International Journal of Agricultural and Food Research

ISSN 1929-0969 | Vol. 3 No. 3, pp. 1-9 (2014) www.sciencetarget.com

In Vitro Antibacterial Efficacy of (Roth) Spreng. () against some Gram Positive and Gram Negative Human Pathogens

Abdul Viqar Khan 1*, Qamar Uddin Ahmed 2, Athar Ali Khan 1 and Indu Shukla 3

1 Department of Botany, Faculty of Life Sciences, Aligarh Muslim University (A.M.U.), Aligarh- 202002, State of Uttar Pradesh (U.P), 2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, IIUM, 25200-Kuantan, Pahang Darul Makmur, Malaysia 3 Department of Microbiology, Jawaharlal Nehru Medical College, A.M.U., Aligarh-202002, U.P., India

Abstract: Leucas cephalotes (Roth.) Spreng (family: Lamiaceae) is a rainy season weed widely distributed in tropical regions of at elevation up to 1,700 m, medicinally employed for the treatment of skin diseases, fever, hepatic disorders, urinary complaints cough and cold. The traditional uses of this strongly suggest its possible antibacterial properties, but its efficacy has not been examined in broad scenes, in present communication, its antibacterial efficacy has been explored. Polar and non-polar extracts (ethyl acetate, methanol, aqueous, benzene, and petroleum ether) at five different concentrations (0.5, 1, 2, 5, and 10 mg/mL) were evaluated for their antibacterial efficacy against seven G+ and eleven G– hospital isolated bacteria. Disc diffusion method was followed to determine antibacterial activity. Phytochemical analysis of plant revealed presence of tannins, glycosides, saponins, steroids, phenolic compounds and flavonoids as secondary metabolites. Maximum antibacterial activity was demonstrated by ethyl acetate and methanol extracts at a minimum concentration (0.5 mg/mL/disc). Extracts were effective on both types of test pathogens. From the present findings, it may be concluded that the plant could be formulated in broad spectrum antibiotic and confirms the traditional uses in pathogenic diseases. Keywords: Leucas cephalotes (Roth) Spreng, Antibacterial efficacy, crude extracts, traditional uses

1. Introduction Herbal are chief sources of potentially pulation through amalgamations with molecular useful chemotherapeutic agents to help health care modelling and synthetic chemistry. These meta- system. The medicinal value of plants lies in a bolites have been found in vitro as well as in vivo wide variety of secondary metabolites, such as, to have antibacterial characteristics. People’s terpenoids, alkaloids, and flavonoids. These natural interest in medicinal plants has greatly increased in compounds often serve as lead compounds whose recent years. This interest has eventually led to the activities can be increased by structural mani- discovery of new biologically-active substances by

* Corresponding author: [email protected] 2 © A.V. Khan, Q.U. Ahmed, A.A. Khan and I. Shukla 2014 | In Vitro Antibacterial Efficacy the pharmaceutical based industries and the has been reported to act as an antibilious in herbal adoption of herbal extracts for self-medication by therapy for jaundice, and helps cure induced the general public (Nahrstedt and Butterweck, filariasis. The whole plant’s powder has 70% 2010; Vogel, 1991). herbal composition and is patented to cure epileptic convulsions and cerebral function India is one of the World’s top 12 mega diversity disorders, it acts as antipyretic, stimulant, expect- countries with 10 biogeographic regions and over rant, aperient, diaphoretic, insecticidal, emmena- 40 sites which are known for their high endemism gogue, antioxidant, anti-inflammatory and anti- and genetic diversity. India has more than one diabetic (Mishra, 2002; Qamaruddin et al., 2002). fourth (8000) of the world’s known medicinal Various classes of phytochemicals have been plant (30,000) of which 90% are found in isolated from different parts of L. cephalotes i.e. forests (Khan et al., 2008; 2010; 2011; 2011a; labdane, norlabdane, flavones, laballenic acid 2012; 2013). Plant species still serve as rich (octadeca-5, 6-dienoic acid), luaric acid, glutaric sources of many novel biological active acid, adipic acid, tridecanoic acid, β- sitosterol and compounds, as very few plants species have been its glucoside. The volatile compounds of thoroughly investigated for their medicinal inflorescence and seeds were found to contain properties (Abubakar et al., 2011; Caius,1986; caryophyllene oxide 26.56%; δ-fenchene 12.02%; Habbal et al., 2011; Khan et al., 2008; Paul et al., α-cardinal 2.13%; 1-hepten-3-ol 6.53%; menthol 2011). Even though pharmaceutical industries have 6.30%; deca hydronaphthalene 5.15% and trans- produced a number of new antimicrobial drugs in caryophyllene 4.05% (Singh et al., 1978; Kamat the last years (Jila, 2011; Karuppiah and Rajaram, and Singh, 1994; Miyaichi et al., 2006). 2012; Parekh and Chanda, 2010; Ruban and Gajalakshmi, 2012; Srinivasan and Balakrishnan, L. cephalotes is traditionally employed for the 2012), resistance to present chemically synthesised treatment of skin diseases, urinary complaints, drugs by microorganisms has inevitably increased fever, liver disorders, cough and cold which are (Ravikumar, 2010; Sati et al., 2011). In general, suggestive of its possible antimicrobial properties, bacteria have the genetic ability to transmit and however, its antibiotic efficacy has not been acquire resistance to drugs used as therapeutic examined in broad scenes. Hence, in present agents (Goossens et al., 2005; Mathew et al., communication, its antibacterial analysis has been 2007). carried out. Leucas cephalotes (Roth.) Spreng. is a rainy season weed and commonly found ascending up to

