“Anti-fungal of Marigold (Calendula officinalis) flower extract” I. INTRODUCTION

Background of the Study
From hundreds of centuries ago, in ancient times, and from times when the human being was active as today on the scene of life and fighting for life, along with his struggles to provide food and clothing, he was thinking about his health, finding ways to live healthy and relieving physical ailments and illness which was later called the medicine and of course the herbal medicine which is now applicable for us.
Flowers always make people better, happier, and more helpful; they are sunshine, food and medicine for the soul. A popular garden plant, Marigold has been valued for many centuries for its' exceptional healing powers and is particularly remarkable in the treatment of wounds. When used for medicinal purposes, it is commonly referred to as 'Calendula'. (Sunset Western Garden Book)
The “marigold” (Calendula officinalis) also called “ruddles”, common marigold, garden marigold, English marigold, or Scottish marigold is a plant in the genus Calendula of the family Asteraceae. It is probably native to southern Europe, though its long history of cultivation makes its precise origin unknown, and it may possibly be of garden origin. It is also widely naturalized further north in Europe (north to southern England) and elsewhere in warm temperate regions of the world. “Marigold” is well known as a wound-healing, antiseptic and stimulating remedy, but in fact modern herbalists rely upon this plant for a wide range of uses. Only Marigold's flower heads are used medicinally. The bright petals contain powerful natural chemicals that give the herb its' amazing healing properties, so always ensure that you use the most vibrant orange flowers available. Mindy Green in her book Calendula, has compiled an impressive list of some of Marigold's other potential actions, including alterative, analgesic, anthelmintic, anti-inflammatory, antispasmodic, aperient, astringent, bactericide, carminative, styptic and tonic. Marigold has a long history of medicinal use, stretching that one of its effective uses is to treat skin allergies which is the focus of the study in ensuring the fast cure of the ointment on the skin of the affected organisms particularly dogs.
“Marigold” is a genus of about 12 to 20 species of annual or perennial herbaceous plants in the daisy family Asteraceae, native to the area from Micronesia East through the Mediterranean region to Iran. Calendula should not be confused with other plants that are also known as marigolds, such as corn marigold, desert marigold, or marsh marigold or plants of the genus Tagetes.(http://hmtjournals.com/vol2_2/13)
Allergic skin reactions are very common, and it can be difficult to figure out what causes them. There are hundreds of different kinds of rashes that can be caused by many things, such as plants like poison ivy, allergic reactions to a medication or a food, or a response to an illness. (Daniel More, MD)
Allergic eczema (atopic dermatitis) and hives (urticarial) are two of the most common skin rashes. Eczema affects 10 percent to 20 percent of children and 1 percent to 3 percent of adults. If you have eczema, your skin may become red, irritated and itchy. Sometimes there are small, fluid-filled bumps that ooze. Hives are red bumps or welts that appear on the body. About 20 percent of Americans have hives at some time in their lives. (Skin MD natural) A common cause of hives is skin allergies. Hives may present themselves after contact with animal dander, insect stings or bites, medications, and foods. Skin MD Natural works to add extra protection to the skin barrier, helping to decrease the occurrence of skin irritations. This increased level of protection can help prevent skin allergies from developing into hives. (Skin MD natural) The management of Skin Allergy without any side effects is still a challenge to the medical system. Year-round or seasonal itching is likely due to allergies. Signs of allergies often start as itching, and the first signs may be to chew, bite, scratch or lick. Red skin and hair loss are common. Blackened and thickened skin that looks like elephant skin is symptoms of a long duration of uncontrolled disease. Infections from bacteria and yeast are common with allergies as the immune system is not responding properly to fight the organisms normally found on the skin. In addition, dogs that chew, bite, scratch and lick disturb the top layer of skin that protects against infection. Licking and chewing can also lead to a warm moist environment, which bacteria and yeast thrive in. Experimental animal’s models are one of the best strategies for the understanding of pathophysiology of any disease in order to design and develop the drugs for its treatment. Numerous animal models have been developed for the past few decades for studying skin allergies and testing for the anti-itchy. On this premise, this research was undertaken to determine the possible therapeutic effects of “marigold” (Calendula officinalis) extracts on allergic dogs. If the result of the study is favorable, this could give a cheaper alternative for the treatment of skin allergies instead of buying expensive medicines. The importance of allergy testing is to rule out possible allergens, especially when multiple allergens are present in daily life. It makes no sense to begin avoiding one potential allergen when the reaction is actually caused by something entirely different.

Objectives This study primarily aims to test the Antifungal Activities of Marigold (Calendulaofficinalis)flower Extract. Specifically, it aims to: 1. To produce an alternative source of skin ointment from the extract of marigold flowers. 2. To reduce body scars with the use of marigold extract. 3. To differentiate the effectivity of a commercialized ointment with that of an alternative one.

Importance of the Study

The researcher came up with the interest to focus on the effectivity of Marigold extract on the reduction of Mold growth. At present the cost of synthetic drugs is still high and it continues to rise causing burden for allergic organisms considering the fact that this allergies requires serious treatment. Thus, there is a need to seek for the treatment of such disease to decrease the mortality and morbidity due to simple skin allergies.

