K.Pallaviet al. / Journal of Pharmacy Research 2011,4(10),3355-3359 Research Article Available online through ISSN: 0974-6943 www.jpronline.info

Pharmacognostic investigation and antibacterial activity of Triticum aestivum K.Pallavi1, G.KumaraSwamy2*, Shruthi1 Ssj College of Pharmacy, vattinagulapalli, Hyderabad. A.P., India. Department of Pharmaceutical Analysis, Trinity College of Pharmaceutical sciences, Peddapalli, Karimnagar (dist) - 505172.A.P., India. Received on: 19-05-2011; Revised on: 08-06-2011; Accepted on:01-07-2011

ABSTRACT is also called botanical medicine or phytotherapy (from Greek phytos = ). Herbal medicine is a medicinal preparation made from a plant and can include the fresh or dried herb or herb part, whole, chopped or powdered or an advanced form of the herb usually made via extraction by a solvent such as water, ethanol, or an organic solvent (e.g., acetone). Such advanced herbal preparations, often processed in a way that establishes relatively fixed chemical be chosen either because they are “marker compounds”, specific or characteristic of a particular genus or species of plant or because they are scientifically active compounds, as they contribute significantly to the preparation’s specific physiological activity. Marker compounds may be active or inert, and can contribute to physiological activity other than the primary intended use of the extract. Advanced herbal products, usually, but not always, as standardized extracts, are often referred to as phytomedicines.

Key words: Pharmacognostic investigation; antibacterial activity; triticum aestivum.

INTRODUCTION Staphylococcus aureus Staphylococci (staph) are Gram-positive spherical bacteria that occur in WHEATGRASS microscopic clusters resembling . Staphylococci are perfectly spherical Triticum aestivum is also called as grass, belonging to the family Graminae. cells about 1 micrometer in diameter. S. aureus(yellow) colonizes mainly the Triticum is a genus of annual and biennial grasses, yielding various type of nasal passages, but it may be found regularly in most other anatomical locales, wheat, native to southwest Asia and the Mediterranean region. Triticum including the skin, oral cavity and gastrointestinal tract. Taxonomically, the aestivum, common or bread wheat, is widely cultivated almost all over the genus Staphylococcus is in the Bacterial family Staphylococcaceae. world. Generally, 15-20 species are recognized, of which about 8 have been Staphylococcus aureus forms a fairly large yellow colony on rich medium. reported to occur in India. Wheatgrass is the YOUNG GRASS sprouted from Staphylococci are facultative anaerobes that grow by aerobic respiration or by wheat grains, which has been allowed to grow for 7 to 8 days. Wheat grains, on fermentation that yields principally lactic acid. The bacteria are catalase- the other hand, are the seeds of the wheat plant.Wheatgrass is juiced or positive and oxidase-negative. S. aureus can grow at a temperature range of 15 powdered and taken because of its huge amount and high level of nutrients to 45 degrees and at sodium chloride concentrations as high as 15 percent. which include , , amino acids, fatty acids, photochemical and Nearly all strains of S. aureus produce the enzyme coagulase. S. aureus should more than 100 types of enzymes. Wheat grains are eaten or added to food always be considered a potential pathogen. (like bread, biscuits, cookies, cakes) to increase its fiber content.Wheatgrass or sprout does not contain gluten thus will not cause gluten-related allergies. In Bacillus subtilis contrast, some people are allergic to wheat grains because it contains gluten. Bacillus subtilis is a Gram-positive, rod-shaped and endospore-forming About The Plant Selected For Investigation aerobic bacterium. It is found in soil Wheatgrass have been of keen interesting phytochemical and pharmacological and rotting plant material and is non- research due to their excellent medicinal values. Different classes of natural pathogenic. It is one of the most products, processing potent physiological and pharmacological activities have studied gram-positive bacteria. One been isolated from wheatgrass and they include chlorophyll, flavonoids and feature that has attracted a lot of polysaccharides. Some of these compounds have been shown to posses’ interest in B. subtilis is its ability to considerable anti-oxidant activity. Wheatgrass is well known in folk medicine differentiate and form endospores. for their laxative property. B. subtilis forms colonies that are dull and may be wrinkled, cream to brown Fig. 1.Staphylococcus aureus Vernacular Names: in colour and when grown in broth S.No. Languages Names have a coherent pellicle; usually with a 1. Telugu Godhuma gaddi single arrangement. Bacillus subtilis is 2. Hindi Kanak ki ghans a bacterium that is used as a fungicide on flower and ornamental seeds, and Scientific name: on agricultural seeds including seeds for Triticum aestivum cotton, vegetables, peanuts, and soybeans. The bacterium colonizes the Wheatgrass Pseudonym developing root system of the plant Because of the exceptional nutritional value and phenomenal benefits of and thus competes with certain fungal wheatgrass, it has been given the name The Green Blood. disease organisms. Use of the fungicide is not expected to harm humans or the DISTRIBUTION: environment. Fig 2.Bacillus subtilis Wheatgrass is native to southwest Asia and the Mediterranean region. Wheat is world’s leading cereal crop, cultivated over an area about 215 million hectares with a production of 584 million tonnes of grain. Maximum area under wheat *Corresponding author. is in China followed by India, while the production China stand first and India K.Pallavi ranks second. Ssj College of pharmacy, vattinagulapalli, Hyderabad. Chemical Constituents: Tel.: +91 9581972289 Vitamins: A, B complex, C, D, E, F, K, and Folic Acid. E-mail:[email protected].

