Friday, 27 May 2016

COMPARATIVE STUDY OF ANTIBACTERIAL ACTIVITY OF ALOE VERA EXTRACT AND CITRUS LEMON ON ACNE AND PUS SAMPLE

The spread of multiple antimicrobial-resistant pathogenic bacteria has been recognized by the World Organization for Animal Health (OIE), the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) as a serious global human and animal health problem. The development of bacterial antimicrobial resistance is neither an unexpected nor a new phenomenon. It is, however, an increasingly troublesome situation because of the frequency with which new emerging resistance phenotypes are occurring among many bacterial pathogens and even commensal organisms. Acne vulgaris is a most common skin disorder of Pilosebaceous unit. That affects areas containing the largest oil glands, including the face, back, and trunk(Leyden,1997)It is generally characterized by formation of seborrhea, come done, inflammatory lesions and presence of bacteria Propionibacterium acnes, Staphylococcus epidermidis and Malassezia furfur in the follicular canal and sebum production (Leyden et al,2001). Propionibacterium acnes have been described as an obligate anaerobic organism. It is implicated in the development of inflammatory acne by its capability to activate complements and by its ability to metabolize sebaceous triglycerides into fatty acids, which chemotactically attract neutrophils. On the contrary, Staphylococcus epidermidis, an aerobic organism, usually involves in superficial infections within the sebaceous unit (CG.et al, 1999).These factors provide a potential target for treatment. Propionibacterium acnes and Staphylococcus epidermidis, aureus are the target sites of anti-acne drugs (Leyden et al, 2001; Hamnerius et al, 1996).
Historically, many infections could be treated successfully according to the clinician’s past clinical experience (i.e. empirical therapy); however, this is becoming more the exception than the rule (Walker, 2007). Resistance has been observed to essentially all of the antimicrobial agents currently approved for use in human and veterinary clinical medicine. This combined with the variety of antimicrobial agents’ currently available, makes the selection of an appropriate agent an increasingly more challenging task. This situation has made clinicians more dependent on data from in-vitro antimicrobial susceptibility testing, and highlights the importance of the diagnostic laboratory in clinical practice.
A number of antimicrobial susceptibility testing (AST) methods reproducibility, accuracy, and individual preference. Standardization and harmonization of AST methodologies, used in epidemiological surveillance of antimicrobial drug resistance, are critical are available to determine bacterial susceptibility to antimicrobials. The selection of a method is based on many factors such as practicality, flexibility, automation, cost, if data are to be compared among national or international surveillance/monitoring programs of OIE Members. It is essential that AST methods provide reproducible results in day-to-day laboratory use and that the data be comparable with those results obtained by an acknowledged ‘gold standard’ reference method. In the absence of standardized methods or reference procedures, susceptibility results from different laboratories cannot be reliably compared. The method used to select samples for inclusion in antimicrobial resistance surveillance programmes, as well as the methods used for primary bacterial isolation, are also important factors that should be standardized or harmonized to allow direct comparison of data between different regions; consideration of these issues is addressed in an OIE document (Dehaumont, 2004). Because of available antimicrobials failure to treat infectious diseases, many researchers have focused on the investigation of natural products as source of new bioactive molecules (Recio and Rios, 1989; Silver and Bostian, 1993).Herbs and other natural remedies can be as effective as traditional treatments, often without the same negative side effects, here are some super healers to add to the all-natural section of medicine cabinet.

 2.1 Natural remedies for skin wounds and infection:

2.1.1 Turmeric (Ease arthritis):
A heaping helping of curry could relieve your pain. That's because turmeric, a spice used in curry, contains curcumin, a powerful anti-inflammatory that works similarly to Cox-2 inhibitors, drugs that reduce the Cox-2 enzyme that causes the pain and swelling of arthritis, it might also: Prevent colon cancer and Alzheimer's disease. Maximize the benefits for general health, adding the spice to your cooking whenever possible for a therapeutic dose.