Scope and Delimitation of the Study

This study will be delimited to the effectivity of the phytochemical components of Marigold (Calendula officinalis) extracts in dogs suffering from skin allergies. To determine the anti-allergic effects of the flower, Marigold extract is applied to the allergic dog together with the application of the commercialized ointment to a respondent with the same situation. The materials and the plant are readily available assuring to produce cheaper ointment in the market.

Place and Time of the Study

The phytochemical analysis of the “marigold” (Calendula officinalis ) extract was conducted at the Industrial Technology Development Institute-Department of Science and Technology, Urdaneta City, on April 9, 2013 with the technical assistance of ITDI research specialists.

REVIEW OF LITERATURE
The Plant Material-“Marigold”- Calendula officinalis
Botanical Description “Marigold”- Calendula officinalisalso called “ruddles” or “spottish” marigold is a plant in the genus Calendula of the family Asteraceae. It is probably native to southern Europe, though its long history of cultivation makes its precise origin unknown, and it may possibly be of garden origin. It is also widely naturalized further north in Europe (north to southern England) and elsewhere in warm temperate regions of the world. “Marigold” is widely cultivated and can be grown easily in sunny locations in most kinds of soils. Although perennial, it is commonly treated as an annual, particularly in colder regions where its winter survival is poor, or in hot summer locations where it also does not survive. Calendula are considered by many gardening experts as among the easiest and most versatile flowers to grow in a garden, especially since they tolerate most soils. In temperate climates, seeds are sown in spring for blooms that last throughout the summer and well into the fall. In areas of little winter freezing (USDA zones 8-11), seeds are sown in autumn for winter color, plants will wither in subtropical summer. Seeds will germinate freely in sunny or half-sunny locations, but plants do best if planted in sunny locations with rich, well-drained soil. Pot marigolds typically bloom quickly from seed (in under two months) in bright yellows, golds, and oranges. (http://en.wikipedia.org/wiki/Calendula_officinalis)

Chemistry This study aims to quantify the active biological compounds in C. officinalis flowers. Based on the active principles and biological properties of marigolds flowers reported in the literature, we sought to obtain and characterize the molecular composition of extracts prepared using different solvents. The antioxidant capacities of extracts were assessed by using spectrophotometry to measure both absorbance of the colorimetric free radical scavenger 2,2-diphenyl-1-picrylhydrazyl (DPPH) as well as the total antioxidant potential, using the ferric reducing power (FRAP) assay.
Folkloric Uses: “Marigold” is used to treat wounds and internally to reduce fever, treat cancer had control dysmenorrhea. Extracts have proved antibacterial, antiviral and immunostimulation in vitro. Petals are consumed as a seasoning. The plant has been used to repel insects. (http://www.krishnaherbals.com/calendula-flowers.html)
Other Folkloric Uses:
• Used for anemia. Irregular menstruation, abdominal pain during menstrual period, rheumatic muscular and bone pain.
• Leaves applied to boils and carbuncles; juice used for earaches.
• Internally, used for indigestion, colic, severe constipation, coughs and dysentery.
• Externally, used for sores, ulcers, eczema, sore eyes and rheumatism.
• Decoction of flowers used for colds, conjunctivitis, mumps, sore eyes.
• Decoction preparation for coughs: Boil a handful of the herb in a liter of water and drink a glass 3x daily.
• In India, juice of the flowers occasionally used as blood purifier and as remedy for piles.
• Infusion or decoction of plant used for colds, rheumatic pains, bronchitis.
• In Brazil, infusion of leaves and flowers used as vermifuge.
• In Mexico, decoction of flowers and leaves used as diuretic and carminative.
• In Aztecs used for carbuncles and eye infections.
• In Brazil and Mexico, used for joint pains and muscle spasms. Also, used for allergic contact dermatitis.
• Elsewhere, used in folk medicine for eye diseases, colds, coughs, conjunctivitis, hemorrhoids and ulcers. (http://www.stuartxchange.org/Ahito.html)
Studies on “Marigold”
• Antioxidant / Analgesic effect: A study showed pronounced antioxidant potential in Aztec marigold flowers and dose-dependent analgesic effect in keeping with its folkloric medicinal use as antiinflammatory and analgesic
• Wound Healing Activity: Ethanolic extract of Tageteserecta leaves was evaluated on adult albino rats. Results showed significant wound healing activity, comparable to the nitrofurazone control. The study supports the wound healing properties of the leaves as claimed in folkloric literature.
• Carotenoid / Nutritional Supplement: Flower extract was found to contain biologically useful lutein compounds and studied for use as nutritional supplement and as poultry food colorant.
• Keratolytic / Anti-verrucae / Podiatric Use: Its use for plantar hyperkeratosis was first described in the early 80s. Marigold paste has been used for painful hyperkeratotic lesions in the UK. Studies yielded tagetone, d-limonene, acimene, linalyl-acetate, linalol, and other terpenes. Tagetone acts as catalyst in inhibiting the production and transmission of keratinocytes. Marigold also showed benefit in the treatment of topical verrucae. Study shows promised for continued investigation for use of marigold therapy for various podiatric conditions.
• Larvicidal: Study of T patula essential oil on fourth instar larvae of mosquito species showed greatest activity against Aaegypti, followed by An. stephansi and C quinquefasciatus. Results were compared to synthetic insecticide, malathion.
• Toxicological Study: Study showed the chloroform extract of T erecta had no toxic effects and validates its traditional use in indigenous systems of medicine.
• Essential Oil: Study of essential oils of T erecta yielded piperitone (50.7%), piperitenone, (E)-ß-ocimene from the leaf oil and 1,8-cineole (23.1%), a-pinene, a-terpineol, piperitone and sabinene from the flower oil.
• Antinociceptive / Anti-Inflammatory: Study of T. erecta alcoholic extracts showed significant antinociceptive activity and significant anti-inflammatory activity comparable to the Diclofenac sodium.
• Insecticidal: Study of crude extracts and fractions of flower of T erecta was tested for insecticidal activity against stored product insect pest, Trilobiumcastaneum. The chloroform extract showed highest toxicity against both larvae and adults of T castaneum.
• Essential Oil / Antioxidant / Camphor and Methyleugenol: Study of essential oil for antioxidant activity using a DPPD assay, free radical scavenging activity and oxidation of deoxyribose assay. Analysis yielded 18 components; b-caryophyllene, limonene, methyleugenol, (E)-ocimene, piperetone, piperitenoneand ?-terpinolene were the main components. Results demonstrated significant antioxidant activity, less than a-tocopherol. The activity was attributed to the presence of camphor and methyleugenol, both naturally occurring in a variety of herbs and spices.
• Antioxidant / Flowers: An ethanolic extract of flowers was studied for antioxidant activity. Phytochemical yielded alkaloids, flavonoids, proteins, steroids, and tannins. Results showed antioxidant activity in all in vitro assays -- DPPH, reducing power, and superoxide radical scavenging activity , with better reducing power than standard ascorbic acid.
• Antidiabetic: Study of hydroalcoholic extract in peritoneallystreptozotocin-induced diabetic rats showed increased glucose levels after 30 minutes, and hypoglycemia only after 120 minutes.
• Antihyperlipidemic Effect: Study of hydroalcoholic extract in induced hyperlipidemic showed a significant decrease in all hyperlipidemic parameters. Lovastatin was sued as standard.
• Antimicrobial: Study of various extracts evaluated the antimicrobial activity of T. erecta and T. pistula flowers. Methanol extract of T. erecta was found to have better inhibitory activity than cold and hot aqueous extracts. Results showed the extracts of both species possess potential broad spectrum antibacterial activity.
• Antibacterial: Study showed the leaf and flower of T. erecta to show a broad spectrum of antibacterial activity. The flower extract showed significant activity against S. lutea, E. coli, B. circulence. (http://www.stuartxchange.org/Ahito.html)

