the treatment of certain diseases of oral cavity including oral cancer, precancerous lesions and periodontal diseases. Oral submucous fibrosis, leukoplakia, and oral lichen planus are also successfully treated with lycopene supplements.
Review studies of lycopene and leukoplakia
Jyotsna Patel et al (2014) conducted a randomized control trial to evaluate the efficacy of lycopene in combination with vitamin E and selenium in the treatment of oral leukoplakia. Patients receiving lycopene in combination with vitamin E and selenium showed statistically significant improvements both clinically and histologically as compared to those receiving placebo. 
T.N. Uma Maheswari (2013) carried out a systematic review to evaluate all the clinical trials using antioxidants namely Vitamin A, E, C, and lycopene as they are the most commonly used antioxidants in the treatment of oral leukoplakia. It was concluded that lycopene is a potential treatment option for the treatment of Oral leukoplakia. 
Singh et al (2004) conducted a double-blind, placebo-controlled randomized controlled trail to evaluate the efficacy of lycopene in the treatment of oral leukoplakia. Comparison was carried out using two different doses of lycopene with placebo. Lycopene supplementation in the doses of 4mg and / or 8mg per day demonstrated reduction in hyperkeratosis in 80% of cases in the duration of three months. Complete remission of the lesion was noted in 55% of cases in the doses of 8mg/day and in 25% of cases in the doses of 4mg/day. 
Zakrzewska et al (2005) conducted a randomized clinical trial and demonstrated improvement in the patients of oral leukoplakia by the treatment with 4 mg and / or 8 mg lycopene per day over a period of 3 months. In patients treated with 8mg lycopene per day, positive histologic changes were also noted. 
Nagao et al (2000) conducted a case-control study to investigate the association between serum micronutrient levels and oral leukoplakia. The present study was conducted in 48 patients of oral leukoplakia and also in 192 control patients. Along with other antioxidants, the serum level of lycopene was also found to be significantly lower in cases as compared to control group. Thus, it was concluded that improvement of micronutrient levels of lycopene and other antioxidants may protect against the relative risk of oral leukoplakia. 
Gupta et al (1998) investigated the relationship of specific nutrients and food items with oral precancerous lesions among tobacco users. While investigating the relationship of specific nutrients and food items with oral precancerous lesions, it was noted that consumption of tomato the main source of lycopene – has protective effect in oral leukoplakia. 
Calendula officinalis (Pot Marigold or English Marigold) is a plant in the Calendula genus (Table 2) which belongs to the Asteraceae family and is commonly known as Zergul (Hindi). 
Taxonomic classification of calendula officinalis  [Table 2]
- Kingdom Plantae
- Subkingdom Tracheobionta
- Division Magnoliophyta
- Class Magnolopsida
- Subclass Asterridae
- Order Asterales
- Family Asterceae
- Tribe Calenduleae
- Genus Calendula
- Species Calendula officinalis
Calendula officinalis is an annual plant, seldom biennial. It grows 30 – 50 cm high, and has about 20 cm long tap root and numerous thin, secondary roots. (Fig 7) The stem is erect, angular, downy and branched from the base up or higher.  The petals and pollen contain triterpenoid esters which has an anti-inflammatory property while the carotenoids, flavoxanthin and auroxanthin has got the antioxidant properties. The leaves and stems contains carotenoids, mostly lutein (80%) and zeaxanthin (5%), and beta-carotene. 
fig. 7 – Calendula officinalis plant
Chemical constituents of Calendula officinalis 
Flavonoids – Various flavonoids have been isolated from the ethanol extract of the inflorescence of Calendula officinalis which are responsible for antioxidant and wound healing properties. They include quercetin, isorhamnetin, isoquercetin, narcissin, calendoflaside, calendoflavoside, calendoflavobioside, rutin, isoquercitrin neohesperidoside, isorhamnetin-3-Orutinoside, quercetin-3-O-glucoside and quercetin-3-O-rutinoside. [65,66]
Carotenoids – The methanol extract of leaves, petals and pollens of C. officinalis flowers showed a number of carotenoids which are responsible for antioxidant (Table 3) and wound healing properties. 
Carotenoid composition of Calendula officinalis  [Table 3]
Carotenoid Constituents % of total carotenoids
- (8’R) – Luteoxanthin 11
- Flavoxanthin 28.5
- (8R,8’R) – Auroxanthin 7.1
- (9’Z) – Lutein-5,6-Epoxide 5.0
- Lutein 2.0
- Antheraxanthin 1.0
- (9Z) – Lutein 0.6
- (5’Z,9’Z) – Rubixanthin 4.0
- Alpha – Carotene 0.8
- Beta – Carotene 3.4
- (5’Z) – Rubixanthin 3.0
- Delta – Carotene 1.4
- (5Z,9Z,5’Z,9’Z) – Lycopene 4.1
- Gamma – Carotene 2.0
- (5’Z) – Gamma – Carotene 4.4
- (5Z,9Z,5’Z) – Lycopene 3.5
- (5Z,9Z) – Lycopene 4.1
- (all -E) – Lycopene 8.7
- Lutein-5,6 -epoxide 1.6
- Terpenoids – Various terpenoids have been reported from the petroleum ether extract of Calendula officinalis flowers. They include sitosterols, stigmasterols, diesters of diols, 3-monoesters of taraxasterol, lupeol, erythrodiol, brein, ursadiol, faradiol-3-O-palmitate, aradiol-3-O-myristate, faradiol-3-O-laurate, arnidiol-3-O-palmitate, arnidiol-3-O-myristate, arnidiol-3-O-laurate, calenduladiol-3-O-palmitate. [68,69]
- Coumarins -The ethanol extract of the inflorescence of the Calendula officinalis has been reported to contain coumarins – scopoletin, umbelliferone and esculetin. 