 In a recent German study of type 2 diabetics, taking cinnamon extract daily successfully reduced blood sugar by about 10%.It might also: Lower cholesterol. Cinnamon packs a one-two punch for people with type 2 diabetes by reducing related heart risks.
2.1.3 Rosemary (Avoid carcinogens):
Frying, broiling, or grilling meats at high temperatures creates HCAs (heterocyclic amines), potent carcinogens implicated in several cancers. But HCA levels are significantly reduced when rosemary extract (a common powder) is mixed into beef before cooking, it might also stop tumors. Rosemary extract helps prevent carcinogens that enter the body from binding with DNA.
2.1.4 Ginger (Avert nausea):
Ginger can prevent stomach upset from many sources, including pregnancy, motion sickness, and chemotherapy. A powerful antioxidant, ginger works by blocking the effects of serotonin, a chemical produced by both the brain and stomach when you're nauseated, and by stopping the production of free radicals, another cause of upset in your stomach, it might also decrease your blood pressure, arthritis pain, and cancer risk. Ginger helps regulate blood flow, which may lower blood pressure and its anti-inflammatory properties might help ease arthritis.
2.1.5 Garlic (Lower cancer risk):
High consumption of garlic lowered rates of ovarian, colorectal, and other cancers; it might also provide cardiovascular benefits. Garlic contains more than 70 active phytochemicals, including allicin, which many studies have shown decreases 
high blood pressure by as much as 30 points. Garlic may help prevent strokes as well by slowing arterial blockages. Crushed fresh garlic offers the best cardiovascular and cancer-fighting benefits.( NANCY,2011)

2.1.6 LEMON JUICE:

According to world health organization (WHO), any plant which contain substances that can be used for therapeutic purpose or which are precursor of chemo-pharmaceuticals semi synthetic new drugs is referred as medicinal plant (salih and Abass 2003).The antimicrobial activity of plants had been received attention many years ago as one of the most effective mechanism for the control of microorganisms (saadi et al, 2003). Crude extracts of some plants could play roles in hastening the haemostatic activities in a damaged tissue or vessels when applied topically on the wound or cut, to arrest bleeding and hasten healing of wound (Ligha et al 2008). Lemon is one of those super foods with a myriad health and cosmetic benefits, being a citrus fruit fights against infection. It helps in production of WBC’s and antibodies in blood which attacks the invading microorganism and prevents infection. It is an antioxidant which de-activates the free radicals preventing many dangerous diseases like stroke, cardiovascular diseases and cancers. It lowers blood pressure and increases the levels of HDL (good cholesterol).Lemon are found to be anti-carcinogenic which lower the rates of colon, prostate and breast cancer. They prevent faulty metabolism in the cell, which can predispose a cell to becoming carcinogenic. Also blocks the formation of nitrosamines in the gut.A few drops of Lemon juice in hot water are believed to clear the digestive system and purify liver as well. The skin of lemon dried under the sun and then ground to make powder can be applied to the hair for a few minutes before bath which relieves head ache and cools the body. Lemon juice is given to relieve gingivitis, stomatitis and inflammation of the tongue, prevent common cold and treat urinary tract infection and gonorrhea. Lemon juice helps to prevent and cure osteoarthritis.