Phytochemical Researches
Phytochemical is chemical compounds that occur naturally in plants (phyto means "plant" in Greek). Some are responsible for color and other organoleptic properties, such as the deep purple of blueberries and the smell of garlic. The term is generally used to refer to those chemicals that may have biological significance, for example antioxidants, but are not established as essential nutrients. Scientists estimate that there may be as many as 10,000 different phytochemicals having the potential to affect diseases such as cancer, stroke or metabolic syndrome.
There is evidence from laboratory studies that phytochemicals in fruits and vegetables may reduce the risk of cancer, possibly due to dietary fibers, polyphenol antioxidants and anti-inflammatory effects. Specific phytochemicals, such as fermentable dietary fibers, are allowed limited health claims by the US Food and Drug Administration (FDA). An important cancer drug, Taxol (paclitaxel), is a phytochemical initially extracted and purified from the Pacific yew tree.
Some phytochemicals with physiological properties may be elements rather than complex organic molecules. For example, selenium, which is abundant in many fruits and vegetables, is involved with major metabolic pathways, including thyroid hormone metabolism and immune function. Particularly, it is an essential nutrient and cofactor for the enzymatic synthesis of glutathione, an endogenous antioxidant.

Phytochemical Substances in Plants
Saponins
Saponins are a class of chemical compounds, one of many secondary metabolites found in natural sources, with saponins found in particular abundance in various plant species. More specifically, they are amphipathic glycosides grouped, in terms of phenomenology, by the soap-like foaming they produce when shaken in aqueous solutions, and, in terms of structure, by their composition of one or more hydrophilic glycoside moieties combined with a lipophilic triterpene derivative. (Annals of Biological Research, 2010)
Carotenoids
Carotenoids are organic pigments that are found in the chloroplasts and chromoplasts of plants and some other photosynthetic organisms like algae, some bacteria, and some fungi. Carotenoids can be produced from fats and other basic organic metabolic building blocks by all these organisms. Carotenoids generally cannot be manufactured by species in the animal kingdom so animals obtain carotenoids in their diets, and may employ them in various ways in metabolism. (FASEB J. 10)
Isorhamnetin
Isorhamnetin is an O-methylated flavonol, a type of chemical compound. It can be found in Tageteslucida, a psychedelic plant from Mexico and Central America.Isorhamnetin is a flavonoid, which occurs naturally in plants, but is also a metabolite of quercetin (isorhamnetin is methylated quercetin). (Bohm, Bruce A.; Tod F. Stuessy (2007)
Cadinol
Cadinol or 10α-hydroxy-4-cadinene is an organic compound, a sesquiterpenoidalcohol.Cadinol was said to act as anti-fungal and as hepatoprotective, and was proposed as possible remedy for drug-resistant tuberculosis.
Triterpenes
Triterpenes are terpenes consisting of six isoprene units and have the molecular formula C30H48. (Melanie (2009) )
The pentacyclictriterpenes can be classified into lupane,oleanane or ursane groups.
A notable pentacyclictriterpene is Boswellic acid.
Animal- and plant-derived triterpenes exist, such as: squalene ambrein (a tricyclic triterpene alcohol) ganoderic acid (quad cyclic)
Triterpenoids are thought of as modified triterpenes, such as lanosterol
Sesquiterpenes
Sesquiterpenes are a class of terpenes that consist of three isoprene units and have the molecular formula C15H24. Like monoterpenes, sesquiterpenes may be acyclic or contain rings, including many unique combinations. Biochemical modifications such as oxidation or rearrangement produce the related sesquiterpenoids.
Sesquiterpenes are found naturally in plants and insects, as semiochemicals, e.g. defensive agents or pheromones. (http://en.wikipedia.org/wiki/Sesquiterpene)
Scopoletin
Scopoletin is a coumarin found in the root of plants in the genus Scopolia like Scopoliacarniolica or Scopolia japonica, in chicory, in Artemisia scoparia, in the passion flower, in Brunfelsia, in Viburnum prunifolium or Kleinhoviahospita. It can also be found in vinegar, some whiskies or in dandelion coffee. A similar coumarin is scoparone. (Miguel CarreroGálvez )

