International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954 Antibacterial activity and High performance liquid chromatography (HPLC) analysis of adscendens Roxb. root extracts

Sapna Thakur@ and DR Sharma School of Biotechnology, Shoolini University, Solan-173229, HP.

ABSTRACT The aim of the present study was to study medicinally active substances present in various root extracts of Asparagus adscendens Roxb. The material was successively extracted with four solvents namely petroleum ether, chloroform, ethyl acetate and n-butanol in a soxhlet extractor. Roots of Asparagus showed the presence of sterols, tri-terpenoids, phenols, ketones and saponins. Sterols and tri-terpenoids play a major role in functional deviations responsible for infertility. The presence of these bioactive constituents is associated with the antibacterial activity of the . The root extracts of Asparagus adscendens extracted by different solvents, showed the spectrum of inhibition on Gram positive bacteria like Staphylococcus aureus, Bacillus subtilis and Gram negative bacteria Escherichia coli, and Pseudomonas aeruginosa by agar well diffusion method. This study scientifically revealed significant zone of inhibition supports the usage of roots of plant as a remedy for various superficial bacterial infections in traditional medicine. Root samples in different seasons were collected followed by their comparative phytochemical fingerprinting studies through HPLC. The analysis reveal phytochemical variationIJST among the season. Future prospects in case of roots of Asparagus adscendens and clinical studies on such plants and their formulations. Key words: Root extract, pathogenic bacteria, antibacterial activity, HPLC, Asparagus adscendens Roxb.

