Prevalence of antifungal activity of Argemone …jbsr.pharmainfo.in/documents/vol2issue3/2010020303.pdfPrevalence of antifungal activity of Argemone mexicana poppy Mohammad Imran*1

Prevalence of antifungal activity of Argemone mexicana poppy Mohammad Imran*1 and ER.Ashok Kumar2

1 Department of microbiology, S.G.P.G Institute of Medical Sciences,Raebareli Road,Lucknow .UP,India India; 2. Dept.of Biotechnology,Himachal Institute Of Life Sciences,Paonta Sahib,Distt.

Sirmour,(H.P.),India. Abstract: The poppy of Argemone Mexicana are high in protein, carbohydrates, lipids and minerals. They are also rich in novel alkaloids, and sterols The poppy of Argemone species contain a high concentration of berberine and sanguinarine alkaloids The alkaloids of argemone are widely used as Analgesic; Antispasmodic; Demulcent; Emetic; Expectorant; Hallucinogenic; Purgative; Sedative; Skin; WartsThe root is alterative and has been used in the treatment of chronic skin diseases. The protein part was extracted and tested against four different fungal strains viz. Aspergillus niger, Aspergillus terreus, Aspergillus flavus, Rhizopus stolonifer etc. A good antifungal activity was observed at temperature 20’C-25’C, the activity was lost completely at temperature 50’C and above. The protein exhibited good inhibition of fungal growth at pH 3 and pH 5 but the activity completely lose at pH 8. . Lower concentration did not show significant antifungal activity. Aspergillus terrus and M. lepray was found to be the most sensitive to its alkaloids. The extracts showed activity and methanol extract showed maximum potency compared to other extracts. Keywords: Argemone, terpenoid, Flavonoid, Sanguinarine, Tannins.

Introduction: Fungi are significant destroyers of foodstuffs and grains during storage, rendering them unfit for human consumption by retarding their nutritive value and often by producing mycotoxins (Marin et al., 1999; Janardhana et al., 1998). A significant portion of the agricultural l produce in the country and the world over become unfit for human consumption due to mycotoxins contamination of grains, especially those produced by species of Aspergillus (Janardhana et al., 1998; Chandra and Sarbhoy, 1997; Devi et al., 2001). More then 25% of the world cereals are contaminated with known mycotoxins and more than 300 fungal metabolites are reported to be toxic the the man and animals( Galvano et al., 2001). The main toxic effects are carcinogenicity, genotoxicity, terratogenicity, nephrotoxicity, hepatotoxicity , reproductive disorders and immunosuppression(Lacey, 1988; Desjardins et al., 2000). Thus , there is a need to search for alternative approaches to store grains/ cereals for human consumption without toxicity problems that are ecofriendly and not capital intensive. Plant extracts of many higher plants have been reported to exhibit antibacterial, antifungal,

and insecticidal properties,under laboratory trails(Satish et al., 1999; Okigbo et al 2006; Shariff et al., 2006 ;Bouamama et al.,2006; Ergene et al., 2006; Kiran et al , 2006; Mohana et al,2006). Medicinal plants are natural, non-narcotic having no side effect preventive and curative therapies which could be useful in achieving the foal of “Health for all” in a coat effective manner. Medicinal plants, since times immemorial, have been used in virtually all cultures as a source of medicine. The widespread use of herbal remedies and health care preparations, as those described in ancient text such as the Vedas and bible, and obtained from commonly used traditional herbs and medicinal plants, have been traced to the occurrence of natural products with medicinal properties. The use of traditional medicine and medicinal plants in most developing countries as a normative basis for the maintenance of good health has been widely observed (UNESCO 1996). Medicinal plants are mostly herbs. The herb is a plant or plant part valued for its medicinal, aromatic or savory qualities. Herb plants produce and contain a variety of chemical substance that act upon the body. No comparisons of herbal drugs can be made with synthetic allopathic

Mohammad Imran et al / J Biomed Sci and Res., Vol 2 (3), 2010,132-142

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drug particularly in suspect of identification, procurement processing, quality control, storage and shelf life.(Siegers C.P. et al 1992). Many drugs commonly used today are of herbal origin indeed, about 25 % of the prescription drugs dispensed in the United States contain at least one active ingredient derived from plant matieral. Some are made from plant extract others are synthesized to anemic a natural plants compound. Many plants used today were know to people of ancient culture throughout the world and they were valued their preservative and medicinal powers (Lawson LD. Et al, 1998). Argemone Mexicana is an American originated plant but now it found in whole South Asia especially in tropical countries eg. India, China, Afghanistan, Pakistan and Bangladesh etc. Argemone Mexicana is commonly known as Mexican poppy, Mexican prickly poppy cardosanto in foreign but in India is known as in Hindi Shialkanta, Satyanashi; in Gujrati: Darudi, Danarese: Balurakkisa, Datturi, Pirangi, datturi, Marathi: Daruri, Firangi-kote-pavola,dhotara., Sanskrit: Brahmadandi, Pitopushpa, Srigalkanta, Svarnakshiri, Malyalam: Ponnummattu, Kantankattiri, Tamil: Kutiyotti, Ponnummuttai, Telugu: Brahmadandicettu etc. Argemone mexicana (Mexican Poppy, Mexican Prickly Poppy or Cardosanto) is a species of poppy found in Mexico and now widely naturalized in the United States, India and Ethiopia. An annual herb with bright yellow sap, it has been used by the Natives of the western US and parts of Mexico. The seed-pods secrete a pale-yellow latex substance when cut open. This argemone resin contains berberine and protopine, and is used medicinally as a sedative. Satyanashi, if cut at any place in plant it should secrete like gold, pale yellow secretion so its name given Svarnakshiri. Its fruit like cup shaped surrounded by pointed