Flavonoids
Flavonoids (or bioflavonoids) (from the Latin word flavus meaning yellow, their colour in nature) are a class of plant secondary metabolites.
Flavonoids were referred to as Vitamin P (probably because of the effect they had on the permeability of vascular capillaries) from the mid-1930s to early 50s, but the term has since fallen out of use. ("Research for Vitamin P")
Quercetin
Quercetin /ˈkwɜrsɨtɨn/, a flavonol, is a plant-derived flavonoid found in fruits, vegetables, leaves and grains. It also may be used as an ingredient in supplements, beverages or foods.Quercetin is a flavonoid widely distributed in nature. The name has been used since 1857, and is derived from quercetum (oak forest), after Quercus. (The Texas Journal of Agriculture and Natural Resource)
Kaempferol
Kaempferol (/'kiːmpfərɒl/) is a natural flavonol, a type of flavonoid, that has been isolated from tea, broccoli, Delphinium, Witch-hazel, grapefruit, cabbage, kale, beans, endive, leek, tomato, strawberries, grapes, brussels sprouts, apples and other plant sources. Kaempferol is a yellow crystalline solid with a melting point of 276-278 °C. It is slightly soluble in water but soluble in hot ethanol and diethyl ether. (C28-35, 2010 Jan-Feb.)
(Krivenko 2008) According to him Calendula is used to aid the healing of wounds and internal and external ulcers. It is an anti-septic and improves blood flow to the affected area. Some clinical studies validate the early treatment of stomach ulcers, although further research is needed.
According to (FedkovichChernivtsiState University in the Ukraine, 2010) Calendula today, is being investigated for its anti-cancer properties. In conjunction with other herbs such as Echinacea purpurea, Scorzonerahumilis L., and Aconitum moldavicum, there has been evidence of success in treating certain cancers (Heren's carcinoma)
(Ebrahimzadeh MA, 2011) He concluded that Calendula tinctures, ointments, and washes are often applied to the skin to help burns, bruises, and cuts heal faster, and to fight the minor infections they cause. Calendula cream is also used to treat hemorrhoids. Animal studies show that calendula does seem to help wounds heal faster, maybe by increasing blood flow to the wounded area and by helping the body make new tissue.

(MaticRao 2006) He Observed the reduction of epithelialization time, an increase in wound strength, and improvement of wound contraction in rats with experimental incision wounds that were topically treated with calendula. The effects may also be mediated by the stimulation of phagocytosis, by increased granulation, and via effects on metabolism of glycoproteins, nucleoproteins, and collagen proteins in tissue regeneration. It has been proposed that Calendula officinalis extract may aid in wound healing by promoting epithelial growth and by enhancing immune responses.
(WaseemSafdar, Hamid Majeed, IshratNaveed, Waqas Khan Kayani, Haroon Ahmed, SaqibHussain, Atif Kamal) They identify bioactive chemical compounds from flower of Calendula officinalis, their antimicrobial activity and setting up the standards specification. C. officinalis L. (Family Compositae) commonly known as Marigold or Calendula is an annual plant upto 1.5 feets tall. The percentage of moisture content was 18.79% per gram and total ash content was 98.74 mg per gram of air-dried flowers indicating the presence of organic compounds. Calendula is perhaps best known for its effectiveness in healing skin problems such as wounds, burns, insect bites, eczema, skin ulcers, and rashes. It has also been used internally to soothe and heal gastric and duodenal ulcers, as a wash for varicose veins and hemorrhoids, as a rinse for toothaches, and as an eyewash for conditions like conjunctivitis. In vitro (test tube) research has shown that calendula contains antimicrobial compounds that inhibit certain strains of Staphylococcus and Candida, as well as E. coli and some protozoa, such as Trichomonas. (The Herb Research Foundation, Boulder, CO, USA) (J ClinBiochemNutr. 2008) He concluded that the effect of Calendula officinalis flower extract was investigated against experimentally induced thermal burns in rats. Burn injury was made on the shaven back of the rats under anesthesia and the animals were treated orally with different doses of the flower extract (20 mg, 100 mg and 200 mg/kg body weight). According to Gesch, Russell he discovered that the oil of its seed contains high levels of calendic acid that can be used as a drying agent to substitute for harmful volatile organic compounds (VOC) in products such as paints and adhesives. Crop breeders from Europe have developed calendula varieties specifically for the production of their seeds for oil. Calendula has good potential to be produced in the northern U.S. because it is well adapted to cool temperate climates. Furthermore, there is considerable pressure being placed on chemical manufactures to replace VOCs in many types of products with less harmful and more environmentally-friendly alternatives such as calendula seed oil. However, little information exists on how well calendula performs and how best to produce it in the northern U.S.
Therefore, an experiment was designed to determine the best time to plant and harvest calendula, while determining the performance of three different varieties. It was discovered that to optimize seed yield, calendula should be planted in early May and perhaps even as early as mid-April when spring wheat is normally planted.
Professor Elie Barbour and co-workers, members of AUB Nature and Conservation Center, found an interesting potential application of Calendula officinalis in vaccination of poultry against 3 different viruses.
It is not economically viable for the farmers to vaccinate their chickens with needles because it is stressful for the poultry and would take days and days to jab thousands of birds one by one. On the other hand, inoculation by spray or in drinking water enables mass vaccination but the virus has to be alive to work as a vaccine, which results in unwanted side effects such as respiratory inflammations and decreases in body weights. Professor Barbour and co-workers showed that Calendula officinalis alleviated the side injuries of the 3 live vaccine viruses.

