ABSTRACT

AloeVera leaf gel is widely used as a traditional folk medicine for the treatment of different infectious diseases. Many of the health benefits associated with AloeVera have been attributed to the polysaccharides present in the gel of leaves. The study is aimed to investigate the phytoconstituents and antimicrobial activity of aqueous, ethanol and acetone extracts of the AloeVera gel against some human and plant pathogens by disc diffusion method. The extracts were screened for the presence of endotoxin contamination by Limulus amebocyte lysate test. The study showed that among three extract ethanol and acetone extracts recorded significant antimicrobial activity against all test pathogens. Antibacterial and antifungal activity of the acetone extract was found to be quite impressive as compared to ethanol and aqueous extracts. Ethanol extract showed high inhibition activity against Bacillus Subtilis(15.10 0.01), Micrococcus kristinae(15.220.02) compared to acetone extract and low inhibition activity against Candida albicans(9.960.2), Microsporum canis(7.270.02) surprisingly extract failed to show significant activity aqueous against all Bacillus Subtilis (7.960.01), Bacillus

Cereus (6.890.03), Staphylococcus aureus (7.890.02), Staphylococcus pyogenes (8.00.01), Proteus mirabilis (7.230.01), Aspergillus fumigates (6.930.13), Aspergillus niger (7.160.2), Aspergillus glaceaus (7.250.02),Tricophytom mentagrophyte (6.690.20), it showed very low inhibition activity indicating that the active principle responsible for antimicrobial activity is more soluble in organic solvents. The results suggested that AloeVera gel extract with acetone can be used as antimicrobial agent against human and plant pathogens for medication, cosmetic and food purposes. Key words: AloeVera gel, Phytoconstituents, antimicrobial activity, human and plant pathogens, Limulus amebocyte lysate test.

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INTRODUCTION
Herbal medicines represent one of the most important fields of traditional medicine all over the world. Promoting the proper use of herbal medicine and essential to study medicinal plants, which have folklore repution in a more intensified way, is required [1]. Contrary to the synthetic drugs, antimicrobials of plants orgin are not associated with side effects and have an enormous therapeutic potential to heal many infectious diseases for example, digitalis, ephedrine and vincristine were all originally discovered through research on plants. The potential for developing antimicrobials from higher plant appears rewarding as it will lead to the development of a Phyto medicine to act against microbes. associated with synthetic anti microbials [2]. The use and search of drugs and dietary supplements derived from plants have accelerated in recent years. Ethano pharmacologists, botanists, micro biologists,and natural product chemists are combining the Earth for phyto chemicals and leads which could be developed for the treatment of infectious diseases. Plants are rich in a wide variety of secondary metabolites,such as tannins, terpenoids, alkaloids and flavanoids, which have been found inviter to have antimicrobial properties(3). Currently, of the one-quarter of the pharmaceuticals dispensed in the United States having higher plant origins, very few are intended for the use as anti microbial, since we have relied on bacterial and fungal sources for these activities.Clinical microbiologists have two reasons to be interested in the topic of antimicrobial plant extracts. First , it is very likely that the that these phyto chemicals will find their way into the arsenal of antimicrobial drugs prescribed by physicians; several are already tested in humans.Second, the public health is becoming increasingly aware of problems with the over prescription and misuse of traditional antibiotics. The use of plant medicine and other alternative forms of medical treatments. Plant-based antimicrobials have enormous therapeutic potential as they can serve the purpose with lesser side effects that are often

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Plants have been an important source of medicine for thousands of years. Even today, the World Health Organization estimates that up to 80 percent of people still rely mainly on traditional remedies such as herbs for their medicines. Its civilization is very ancient and the country as a whole has long been known for its rich resources of medical plants. Today, Ayurvedic, Hoemoeo and Unaniphysicians utilize numerous species of medicinal plants that found their way a long time ago into the Hindu Material Media. Bacterial resistance to antibiotics is increasingly becoming a concern to public health. Currently used antibiotic agents are failing to bring an end to many bacterial infections due to super resistant strains. For this reason the search is ongoing for new antimicrobial agents, either by the design and synthesis of new agents or through the search of natural sources for as yet undiscovered antimicrobial agents. Herbal medications in particular have seen a revival of interest due to a perception that there is a lower incidence of adverse reactions to plant preparations compared to synthetic pharmaceuticals. Coupled with the reduced costs of plant preparations, this makes the search for natural therapeutics an attractive option. The use of herbs and medicinal plants as the first medicines is a universal phenomenon. Every culture on earth, through written or oral tradition, has relied on the vast variety of natural chemistry found in healing plants for their therapeutic properties .All drugs of the past were substances with a particular therapeutic action extracted from plants. Medicinal plants may be defined as any plant that can be put to culinary or medicinal use such as fox glove, opium poppy, and garlic. More and more researchers find that food and their individual constituents perform similar fashion to modern drugs and sometimes better without the dreaded side effects. Plant extracts have great potential as antimicrobial compounds against microorganisms and they can be used in the treatment of infectious diseases caused by resistant microbes. The synergistic effect enables the use of the respective antibiotic when it is no longer effective by itself during therapeutic treatment. Resistance of microbes to antimicrobial agents has resulted in morbidity and mortality from treatment failures and increased health care costs. Although defining the precise public health risk and estimating the increase in costs is not a simple undertaking, there is little doubt that emergent antibiotic resistance is a serious global problem.

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Appropriate antimicrobial drug use has unquestionable benefit, but physicians and the public frequently use these agents inappropriately. Inappropriate use results from physicians providing antimicrobial drugs to treat viral infections, using inadequate criteria for diagnosis of infections that potentially have a bacterial aetiology, unnecessarily prescribing expensive, broadspectrum agents, and not following established recommendations for using chemo prophylaxis. The availability of antibiotics over the counter, despite regulations to the contrary, also fuel inappropriate usage of antimicrobial drugs in India. The easy availability of antimicrobial drugs leads to their incorporation into herbal or "folk" remedies, which also increases inappropriate use of these agents. Widespread antibiotic usage exerts a selective pressure that acts as a driving force in the development of antibiotic resistance. The association between increased rates of antimicrobial use and resistance has been documented for nosocomial infections as well as for resistant community acquired infections. As resistance develops to "first-line" antibiotics, therapy with new, broader spectrum, more expensive antibiotics increases, but is followed by development of resistance to the new class of drugs. Resistance factors, particularly those carried on mobile elements, can spread rapidly within human and animal populations. Multidrug-resistant pathogens travel not only locally but also globally, with newly introduced pathogens spreading rapidly in susceptible hosts. Antibiotic resistance patterns may vary locally and regionally, so surveillance data needs to be collected from selected sentinel sources. Patterns can change rapidly and they need to be monitored closely because of their implications for public health and as an indicator of appropriate or inappropriate antibiotic usage by physicians in that area. Clinical microbiologists have two reasons to be interested in the topic of antimicrobial plant extracts. First, it is very likely that these phytochemicals will find their way into the arsenal of antimicrobial drugs prescribed by physicians; several are already being tested in humans. It is reported that, on average, two or three antibiotics derived from plants are launched each year.