Journal of Pharmacy Research Vol.4.Issue 10. October 2011 3355-3359 K.Pallaviet al. / Journal of Pharmacy Research 2011,4(10),3355-3359 Minerals: , , , Sodium, , Sulphur, current knowledge, repletion may play a role in delaying type 2 Phosphorus, Cobalt, Magnesium, . diabetes onsets and potentially in warding off its devastating complications - cardiovascular disease, retinopathy, and nephropathy. Other nutrients in Amino Acids: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, wheatgrass support energy level and overall health of diabetic Phenylalanine, Threonine, Tryptophan, Valine, Alanine, Proline, Tyrosine, Aspartic Acid, Cysteine, Glutamic Acid, Glycine. Ø Kulkarni SD et.al. reported that the aqueous and ethanolic extracts of wheatgrass were found to inhibit significantly ascorbate-Fe2+ induced lipid Enzymes: Superoxide Dismutase, Peroxidase, Phosphatase. Catalase, peroxidation in rat liver mitochondria. Cytochrome oxidase,DNAse, RNAse, Hexokinase, Malic dehydrogenase, ØR.K. Marwaha et.al. reported beneficial effect of wheat grass juice as decrease protease, Nitrate reductase,Nitrogen oxyreductase, Fatty acids in the requirement of packed red cells by 25% or more thalassemia patients. transhydrogenase, Phospholipase, Polyphenoloxidase, lipase. Ø Padalia S et.al. reported various studies and reasons emphasizing the multi•tude potentials of wheatgrass. Wheatgrass contains chlorophyll and flavonoids in good amount. Wheatgrass is called the Green Blood. Chlorophyll is the green pigment of the plant that Reasons for taking up the present work resembles the structure of the hemoglobin of the red blood cells. Dr. Hans Survey of literature revealed that wheatgrass is medicinally important. To Fischer, a German chemist and a group of associates won a Nobel Prize for this quote some of its medicinal uses-The whole plant and grains are used to treat discovery. Like the human blood, chlorophyll carries energy, nutrients and blood related disorders. Wheatgrass contain different classes of compounds of oxygen to the different parts of the . It is the blood of the plant. pharmacological importance such as flavonoids, chlorophyll. In view of the Interestingly, chlorophyll in the human body also delivers and oxygen above claims and facts the present investigation was undertaken to explore to the blood and more so, studies have shown that it can stimulate production the possible anti-bacterial activity of wheatgrass.This thesis embodies of healthy red blood cells and prevents anemia and different blood disorders. pharmacognostic investigation and anti-bacterial studies carried out on the different extracts of the wheatgrass.