Studies on Anti-fungal Botanicals
Oregano
Oregano (Origanumvulgare) is also known as Wild Marjoram, Mountain Mint, Origanum, Wintersweet and Winter Marjoram. This erectly spreading plant has strong aromatic characteristics, with leaves and stems that are fleshy. The leaves of oregano are heart-shaped, with toothed edges, and which, grow for up to 9 meters in length. In other countries, the plant is primarily used as a culinary ingredient. However, in countries like the Philippines, Oregano is a known herbal medicine for its strong anti-oxidant properties. Oregano contains a rosmarinic acid compound, thymol, and carvacrol that are responsible for its anti-inflammatory, anti-bacterial, anti-oxidant, anti-fungal and anti-viral properties. Oregano also contains flavinoids, triterpenoids, sterols, vitamin C, and vitamin A. Its anti-bacterial properties have been proven by recent studies to treat infections of the reproductive tracts, and which make it ideal to be given to women who have just given birth. The volatile oils in oregano and its properties are believed to be responsible for slowing the process of spoilage of food and thus minimizing the risk of ingesting harmful bacteria, parasites and fungi.
Echinacea
Echinacea purpurea (eastern purple coneflower or purple coneflower) is a species of flowering plant in the genus Echinacea of the family Asteraceae. Its cone-shaped flowering heads are usually, but not always, purple in the wild. It is native to eastern North America and present to some extent in the wild in much of the eastern, southeastern and midwest United States. Echinacea is an herb. Several species of the echinacea plant are used to make medicine from its leaves, flower, and root.
Echinacea is widely used to fight infections, especially the common cold and other upper respiratory infections. Some people take echinacea at the first sign of a cold, hoping they will be able to keep the cold from developing. Other people take echinacea after cold symptoms have started, hoping they can make symptoms less severe. The people who use Echinacea to treat symptoms have the right idea. Research to date shows that echinacea probably modestly reduces cold symptoms, but it’s not clear whether it helps prevent colds from developing. Echinacea is also used against many other infections including the flu, urinary tract infections, vaginal yeast infections, genital herpes, bloodstream infections (septicemia), gum disease, tonsillitis, streptococcus infections, syphilis, typhoid, malaria, and diphtheria.
Sea kelp
Sea kelp contains over sixty essential vitamins, minerals, and trace elements. It is the richest natural source of iodine, which is vital for preventing and treating iodine deficiencies. Iodine is also important for the function of the thyroid gland and is a key part of thyroid hormones, which are necessary for the body’s normal development and metabolism. In this way, sea kelp can help to regulate weight, as the regulation of metabolism can be important for this.
As well as iodine, sea kelp contains magnesium, calcium, iron, copper, potassium, zinc, and natural sea salt. Sea kelp has the highest natural concentration of calcium of any food (it has ten times more than milk!) and it is also high in fibre.
Sea kelp also contains protein, vitamins A, B, C, and E, and beta-carotene. They are also high in lignans, plant compounds that have cancer-protective properties. Sea kelp takes the minerals and trace elements that are found in sea water and concentrates these in its leaves and stems in almost the exact proportions that are found in healthy human blood.
Citric acid
Citric acid is a weak organic acid with the formula C6H8O7. It is a natural preservative/conservative and is also used to add an acidic or sour taste to foods and soft drinks. In biochemistry, the conjugate base of citric acid, citrate, is important as an intermediate in the citric acid cycle, which occurs in the metabolism of all aerobic organisms.
Citric acid is a commodity chemical, and more than a million tonnes are produced every year by fermentation. It is used mainly as an acidifier, as a flavoring, and as a chelating agent.

Studies on Dog- Bull Terrier Skin Allergies
The Bull Terrier is a breed of dog in the terrier family. They have large, egg-shaped heads, and small triangular eyes. There is also a miniature version of this breed which is officially known as the Miniature Bull Terrier.