2.1.6.1 Medicinal Value of Lemon:

Lemon is an important medicinal plant of the family Rutaceae. It is cultivated mainly for its alkaloids, which are having anticancer activities and the antibacterial potential in crude extracts of different parts (viz., leaves, stem, root and flower) of Lemon against clinically significant bacterial strains has been reported (Kawaii et al, 2000). Citrus flavonoids have a large spectrum of biological activity including antibacterial, antifungal, antidiabetic, anticancer and antiviral activities (Burt, 2004; Ortuno et al, 2006). Flavonoids can function as direct antioxidants and free radical scavengers, and have the capacity to modulate enzymatic activities and inhibit cell proliferation (Duthie and Crozier, 2000). In plants, they appear to play a defensive role against invading pathogens, including bacteria, fungi and viruses (Sohn et al., 2004). Flavonoids are generally present in glycosylated forms in plants, and the sugar moiety is an important factor determining their bioavailability. Preparation from peel, flowers and leaves of bitter orange (Citrus aurantium L.) are popularly used in order to minimize central nervous system disorders (Pultrini et al, 2006).Lemon is an antioxidant which deactivates the free radicals preventing many dangerous diseases like stroke, cardiovascular disease and cancers. It also fights against infection. It helps in production of WBC’s and antibodies in blood which attacks the invading micro organisms and prevents infection. Its healing ability could be due to its bacteria ridding and astringent properties. Lemon juice when applied in the site of bites and stings of certain insects relieves its poison and pain. It relieves chilblains and itchy skin. Its application to acne (pimples) dries the existing ones and prevent from getting more. Chemical contents of citrus limonum risso: its main active ingredients are flavonoids, Ascorbic acid (vitamin c), essential oil (Lignan, 2005), caffeine, pectin, minerals especially potassium and calcium, water fibers and sucrose, (Wikipedia 2013). Pharmacologically, lemon is primarily important for its vitamin C & potassium content. German studies in the late 1980s related to effect of peel lemon oil also posses antioxidant activity clinical trials are lacking. Lemon juice may increase iron absorption.

2.1.6.2 Mechanism of Lemon juice in agar well diffusion method:

Antibacterial analysis method was performed to evaluate the antimicrobial properties of plant extract with the help of agar well diffusion method (Hadacek and Greger, 2000). Screening of bacterial strains for susceptibility to C.limonum fruit juice extract was by a modification of agar well diffusion technique (Okeke et al., 2001).

2.1.7 Aloe Vera gel:

The semi-tropical plant, Aloe Vera, has a long and illustrious history dating from biblical times. It has been mentioned throughout recorded history and given a high ranking as an all-purpose herbal plant. Aloe’s thick, tapered, spiny leaves grow from a short stalk near ground level. It is not a cactus, but a member of the tree lily family, know as Aloe barbadensis. Aloin and its gel are used as skin tonic against pimples. Aloe vera is also used for soothing the skin, and keeping the skin moist to help avoid flaky scalp and skin in harsh and dry weather. The Aloe sugars are also used in moisturizing preparations [Barcroft and Myskja, 2003]. Mixed with selected essential oils, it makes an excellent skin smoothening moisturizer, sun block lotion plus a whole range of beauty products. Due to its soothing and cooling qualities, Maharishi Ay- urveda recommends Aloe vera for a number of skin problems [Joseph et al, 2010]. Aloe vera extracts have antibacterial and antifungal activities, which may help in the treatment of minor skin infections, such as boils and benign skin cysts and have been shown to inhibit the growth of fungi that cause tinea [Sumbul et al, 2004]. Aloe juice is helpful in smooth functioning of the body machinery [Saroj et al, 2004]. It reduces cell-damaging process during stress condition and minimizes biochemical and physio-logical changes in the body (Foster, 1999).