—Chlorophyll can be extracted from many plants, but the wheatgrass is superior because it has been found to have over 100 elements needed by man, if grown in organic soil, it can absorb 92 out of 102 known minerals. —Taste of barley grass is bitter —Alfalfa also contains abundant amount of chlorophyll, it is difficult to grow it in the home as its roots spread deeper in the ground. —Paddy grass is generally very dry. —Palak () leaves juice develop diarrhea. —Other green-leaf vegetables such as dill leaves and fenugreek leave also contains medicinal properties, but their taste is generally unpalatable.

Production of Plant Material The Seeds of Triticum aestivum Linn were soaked for twelve hours and sown in place containing red soil. After 7 days when the wheatgrass is about 7-8 inches that is when it is at its nutritional peak it is trimmed with a pair of scissors above the soil surface. Fresh plant material were collected, washed under running tap water to remove adhering dust, dried under shade for 2 days and figure 10. Difference between hemoglobin and chlorophyll pulverized in a mechanical grinder. The coarse powder was used for further studies. Fig 3 Wheatgrass & Health Problems Constituents per 100gm powder: Proteins 32.15gm Pharmacognostic Study Anemia, Thalassemis & Other Blood Carbohydrates 34.97gm Disorders Sugars 13.65gm Fat 0.11gm Organoleptic evaluation: Chlorophyll in wheatgrass, having almost Fiber 3.65gm In this evaluation, various sensory parameters of the plant material are identical molecular structure with the “heme” Sodium 110mg observed with sense organs. The leaf powder of the plant was studied for their of the blood, has been shown to intensify Beta carotene 105mg organoleptic characters like colour, odour, and taste. production of healthy red blood cells. More Chlorophyll 1170mg recent research indicates that some porphyrins (ringed structures in chlorophyll) Transverse section: stimulate the formation of the protein portion of the hemoglobin molecule. The microscopic evaluation is essential for powdered crude drugs. Powder of This explains the effect of chlorophyll in blood production.Other than the crude drug consists of the fragments of cells in the form of recognizable chlorophyll, Iron, (B3), (B12), folic acid, copper, tissues. Another important aspect of microscopical evaluation is the study of potassium and protein in wheatgrass, which are all involved in healthy red transverse section of leaves. These constants are of diagnostic significance blood cell production, make it an excellent remedy for anemia and other blood and are used for authentification of leaf drug or for the detection of adulterants. disorders. Powder analysis: Wheatgrass contains an enzyme called P4D1 and Abscissic Acid (ABA), which Powder analysis was done by placing a little amount of powder on a neat glass strips cancer, cells off their protective covering so that our body’s immune slide and then 1-2 drops of phloroglucinol and drops of concentrated system can attack and easily destroy them. According to some studies, abscissic hydrochloric acid were added and properly mixed with brush. The slide acid is a formidable anti-cancer agent that is proven to be deadly against any preparation was covered with a cover slip, 1 drop of glycerin water was added form of cancer even in small amounts. Cancer cells cannot thrive in an and the slide was then examined under microscope with suitable magnification. alkaline and well-oxygenated environment. As such, wheatgrass is a powerful food for cancer a patient since it helps provide an alkaline environment to the Physicochemical parameters: cells while chlorophyll oxygenates the body. Antioxidants, e.g. SOD, Vitamins Physicochemical parameters i.e. moisture content, Total ash, Acid insoluble C, E, bioflavonoid, carotenoids and other photochemical and enzymes, are ash, Water soluble extractive value, Alcohol soluble extractive value also abundant in wheatgrass. determination were carried out as per WHO Guidelines.