Appearance
The Bull Terrier's most recognizable feature is its head described as 'egg shaped' when viewed from the front, the top of the skull is almost flat from ear to ear. Profile curves gently downwards from top of skull to tip of nose which should be black and bent downwards at tip. Nostrils are well developed and under-jaw deep and strong snout. The unique triangle-shaped eyes are small, dark, and deep-set. The body is full and round, while the shoulders are robust and very muscular and the tail is carried horizontally. They are generally white in colour and it walks with a jaunty gait, and are popularly known as the 'gladiator of the canine race.
Temperament
Bull Terriers are known to be courageous and active. They enjoy being around people but are strong willed and thus require an assertive owner; as such the Bull Terrier is not recommended for households with small children if the owner is a first time Bull Terrier owner. Bull Terriers need the companionship of their owners and should not be kept outside in a kennel. Bull Terriers can be both independent and stubborn and for this reason are not considered suitable for an inexperienced dog owner. A firm hand and an assertive demeanour are essential if the Bull Terrier is not to run riot. They are protective of their family, although comprehensive socialization at an early age will prevent them from becoming over-protective and neurotic.
When it comes to other animals, caution should be advised. Bull Terriers have a strong prey instinct and, like any strong dog breed, can cause injury or death to people or other animals, especially cats. That said, puppies brought up with cats and other animals get on well with the animals they know; however, they can never be completely trusted with other animals. Uncastrated males often do not get along with other male dogs. Males and females however can live together happily and two females can also be a good combination with care and supervision. Introducing a Bull Terrier of the same sex as the dog in residence is considered unwise, and some Bull Terriers won't countenance other dogs of either sex.
Health:
All puppies should be checked for deafness, which occurs in 20% of pure white dogs and 1.3% of dogs and is difficult to notice, especially in a relatively young puppy. Many Bull Terriers have a tendency to develop skin allergies. Insect bites, such as those from fleas, and sometimes mosquitoes and mites, can produce a generalized allergic response of hives, rash, and itching. This problem can be stopped by keeping the dog free of contact from these insects, but this is definitely a consideration in climates or circumstances where exposure to these insects is inevitable. A UK breed survey puts their median lifespan at 10 years and their mean at 9 years (1 s.f., RSE = 13.87% 2 d. p.), with a good number of dogs living to 10–15 years.
History:
Early in the mid-19th century the "Bull and Terrier" breeds were developed to satisfy the needs for vermin control and animal-based blood sports. The "Bull and Terriers" were based on the Old English Bulldog (now extinct) and one or more of Old English Terrier and "Black and tan terrier", now known as Manchester Terrier. This new breed combined the speed and dexterity of lightly built terriers with the dour tenacity of the Bulldog, which was a poor performer in most combat situations, having been bred almost exclusively for fighting bulls and bears tied to a post. Many breeders began to breed bulldogs with terriers, arguing that such a mixture enhances the quality of fighting. Despite the fact that a cross between a bulldog and a terrier was of high value, very little or nothing was done to preserve the breed in its original form. Due to the lack of breed standards—breeding was for performance, not appearance—the "Bull and Terrier" eventually divided into the ancestors of "Bull Terriers" and "Staffordshire Bull Terriers", both smaller and easier to handle than the progenitor.
About 1850, James Hinks started breeding "Bull and Terriers" with "English White Terriers" (now extinct), looking for a cleaner appearance with better legs and nicer head. In 1862, Hinks entered a bitch called "Puss" sired by his white Bulldog called "Madman" into the Bull Terrier Class at the dog show held at the Cremorne Gardens in Chelsea. Originally known as the "Hinks Breed" and "The White Cavalier", these dogs did not yet have the now-familiar "egg face", but kept the stop in the skull profile.
( http://en.wikipedia.org/wiki/Bull_terrier)
Anti-fungal Drugs on the Bull Terrier
Amphotericin B
Treats infections caused by a fungus. Belongs to a class of drugs called antifungals.