600-1,800 m particularly in the hilly regions of Nepal, India, Pakistan, Bangladesh and Myanmar. In the East Asia this plant is found in Afghanistan and Western in open areas at elevation of 1,700 m (Khan, 2002). Traditionally, its decoction is used in the treatment of malarial fever and for snake bite. The dried inflorescences are smoked Leucas cephalotes and the smoke exhaled through the nose to treat (Inflorescence) nose bleeds. Dried leaves along with tobacco (1:3) are smoked to treat bleeding as well as itching piles and fresh leaves eaten as a potent herb. The 2. Materials and Methods juice of leaves is used topically in psoriasis, skin 2.1 Plant Material eruptions, scabies and internally for the treatment of urinary complaints. The flowers are adminis- Fresh plants’ material was collected after the rainy tered in the form of syrup or with honey for cough season in the month of September 2010 from the and colds (Baburao et al., 2010; Manimekalai et wild localities of its natural distribution from rural al., 2011). Pharmacologically, L. cephalotes has areas of Aligarh and was authenticated by Dr. been reported to exert hepatoprotective action in Athar Ali Khan (Taxonomist) Department of CCl 4 induced hepatotoxicity in animals, its juice Botany, Faculty of Life Sciences, Aligarh Muslim

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University, Aligarh, 202002, India and was petroleum ether soluble extract (13.58 g). deposited at the same department (Voucher Petroleum ether insoluble fraction of methanolic Specimen No. AVKLC: 013). extract was refluxed with benzene for 1 hr, filtered and refluxed again with benzene for 1 hr and 2.2 Preparation of Extracts and Fractions filtered. The process was repeated five times until Plant extracts were prepared according to the there was no coloration. Benzene was evaporated following protocol: Pulverized plant material (500 under reduced pressure to obtain benzene soluble g) of whole plant was refluxed with 95% methanol extract (13.83 g). Benzene insoluble fraction of the (MeOH) on hot water bath for 1 hr. Mother liquor methanol extract was further refluxed with ethyl (crude MeOH extract) was filtered out and residual acetate for 1 hr, filtered and refluxed again with plant material was again refluxed with 95% ethyl acetate for 1 hr and filtered, the whole methanol for 1 hr, the whole process was repeated process was repeated 5 times to ensure maximum four times to obtain maximum yield of methanol yield. Ethyl acetate was evaporated under reduced soluble compounds from the plant material. The pressure to obtain ethyl acetate soluble extract extract was evaporated to dryness at 35°C under (17.05 g). Ethyl acetate insoluble fraction of the reduced pressure (Harborne, 1988). Dried methanolic extract was again refluxed with methanolic extract (67.5 g) was further refluxed methanol (95%) for 1 hr, filtered and was then with petroleum ether (60-80 °C) for 1 hr and repeatedly refluxed five times with methanol. The filtered. Residual methanolic extract was again methanol soluble fraction was evaporated under refluxed with petroleum ether for 1 hr and the reduced pressure to obtain methanolic extract entire process was repeated five times until there (11.09 g), while methanol insoluble residue was was no coloration. Petroleum ether extract was discarded. (Figure 1) evaporated under reduced pressure to obtain

Dry pulverized whole plant L. cephalotes , 500g

treated with methanol

MeOH extract, 67.5 g

treated with petroleum ether

petroleum ether insoluble extract petroleum ether soluble extract

treated with benzene

benzene soluble extract, benzene insoluble extract 13.83g

treated with ethyl acetate

ethyl acetate insoluble extract ethyl acetate soluble extract , 17.05g

treated with methanol

methanol soluble extract, methanol in soluble extract, 11.09g discarded

Figure 1: Strategic preparation of non-polar and polar extracts of whole plant L. cephalotes based on different polarities of organic solvents.