Corresponding Author@ Email id- [email protected], Phone no: +919459506118

______R S. Publication, [email protected] Page 359 International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954 INTRODUCTION The use of plants as a medicine is as old as human civilization; About 1,100 plants species are frequently used in traditional system of healthcare and medicines. Out of these medicinal plants, 500 plants are commonly used in preparation of Ayurvedic, Unani and Homeopathic drugs [1]. Several reports on the antimicrobial activity of different herbal extracts have been reported from different regions of the world [2-5]. Recently much attention has been paid to plant extracts and biologically active compounds isolated from plant species used in herbal medicine because of the ease of availability, no side effects, cost efficient and possess good antimicrobial activity against pathogenic microorganisms [6] and having a good response in the treatments. Asparagus adscendens Roxb. is a flowering perennial plant species in the genus Asparagus, a large genus of herbs and under shrubs with stout, creeping, tuberous root-stocks and erect or climbing stems and classified in the family [7]. Asparagus adscendens was initially grown in thick forest in natural form, and is a customary medicinal plant. It is an herb with sub-erect lanceolate leaves and tuberous root system and can grow up to an utmost height of 1.5 feet while Tubers can grow up to a depth of 10 inch. The tuberous roots are white that grows well in tropical and sub-tropical climates with heights up to 1,500 meters. Active compounds present in Asparagus adscendens are well known for their multiple health benefits. The powdered dried root exhibits galactogogic properties and it is reported to be useful against diarrhea, dysentery and inIJST general debility [8]. , diosgenin and sitosterol have been isolated earlier from the fruits, whereas some steroidal saponins were reported from fruits, roots and leaves and several lipid constituents has identified from the roots [9-11]. Asparagus adscendens is known for its activities like anti-cancer, anti-filarial [12], anti- stress and anti-inflammatory [13]. Geri forte is herbal formulation with Asparagus adscendens as one of the main constituents and has been widely studied for stimulation of insulin secretion [14]. Plants are used medicinally in different countries and are a source of many potent and powerful drugs. Medicinal plants represent a rich source of antimicrobial agents [15]; a wide range of medicinal plant parts is used for extract as raw drugs and they possess varied medicinal properties. There is a continuous need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases [16]. Reports are available on the use of several plant by-products, which posse’s antimicrobial ______properties, on several pathogenic bacteria and fungi [17, 18]. Here, we evaluate the potential of R S. Publication, [email protected] Page 360 International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954 Asparagus adscendens extracts for antibacterial activity against important pathogenic Gram positive and Gram negative bacteria’s. Saponins and Sarsasapogenin (saponin glycoside) have been isolated from fruits of Asparagus curillus and Asparagus officinalis and the leaf, root and fruits of Asparagus racemosus and analysed for quantification [19-22]. The defatted roots of Asparagus adscendens Roxb. yielded two new spirostanol glycosides (asparanin C and asparanin D) and two new furostanol glycosides (asparoside C and asparoside D) [11]. Spirostanol glycosides (asparanin A and asparanin B) and two furostanol glycosides (asparoside A and asparoside B) have been isolated from the methanol extract of the roots of Asparagus adscendens [23]. Saponins were successfully separated on C18 column [24-26] and C8 column [27, 28]. The HPLC analysis revealed polyphenolic components like rutin, quercetin, kaempferol, and isorhamnetin in the green Asparagus [29] and diosgenin in Asparagus officinalis determine by RP-HPLC analysis [30]. This paper reports the marker drug sarsasapogenin in Aaparagus adscendens Roxb. bring to focus the quality of drug grown in different seasons of Himachal Pradesh, India. MATERIALS AND METHODS Plant material The plant material used in this study fresh roots of Asparagus adscendens were collected in the month of January, from site Kumarhati,IJST Distt. Solan of Himachal Pradesh. The healthy and matured roots were selected, washed thoroughly. The epidermis of roots was peeled off with the help of a scalpel and the pith was separated with the help of forceps to get the cortex; followed by surface sterilization and then washed with distilled water. Surface sterilized plant material shade-dried, powdered and used for extractions. Extraction Thoroughly washed dried roots of Asparagus adscendens were dried in shade for five days and then powdered with the help of mechanical grinder. 500 g of shade-dried powder was filled in the thimble and extracted successively with 250 ml each of petroleum ether then with methanol. After that extract was fractionate with chloroform, ethyl acetate and n-butanol solvents respectively. The solvent extracts were concentrated under reduced pressure and each of these solvent extract was weighed and preserved at 4ºC in airtight bottles until further use. 1mg of each ______R S. Publication, [email protected] Page 361 International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954 solvent residue was dissolved in 1 ml of respective solvents were used as the test extracts for antimicrobial activity assay. Phytochemical screening A systematic and complete study of crude drugs includes a complete investigation of both primary and secondary metabolites derived from plant metabolism using different qualitative test were performed for establishing profiles of various extracts for their nature of chemical composition as per the methods given by Harborne [31]. The powdered roots extracted with petroleum ether, chloroform, ethyl acetate and n-butanol solvents were evaluated for qualitative determination of major phyto-constituents i.e. Sterols, Tri-terpenoids, Alkaloids, Phenols, Saponins, Flavonoids, Ketones, Resin and Starch. 