These prevent cell damage and mutation that may lead to cancer or hasten Determination of Moisture content: About 1.5gm of powder is weighed spread of cancer cells in the body. In fact, enzymes and amino acids in and taken into porcelain dish. It is dried in oven at 105OC, cooled and weighed. wheatgrass have been shown to deactivate the carcinogenic benzopyrene found The loss in weight is calculated. in charcoal broiled meat and smoke fish. When taken before a meal, Fiber in wheatgrass regulates absorption of sugar and cholesterol from the food. This Determination of Total ash: will prevent sudden rise of blood sugar level, which is commonly experienced About 2gm of powder is weighed and taken into dish and heated with a burner by diabetic patients after a meal. Wheatgrass contains high level of Magnesium till all the carbon is burnt off and cooled. The ash is weighed and the percentage since this is the central element in chlorophyll. Magnesium of total ash is calculated with reference to air-dried drug. supplementation has been shown to improve insulin sensitivity. Based on

Journal of Pharmacy Research Vol.4.Issue 10. October 2011 3355-3359 K.Pallaviet al. / Journal of Pharmacy Research 2011,4(10),3355-3359 Determination of Acid insoluble ash: epidermis. Midrib represents a flat ventral surface and convex dorsal surface. To the above formed ash 25ml of hydrochloric acid is added and burnt for five The epidermal layers are continuous over the midrib, collateral and conjoint minutes. It is then filtered and the residue is washed with hot water. The residue vascular bundle is prominent occupying the central portion of the midrib. is burnt until all the carbon burnt off and cooled. The residue is weighed and the Vascular bundle is surrounded by sclerenchymatous tissue. acid insoluble ash is calculated with reference to air-dried drug. Powder analysis Determination of Water soluble ash: The wheatgrass powder has shown the presence of following plant tissue Ash was boiled for 5minutes with 25ml of water; insoluble matter was collected systems under microscope: on a filter paper and washed with hot water and ignited for 15 minutes. The Starch grains : Oval shaped insoluble ash is weighed and the water soluble ash is calculated with reference Vascular tissue : Xylem and Phloem to air-dried drug. Trichomes : Unicellular covering trichomes along with epidermal cells Stomata : Dumble shaped Determination of Water soluble extractive value: Fibers : Lignified About 5gm powder is taken into conical flask and about 100ml chloroform is Calcium oxalate crystals: Prismatic type added. The flask is corked and kept aside for 24hrs. It is filtered and 25ml of the filtrate is taken into dish and dried at 100OC, cooled. The extractive value Transverse section of wheatgrass is calculated with reference to air-dried drug.

Determination of Alcohol soluble extractive value: About 5gm powder is taken into conical flask and about 100ml alcohol is added. The flask is corked and kept aside for 24hrs. It is filtered and 25ml of the filtrate is taken into dish and dried at 100OC, cooled. The extractive value is calculated with reference to air-dried drug.

Extraction of Plant Material Extraction is the process of obtaining the constituents by separating it from crude drug by the use of solvents. Powdered material is extracted with suitable solvent or mixture of solvents for extracting the various phytoconstituents present in the crude drug.

Xy- xylem; Ph-phloem; Tr-trichome; Scl-sclerenchyma; Me-mesophyll cells; LE-lower epidermis; UE- upper epidermis Fig.4. Tranverse section of wheet grass showing vascular bundle

Fig 3.cultivation of wheatgrass Powder analysis

Maceration: About 50gm powder was weighed and extracted by maceration using the solvents petroleum ether, chloroform, acetone, methanol at room temperature in a conical flask for 3 days. The material was stirred from time to time to ensure proper extraction. After 3 days, the contents of the flask were filtered through vacuum filter and the filtrate was concentrated under reduced pressure below 50ÚC, until a soft mass obtained and then preserved in a desiccator.

Anti-bacterial assay Anti-bacterial activity is the process of killing or inhibiting he growth of Fig 4.Unicellular trichomes bacteria. Anti-bacterial agent either kills (bactericidal) the bacteria or inhibits the growth (bacteriostatic) of bacteria.