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2.3 Preparation of Aqueous Extract b) Lead acetate Test : A 10% w/v solution of basic lead acetate in distilled water was added Shade dried 500 g pulverized plant material was to test sol. As a result precipitation occurred. poured with double distilled water, and left for 72 hrs at room temperature. The flask was then 2.4.6 Test for Flavonoids refluxed over hot water bath for about 2 hrs and the a) Shinoda Test : Test residue was dissolved in 5 mother liquor was filtered, the entire process was mL ethanol (95% v/v) and reacted with few repeated 4 times to obtain maximum yield of water drops of conc. HCl and 0.5 g of Mg metal. soluble components (43.12 g). The filtrate, thus Pink, crimson or magenta colour was obtained, was evaporated to complete dryness developed. under reduced pressure by using rotary evaporator and eventually freeze dried. The plant extracts thus b) Ammonia Test : Filter paper strips were dipped obtained were kept carefully in closed bottle at - in the alcoholic extract and ammoniated Strips 20 °C until further use. turned yellow. 2.4 Phytochemical Analysis 2.4.7 Test for Amino Acids and Proteins 2.4.1 Test for Alkaloids a) Ninhydrin Test : 0.1% w/v solution of Ninhydrine was dissolved in n-butanol and a) Dragendroff’s Test : Dragendroff’s reagent then added to test sol. A violet or purple colour (potassium bismuth iodide) did not give was developed. reddish brown precipitate indicative of alkaloids presence. b) Cysteine Test : 2 ml of 40% w/v NaOH and 2 drops of 10% w/v lead acetate solution were b) Mayer’s Test : Mayer’s reagent (potassium added to 5ml of test sol, resulting solution is mercuric iodide) did not give cream precipitate boiled for few min to obtain black precipitate. indicative of alkaloids presence. c) Biuret Test : Residue is taken in water, 4% 2.4.2 Test for Glycosides NaOH solution is added then few drops of 1% a) Keller-killiani Test (for deoxy sugar): 1 mL of CuSO 4 sol were added, violet or pink colour glacial acetic acid containing traces of FeCl 3 was obtained. and 1 mL of conc. H SO were added. A 2 4 2.4.8 Test for Sugars reddish-brown colour formed at the junction of two layers and the upper layer turned bluish a) Molisch Test : A few drops of α-naphthol and green. then conc. H 2SO 4 were added to test solution. Violet colour ring was formed at the junction 2.4.3 Test for Saponins of two layers. a) Foam Test : Powdered drug residue was taken b) Fehling’s Test : Fehling A and Fehling B in a test tube and shaken vigorously with a solution were mixed in equal amount and small amount of NaHCO and water. 3 boiled then added with test solution and again Characteristic honeycomb like froth was heated. Yellow to brick red colour was obtained. obtained. 2.4.4 Test for Steroids c) Benedict’s Test : Test solution and Benedict’s a) Salkowaski reaction : Residue of extract was reagent were mixed and heated in water bath taken in 2 mL of CHCl 3 and 2 mL of conc. for 5-10 minutes. Yellow to red or green H2SO 4 added from the side of the test-tube and colour were obtained. then shaken for a few minutes. Red colour 2.4.9 Test for Total Flavonoids Content developed in the CHCl 3 layer. Twenty grams of plant powder was extracted 2.4.5 Test for Tannins and Phenolic Compounds repeatedly with 250 mL of 80% aqueous methanol ° a) Ferric chloride reagent : FeCl 3 sol. was added (MeOH) at room temperature (29 C). The whole to test sol. Dark green or deep blue colour was solution was filtered through Whatman filter paper obtained. No: 42 (125 mm). The filtrate was evaporated to