1. Test for Sterols Different extracts were dissolved in chloroform, filtered and the filtrate was tested for sterols and tri-terpenes [32]. a. Salkowski test: Few drops of concentrated sulphuric acid was added to the chloroform solution, shaken and allowed to stand, appearance of red color in lower layer indicates the presence of sterols. 2. Test for Tri-terpenes a. Salkowski test: Few drops of concentrated sulphuric acid was added to the chloroform solution, shaken and allowed to stand, appearanceIJST of golden yellow color indicates the presence of tri-terpenes [33]. 3. Test for Alkaloids The various extracts were basified with ammonia and extracted with chloroform. The chloroform solution was acidified with dilute hydrochloric acid. The acid layer was used for testing the alkaloids. a. Dragendorff’s reagent (Potassium Bismuth Iodide): The acid layer was treated with few drops of Dragendorff’s reagent. Formation of reddish brown precipitate indicates the presence of alkaloids [34]. 4. Test for Phenols a. Ferric chloride test: Few drops of neutral ferric chloride solution were added to little quantity of alcoholic extract. Formation of red, blue, green color indicates the presence of phenolic nucleus [35, 36]. ______R S. Publication, [email protected] Page 362 International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954 5. Test for Saponins a. Foam test: Small amount of extract was shaken with little quantity of water, if foam produced persists for 10 minutes; it indicates the presence of saponins. b. Froth test: To 5 ml of extract of the drug added a drop of sodium bicarbonate solution. Shaken the mixture vigorously and left for 3 minutes. Honey comb like froth is formed [33]. 6. Test for Flavonoids a. Shinoda test: To the alcoholic solution of extract a few fragments of magnesium ribbon and concentrated hydrochloric acid was added. Appearance of red to pink color after few minutes indicates the presence of Flavonoid [35, 36]. 7. Test for Ketones a. Benedict test: Small amount of extract was shaken with little quantity of water, and a small amount of Benedict's reagent is added and kept in a water bath, usually 4–10 minutes, the solution should progress in the colors of blue green, yellow, orange, red, and then brick red or brown (with high glucose present). A color change would signify the presence of ketones. 8. Test for Resin Dissolved the extracts in acetone and pour the solution in to distilled water. Turbidity indicates the presence of resins [32]. 9. Test for Starch Dissolved 0.015 gm of iodine and 0.075 gm of potassium iodide in 5 ml of distilled water and add 2-3 ml of an extract of drug, blue colorIJST is produced indicates the presence of starch. AntibacterialThe antibacterial screening activities of different extracts were studied by agar well diffusion method against Gram positive bacteria Staphylococcus aureus, Bacillus subtilis and Gram negative bacteria Escherichia coli, and Pseudomonas aeruginosa. The bacterial cultures were obtained from Department of Microbiology, School of Biotechnology, Shoolini University, Solan, Himachal Pradesh, India. Bacterial strains grown on nutrient agar at 37°C for 18 hr were suspended in a saline solution (0.85% NaCl) and adjusted to a turbidity of 0.5 MacFarland standard (108 CFU/ml). Well - Diffusion assay Mueller –Hinton agar media was used to perform sensitivity assay [37] was prepared and transferred to sterilized petri plates and allowed to solidify. The suspension was used to inoculate on petri plates and swabs the entire surface of the agar media. The inoculums were uniformly ______R S. Publication, [email protected] Page 363 International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954 distributed to the surface of the media by rotating the petri plates. Wells were punched in the surface of agar plates and filled with 50µl of 1mg/ml of each extract. The experiment conducted in triplicate. The plates were left for 1h at room temperature as a period of pre incubation to minimize the effect of variation in time between the applications of different solutions. Now all the plates were incubated at 37°C for 18 hr and observed for antibacterial activity. The diameter of zone of inhibition produced by the extract compared with those produced by the commercial control antibiotics Ampicillin (25µg/ml). Preparation of samples for HPLC Samples of roots of Asparagus adscendens were collected in two different seasons from the same site for quantitative analysis of marker compound. One sample was collected in the month of January and the second was collected in the month of May. 2g of the powdered root material was weighed into 50ml flasks and 20ml of the solvent (100% methanol) was added into the flask. It was allowed to soak at room temperature for overnight after that the mixture was stirred for 12 hours at room temperature. The mixture was filtered through a Watts-man filter paper and the filtrate was collected and repeat the procedure with the residue. After collecting the filtrate both the filtrates were then combined and concentrated using a rotary evaporator under vacuum at 50°C. The pale yellow material is collected and stored in air tight container at 4°C for further use. HPLC Method IJST The extracts obtained from solvent extractions were analysed using high performance liquid chromatography (HPLC). The sample to be analysed was dissolved in 100% dimethyl sulphoxide (DMSO) at concentration of 1mg/ml. The solution was centrifuged at 10,000 rpm and the supernatant was filtered through 0.45μm Millex filter unit (Millipore). 10μl of the above solution was injected in HPLC connected to DAD UV-vis detection system at 190-800 nm in gradient mode. Marker compound in Asparagus adscendens Roxb. The markers of our interest in Asparagus adscendens was Sarsasapogenin. To correlate the retention time of the marker compounds with Asparagus adscendens root extract, standard markers Sarsasapogenin were injected in HPLC using same conditions as that of the extract. From chromatograms 1 it was evident that the peak at 15.2 minutes corresponds to Sarsasapogenin. ______R S. Publication, [email protected] Page 364 International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954 RESULTS AND DISCUSSIONS Percentage yield The yield of the petroleum ether, chloroform, ethyl acetate and n-butanol extract for, roots of Asparagus adscendens was found out to be 0.16%, 0.37%, 13.03% and 10.44% respectively ethyl acetate shows maximum yield (Table 1& Figure 1).