spikes, it contain very small like mustard seed filled seed. When its send threw on the heated coal, it makes sound like bharbhard. In whole Uttar Pradesh it is famous as its name bharbhard. Materials and Methods: 1. Materials 1.1. Plant samples:- S. No. Plants Part used 1. Argemone

Mexicana Poppy+leaves

2. Argemone platyceras

poppy

1.2. Chemicals and Reagents S. No.

Chemicals/ reagents Name

Uses

1. Acetic anhydride

Phytochemical screening

2. Acetone Protein estimation 3. Alkaline

Na2CO3 Protein estimation

4. Antibiotic Antifungal activity 5. Antifungal

agents Antifungal activity

6. Ammonium persulfate

Protein estimation

7. Bovine serum albumin(BSA)

Protein estimation

8. Chloroform Phytochemical screening

9. Folin’s reagent Protein estimation

10 Copper sulphate reagent

Protein estimation

11 HCl Phytochemical screening

12 Mayer’s reagent

Alkaloid screening


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S. No.

Chemicals/ reagents Name

Uses

13 H2SO4 Phytochemical screening

14 Dragencloffs reagent


15 Methanol Antifungal activity

16 Ethanol Antifungal activity

17 Fehling solution


18 Sodium bicarbonate

As buffer

19 Ferric chloride Phytochemical screening

20 NaOH Protein estimation

21. Trichloroacetic acid

Protein estimation

1.3. Equipments S. No.

Equipments name

Uses

1. Autoclave Sterilization

2. Balances Weighing chemicals

3. Beakers Storage the chemicals

4. Centrifuge Separation of sample particles

5. Conical flasks Mixing chemicals

6. Forceps Put up the test samples

7. Funnels Filtration of the samples

S. No.

Equipments name

Uses

8. Incubator Test, culture microorganism

9. Laminar air flow Inoculation of microorganisms in Petri plate

10. Oven Drying glass ware

11 Petri dishes Cultivation of microorganism

12 pH meter Determine the pH of samples

13 Pipette Take sample volume

14 Micro-piptte Take small volume of sample

15 Spectrophotometer Determine O.D.(optical density)

16. Water bath Heat the sample

17. Colony counter Count the colony of microorganism

18. Filter paper Separate the supernatant

19. Inoculums loop Inoculate small amount of microorganism on plate

1.4. Microbiological media:- Nutrient agar (NA) Nutrient broth (NB) Sabouraud dextrose agar (SDA) Sabouraud dextrose broth (SDB) Potato dextrose agar (PDA)


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1.5. Fungal strains:- Aspergillus niger Aspergillus terreus Aspergillus flavus Aspergillus stolonifer 1.6. Bacterial strain:- Mycobacterium leprey Streptococcus aureus, Escherichia coli 2. Procedure:- 2.1. Phytochemical screening tests:- The tests were done to find the presence of the active chemical constituents such a alkaloids, glycosides, trepenoids and steroids, flavonoids, reducing sugar and tannin by the following procedure:- Alkaloids:-alkaloid solution produces white yellowish precipitate when a few drops of Mayer”s reagents are added (Siddiqui and Ali, 1997).Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer”s reagent(Evabs in 2002) The alcoholic extract was evaporated to dryness and the residue was heated on a boiling water bath with 2% hydrochloric acid. After cooling, the mixture was filtered and treated with a few drops of Mayer’s reagent. The samples were then observed for the presence of turbidity or yellow precipitation. Glycoside: Take 0.2 gm the solution of the extract in glacial acetic acid, few drops of ferric chloride and concentrated sulphuric acid are added, and observed for a reddish brown coloration at junction of two layers and the bluish green color in the upper layer (Siddiqui at el,1997). Terpenoid and steroid: Take 4mg of extract was treated with 0.5 ml of acetic anhydride and 0.5 ml of chloroform. Then concentrated solution of sulphuric acid was added slowly and red violet color was observed for