Science Target Inc. www.sciencetarget.com International Journal of Agricultural and Food Research | Vol. 3 No. 3, pp. 1-9 5 dryness using rotary evaporator at standard the agar surface with the help of sterile forceps. temperature. Quantitative Result : Flavonoids Finally, the sensitivity discs were placed with ≤8.09%. forceps carefully to make complete contact with the surface of the medium on the plate. Plates were 2.4.10 Test for Total Tannins Content kept at room temperature for 30 minutes (Pre- One gram of plant powdered drug was extracted diffusion time). Inoculated petri dishes were with 100 mL of distilled water by shaking, using incubated at 37 °C overnight and at the end of the shaker for an hour and left overnight, solid period, inhibition zones formed on the medium material was allowed to settle and the liquid was were evaluated in mm. The experiments were filtered through a Whatman no. 1 filter paper, the repeated thrice. The mean of the triplicate of the first 20 mL of filtrate was discarded and then 10 results has been summarized in table 1. mL of the filtrate was put into a conical flask. To it 2.7 Statistical Analysis 750 mL of water and 25 mL of Indigo sulphuric acid sol. was added and titrated with 0.1 N KMnO 4 All values are expressed as the mean ± the solutions. The flask was shaken vigorously till a standard error of the mean (SEM); linear golden yellow end point was reached. A blank regression analyses and correlation coefficients to determination was also performed and percentage determine the relationship between two variables, calculated. Each mL of KMnO 4 solution is they were calculated using MS-DOS software equivalent to 0.004157 gm of total tannins. (GraphPad InStat statistical program). Quantitative Result : Total Tannins ≤0.1% (Harborne, 1988). 2.5 Microorganisms 3. Results The whole plant extracts of L. cephalotes were Results of in vitro antibacterial study are presented tested for possible antibacterial activity against in table 1 and figure 2. It was observed that non eighteen (18) human pathogenic bacteria listed in polar extracts (Petroleum ether (PE) and benzene table 1. The authenticated microorganisms were extracts (BZ)) had lower range of antimicrobial procured from stock culture, Department of inhibition against test pathogens as compared to Microbiology, Jawaharlal Nehru Medical College, polar extracts (Ethyl acetate (EtOAc), methanol Aligarh-202002, U.P., India. The bacterial strains (MeOH) and aqueous (AQ) extracts). BZ extract were sub-cultured at 37 °C for 24 hrs and was able to inhibit the growth of two Gram- maintained on nutrient agar media. positive and four Gram-negative pathogens, while PE extract revealed antibacterial activity against 2.6 Antibacterial Activity five Gram-positive and three Gram-negative National Committee for Clinical Laboratory pathogens. On the other hand EtOAc, MeOH and Standards (NCCLS, 2003; 2004) were strictly AQ extracts showed higher antibacterial activity as followed to perform antimicrobial disc they were able to restrict the growth of eleven susceptibility testing using disc diffusion method bacterial strains (five Gram-positive and six Gram- to test plant extracts against test strains. negative pathogens). EtOAc fraction showed the Standardized inoculums agar plates were highest antibacterial activity against S. aureuus, S. inoculated using a sterile swab dipped into culture albus and P. shigelloides. MeOH extract also inoculums adjusted to 1.5 x 10 8 bacterial/mL using demonstrated good antibacterial activity; however, 0.5 Farland turbidity standard, the agar was the zone of inhibition was lower than the EtOAc streaked in three directions turning the plates by extract, while moderate sensitivity was observed 60 ° by each streak. All the extracts were sterilized for AQ extract. As seen in the Table 1; EtOAc, by filtration through 0.045 µ/m membrane filter MeOH and AQ plant fractions showed greater and then autoclaved at 122 °C. The paper disc inhibition effects then PE and BZ. This showed the (Whatman filter paper no 1) with 0.5, 1, 2, 5, and presence of some moderate or strong polar 10 mg/mL plant extracts were dried and placed on components (secondary metabolites) .