- Quinones – Quinones reported from Calendula officinalis are plastoquinone, phylloquinone, alpha – tocopherol in the chloroplast, ubiquinone, phylloquinone, alpha – tocopherol in mitochondria, and phylloquinone in the leaves. 
- Amino acids – The ethanol extract of flowers of the plant has been reported to show the presence of 15 amino acids in free form: alanine, arginine, aspartic acid, asparagine, valine, histidine, glutamic acid, leucine, lysine, proline, serine, tyrosine, threonine, methionine and phenylalanine. Amino acid content of the leaves is about 5 %, stems 3.5 % and flowers 4.5 %. 
Calendula officinalis and its properties 
The plants of Calendula officinalis are rich in flavonoids, especially aglycons and glycosides of flavonols (isorhamnetin, quercetin), saponosides, lipids (including sterols and carotenoids), organic acids and saccharides.  Other flavonoids include lupeol, quercetin, protocatechuic acid, many alkaloids and triterpenoids, flavoxanthin, luteoxanthin, lycopene, auroxanthin, lutein, beta carotene and phenolic compounds are present in this flower.  The residual aqueous extract was assayed for antioxidant activity by liposomal lipid peroxidation – induced Fe2+ and ascorbic acid. Antioxidant activity was demonstrated by ether, butanol and water extract which contained flavonoids. Antioxidant activity assay for propylene glycol extract of petal and flower heads showed that the extracts of petals were potent than the head extracts.
Based on in vitro granulocyte test, immunostimulant property was demonstrated by polysaccharide(PS) fraction of calendula officinalis. highest phagocytosis (54-100%) was seen at a concentration of 10-5 -10-6 mg/mL for PS I, while PS-I and PS-II exhibited 40-57 and 20-30% phagocytosis respectively. 
Activity-guided isolation of fraction of methanolic extract of C. officinalis has shown cytotoxic activity in vitro and the active compounds isolated are calenduloside F6′-O-n-butyl ester, which is active against leukemia, colon cancer and melanoma, cell lines with GI50 values of 0.77-0.99 μmole, except for leukemia, renal cancer and breast cancer cell lines; and calenduloside G6′-O-methyl ester, which is active against all the cancer cell lines mentioned above with GI50 of 20 μmole except for ovarian cancer and renal cancer cell lines. 
Aqueous laser-activated calendula flower extract (LACE) showed potent in vitro inhibition of tumour cell proliferation when assayed against a wide variety of human and murine tumour cell lines.
Wound healing Activity
The ethanol extract of the plant’s flowers was investigated in experimentally induced thermal burns in rats. The level of acute phase proteins (heptaglobin, orosomycid) and tissue damage marker enzymes – alkaline phosphatase, alanine and aspartate transaminase were found to be significantly decreased. Decrease in the levels of lipid peroxidation was attributed to its antioxidant property. 
The hydroalcohol extract of the flowers, when given to CCl4-intoxicated liver in albino male Wistar rats at a dose of 10 mL/kg, resulted in a reduction of hepatocytolysis by 28.5 % due to reduction in glutamo-oxalate-transaminase (GOT) and glutamo-pyruvate-transaminase (GPT).  However, histoenzymology showed reduction of steatosis of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), cytochromoxidase (Cyox) and Mg2+ dependant adenosine triphosphatase (ATPase).
Anti-inflammatory and Anti-oedematous Activities
Activity-guided isolation of calendula officinalis flowers has demonstrated that the anti-inflammatory activity of the flower is mainly due to oleananetype triterpene glycoside, presence of triterpenoids, the three most active compounds of which were the esters of faradiol-3-myristic acid, faradiol-3-palmitic acid and 4-taraxasterol. Dichloromethane extract of the plant’s flower heads inhibited croton oil-induced oedema and further isolation showed that the esters of faradiol-myristic acid, faradiol-palmitic acid had antioedematous activity with an oedema inhibition of nearly 50 % at a dose of 240 μg/cm2.
Dichloromethane-methanol extract of C. officinalis flowers exhibited potent anti-HIV activity in in vitro MTT/tetrazolium-based assay. This activity was attributed to inhibition of HIV1-RT at a concentration of 1,000 μg /mL as well as suppression of the HIV mediated fusion at 500 μg/mL. 