Uses of This Medicine
Amphotericin B is an antifungal. It is used to help the body overcome serious fungus infections. It may also be used for other problems as determined by your doctor.
Amphotericin B is available only with your doctor's prescription. (http://www.ncbi.nlm.nih.gov/pubmedhealth/PMHT0008975/?report=details)
Caspofungin
Caspofungin acetate belongs to a new class of antifungals referred to as the echinocandins. The first of these, echinocandin B, was originally isolated from Aspergillus spp. in 1974 and structurally modified to yield cilofungin in the 1980s and subsequently LY303366 (Lilly) in the 1990s. Because of vehicle-related nephrotoxicity, further clinical development of cilofungin was terminated, but LY303366 is still under clinical development. Similar in structure, caspofungin acetate is a semisynthetic derivative of pneumocandin Bo. (http://www.medscape.com/viewarticle/406984_3)
Fluconazole
Fluconazole is an antifungal drug used in the treatment and prevention of superficial and systemic fungal infections which can invade parts of the body including the mouth, lungs, throat, bladder, esophagus, genitals, and the blood. It is also used to prevent fungal infection in people with weak immune systems which can be caused by cancer treatment, bone marrow transplants, or diseases such as AIDS. Fluconazole is not for treating viral infections such as the common cold or flu.
Flucytosine
Flucytosine (5-fluorocytosine) is a fluorinated pyrimidine related to fluorouracil that was initially developed as an antineoplastic agent. It should be stored in airtight containers protected from light. Solutions for infusion are unstable and should be stored at 15–20°C. Usually, it is given PO in capsules.
ANTIFUNGAL ACTIVITY
Mode of Action: Flucytosine is converted by cytosine deaminase in fungal cells to fluorouracil, which then interferes with RNA and protein synthesis. Fluorouracil is metabolized to 5-fluorodeoxyuridylic acid, an inhibitor of thymidylate synthetase. DNA synthesis is then halted. Mammalian cells do not convert large amounts of flucytosine to fluorouracil and, thus, are not affected at usual dosage levels.
Fungal Resistance
Resistance to flucytosine can develop rapidly even during the course of treatment; this precludes its use as the sole treatment for mycotic infections. The mechanisms of resistance are not completely understood.
Itraconazole
Itraconazole falls under the category of antifungal drugs called triazoles. Itraconazole is used to treat fungal infections in the lungs or in fingers, toes, nails and other body parts. This drug is also prescribed for fungal infection in HIV patients. Oral yeast infections can also be treated with the help of this medicine. You can follow the FAQ page thoroughly to know more about this medication. This drug is approved by the FDA. traconazole (Generic Sporanox) - Antifungal medication)
Ketoconazol
Ketoconazole is used topically for the treatment of tinea corporis, tinea cruris, and tinea pedis caused by Epidermophyton floccosum, Trichophyton mentagrophytes, or T. rubrum. The drug also is used topically for the treatment of cutaneous candidiasis caused by Candida albicans. (http://www.irannajo.com/en/products/ketoconazole.html)
Nystatin
Nystatin is a topical and oral antifungal agent with activity against many species of yeast and candida albicans, which is used largely to treat skin and oropharyngeal candidiasis. Nystatin is a polyene macrolide antibiotic that acts by binding to sterols in the plasma membranes of fungi causing the cells to leak, eventually leading to fungal cell death. Nystatin is indicated for the treatment of yeast and candidal infections of the skin, mucous membranes and gastrointestinal tract. (http://livertox.nih.gov/Nystatin.htm)
Posaconazole
Posaconazole is the newest triazole antifungal agent. It is structurally related to itraconazole and has activity against Candida species, Aspergillus species, Cryptococcus neoformans, the zygomycetes, and other filamentous fungi. Randomized, double-blind trials have shown posaconazole to be at least as efficacious as fluconazole for the prevention of invasive fungal infections in immunocompromised patients. It has also shown promising results in the treatment of various fungal infections refractory to other antifungal therapy.
Voriconazole
Voriconazole is a triazole antifungal agent used primarily in the treatment or prevention of aspergillosis and candidal infections. Voriconazole therapy is associated with transient, asymptomatic serum aminotransferase elevations and is a rare cause of clinically apparent acute drug induced liver injury. (http://livertox.nih.gov/Voriconazole.htm)

On the Experimental Animal
Strong and handsome, the Bull Terrier is a dog of muscular symmetry and sweet disposition. The head is long and deep, curving from nose to the top of the skull; when viewed from the front, the head should resemble an oval. Eyes are close and deep set, small and triangular. Ears are small, erect and close set; teeth meet in a scissors bite. The body is muscular and big boned, though never coarse. The tail is short and carried horizontally. Feet are round, compact and catlike. The Bull Terrier’s coat is short, glossy and harsh to the hand, and the skin should be tight throughout. Color is either solid white with our without markings on the head, or any color other than white (often brindle) with white markings on the head.
The pit bull terrier is a powerful and athletic breed, originated from the crossbreeding of bulldogs and terriers during the 19th century. According to United Kennel Club, the characteristics of a pit bull terrier include a broad head, a well-developed lower jaw, a strong and muscular body, as well as a friendly and nonaggressive temperament.

MATERIALS AND METHODS
Materials
This section presents the materials used in the phytochemical screening and anti-fungal assay of Marigold (Calendula officinalis) flower extract.
A. Marigold Content:
Etahnolic extract of shade dried petals of Marigold Flowers was prepared and used for pythochemical screening and anti-bacterial activity. The in-vitro anti-bacterial activity of crude ethanolic extract was evaluated by agar well diffusion method against Gram positive S. Aureus and Gram negative E.coli as compared to standard anti-bacterial drug ampicillin ( 10 micro gm/ ml).
B. Pytochemical Screening
The extract was found to contain flavonoids, glycocides and pseudotannins. Lutein was isolated and its presence confirmed by TLC using solvent system- petroleum ether; benzene (90:10) moderate anti-bacterial activity as compared to ampicillin.