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ALOEVERA PLANT PROFILE

Bionomical name Scientific classification: Kingdom Division Class Series Family Genus Species Synonyms:

Aloe barbadensis

: : : : : : :

Plantae Phanerogamae Monocotyledonae Coronariae Liliaceae Aloe A. Vera Kumari,Kalabandha

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Habitat: It a grows in the tropical region of India, especially in the Western parts of the country. It is originated in African Continent, AloeVera is a member of Liliaceae family. AloeVera (L.)Burm.Fil(Synonym Aloe berbadensis Miller)(Thelugu-Kalabandha, Tamil-Southakathalai, Hindi-Gikanvar) is a cactus like plant with green, dagger-shaped leaves that are fleshy, tapering, spiny, marginated and filled with a clear viscous gel[4]. The name was derived from the Arabic alloeh meaning bitter because of bitter liquid found in the lives. It is also known as lily of the desert the plant of immortality and the medicine plant with qualities to serve as alternate medicine. AloeVera is as old as civilization and throughout history it has been used as a popular folk medicine. It is present in the arde regions of India and is believed to be effective in treating stomach ailments, gastrointestinal problems, skin disease, constipation, radiation injury, inflammatory effect, healing wounds and burns, ulcer and diabetes. Currently the plant is widely used in skin care, cosmetics and as nutraceuticals [5]. In this present study the gel of AloeVera leaves were investigated for their Phyto chemical compounds and antimicrobial activity against some human and plant pathogens. Botanists agree that AloeVera plant originated in a dry, warm climate of Africa. Reliable sources, however, indicate that the Aloe originated in South Africa and long before the Christian era spread along the routes into Egypt, crossed the Mediterranean and Red Sea and then into Spain, East India, China, West Indies, South America -into all of the warmer areas of the Western Hemisphere., (6)AloeVera continues to growing in many tropical areas of the world today, but it can only flourish in colder climates if brought inside during cold weather. It can not survive freezing ground temperatures. Vital nutrients can be lost and damage can occur at temperatures of 40 degrees Fahrenheit or below. It can flourish in temperatures as great as 104 degrees F. It even handles severe drought conditions. But whatever you do, do not over water and drown your plants. The roots should not stand in the water. In the United States, AloeVera grows commercially in the Rio Grande Valley of Texas, in California and Florida, and in unique greenhouses in Oklahoma.(7) Internationally it grows in Mexico, India, South America, Caribbean, Australia, and Africa.Because Aloevera grows best in warmer climates, the colder parts of Europe were slower to learn of its medicinal values. It grows nicely in Spain, Portugal, and Italy.

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The Aloevera plants appear like succulent cactus plants, belonging to the family Liliaceae .It is a perennial plant that grows readily in hot, dry climates and currently, because of demand, is cultivated in large quantities. The firm, sturdy, sessile three sided leaves can grow up to 20 inches long and 5 inches across. The leaves grow in a rosette pattern and are broad at the base,color pea green,bright yllow tubular flowers 25-35cm in length arranged in a slender loose spike, stamens frequently project beyond the perianth tube. A full-grown plant may take 3 to 4 years to reach maturity. The AloeVera has the ability to stop any wounded leaf from losing water and nutrients. It is a self healer due to the special chemicals and physical properties involved. The hard green outer leaf has the strongest potency of nutritional content in the gel. The Aloevera leaf consists of four layers. It includes the rind, sap, mucilage, and parenchyma or gel. The newer growth in the center of the plant is protected by the more mature leaves. The Aloes produce tiny miniature plants called pups.(9) The generations of past mention the healing methods of AloeVera plants being handed down through the centuries by word of mouth. We find that the use of AloeVera appears throughout history with many testimonials of its medicinal values. The earliest record of AloeVera use comes from the Egyptians. There are records of the Egyptians drawing pictures of AloeVera plants on the walls of the temples. Many cultures such as the Egyptians would have even elevated the plant to a god-like status. The healing properties of the AloeVera were utilized for centuries earning the name Plant of Immortality.(10) The Mahometans of Egypt thought of AloeVera as a religious symbol, and. they believed that the holy symbol hanging in the doorway would protect them from slanderous and evil influence.The Egyptians used the AloeVera to make papyrus like scrolls as well as for treatment of tuberculosis.(11) AloeVera lost its potency for healing when it started being imported. The pulp worked best when fresh. This hindered Aloe veras reputation in the medical community. Europe and North Americas medical profession quit using AloeVera and replaced it with drugs. The scientists determined that the oxidation process hindered the healing properties of Aloe vera. It caused the plant to loose quality and effectiveness, gradually leading to its loss of popularity in areas where it is not grown. By the 1970s there was a breakthrough in processing techniques and they successfully; stabilized the leaf gel by using natural ingredients and cold pressing.
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They also found a way to separate the rind and aloin. These new found processing techniques have created a new market for Aloe vera.(12) Aloe sales currently supports a multi-billion dollar business world wide. For thousands of years AloeVera was part of myths and legends but today it plays a role to help improve health and nutrition. Some say that Aloe existed as a predecessor to cortisone on the island of Hawaii in Kona. The Hawiaan people would mash the leaves and stems of Aloe to make a poultice for arthritic conditions. It was quite successful.(13)

CHEMICAL CONSTITUENTS OF ALOE VERA:

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AloeVera has marvelous medicinal properties. Scientists have discovered over 150 nutritional ingredients in Aloe vera. There seems to be no single magic ingredient. They all work together in a synergistic way to create healing and health giving benefits. The ten main areas of chemical constituents of AloeVera include: amino acids, anthraquinones, enzymes, minerals, vitamins, lignins, monosaccharide, polysaccharides, salicylic acid, saponins, and sterols.(14) The amino acids in AloeVera are the building blocks of protein and influence our brain function. Humans require 22 amino acids and the body will make all of them except for eight essential amino acids which our body gets from the food/drinks that we take in. Every one of the essential amino acids are available in AloeVera and they include isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine,and tryptophan. Some of the other non-essential amino acids found in AloeVera include alanine, arginine, asparagine, cysteine, glutamic acid, glycine, histidine, proline, serine, tyrosine, glutamine, and aspartic acid.