Antibacterial and Antifungal properties
The methanol extract and 10 % decoction of the plant’s flowers were assessed for their activity against anaerobic and facultative aerobic periodontal bacteria. The results showed marked inhibition against all tested microorganisms with MIC >2048 mg/L.When the essential oil of the flowers was tested (using disc diffusion technique) against various fungal strains, it showed good potential antifungal activity. 
Bogdanova NS et al in the study of antiviral properties of calendula officinalis concluded that a tincture of the flowers suppressed the replication of herpes simplex, influenza A2 and influenza APR-8 viruses in vitro. 
Other activities –
Plant extract has shown to have hypoglycemic property due to their antioxidant potential which can be utilized for diabetes patients as they are associated with low level of antioxidants and  Calendula could be cardioprotective against ischemic heart disease. Calendula officinalis also can be used for the treatment of fever, conjunctivitis, pharyngitis, aphthous stomatitis and gingivostomatitis, abrasions and minor burns of skin.  It may be helpful as a remedy for jaundice, constipation, to treat varicose veins.
Review of studies involving calendula officinalis extract
V. Sambale et al (2014) isolated calendula mucilage to be used as mucoadhesive agent. They also formulated controlled release buccoadhesive tablets in order to avoid hepatic first pass metabolism. Tablets containing 75mg of calendula mucilage demonstrated controlled drug release. Thus, the authors demonstrated successful formulation of buccoadhesive tablets containing calendula mucilage. 
Neda Babee et al (2013) evaluated the effect of Calendula officinalis flowers extract on radiation-induced oropharyngeal mucositis (OM) in patients with head-and-neck cancer. Patients were randomly divided into two groups, Group I received drug (2% Calendula extract) and the Group II received placebo. The results of the study demonstrated significant decrease in the intensity of oral mucositis in the patients receiving test drug. Thus, they concluded that the extract of Calendula effectively decreases the intensity of radiotherapy induced mucositis and the antioxidant capacity of the flower may be accountable for the outcome. 
Mayur Sudhakar Khairnar et al (2013) evaluated the efficacy of calendula officinalis in reducing plaque and gingival inflammation. 240 patients were included in the study who were divided into test group (120 patients) and control group (120 patients). Patients in the test group were instructed to rinse their mouth with diluted calendula tincture (2 ml calendula tincture in 6 ml of distilled water) twice daily for 6 months. Patients in the control group were advised to rinse their mouth with 8 ml distilled water. In the II visit, oral prophylaxis was carried out for all the patients after recording the clinical parameters. The results of the study showed a statistically significant difference in plaque index, gingival index and sulcus bleeding index of the test group while no significant results were reported in the control group. Hence, it was concluded that calendula officinalis mouthwash effectively reduces plaque and gingival inflammation and can be used as an adjunct to scaling. 
B. Amoian et al (2010) investigated the effectiveness of calendula officinalis toothpaste in reducing plaque formation and gingival inflammation. 40 patients were enrolled for the study who were divided into two treatment groups. Group I (20 patients) were treated with base dentrifice while patients in Group II were treated with calendula officinalis toothpaste. The authors reported a significant reduction in bleeding on probing and plaque index in Group II. Thus they concluded that toothpaste prepared from Calendula officinalis extract can be successfully used as an adjunctive treatment for reducing gingival inflammation. 
Lucia Helena Denardi Roveroni-Favaretto (2009) reported a case of recurrent exfoliative cheilitis that was treated with topical preparation of calendula officinalis L. earlier the patient was on corticosteroid therapy (0.1% triamcinolone cream) which was suspended before initiating the treatment with 10% topical Calendula officinalis. the therapy was instituted for 15 days following which the lesion healed. The results obtained by the authors led them to conclude that calendula officinalis L. can be considered as a potential therapy for the cases of exfoliative cheilitis. 
Eva Jimenez-Medina (2006) evaluated cytotoxic anti-tumor and immunomodulatory activities of laser activated calendula oficinalis extract (LACE) in vitro. They demonstrated a potent inhibition in the growth of tumour cell lines; whereas the proliferation and activation of peripheral blood lymphocytes was increased. The mechanism behind the inhibition of cell lines was identified as the induction of apoptosis and cell cycle arrest. In addition to this, the studied extract also demonstrated anti-tumor activity in nude mice in vivo. 
K.C. Preethi (2006) evaluated the antioxidant potential of Calendula officinalis flowers both in vitro and in vivo. It was reported that Calendula officinalis scavenges superoxide radicals by inhibiting lipid peroxidation. Apart from this, the extract also produced a dose dependent scavenging of nitric oxide. On oral administration of the extract, generation of superoxide in macrophages was inhibited in vivo by 12.6% and 38.7% at doses of 100 and 250 mg/kg by weight. The results of the study demonstrated significant antioxidant activity of Calendula officinalis in vitro and in vivo. 
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