Paper disc diffusion method: The four different leaf extracts were tested for antibacterial activity using paper disc diffusion method. Nutrient agar medium was prepared, sterilized and used as the growth medium for bacterial culture. 20ml of sterilized medium was poured into each sterilized petridish, covered and allowed to solidify. The plates were then seeded with the test microorganisms i.e., bacterial culture by using sterile cotton swabs. The sterilized paper discs were soaked in the prepared solution of different solvent extracts (1µg/ml) and were dried at 50ÚC. The dried discs were placed on medium plates seeded with test micro-organisms. The plates were then incubated at 37ÚC. The zone of inhibition was measured Fig 5.Fibers Fig 6.Calcium oxalate crystals after 24hrs. In the present study Amoxicillin 1µg/ml was used as standard anti- bacterial drug.

Production of Wheatgrass About 500gm of wheat grains were sown in red soil. After a week about 2kgs of wheatgrass was collected.

Transverse section Transverse section of leaflet shows an upper epidermis covered with cuticle. Only covering trichomes emerge from epidermal layer. Dumble shaped types of stomata are seen in the upper epidermis. Mesophyll is made up of uniform parenchyma cells, loosely arranged. Lower epidermis is very similar to upper Fig 7.Xylem and Phloem Fig 8.Starch grains Journal of Pharmacy Research Vol.4.Issue 10. October 2011 3355-3359 K.Pallaviet al. / Journal of Pharmacy Research 2011,4(10),3355-3359 Physicochemical parameters of wheatgrass: The moisture content, total ash, acid insoluble ash, water soluble ash, water soluble extractive value, alcohol soluble extractive value were determined. Wheatgrass contains about 4.05% moisture content, 2.3% acid insoluble ash, 4.0% water soluble ash. Extractive values indicate the nature of chemical constituents present in the drug. The alcohol and water soluble extractive of wheatgrass were found to be 3.75% and 2.5% respectively.

Loss on drying: Sample content Moisture content %

Leaves 4.05

Ash values: Sample Total Acid insoluble Water soluble content ash % ash % ash % Leaves 7.5 2.3 4.0

Extractive values: Fig 10.Zone of inhibition of Staphylococcus aureus Sample Water soluble Alcohol soluble content extractive value % extractive value % DISCUSSION Leaves 2.5 3.75 In ethnomedicinal practices, the medicinal healers use wheatgrass as “Green Blood” in the treatment of anaemia and other blood related disorders.In the present study, microscopic evaluation has been documented such as transverse Extraction of Plant Material section of wheatgrass showed an upper epidermis with cuticle, unicellular trichomes, dumble shaped stomata, vascular bundles surrounded by Preparation of extracts: sclerenchymatous tissue. Macroscopic evaluation has also been documented. Various leaf extracts of wheatgrass were prepared by successive solvent Powder analysis showed oval shaped starch grains, vascular bundles, unicellular extraction by maceration. The extracts obtained were evaporated under reduced trichomes, dumble shaped stomata, lignified fibers, and prismatic type calcium pressure and dried. The color, nature and percentage yield of all the extracts oxalate crystals.The powdered drug was subjected to extraction with various were recorded and indicated that wheatgrass powder upon maceration produced solvents such as petroleum ether, chloroform, acetone, methanol by successive highest percentage of methanol. Whereas, lowest percentage of petroleum maceration based on polarity and the concentrated extracts (1µg/ml) were ether extract was obtained. used for anti-bacterial assay.