Located in the sap of the leaves you will find twelve anthraquinones, a phenolic compound that has stimulating effects on the bowels and antibiotic properties. In small amounts the anthraquinones do not have a purgative effect. They help with absorption from the gastro intestinal tract and have anti-microbial and pain killing effects. Too many anthraquinones can produce abdominal pain and diarrhea. The most important anthraquinones are aloin and emodin. They are anti-bacterial, anti-viral, and analgesic. (15)The anthraquinones in AloeVera breakup residue, pus and lifeless cells, bring blood to the region and flush out material from the wounds and ulcers.36 Enzymes act as biochemical catalysts that break down the proteins we eat into amino acids. The enzymes turn the food we eat into fuel for every cell in our body, enabling the cells to function and work efficiently. The main enzymes found in AloeVera include Amylase (breaks down sugars and starches), Bradykinase (stimulates immune system, analgesic, antiinflammatory), Catalase (prevents accumulation of water in the body), Cellulase (aids digestion cellulose), Lipase, Oxidase, Alkaline Phosphatase, Proteolytiase (hydrolyses proteins into their constituent elements), Creatine Phosphokinase (aids metabolism), andCarboxypeptidase.(16) The next thing we need to ask ourselves is what fuels the enzymes? The key is the vitamins and minerals we take in. For instance if we lack in zinc and/or Vitamin B6, our body
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will not be able to break down or use protein. Because of the healing properties of AloeVera and its synergistic action, the body receives what it needs to work properly. Aloe vera, an antioxidant rich plant, contains vitamins such as A, C, and E plus the minerals, zinc, and selenium. Anti-oxidants help boost the immune system and combat free radicals in the body.(17) It also contains Vitamins B1, B2, B3, B5, B6, and B12 along with choline, calcium (teeth and bone formation, muscle contractions and heart health), magnesium(strengthens teeth and bones, maintains healthy muscles and nervous system, activates enzymes), zinc (speeds up wound healing, mental quickness assists with healthy teeth, bones, skin, immune system, and digestive aid), manganese (activates enzymes, builds healthy bones, nerves and tissues), chromium (assists with protein metabolism and balancing of blood sugars), selenium which all influence our brain performance.(1)

Additional minerals found in AloeVera include copper (important for red blood cells, skin and hair pigment), iron (involved in oxygen transportation and making of hemoglobin in red blood cells), potassium (helps with fluid balance), phosphorus (helps build bones and teeth, assists with metabolism and body pH), and sodium (regulates body liquids, helps with nerve and muscle performance, and helps deliver nutrients into body cells).(19) AloeVera also contains the trace minerals of rhodium and iridium used in cancer and tumor research experiments.41 Another component of AloeVera consists of the lignins, a major structural material of cellulose content, that allows for penetrative properties.(20) AloeVera can soak into the skin up to seven layers deep. Lignins penetrate the toughened areas of the skin being beneficial for skin problems such as eczema and psoriasis. The next elements of AloeVera we will discuss include monosaccharides and polysaccharides. Monosaccharides contain the simple sugars which include glucose. The polysaccharides are the more complex long-chain sugars involving glucose and mannose or the gluco-mannans. These sugars are ingested whole from the stomach. They do not get broken down like other sugars, and appear in the bloodstream in exactly the same form. This process is known as pinocytosis. Once in the blood stream, they exert their healing and immuno-regulating effect. Some of these polysaccharides are not absorbed but stick to certain
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cells lining the gut and form a barrier preventing absorption of unwanted material so helping to prevent a leaking gut syndrome. The sugars are also used in moisturizing preparations.44 One polysaccharide, acemannan, is known for its ability to restore and boost the immune system by stimulating the production of macrophages and improving the activity of TLymphocytes by up to 50 %. Acemannan produces immune agents such as interferon and interleukin which help to destroy viruses, bacteria, and tumor cells. Acemannan improves cellular metabolism by normalizing cellular function and regulating the flow of nutrients and wastes in and out of the cells. It knows how to destroy parasites and fungus. In some AIDS patients, it even protected the immune system from the toxic side effects ofAZT. Many sources stated that AloeVera has mucopolysaccharides, nitrogen containing polysaccharides, found in animals and bacteria. A regulation and testing board for AloeVera products known as the International Aloe Science Council concludes that some people are misinformed and confused on terminology. AloeVera contains salicylic acid which is an aspirin-like compound with anti -inflammatory, analgesic, and anti-bacterial properties. It has anti-pyretic properties for reducing fevers. Other constituents of AloeVera would include prostaglandins, tannins, magnesium lactate, resins, tannins, proteins such as lectins, monosulfonic acid and gibberlin. Another constituent of AloeVera includes saponins. These are soapy substances from the gel that is capable of cleansing and having antiseptic properties. The saponins perform strongly as antimicrobial against bacteria, viruses, fungi, and yeasts. The plant sterols or phyto-steroids in AloeVera include Cholesterol, Campesterol, Lupeol, and B (Beta sign) Sitosterol. The plant steroids have fatty acids in them that have antiseptic, analgesic, and anti-inflammatory properties.

Without limiting the scope of the invention, its background is described in connection with the identification of novel anti-microbial agents isolated from Aloe vera, as. Heretofore, in this field, organisms that cause infectious disease, namely, viruses, bacteria, fungi and multicellular parasites, humankind has sought to control their morbidity and mortality. With the
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isolation and characterization of powerful antibiotics, beginning over half a century ago, the balance of power between humans and microbes has been shifted toward humankind. For several decades after the introduction of penicillin in the 1940's, for example, the conquest of infectious disease appeared imminent. The widespread use of antibiotics, added to the evolutionary flexibility of microbes, has made that victory less than certain. An increasing number of bacteria, fungi and other microbes are developing resistance to antibiotics. A number of factors have contributed to the increase in microbes that are resistant to antimicrobial agents. The use of combinations of anti-microbial agents to treat nosocomial infections, particularly among patients whose immune systems are compromised by AIDS, chemotherapy, or immunosuppressive drugs, has led to a dramatic increase in multiple drug resistant (MDR) infections. Unfortunately, the future does not look bright in the war against infectious disease, as MDR strains of microbes continue to proceeding at an alarming rate. In fact, MDR strains are adapting faster than the introduction of new, more potent antibiotics. Microbes have been developing strategies to cope with change for hundreds of millions of generations. In fact, some bacteria have generation cycles of 20 minutes, with each cycle providing the opportunity to evolve and adapt. Bacteria have adapted to an extraordinary range of conditions and developed defenses against all sorts of environmental threats, environmental and artificial. To a microbe the human body is just another environment to colonize. While antibiotics are just another toxic environmental agent against which the microbe must develop an escape strategy. For organisms with populations that have already adapted to such extreme environments as boiling underwater hot-springs, learning to cope and evade antibiotics was only a matter of time and evolution.