terpenoid and green bluish color for steroids ( Siddiqui at el, 1997). Flavonoid: Take 4ml of extract solution was treated with 1.5ml of 50% methanol solution. The solution was warmed and the metal magnesium was added. To this solution, 5-6 drops of concentrated hydrochloric acid was added and the red color was observed for flavonoids (Siddiqui at el,1997 ). 2.2 Tannins;- To 0.5 ml of extract solution 1 ml of water and 1-2 drops of ferric chloride solution was added. Blue color was observed for Gallic tannins and green black for catecholic tannins(Iyengar,1995). 2.3 Reducing sugar:- To 0.5 ml of extract solution, 1ml of extract solution, 1 ml of water and 5-8 drops of Fehling’s solution was added at hot and observed for brick red precipitate. 2.4. Antifungal activity of alkaloid extract:- Antifungal assay: Fungal infections are the most common among the human population and a number of severe disease caused by the fungus in human body especially skin and then enter into internal tissue organ. For the prevention of these disease , a number of therapeutic agents are also available in the market but most of them are effective as topical application. Rare drugs available for deep mucosal infection. In this study, the antifungal activity was studied against following cultures- -Aspergillus niger - Aspergillus terreus - Aspergillus stolonifer - Candida albicans 2.5. Procedure of alkaloid extraction: Preparation of extract: For extraction, methanol and ethanol were use as solvents. An amount of plants part’s paste was extracted from 250 gm of plant material of each plants by using mortar for making paste.


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Plant sample (weight and paste in mortar) (Add CH3OH) Filter the paste Aqueous phase (evaporate at 40’C) Organic phase Extract (add CH3COOH 5%) Filter Aqueous phase (discard) Organic phase (add CH2Cl2) Add Na2 CO3 10% Adjust pH 10 Extract Add CH2Cl2

Aquous phase discard organic phase Evaporate at 40’C Alkaloids extracted3. Procedure 3.1 Alkaloid test: Alkaloid, a chemical substance of plant origin composed of carbon, hydrogen, nitrogen, and (usually) oxygen. The

alkaloids are organic bases similar to the alkalies (inorganic bases); the name means alkali-like. Most alkaloids have pronounced effects on the nervous system of humans and other animals. Many are used as drugs.


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Some familiar alkaloids are caffeine, nicotine, quinine, cocaine, and morphine. Alkaloids solution produces white yellowish precipitate when a few drops of Mayer’s reagents are added (Siddiiqui and Ali, 1997). Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer’s reagent (Evans, 2002). The alcoholic extract was evaporated to dryness and the residue was heated on a boiling water bath with 2%hydrochloric acid HCl. After cooling, the mixture was filtered and treated with a few drops of Mayer’s reagent. The samples were then observed for the presence of turbidity or yellow precipitation. 4. Result and Discussion: The result of the effect of alkaloids on fungus and bacteria by showing the zone of inhibition can shown in table below: According to the observation of effect of alkaloids on fungus and bacteria shows different type of effects of alkaloids. We isolate the alkaloids from the Argemone Mexicana. And perform antifunal activity against Candida albicans, Aspergillus nigers and S. aureus etc. observe the zone of inhibition against these micro-organism and also compare with antibacterial and antifungal drugs. The alkaloids isolated from the Argemone Mexicana has both harmful and beneficial activity. It alkaloids are mostly beneficial when it is used superficially, it shows active inhibitor against bacteria and fungus. It inhibit the bacterial cell wall permeability and leak out its cytoplasm from the cell and bacterial cell unable to survive. The alkaloid isolated from the Argemone Mexicana has harmful effect when it is taken orally, it will accumulated in the kidney and other organ where it function as inhibitor of the function of the organ. Argemone mexicana is used by traditional healers in Mali to treat malaria. And, the seed oil itself is used medicinally to treat dropsy, jaundice and skin diseases. Katkar

oil poisoning causes epidemic dropsy, with symptoms including extreme swelling, particularly of the legs. Sanguinarine is a quaternary ammonium salt from the group of benzylisoquinoline alkaloids. Sanguinarine is a toxin that kills animal cells through its action on the Na+-K+-ATPase transmembrane protein. Epidemic dropsy is a disease that results from ingesting sanguinarine. Berberine is considered an ineffective antibiotic. However, when applied in vitro and in combination with methoxyhydnocarpin, an inhibitor of multidrug resistance pumps, berberine inhibits growth of Staphylococcus aureus. Among all elements found in the plants, alkaloids are the most powerful as well as very effective. Hence, it is little surprising that the alkaloids have been researched and examined the most by the modern day scientists. The strength or effectiveness of the alkaloids commonly includes everything or all substances that are poisonous in the plants. Therefore, it would be right to state that the exploits of any kind of alkaloids on the human body is naturally traumatic and painful. Alkaloids often contain one or more rings of carbon atoms, usually with a nitrogen atom in the ring. The position of the nitrogen atom in the carbon ring varies with different alkaloids and with different plant families. In some alkaloids, such as mescaline, the nitrogen atom is not within a carbon ring. In fact, it is the precise position of the nitrogen atom that effects the properties of these alkaloids. Although they undoubtedly existed long before humans, some alkaloids have remarkable structural similarities with neurotransmitters in the central nervous system of humans, including dopamine, serotonin and acetylcholine. The amazing effect of these alkaloids on humans has led to the development of powerful pain-killer medications, spiritual drugs, and serious


137

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139

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140

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[2] Langfortoxicityanimal t

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Prevalence of antifungal activity of Argemone …jbsr.pharmainfo.in/documents/vol2issue3/2010020303.pdfPrevalence of antifungal activity of Argemone mexicana poppy Mohammad Imran*1