Table 1: Percentage yield of Asparagus adscendens root extracts using different solvent system Solvent extract % yield obtained Petroleum ether 0.16 %

Chloroform 0.37% Ethyl acetate 13.03% N-butanol 10.44%

14

12

10

8

6

% yield 4

2 0 IJST pet ether chloroform ethyl acetate N-butanol Solvent extracts

Figure 1: Percentage yield of Asparagus adscendens root extracts using four solvent system

Phytochemical screening The petroleum ether, chloroform, ethyl acetate and n-butanol extracts of roots of Asparagus adscendens were subjected to qualitative phytochemical screening for the detection of phyto-constituents like Sterols, Tri-terpenoids, Alkaloids, Phenols, Saponins, Flavonoids, Ketones, Resin and Starch. The results revealed the presence of tri terpenoids in petroleum ether and chloroform extracts whereas sterols, phenols, saponins and ketones are present in ethyl ______acetate and n-butanol extracts of roots of Asparagus adscendens (Table 2). R S. Publication, [email protected] Page 365 International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954 Table 2: Qualitative Phytochemical analysis of Asparagus adscendens root extracts Preliminary Phytochemical Pet ether Chloroform Ethyl acetate N-butanol screening extract extract extract extract Sterols ̶ ̶ + + Tri-terpenoids + + ̶ ̶ Alkaloids ̶ ̶ ̶ ̶ Phenols ̶ ̶ + + Saponins ̶ ̶ + + Flavonoids ̶ ̶ ̶ ̶ Ketones ̶ ̶ + + Resin ̶ ̶ ̶ ̶ Starch ̶ ̶ ̶ ̶ (+): Indicates the presence of phytochemical constituents; (-): Indicates the absence of phytochemical constituents Antibacterial activity The petroleum ether, chloroform, ethyl acetate and n-butanol extracts of roots of Asparagus adscendens were subjected to antibacterial activity against (Sa) Staphylococcus aureus, (Bs) Bacillus subtilis, (Ec) Escherichia coli, and (Pa) Pseudomonas aeruginosa; both gram-positive and gram-negative bacteria were sensitive to the extracts. In this study, zone of inhibition recorded for various organisms was found; in petroleum ether root extract have potent antibacterial activity against Staphylococcus aureus (18mm) whereas chloroform root extract exhibited significant activity against Bacillus subtilis (15mm), Escherichia coli (14mm) and Pseudomonas aeruginosa (16mm). Activity of extracts of the plant was comparable to that of reference standard drug Ampicillin 25μg/ml. Asparagus adscendens root extracts exhibited good antimicrobial activity and results were tabulatedIJST along with figures (Table 3; Figure 2). Table 3: Antibacterial activity of various extracts of Asparagus adscendens root Inhibition Zone (mm) Samples Extracts Sa Bs Ec Pa Pet ether extract 18 12 13 13

Asparagus Chloroform extract 13 15 14 16 adscendens root Ethyl acetate extract 14 12 12 13

N-butanol extract 12 14 11 14 Pet ether 0 0 0 0 Chloroform 0 0 0 0 Control Ethyl acetate 0 0 0 0 N-butanol 0 0 0 0 Standard Ampicillin 27 25 22 26 Antibiotic Sa Staphylococcus aureus, Bs Bacillus subtilis, Ec Escherichia coli, Pa Pseudomonas aeruginosa. ______R S. Publication, [email protected] Page 366 International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954

24 pet ether chloroform 20 ethyle acetate n-butanol 16

12

8

Zone of inhibition (mm) inhibition of Zone 4

0 Sa Bs Ec Pa Gram positive and Gram negative bacteria's

Figure 2: Inhibition Zone of Asparagus adscendens Root Extract against Gram Positive and Gram Negative Bacteria’s.

Molecular markers optimization: The concentration of sarsasapogenin varies from season to season. In the samples studied, roots had higher percentage of sarsasapogenin 2.1%, which were collected in the month of January (Figure 3b), on the other hand samples collected in the month of May had lower percentage of sarsasapogenin i. e. 1.0% (Figure 3c). Which seems to be appropriate season for cultivation and proper harvesting period is January when the desired content of marker drug was higher in roots. IJST

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(a)

(c)

(b) IJST

(c) Figure 3: (a) HPLC Chromatogram showing the peak of standard, (b) Root sample collected in the month of January, (c) Root sample collected in the month of May.

______R S. Publication, [email protected] Page 368 International Journal of Advanced Scientific and Technical Research Issue 5 volume 3, May.-June. 2015 ______Available online on http://www.rspublication.com/ijst/index.html ISSN 2249-9954 Conclusion The scientific paper establishes that Asparagus adscendens root has subjected to qualitative phytochemical screening of the petroleum ether, chloroform, ethyl acetate and n- butanol extracts which revealed the presence of tri terpenoids, sterols, phenols, saponins and ketones. The root extracts shows significant antibacterial activity against Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis and Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and was very well compared with standard reference drug Ampicillin 25 μg/ml .The maximum zone of inhibition with root extract of Asparagus adscendens against Staphylococcus aureus (18mm) whereas extract exhibited significant activity against Bacillus subtilis (15mm), Escherichia coli (14mm) and Pseudomonas aeruginosa (16mm). The analytical method described in this paper is accurate and precise for quantitative determination of sarsasapogenin in Asparagus adscendens. Roots had higher concentration of bioactive in sample which was collected in January. This period seems to be proper harvesting period of Asparagus adscendens.

ACKNOWLEDGEMENTS The author thanks Dr. Kamal Dev, Associate professor of School of Biotechnology, Shoolini University for their continuous support.

IJST

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