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Table 1 Antibacterial activity of Leucas cephalotes (whole plant) inhibition zone (mm) Gram -positive bacteria Gram -negative bacteria 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 PETROLEUM 0.5 ------1 8 8 - - - - 8 ------8 - - - 9 2 11 11 - 11 11 - 15 ------11 11 - - 14 5 15 14 - 15 16 - 19 ------15 16 - - 17 10 15 16 - 19 19 - 23 ------19 19 - - 20 BENZE NE 0.5 ------7 7 - - - 1 - 8 - - - - 8 - - - - 9 - 13 12 - - 9 2 - 11 - - - - 15 - - - - 15 - 19 17 - - 15 5 - 14 - - - - 20 - - - - 18 - 21 21 - - 17 10 - 16 - - - - 22 - - - - 22 - 23 22 - - 22 ETHYL ACETATE 0.5 - - - 7 7 ------7 - - - 7 1 10 12 - 11 11 - 9 - 9 - 10 9 - 13 12 - - 11 2 16 18 - 14 14 - 16 - 13 - 15 16 - 18 17 - - 18 5 20 20 - 17 17 - 20 - 17 - 17 19 - 20 20 - - 20 10 23 23 - 20 21 - 23 - 19 - 20 22 - 22 22 - - 24 METHANOL 0.5 8 7 - - 7 ------7 7 - - 8 1 12 12 - 11 12 - 10 - 9 - 10 11 - 13 12 - - 11 2 17 18 - 15 15 - 16 - 14 - 15 16 - 18 17 - - 19 5 22 20 - 17 18 - 21 - 19 - 19 19 - 22 21 - - 22 10 25 23 - 21 22 - 24 - 22 - 23 24 - 26 23 - - 25 AQUEOUS 0.5 ------1 8 9 - 9 - - 8 ------9 - - 9 2 11 12 - 13 12 - 14 - 8 - 10 11 - 8 12 - - 14 5 15 15 - 16 14 - 18 - 11 - 12 16 - 11 14 - - 17 10 18 17 - 18 16 - 20 - 14 - 16 19 - 13 16 - - 20 CHLORAMPHENICOL 10 µg 18 8 16 - - - 16 18 16 - 16 18 - 16 17 19 18 20

Gram-positive bacteria : 1. Staphylococcus aureus, 2. Staphylococcus aureus ATCC 25953, 3. Staphylococcus albus, 4. Streptococcus haemolyticus Group-A, 5. Streptococcus haemolyticus Group-B, 6. Streptococcus faecalis, 7. Bacillus subtilis.

Gram-negative bacteria: 1. Escherichia coli, 2. Edwardsiella tarda, 3. Klebsiella pneumoniae, 4. Proteus mirabilis, 5. Proteus vulgaris, 6. Pseudomonas aeruginosa, 7. Salmonella typhi, 8. Shigella boydii, 9. Shigella dysenteriae, 10. Shigella flexneri, 11. Plesiomonas shigelloides. Values are expressed as mean of replication of three values. (-): no inhibition.

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Figure 2: Propensity of different plant extracts against Gram-positive and Gram-negative bacteria

4. Discussion serve as antimicrobial agents or it can be further studied to formulate compounds which are less Present communication brought to light an toxic and cheaper to help the ailing community. interesting finding for antibacterial approach of Findings also justify traditional uses of plant in the traditionally important plant L. cephalotes . It curing fever, urinary, chest and GIT infections. was opted that both traditionally practiced solvent that is, Water and Lab practiced EtOAc Medicinal plants constitute an effective source and MeOH (polar) and PE and BZ should be of traditional and modern medicine in many used as non-polar solvents. Aqueous plant countries like China, India, as well as extract also inhibited the growth of five Gram- other Asian nations. Herbal medicine has shown positive and six Gram-negative pathogenic to have genuine utility and about 80% of bacterial strains as demonstrated by EtOAc and population depends on it as a primary health MeOH extracts. The most appropriate method care as WHO advocates. During the last thirty would be that in which the extracts were the years, the development of resistance and various same as that used in folk medicine or phyto- undesirable side effects of present chemically therapy, although in the lab the use of methanol synthesised drugs and unavailability of or ethanol extract is much more common. But antibiotic to the rural populace has prompted the zone of inhibition at the same concentration scientific community to search for new was more pronounced in the later extracts antimicrobials, mainly of plants’ origin to (EtOAc and MeOH) which might be due to two overcome the afore mentioned disadvantages reasons, firstly, the nature of biological active and needs. components like tannins, flavonoids, saponins which could be enhanced in presence of the above polar solvents, it is documented that Conflict of Interest above secondary metabolites are well known for We declare that we have no conflict of interest. antimicrobial activity. Secondly, the stronger extraction capacity of EtOAc and MeOH could have produced greater number of active Acknowledgements constituents. The inference can be drawn that the antibacterial activity observed with the Thanks are due to the Department of Science and EtOAc, MeOH and AQ extracts suggest the Technology SERC Division, New Delhi for presence of bioactive compounds which can financial support to the corresponding author.

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