2.1.7.1 Medicinal value of aloe Vera gel:

A. Vera has been used for many centuries for its curative and therapeutic properties and although over 75 active ingredients from the inner gel have been identified, therapeutic effects have not been correlated well with each individual component [Habeeb et al, 2007]. Many of the medicinal effects of aloe leaf extracts have been attributed to the polysaccharides found in the inner leaf parenchymatous tissue[Tizard et al,2004; Turner et al,2004).But it is believed that these biological activities should be assigned to a synergistic action of the compounds contained therein rather than a single chemical substance(Dagne et al,2000). In the pharmaceutical industry, it has been used for the manufacture of topical products such as ointments and gel preparations, as well as in the production of tablets and capsules (Eshun et al, 2004). Important pharmaceutical properties that have recently been discovered for both the A.vera gel and whole leaf extract include the ability to improve the bioavailability of co-administered vitamins in human subjects (Vinson et al ,2005).The Aloe gel has been used for the treatment of radiation burns and radiation ulcers (Syed et al,1997).The fresh gel was more effective than the cream [Tizard  et al,2004) as Aloe gel-treated lesions healed faster (11.8 days) com-pared to burns treated with petroleum jelly gauze (18.2 days) by Fulton [Fulton,1990]. Aloe vera is anthelmintic, aperients, carminative, deob-struent, depurative, diuretic, stomachic and emmena-gogue. Juice is used in skin care medicine, dyspepsia, amenorrhea, burns, colic, hyperadenosis, hepatopathy, splenopathy, constipation, span menorrhea, abdominal tumors, dropsy carbuncles, sciatica, lumbago and flatu-lence. The elio, a product made by juice of this plant, is used for helminthiasis in children and is a purgative, anthelmintic & emmenagogue. A number of glycoprotein present in Aloe vera gel has been reported to have anti-tumor and antiulcer effects and to increase proliferation of normal human dermal cells [Choi et al 2001; Yagi et al 2003]. Gel is useful in ulcerative colitis and pressure ulcers, respectively [Langmead et al, 2004; Thomas et al, 1998,]. Traditionally, Aloe vera gel is used both, topically (treat-ment of wounds, minor burns, and skin irritations) and internally to treat constipation, coughs, ulcers, diabetes, headaches, arthritis, immune-system deficiencies [Eshun and He, 2004; Vogler and Ernst,1999]. Aloe vera has been used for medicinal purposes in several cultures for millennia: Greece, Egypt, India, Mexico, Japan, and China [Marshall, 1990]. The Egyptians used the Aloe vera to make papyrus like scrolls as well as for treatment of tuberculosis (Baker,1975) Nadkerni [Nadkern,1976] stated various preparations of Aloe barbadensis like confection, lotion and juice, useful remedies for curing various dis-eases. Aloe contains mixture of glycosides collectively called Aloin which is the active constituent of various drugs. Traditionally Aloe is extensively used in treating urine related problems, pimples and ulcers etc. It is also used in gerontology and rejuvenation of aging skin. The juice of Aloe vera leaves is used as stomachic tonic and purgative. Scientific evidence for the cosmetic and therapeutic effectiveness of Aloe vera is limited and when present is frequently contradictory [Nadkerni, 1976; Marshall, 2000].Currently, the plant is widely used in skin care, cosmetics and asnutraceuticals [Gordon and David, 2001]. Aloe vera gel has been reported to have a protective effect against radiation damage to the skin
[Roberts and Travis, 1995; Sato and Ohta,1990]. The antiseptic property of Aloe vera is due to presence of six antiseptic agents namely lupeol, salicylic acid, urea nitrogen, cinnamonic acid, phenols and sulphur. These compounds have inhibitory action on fungi, bacteria and viruses. Though most of these uses are interesting con- trolled trials are essential to determine its effectiveness in all diseases [Zawahry et al, 1973].

2.1.7.2 Mechanism of aloe Vera gel juice in agar well diffusion method:

The activity of A. vera inner gel against both Gram-positive and Gram-negative bacteria has been demonstrated by several different methods [Habeeb et al, 2007].Aloe Vera gel has the tendency to cure many skin infections clear zone shows in agar well diffusion method which means ale gel is effective against bacterial strains. A variety of methods are found for this purpose and since not all of them are based on same principles, results obtained will also be profoundly influenced not only by the method selected, but also by the microorganisms used to carry out the test, and by the degree of solubility of each test-compound (Berghe and Vlietinck, 1991; Rios et al, 1988). The test systems should ideally be simple, rapid, reproducible, and inexpensive and maximize high sample throughput in order to cope with a varied number of extracts and fractions. The complexity of the bioassay must be defined by laboratory facilities and quality available personnel (Hadacek and Greger, 2000; Hostettm et al, 1997).
The currently available screening methods for the detection of antimicrobial activity of natural products fall into three groups, including bioautographic, diffusion, and dilution methods. The bioautographic and diffusion methods are known as qualitative techniques since these methods will only give an idea of the presence or absence of substances with antimicrobial activity. On the other hand, dilution methods are considered quantitative assays once they determine the minimal inhibitory concentration (Berghe and Vlietinck, 1991).