Overuse of antibiotics has contributed to the problem of MDR microbial strains. The indiscriminate use of antibiotics throughout the world contributes to the continued emergence of MDR strains of bacteria such as Pseudomonas, Streptococcus and Staphylococcus . MDR strains have evolved in large part because many patients fail to complete the required course of antibiotic treatment, allowing stronger members of the microbial pool to be selected for in the next round of treatment. Increases in ear and sinus infections in children have been caused by the
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use of antibiotics to treat viral infections, infections that are not susceptible to antibiotic treatment. The current trend in medicine is to prescribe second-line and even last-resort antibiotics in place of first-line antibiotics. Even when there is no reason to suspect resistance to first-line antibiotics, the drive toward using stronger, faster drugs is inevitable when faced with a sick patient. In the case of recurrent lung infections in cystic fibrosis patients, physicians have had no choice but to escalate to antibiotic treatment with second-line antibiotics, eventually causing the infecting bacteria to become resistant to all available antibiotics. The emergence of multi-drug resistant( MDR )microbes has changed the balance between host and parasite, from a position in which the medical community seemed poised to achieve a conquest has lost ground in achieving a permanent conquest of microbial infection. But much has been learned in the process. Using a deeper understanding of microbes and their mechanisms of resistance, the biomedical community can continue to mount a broad array of defenses against them. The microbes growing resistance to traditional antibiotics has renewed the attention to medical basics, such as public health measures, that include a renewed effort to stem infectious diseases by increasing hygiene. For example, in HIV-infected populations, which have become breeding grounds for resistant microbes, renewed educational outreach efforts focus on the use of prophylactics. Nosocomial infections present the greatest threat to immuno-compromised patients, because MDR microbes infect the most vulnerable patients. It is the increase in MDR of microbes, and in particular bacteria, that has led to a resurgence of interest in revitalizing and improving basic techniques (like hand-washing) for preventing the spread of infection. It has also increased the need for alternative, next-generation, anti-microbial agents. These anti-microbial agents, viz., anti-viral, anti-bacterial, anti-fungal and anti-parasitic, must also be safe for use in humans and other animals. Antimicrobial-drug resistance is an increasingly important factor and poses a serious international challenge to public health in community and institutional settings. The list of resistant bacteria of major public health importance includes those causing tuberculosis, gonorrhea, pneumococcal infections, and hospital-acquired enterococcal and staphylococcal infections. Antimicrobial-drug resistance has resulted in prolonged and more serious illness, the
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use of more expensive and often more toxic drugs and drug combinations, and increased fatality rates. While pharmaceutical and biotechnology companies are constantly developing novel products based on presently known antibiotics to overcome resistance, the next-generation of anti-microbial agents must break from the known approaches to isolate and characterize these activities. A better understanding of the microbiology and molecular genetics of microbial resistance is leading to the development of a new generation of anti-microbial agents that use an approach that is intended to attack standard mechanisms of action to kill bacteria or fungi. These so called new approaches to fighting microbial infections rely on variations of existing drugs having longer half-lives and more potent effects, but rely on the existing database of pharmaceutics to attempt to outpace the microbes ability to evolve. Both competition for nutrients and bacteriocin production play a role in determining the establishment of microbial communities in nature. When analyzing symbiotic associations this may be further influenced by the presence of antimicrobial chemicals produced by the host. In the case of AloeVera barbedensis, the plant has been shown for centuries to exert broad spectrum healing activities. The source of the antimicrobial agents isolated herein were determined, as were the distinct populations of bacteria, and their dynamics within the indigenous microflora of Aloe vera. Localization of specific microbial populations was assessed using both direct culture of dissected plant material and immunological detection within tissue sections. Relative size and population diversity were determined through direct culture. Immunological detection demonstrated discrete populations within specific plant structures.

Bacterial identification was accomplished using standard staining and biochemical analysis. To more completely identify the specific species of bacterium isolated and their role in the antibacterial activity isolated herein, the environmental specimens were further analyzed using restriction fragment length polymorphism (RFLP) analysis. RFLP analysis was used to differentiate the various species of Bacillus found within the plant as well as definitive identification of Aeromicrobioum species and Curtobacterium species.

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The efficacy of aloe liquid (see e.g., Coats, Aloe Vera: The Inside Story) as an antimicrobial agent is shown herein to have a wide range of gram negative and gram positive bacteria. The antimicrobial agents of the present invention are shown herein to effectively kill, or greatly reduce or eliminate the growth rate of the following bacteria: Staphylococcus aureus, Streptococcus pneumonia, Streptococcus pyogenes, Pseudomonas aeruginosa, E. coli, Propionibacterium acne, Helicobacter pylori, and Salmonella typhi. The anti-bacteriocidal activity demonstrated herein is not due to the preservatives used in the preparation of the clear gel or the isolation of the antibacterial components, as the antimicrobial agents isolated herein were isolatable from liquid collected directly from freshly cut whole leaves prior to used in the same killing assays. In addition to the antimicrobial activities of its liquid, it also has been shown to be nontoxic even when taken internally. These properties combined provide the impetus for the use of the antimicrobial agents isolated herein when used alone or in combination. The present invention demonstrates the isolation and identification of new, non-toxic, FDA approved antimicrobial agents that have been identified and isolated from the Aloe plant. These agents are efficacious and nontoxic, with a broad spectrum of antimicrobial properties. Generally, and in one form of the invention, a composition isolated from the gel liquid of AloeVera including, at least one antimicrobial agent isolated from the clear gel isolated from the whole leaf of AloeVera , wherein the antimicrobial agent is an agent produced by the AloeVera or indigenous bacteria that colonize the Aloe vera, is disclosed.

Furthermore, a method of decreasing the growth of a broad spectrum of bacteria including the steps of, isolating at least one antibacterial agents from the clear gel of an AloeVera plant and directly contacting the bacteria with at least one antibacterial agent from aloe vera, is also disclosed. The present invention is based on the recognition that AloeVera isolated have been used to treat, and increase the healing rate of, wounds and other infectious diseases. The present invention is also based on the recognition that antibacterial agents secreted by AloeVera and the
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bacteria that grow in the gel and rind of AloeVera and which exhibit a wide range antimicrobial activity when exposed directly to the target microbe can be isolated. The antibacterial agents isolated herein have molecular weights of about: 555,000; 470,000; 240,000; 160,000; 25,000 and 4,000 Daltons. Also, the antibacterial agents isolated herein, are partially secreted by bacteria that grow in the gel and rind of AloeVera . The secreted products of these species of bacteria exhibit a wide range antimicrobial activity when exposed directly to target microbes and each other and include: Aerobacterium, Bacillus, Curtobacterium, Arthrobacter, Sporosarcina, and Clavibacter.

Historically AloeVera has been used to treat human and animal medical problems from A to Z, and many more uses for the leaf have been suggested in contemporary literature: A B allergies, abscesses, abrasions, asthma, acne, acid indigestion, allergic reactions, anemia, arterial insufficiency, arthritis, athlete's foot, AIDS. bad breath, burns, boils, bursitis, baldness, blisters / blistering, bruises, bronchitis, bloody scours in calves, body cleanser, bladder infections, blood pressure.