Percentage yield of wheatgrass extract: All the extracts obtained from wheatgrass powder showed mild to strong S.No Method of Solvent Physical Percentage activity against most of the tested organisms. The results were compared with extraction form yield those of Amoxicillin as standard antibiotic. Of the four extracts, petroleum ether extract did not show any activity against gram positive and gram negative 1. Maceration Petroleum ether Resinous 1.204 2. Maceration Chloroform Resinous 2.148 bacteria. Chloroform extract displayed excellent activity against gram positive 3. Maceration Acetone Resinous 1.674 bacteria Bacillus subtilis whereas acetone extract showed considerable activity 4. Maceration Methanol Powder 3.852 against gram positive bacteria Bacillus subtilis. Methanolic extract showed Anti-bacterial activity: moderate activity against both gram negative bacteria Escherichia coli and Anti-bacterial activity was carried out using disc diffusion method using four gram positive bacteria staphylococcus aureus. On over all consideration, different extracts (petroleum ether, chloroform, and acetone, methanol) and petroleum ether was found inactive compared with other extracts. Amoxicillin as standard against gram positive and gram negative bacteria (Staphylococcus aureus, Bacillus subtilis, E.coli). Out of the four extracts, CONCLUSION petroleum ether was inactive against all the bacteria, chloroform showed excellent activity against Bacillus subtilis, acetone showed considerable activ- Wheat Grass is used to promote healthy living and longevity. ity against Bacillus subtilis and methanolic extract showed moderate activity Wheatgrass is used to treat liver disorders, inflammatory diseases, constipation, against Escherichia coli and Staphylococcus aureus. anaemia, cancer.The work carried was basic approach to findout the anti- bacterial activity in wheatgrass. The bacterial study of various wheatgrass extracts (chloroform, acetone, methanol) were found to be effective against Bacteria Petroleum Chloroform Acetone Methanol Amoxicillin various gram positive and gram negative bacteria and may be attributed to ether (mm) (mm) (mm) (mm) (mm) various phytochemical constituents present in the crude extract. Several phytoconstituents like flavonoids, phenolics and polyphenols, tannins, Escherichia coli _ 2 2 3 12 Bacillus subtilis _ 5 4 _ 9 terpenoids, sesquiterpenes etc are effective anti-bacterial substances against Staphylococcus aureus _ 1 1 4 11 wide range of micro-organisms. The purified components may have even more potency with respect to inhibition of microbes. Therefore, further Graphs works on the types of phytoconstituents and purification of individual groups of bioactive components can reveal the exact potential of the plant to inhibit several pathogenic microbes.

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Fig 9.Zone of inhibition of Escherichia coli Journal of Pharmacy Research Vol.4.Issue 10. October 2011 3355-3359 K.Pallaviet al. / Journal of Pharmacy Research 2011,4(10),3355-3359 9. Murphy, sean, Wheatgrass, healthy for the body, Australia’s National Rural Affairs 14. Rita, The Benefits of Green”, Rita’s Natural Food Market-2009. Weekly-2002 15. M J West, Fight cancer through powerful natural strategies. pg: 54-55-2009. 10. Kohler et.al. , Growth stimulating properties of grass juice, Science-1936. 16. Mukherjee PK, Quality standard of herbal drugs, an approach to evaluation of 11. Valli, Green Blood Therapy:cure for many diseases”, Health Mad, page: 11-14- botanicals”, Business Horizons New Delhi: Pharmaceutical publisher.2002. 2010. 17. Kumar shankul .et.al. “Pharmacognostic investigation on wheatgrass”Indian 12. Padalia S et.al. Multitude potential of wheatgrass juice (Green Blood): An Journal of Pharma and Biosciences, vol-1 pg: 4-7-2010 Overview, Chronic Young Scientist, page: 23-28-2010. 18. Rangari VD ., Pharmacognosy and Phytochemistry I.-2001 13. R.K. Marwaha et.al. Wheat Grass Juice Reduces Transfusion Requirement in Patients 19. Quality standard of Indian medicinal plants; Indian council of medicinal research with Thalassemia Major: A Pilot Study, Indian Pediatrics, vol. 41,716-720, July- New Delhi -2005. 2004. 20. Arul kumar et.al. ,Anti-microbial activity of Cassia rorburgiii”, Indian journal of chemicals and pharmaceuticals, page: 53-57-2010 Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 10. October 2011 3355-3359