The main purpose of this study is to compare the sensitivity against microorganisms with the help of aloe gel and lemon juice extract. For this, we used screening method i.e. agar well diffusion method to check the antibacterial activity of clinical samples(pus and acne samples).This study shows that herbal products are more preferable  for skin infections, wounds and have a great healing quality. As compare to other medicines and antibiotic drugs these herbal products are toxic and chemical free and no side effects after used.
Discussion:

Herbal medicines are a valuable and readily available resources for primary health care and complementary health care system, undoubtedly the plant kingdom still holds many species of plants containing substances of medicinal value that have yet to be discovered, though large number of plants are constantly being screened for their antimicrobial effect, these plant may prove to be a rich source of compounds with possible antimicrobial activities, but more pharmacological investigations are necessary.

7.1.1 Antimicrobial activity of Aloe vera gel:
Except for Vitamin D, Aloe Vera plants are rich in all vitamins, including the antioxidant Vitamins A (beta-carotene), C and E. It is also one of the very few plant sources of Vitamin B12, Many important minerals are found in Aloe Vera Plants, such as Calcium, Sodium Potassium. In the present study aloe vera gel showed antibacterial activity against 3 different bacterial isolates. i.e. (Staphylococcus aureus, Pseudomonas areginosa and Staphylococcus epidermidis). The antibacterial sensitivity check by the zone of inhibition in diameter on MHA media by well diffusion method. Pseudomonas areginosa highest inhibitory zone (23 mm in diameter) against Aloe vera gel extract. While in other studies (Munther and AlKayya, 2015) it was observed that P.areginosa also showed maximum inhibitory zone (up to 22 mm in diameter).In the present study it was also observed that Staphylococcus aureus and Staphylococcus epidermidis show sensitivity against aloe vera gel extract i.e. (24 mm in diameter).While in previous study (Munther and AlKayya, 2015) it was also observed that Staph.aureus and Staph.epidermidis showed ( up to 20 mm in diameter ) on agar well diffusion method. Thus, as a result it was observed that aloe vera gel (juice) shows sensitivity against bacterial isolates.

7.1.4 Antimicrobial activity of Lemon juice:
Lemons are most known for their Vitamin C content. Lemons also contain a range of other vitamins and minerals. As an alkaline fruit, lemons also kill many forms of bacteria that are known to cause acne. As an alkaline fruit, lemons also kill many forms of bacteria that are known to cause acne. Lemon Peel can be effective in lowering cholesterol. In the present study lemon juice show antibacterial activity against 3 different bacterial isolates. i.e. (Staphylococcus aureus, Pseudomonas areginosa and Staphylococcus epidermidis). In which Staphylococcus aureus show maximum inhibitory zone (36 mm in diameter) on MHA media while in the previous studies(Aveen,2015) it was also observe Staphylococcus aureus show (20 mm in diameter ) on well diffusion method. In the present it was observed that Pseudomonas areginosa also showed high inhibitory zone (33 mm in diameter) against lemon juice extract. While in other study (Mathur et al, 2011) showed that lemon juice having maximum zone of inhibition against P. aeruginosa i.e. (28 mm in diameter). Thus, as a result lemon juice shows sensitivity against bacterial isolates.

Comparatively in the present study of lemon juice and aloe vera gel, Lemon is more sensitive as compare to aloe vera gel. Different bacterial clinical samples has been taken i.e. (Staphylococcus aureus , Staphylococcus epidermidis and Pseudomonas areginosa) were found sensitive and  the percentage show high rate of sensitivity of plant extract used on ( Muller-Hinton agar).As a result  lemon is 100 % sensitive against clinical bacterial isolates and aloe vera gel juice 80 % sensitive and 20 % resistant. Therefore, this study shows that herbal plants have the capability for the treatment of skin wounds and infection.

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