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corneal ulcers, contusions, canker sores (aphthous ulcers), cuts (lacerations), cataracts, chapped / chafed skin & lips, coughs, colds, colitis, carbuncles, colic, cradle cap, cystitis, Candida, chemotherapy, constipation. dermatitis, dandruff, dry skin, denture (gum) sores, diaper rash, dishpan hands, dysentery, diabetes, depression. edema, erysipelas, epidermitis, Epstein-Barr virus, exanthema, enteritis in fowl, ear ache, fauves, fissured nipples, fever blisters, fungus. genital Herpes, gingivitis, glaucoma, gangrene. heat rash / prickly heat, headache of all kinds, hemorrhoid, heart burn, high blood pressure. impetigo, inflamed joints, insomnia, ingrown toenails, infertility due to anovulatory cycles, indigestion, insect bites. jaundice, joints, keratosis follicularis, kidney infections. laxation, leprosy, laryngitis, lupus, liver ailments, leukemia multiple sclerosis, mastitis in dairy cattle, mouth irritations, muscle cramps / strains, moles nausea of all kinds onycholysis, odor control of chronic ulcers, oral disorders pin worms, psoriasis, prostatitis, poison ivy / oak, pancreas razor burn, radiation burns, radiation dermatitis, rashes stings, styes, sprains, senile moles, sores of all kind, seborrhea, stretch marks, sore throat, shingles, staph infections, sunburns, sciatic nerve, sickle-cell disease tonsillitis, tendinitis, trachoma, tuberculosis ulcerations of all kinds, urticaria, ulcers (peptic and duodenal) vaginitis, venereal sores, venous stasis, varicose veins wind burns, wheal, wounds of all kinds, warts X-ray burns, yeast infections, zoster (shingles)

D E-F G H I J-K L M N O P R S T U V W X-Y-Z

FIVE UNIQUE BENEFITS TO THE HUMAN BODY 1. Penetration - Aloe has the ability to reach the deepest body tissues, some seven layers deep. 2. Antiseptic - Aloe has six antiseptic agents: lupeol, salicylic acid, urea nitrogen, cinnamic acid, phenol and sulfur. Kills bacteria, viruses and fungus. 3. Stimulates cell growth - Aloe stimulates the birth of new, healthy tissue.
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4. Settles nerves 5. Cleanses - Aloe detoxifies and normalizes your metabolism.

REVIEW OF LITERATURE:
AloeVera is reported, shown to have anti-inflammatory activity immuno- stimulatory activity, and cell growth stimulatory activity. Activity against a variety of infectious agents has been attributed to Aloe Vera; for instance, antibacterial, and antiviral and anti fungal. Acemannan, a polysaccharide component from whole plant material, has been proposed to have indirect antimicrobial activity through its ability to stimulate phagocytic leukocytes.
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Wang et al. (1998) have reported on the effect of the anthaquinone aloe emodin on arylamine Nacetyl transferase activity in Heliobacter pylori, and hence its antimicrobial activity. The anthraquinones in AloeVera breakup residue, pus and lifeless cells, bring blood to the region and flush out material from the wounds and ulcers. Anti-Oxidants help boost the immune system and combat free radicals in the body. The polysaccharides are the more complex long-chain sugars involving glucose and mannose or the gluco-mannans. One polysaccharide, acemannan, is known for its ability to restore and boost the immune system by producing immune agents such as interferon and interleukin which help to destroy viruses, bacteria, and tumor cells. In some AIDS patients, it even protected the immume system from the toxic side effects AZT. Carrington Laboratories in the United States have separated the acemannan from Aloe Vera. The product is sold as Carryisym and is being used for treatment of AIDs and Feline Leukemia. Other constituents of AloeVera would include prostaglandins, tannins, magnesium lactate, resins, mannins, proteins such as lectins, monosulfonic acid and gibberlin. AloeVera includes saponins that is capable of clensing and having antiseptic properties. Phyto-steriods in AloeVera include Cholesterol, Campesterol, Lupeol, and B (Beta Sign) Sitosterol which have antiseptic, analgesic and anti-inflammatory properties .

OBJECTIVE AND SCOPE OF WORK:

Bacterial resistance to antibiotics is increasingly becoming a concern to public health. Currently used antibiotic agents are failing to bring an end to many bacterial infections due to super resistant strains. For this reason the search is ongoing for new antimicrobial agents, either by the design and synthesis of new agents or through the search of natural sources for as yet undiscovered antimicrobial agents. Herbal medications in particular have seen a revival of interest due to a perception that there is a lower incidence of adverse reactions to plant

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preparations compared to synthetic pharmaceuticals. Coupled with the reduced costs of plant preparations, this makes the search for natural therapeutics an attractive option. Bacterial resistance to antibiotics is increasingly becoming a concern to public health. Currently used antibiotic agents are failing to bring an end to many bacterial infections due to super resistant strains. Search is ongoing for new antimicrobial agents, either by the design and synthesis of new agents or through the search of natural sources. Herbal medications have shown a lower incidence of adverse reactions compared to synthetic pharmaceuticals.
Coupled with the reduced costs of plant preparations, this makes the search for

natural

therapeutics an attractive option.

PLAN OF WORK: Collection and Authentication of Plant.

Selection and collection of plant material and Processing of the plant material

Preparation of extracts using different solvents.

Phytochemical screening 20 | P a g e

 In-Vitro anti microbial assay using Agar Diffusing.

MATERIALS AND METHODS

Collection of Plant Material: Leaves of AloeVera were collected from herbal garden of Osmania University, Hyderabad. The plant was authenticated by taxonomist in Department of Botany, Osmania University, Hyderabad.

Preparation of AloeVera gel Extracts:

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Mature, healthy and fresh leaves of AloeVera having a length of approximately 2-3 feet were washed with hot water(40OC), dissected longitudinally and the colorless parenchymatous tissue was scraped out, liquefied in a food blender to remove the fibers .The resultant solution was lyophilized and stored at 4oC [21]. Known amounts of the lyophilized powder were extracted with Distilled water, Ethanol (95%) and Acetone at a temperature not exceeding the boiling point of the solvent [23].The solvent was recovered by distillation over the boiling water bath at atmospheric pressure and the remaining under reduced pressure in rotavapor .The resultant residue was stored in a freezer at -80OC until use. Known amount of solvent free sample was dissolved in DMSO to obtain the desired concentration and subjected to antimicrobial studies. Endotoxin assay (Limulus amebocyte lysate test- LAL) Presence of bacterial endotoxin in all three extracts (Aqueous, Ethanol and Acetone) were measured by LAL assay using an E-TOXATE kit (Sigma, St. Louis, USA) as per manufacturer's instructions. Briefly, each extracts were incubated serially with LAL and
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chromogenic substrate, detection of endotoxin was measured by generation of p-nitroaniline at 405 nm and quantified against a standard curve of supplied bacterial endotoxin.(24) Human pathogenic Microbial Cultures: Bacterial Cultures: Bacillus Subtilis, Bacllus Cereus, Bacllus Megaterium, Entero coccus facalis, Staphylococcus aureus, Staphylococcus pyogenes, Staphylococcus epidermidis, Micrococcus kristinae, Agrobacterium tumefaciens, Escherichia coli , Entrobacter aerogenes, Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Salmonella typhi, Shigell sonnei were obtained from the Department of Microbiology, Nizam Medical college, Hyderabad, India. All the test strains were maintained on nutrient agar slope (Hi-Media Laboratories Pvt. Limited, Mumbai, India) at room temperature and were sub-cultured into newly prepared nutrient agar slants, every two-week. Fungal Culture: Aspergillus fumigatus, Aspergillus Niger, Aspergillus flavus, Aspergillus glaceaus, Candida tropicalis, Candida albicans, Fusarium oxysporum, Micro sporum canis, Tricophytom mentagrophyte were obtained from the Department of Microbiology, Nizam Medical College, Hyderabad, India. All the fungal strains were maintained on malt extract-agar, while Candida tropicalis, Candida albicans are maintained on Sabouraud-dextrose-agar (HiMedia Laboratories Pvt. Limited, Mumbai, India) at room temperature and were sub cultured, every two -weeks.

Plant pathogenic microbial Cultures: Pseudomonas syringe, Xanthomonas camestris, Colletotrichum coccodes, Fusariumoxysporum, Rhizoctonia solani these test strains were isolated from tomato, potato crops by the hyphae point and monoscopic techniques. The isolate strains were maintained on potato-dextrose-agar (Hi-Media Laboratories Pvt. Limited, Mumbai, India) at room temperature. All the microbial cultures assay. Disc preparation: The 6mm (diameter) discs were prepared from what man No.1 filter paper and the discs were sterilized by autoclave at 121OC. After sterilization the moistened discs were kept in hot air oven at 50OC [25].Then disc were impregnated with suitable concentration of the AloeVera gel extracts from stock of 1mg/ml. Each disc contained 50g of extract.
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were served as test pathogens for the

Determination of Antibacterial Activity: Antibacterial activity was carried out by diskdiffusion method using nutrient Agar medium [26]. Hundred micro liter of suspension containing 108 colony forming units mL-1 of bacteria spread over the nutrient agar medium plates by using separate sterile cotton buds. After the microbial lawn preparation three different extract of plant discs(aqueous, ethanol and acetone extract) were firmly pressed on to the agar surface of each seeded plate. Petri dishes were incubated at 37OC for 24 h and the average diameter of the inhibition zone surrounding the wells was determined visually. Determination of Antifungal activity: Antifungal activity was carried out by disk-diffusion method [27]. Hundred micro liter of suspension containing 104 spores mL-1 of fungi was spread on Potato Dextrose agar (PDA) medium. The plates were allowed to dry for 10-15 min. After the microbial lawn preparation three different extract of plant discs were placed on the organism inoculated plates. The plates were incubated aerobically at 25OC for 72 h for fungi. Anti fungal activities were measured and indicated by clear zones of inhibition against the test organism. The diameter of the minimum zone of inhibition was measured in mm. For each test, three replicates were performed.

Phytochemical Analysis: Phytochemical screening can be done by performing various chemical identification tests which not only give information about the chemical nature of the phytoconstituents but also serves for the quantitative estimation. The various chemical identification tests performed are as follows: 1. Detection of carbohydrates:

Molischs test Solution of extract is added with alcoholic - naphthol followed by concentrated H2SO4, a violet colored ring appears at the junction. Barfoeds test 1ml of solution of extract is added with Barfoeds reagent and heated. If red cupric oxide is formed, monosaccharide is present.
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Selivanoffs test To the solution of extract add crystals of resorcinol and equal volume of concentrated HCl and heat on a water bath, rose color is produced. Osazone formation test Heat the solution of extract with solution of phenyl hydrazine hydrochloride, sodium acetate and acetic acid. Yellow crystals formed are examined under microscope which are of characteristic shape for particular sugars.

2. Detection of coumarin glycosides: Place a small amount of sample in test tube and cover the test tube with a filter paper moistened with dilute sodium hydroxide solution. Place the covered test tube on water bath for several minutes. Remove the paper and expose to ultraviolet light, the paper shows green fluorescence[16].

3. Detection of triterpenoids:

Libermann Burchard test Treat the extract with few drops of acetic anhydride, boil and cool. Then add concentrated sulfuric acid from the side of the test tube, brown ring is formed at the junction of two layers and formation of deep red color indicates presence of triterpenoids.

Salkowski test Treat the extract with few drops of concentrated sulfuric acid, formation of yellow colored lower layer indicates presence of triterpenoids.

4. Detection of essential volatile oils: To the thin section of the drug, add alcoholic solution of Sudan III. Red colour obtained by globules indicate the presence of volatile oil. To the thin section of the drug, add a drop of tincture alkane. Red colour indicates the presence of volatile oil.

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Phytochemical analysis of Ethanol, Acetone and Water extracts for presence/absence of metabolites such as carbohydrates, alkaloids, glycosides, Flavonoids, tannins, steroids, saponins, triterpenoids, and Phenolic anthraquinones was carried out [28].

Statistical Analysis: Data were expressed as mean standard deviation. The data obtained were subjected to ANOVA test to determine whether there was significant difference between extract used and also between the lengths of incubation.

RESULTS:
Antimicrobial activity of different extracts of AloeVera gel (ethanol, acetone and aqueous) at 50g concentration against some important human and plant pathogenic micro organisms were presented in table 1-4. LAL assay showed the amount of endotoxin to be 0.01 IU/ml in all the three extracts indicating a negative result. This confirms very negligible amount of endotoxins in the extracts which were screened against human and plant pathogens.