Plant Material
Fresh leaves of the plant Calendula officinalis (Linn.) were obtained identified and authenticated from the voucher specimen SPTM/Cal 1.The collected leaves were dried in shade, crushed to coarse powder and used for further studies. Preparation of Extract
The leaves of Calendula officinalis were collected, dried in the shade, powdered, weighed (1 kg) and they were subjected to continuous hot extraction in soxhlet apparatus. The extraction was carried out as per the polarity of the solvents with petroleum ether chloroform, methanol and finally with water for 36 hours. The solebt from each extraction was distilled off and the concentration was carried out on a water bat to appropriate consistency and then evaporated to dryness and used for the phytochemical analysis. The residue for each extract was petroleum ether (120 gm), chloroform (200 gm), methanol (300 gm) and finally water (100 gm)8. The extracts were subjected to preliminary qualitative tests to identify the various phytoconstituents present in leaves.
Amino acids
The amino acids can be detected as per the standard procedure depicted in Harbone. The plant material was mixed with 10 % isopropanol and ground using motor and pestle. The filtrate was centrifuged at 13000 rpm for 3 min and the supernatant was used for the detection of amino acid. Samples were spotted on TLC plate using n-butanol-acetic acid-water (12:3:5) as mobile phase. TLC plates were sprayed with 0.2% Ninhydrin in acetone, dried over at 105°C for 1-2 minutes. The presence of violet colour to pink colour indicates10,11.
Essential oils
They were detected by using methelyne-di-chloride-chloroform –ethyl acetate-n-propanaol (47: 45: 2: 2.5) as mobile phase. TLC plates were sprayed with vanillin sulphuric acid reagent12 and dried at 105°Cfor 2 minutes. Appearance of pink, brown colour spots shows the presence of essential oils.
Triterpenoids
The detection for triterpenoids was done by applying the samples on TLC plate impregnated with silver nitrate and they were developed in butanol-2M ammonium hydroxide (1:1) as mobile phase. The detection was done by spraying antimony trichloride. The purplish spot indicates the presence of triterpenoids.
Alkaloids
They can be detected by Dragendroff’s reagent. The test sample was applied on precoated TLC plates developed in chloroform: Methanol (9:1) as mobile phase, dried sprayed with the reagent. Appearance of orange red spot at room temperature indicates the presence of alkaloid.
Saponin
Sample was applied on TLC plates developed in Chlorofrom-methanol-water (60:35:5) as mobile phase and dried. Plates were then sprayed with 1% vanillin 5% sulphuric acid reagent and dried at 110°C for few minutes glycosides appear as dark bluish to black spot.
Sterols
Generally steroids have a nucleus of cyclopentene perhydrophananthrene found in plant tissues called as phytosterols. In higher plants stigmasterol, sitosterol are the most common sterols found in abundant which are in free and in combined form. For the detection of steroids samples were applied on TLC plates developed in Chloroform- methanol (3:4) as mobile phase and were detected by spraying anisaldehyde. TLC plates were also developed in benzene- methanol (95:5) as another mobile phase and were detected by spraying 5% alcoholic sulphuric acid reagent. Bluish- green spots were observed13.
Fatty acids
For the detection of fatty acids samples were applied on a pre-coated TLC plates, our dried and were developed in Hexane- ethyl acetate (95: 5) as mobile phase, sprayed with 5% alcoholic potassium permanganate solution. The appearance of dark brown spots indicates the presence of fatty acids.

TREATMENT OF DATA
Total phenols determination
Total phenolic contents were evaluated using Folin-Ciocalteu reagent. A dilute solution of the extract (0.5 ml of 1:100 mg ml-1 methanolic extract) or gallic acid (phenolic standard compound) was mixed with the reagent (5 ml, 1:10 diluted with distilled water) and aqueous Na2CO3 (4 ml, 1 M). After 15 min standby period, the total phenol contents were assayed using colorimetry at 765 nm. The standard curve was obtained using 0, 25, 50, 100, 150, 250 and 500 mg l-1 solutions of gallic acid in methanol:water (50:50, v/v). Total phenolic contents were shown as mg/g (of dry mass) of gallic acid equivalent, which is a common reference compound.
Total flavonoids determination
Total flavonoid contents were determined using aluminum chloride colorimetric method. Calendula methanolic extract (0.5 ml of %1 extract) was mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml of distilled water. Then the mixture was left at room temperature for 30 min; the absorbance of the reaction mixture was measured at 415 nm. Different quercetin solutions at concentrations 6.25, 12.5, 25, 50 and 100 mg l-1 in methanol were prepared to obtaining standard curve.

Statistical analysis
Data were analyzed by using t-test, Mann–Whitney U test, one-way ANOVA and repeated measures ANOVA tests. The p values of less than 0.05 were considered as the statistical significant difference.
RESULTS
Statistical analysis was performed on 20 and18 patients in calendula and placebo groups respectively (Additional file 1). Thirty eight patients with mean age 54.1 (range 46–72) completed the study. None of the patients in calendula group underwent medication for OM severity and radiotherapy was not ceased for this reason. In calendula group three patients did not show OM in the whole treatment period. OM intensity was measured according to OMAS scores at the end of each week for both calendula and placebo groups. OMAS scores were significantly lower in calendula group compared to placebo at week 2, 3 and 6 of the study. According to repeated measures ANOVA test, the differences between OMAS of calendula and placebo during the weeks of evaluation were statistical significant (p < 0.001) and OM intensity was significantly decreased in calendula compared to placebo group (p = 0.048). Although OM intensity was higher in women, but in both group of genders calendula had better results compared to placebo (Figure 2). Quercetin concentration as a main flavonoid content was measured. According to the standard curve, the mean ± SD of quercetin concentration in the extract was 19.41 ± 4.34 mg/l (%RSD = 15.33). The flavonoid contents of calendula flowers extract based on quercetin equivalent concentration (the standard curve equation: y =0.007× + 0.3337, r = 0.998) was 76.66 ± 23.24 mg/g.

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