Antibacterial activity (Table 1, 2 and Fig 1&2): Table 1: Antibacterial activity of AloeVera gel extracts against the gram (+) ve bacterial strains tested based on disc diffusion method
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MICRO ORGANISM

AQUEOUS EXTRACT(50g) 7.96 0.01 6.89 0.03 _ _ 7.89 0.02 8.0 0.01 _ _

ETHANOL EXTRACT(50g) 15.10 0.01 9.26 0.04 10.32 0.02 9.34 0.02 11.07 0.07 12.00 0-02 7.98 0.01 15.22 0.02

ACETONE EXTRACT(50g) 10.08 0.01 10.28 0.02 10.9 0.05 10.10 0.01 15.98 0-02 16.46 0.02 14.00 0.03 11.29 0.01

Bacillus Subtilis Bacllus Cereus Bacllus megaterium Entero coccus facalis Staphylococcus aureus Staphylococcus pyogenes Staphylococcus epidermidis Micrococcus kristinae

Antibacterial activity: Zone of Inhibition (mm in diameter) (Mean SD) (n=3), = Negative antibacterial activity (no zone of inhibition),

Fig: Bacillus Cereus

Fig: Bacillus Subtilis

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Fig: Bacillus aureus

Fig: 1 Antibacterial activity of AloeVera gel extracts against the gram (+) ve bacterial strains Table 2: Antibacterial activity of AloeVera gel extracts against the gram (-) ve bacterial strains tested based on disc diffusion method
MICRO ORGANISM AQUEOUS EXTRACT(50g) _ 6.21 0.01 _ _ 7.23 0.01 ETHANOL EXTRACT(50g) 10.56 0.02 9.88 0.01 7.26 0.02 11.98 0.03 11.02 0.03 ACETONE EXTRACT(50g) 11.39 0.05 15.03 0.02 7.34 0.02 16.04 0.03 14.12 0.02

Agrobacterium tumefaciens Escherichia coli Entrobacter aerogenes Pseudomonas aeruginosa Proteus mirabilis

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Proteus vulgaris Salmonella typhi Shigell sonnei

_ _ _

7.98 0.04 9.76 0.04 6.57 0.03

15.09 0.02 10.77 0.03 7.25 0.02

Antibacterial activity: Zone of Inhibition (mm in diameter) (Mean SD) (n=3), = Negative antibacterial activity (no zone of inhibition),

Fig: Escherichia coli

Fig: Pseudomonas aeruginosa

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Fig: Proteus vulgaris

Fig: 2 Antibacterial activity of AloeVera gel extracts against the gram (-) ve bacterial strains Antibacterial activity of AloeVera gel was analyzed against eight gram +ve, eight gramve pathogenic strains. Among three extract ethanol, acetone extracts recorded significant antibacterial activity against all test pathogens, the maximum antimicrobial activities were observed in acetone extract other than ethanol and aqueous extract. Antibacterial activity of the acetone extract of AloeVera gel was found to be quite impressive as compared to ethanol and aqueous extracts. However, high inhibition activity against Staphylococcus aureus (15.98 0.02), Staphylococcus pyogenes (16.46 0.02), Staphylococcus epidermidis (14.0 0.03), Escherichia coli (15.03 0.02), Pseudomonas aeruginosa (16.04 0.03), Proteus mirabilis (14.12 0.02), Proteus vulgaris (15.09 0.04) was observed, moderate inhibition activity against Bacillus Subtilis (10.80 0.01), Bacllus Cereus (10.25 0.02), Bacllus Elaterium (10.90 0.05),Entero coccus facalis (10.10 0.01), Agrobacterium tumefaciens (11.39 0.05), Salmonella typhi (10.77 0.03) and low inhibition activity against Shigell sonnei (7.25 0.02), Entrobacter aerogenes (7.34 0.02). In case of ethanol extract it showed inhibitory activity against all test pathogens but it showing high inhibition activity against Bacillus Subtilis (15.10 0.01), Micrococcus kristinae (15.22 0.02) compared to acetone extract, surprisingly aqueous extract could not showing any inhibitory effect against all pathogens except in Bacillus Subtilis (7.96 0.01), Bacllus Cereus (6.89 0.03), Staphylococcus aureus (7.89 0.02), Staphylococcus pyogenes (8.0 0.01),Escherichia coli (6.21 0.05), Proteus mirabilis (7.23 0.01) it showing very low inhibiton activity.
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Anti fungal activity (Table 3 and Fig 3):Table 3: Anti fungal activity of AloeVera gel extracts against the fungal strains tested based on disc diffusion method.
MICRO ORGANISM AQUEOUS EXTRACT(50g) 7.25 0.02 7.16 0-02 _ 6.93 0.13 _ _ _ _ 6.69 0.20 ETHANOL EXTRACT(50g) 11.12 0.03 10.10 0.21 11.57 0.02 10.63 0.04 11.1 0.04 9.96 0.21 10.49 0.05 7.27 0.02 10.21 0.28 ACETONE EXTRACT(50g) 14.63 0.05 10.20 0.03 15.27 0.02 11.06 0.11 11.94 0.04 10.20 0.03 10.83 0.02 11.20 0.04 11.61 0.01

Aspergillus fumigates Aspergillus niger Aspergillus flavus Aspergillus glaceaus Candida tropicalis Candida albicans Fusarium oxysporum Micro sporum canis Tricophytom mentagrophyte

Anti fungal activity: Zone of Inhibition (mm in diameter) (Mean SD) (n=3), = Negative anti fungal activity (no zone of inhibition).

Fig: 3 Anti fungal activity of AloeVera gel extracts against the fungal strains

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Antifungal activity of AloeVera gel was analyzed against nine fungal pathogenic strains. Among three extract ethanol, acetone extracts recorded significant antifungal activity against all test pathogens, the maximum antifungal activities were observed in acetone extract other than ethanol and aqueous extract. Antifungal activity of the acetone extract of AloeVera gel was found to be quite impressive as compared to ethanol and aqueous extracts. However, high inhibition activity against Aspergillus fumigates (14.63 0.05), Aspergillus flavus (15.27 0.02) was observed, moderate inhibition activity against Aspergillus Niger (10.20 0.03), Aspergillus glaceaus (11.06 0.11), Candida tropicalis (11.94 0.04), Candida albicans (10.20 0.03), Fusarium oxysporum (10.83 0.02), Micro sporum canis (11.20 0.04). In case of ethanol extract it showing inhibition activity against all test pathogens but it showing low inhibition activity against Aspergillus flavus (11.57 0.02), Micro sporum canis (7.27 0.02). Surprisingly aqueous extract could not showing any inhibitory effect against all pathogens except in Aspergillus fumigatus (7.25 0.02), Aspergillus Niger (7.16 0.2), Aspergillus glaceaus (6.93 0.13), Tricophytom mentagrophyte (6.69 0.20) it showing very low inhibiton activity.

Table 4: Antimicrobial activity of AloeVera gel extracts against the plant pathogenic strains tested based on disc diffusion method.
MICRO ORGANISM AQUEOUS EXTRACT(50g) _ _ _ _ _ ETHANOL EXTRACT(50g) 10.12 0.03 6.46 0.02 8.99 0.03 10.07 0.04 9.91 0.03 ACETONE EXTRACT(50g) 13.28 0.02 7.95 0.04 9.73 0.03 11.19 0.01 10.29 0.02

Pseudomonas syringe Xanthomonas camestris Colletotrichumcoccode s Fusarium oxy sporum Rhizoctonia solani

Antimicrobial activity: Zone of Inhibition (mm in diameter) (Mean SD) (n=3), = Negative antibacterial activity (no zone of inhibition),

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Fig: 4

Antimicrobial activity of AloeVera gel extracts against the plant pathogens

The result of AloeVera gel extracts against the plant pathogens showed (Table 4 and Fig 4) that among three extract ethanol, acetone extracts showed an inhibitory effect against all test pathogens. Surprisingly aqueous extract could not show any inhibitory effect against all pathogens.

Phytochemical Evaluation:
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Table 5: Preliminary Phytochemical analysis of AloeVera gel extracted with different solvents Phytoconstituents Carbohydrates Alkal oids Glycosides Flavonoids Tannins Steroids Saponins Triterpenoids Phenolic anthraquinones Test Molish Test Dragnedroft Mayer Wanger Glycoside test Flavonoid test(0.5% KOH alch) Tannins test(1% Pb(OAC)2) Steroid test Saponine test(5% Hgcl2 Triterpenoids test Phenole test(1% FeCl3)
WE AE EE

+ + + + + +

+ + + + + + +

+ + + + + + +

+ Present, - Absent, WE-Water extract, AE-Acetone extract, EE-Ethanolic extract

Phytochemical analysis of all the extracts revealed that Carbohydrates, Glycosides, Flavonoids Phenolic anthraquinones, Tannins and Saponins are generally present in all the extract. Triterpenoids were found in Acetone and Ethanolic extracts. Other metabolites such as Alkaloids, Steroids were absent in all the extracts (Table 5).

DISCUSSION
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Antibiotics provide the main basis for the therapy of microbial infections. However, the high genetic variability of bacterial enables them to rapidly evade the action of antibiotics by developing antibiotic resistance. In recent years development of multidrug resistance in the pathogenic bacteria and parasites has created major clinical problems in the treatment of infectious diseases[29].This and other problems such as toxicity of certain antimicrobial drugs on the host tissue[30,31] triggered interest in search of new antimicrobial substances/drugs of plant origin. Considering the rich diversity of plants, it is expected that screening and scientific evaluation of plant extracts for their anti-microbial activity may provide new anti-microbial substances; hence the present investigation clearly reveals the antimicrobial nature of this plant and suggests that this plant could be exploited in the management of diseases caused by these microbes in both plant and human systems. It is interesting to note that antimicrobial activity was highly pronounced in solvent extracts compared to aqueous extract. It is also important to note that susceptibility of the pathogens was varied to solvent extract and aqueous extract. This indicates the presence of more than one active principle in AloeVera gel. Plants are rich reservoir of antimicrobials[32] it is observed that a single plant is known to contain several active principles of biological significance[33] .The present finding is hence highly encouraging in recognizing a plant of interesting antimicrobial activity. Numerous aloe species around the world are used for conditions ranging from dermatitis to cancer [34]. Furthermore, the fresh gel, juice or formulated products have been used for medical, food and cosmetic purposes, as well as for general health [35]. In the last decade, AloeVera has been used extensively in health drinks, topical creams, toiletries and cosmetics [36] and there are many reported claims of its beneficial properties, encompassing a broad range of conditions [37]. There is a wide range of research from all over the world based upon different species of Aloe for antimicrobial activity. In the study, AloeVera gel had inhibitory effect against some diseases caused pathogens in human and plants. Among three extract (ethanol, acetone and, aqueous extracts) ethanol, acetone extracts recorded significant antimicrobial activity against all test pathogens, the maximum antimicrobial activities were observed in acetone extract other than ethanol and aqueous extract. Antibacterial and antifungal activity of the acetone extract of AloeVera gel was found to be quite impressive as compared to ethanol and aqueous extracts. In case of ethanol extract it showing inhibition activity against all test pathogens but it showed high inhibition activity against Bacillus Subtilis, Micrococcus kristinae compared to acetone extract
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and low inhibition activity against Aspergillus flavus, Microsporum canis surprisingly aqueous extract failed to show any inhibitory effect against all pathogens except in Bacillus Subtilis, Bacllus Cereus, Bacllus Megaterium,Staphylococcus aureus, Staphylococcus pyogenes, Proteus mirabilis,Aspergillus fumigates ,Aspergillus niger, Aspergillus glaceaus,Tricophytom mentagrophyte. Regarding the possibility of presence of endotoxins in the extracts many literatures in both in vitro and in vivo studies revealed below detectable level of endotoxin level in AloeVera gel [38]. As the extracts were significantly active against plant pathogens, it may be employed as a promising pesticide against plant pathogens which were under taken in our study. Further studies are to be under taken to establish the pesticidal activity of AloeVera gel extracts. The mechanism of action of the gel extract on the lysis of bacterial cells may be due to the pore formation in the cell wall and the leakage of cytoplasmic constituent by the active components such as alkaloids present in the gel extract as revealed by Shelton [39]. There are over 75 known ingredients in the AloeVera leaf gel and they are all contained in about 1% of the plant, the rest(99%) being water, so they are obviously present only in small amounts. Their action is thought to arise from the synergistic effect of these substances i.e., they can be likened to work together as a team so that the total effect is greater than would be expected from the individual effect of each substance. Thus it was not surprising that AloeVera leaf gel extract was highly prized just for this reason. Reports suggest that the beneficial effects of AloeVera gel are due to its high molecular weight compounds such as polysaccharides [40], lectin-like proteins [41] and prostaglandins [42]. Aloes anti-inflammatory effects may be due to a bardykinin degrading glycoprotein [43] and mannose-6-phosphate may have a role in the wound healing process [44]. Anthraquinones form various plant extracts have been studied for their possible anti-viral properties [45]. The discovery of a potent herbal remedy that is safe will be a big advancement in fungal infection therapies. The presence of anthraquinones, saponins, tannins, lectins and alkaloids in the extract may be attributed to the antifungal actions of Aloe Vera. The results of the present study also explains the use of this plant in folk medicine for the treatment of various diseases whose symptoms might involve microbial infections and underline the importance of the ethno botanical approach for the selection of AloeVera in the discovery of new bioactive compounds. From the above results, it may be concluded that AloeVera gel extracts possesses compounds with antimicrobial properties which can be used as antimicrobial agents in new drugs for therapy of infectious disease in humans and plants. Further studies are in progress
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to isolation and estimation of active components responsible for the antimicrobial properties of AloeVera leaf gel and to elucidate their molecular mechanism of action.

CONCLUSION
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AloeVera gel extracts possesses compounds with antimicrobial properties which can be used as antimicrobial agents in new drugs for therapy of infectious disease in humans and plants. Further studies are in progress to isolation and estimation of active components responsible for the antimicrobial properties of AloeVera leaf gel and to elucidate their molecular mechanism of action. This investigation shows that the gel of AloeVera are useful and that they can complement one another in their medicinal capabilities.

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