Indian Journal of Natural Products and Resources Vol. 4(3), September 2013, pp. 273-277

Mineral composition and antimicrobial activity of cordata (G. Don) Clarke aerial parts and roots

Shivani Joshi*1, Devendra Mishra2, Ganga Bisht1 and Khadga Singh Khetwal1 1Department of Chemistry, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand, India 2Department of Applied Chemistry, Birla Institute of Applied Sciences, Bhimtal, Uttarakhand, India

Received 30 June 2012; Accepted 24 December 2012

The study aim was to evaluate the concentration of mineral elements and antimicrobial activity of (G. Don) Clarke aerial parts and roots. Macro minerals, viz. sodium, potassium, calcium, lithium were estimated by Flame Photometer while micro minerals, viz. iron, copper, manganese, zinc and cobalt were determined by Atomic Absorption Spectrophotometer. Among all the elements, highest concentration of potassium was recorded in both aerial parts (385.6 mg/100 g) and roots (421.3 mg/100 g) of S. cordata. Methanol extract of both S. cordata aerial parts and roots obtained by Soxhlet extraction were screened for their antimicrobial activity against 6 bacteria and 2 fungi using disc diffusion method. The results showed that both the extracts exhibited moderate antibacterial activity while roots showed good antifungal activity against Candida albicans (ZOI =20 mm).

Keywords: Swertia cordata, Medicinal , Spectrophotometer, Flame Photometer, Disc diffusion. IPC code; Int. cl. (2011.01)−A61K 36/00

Introduction isoorientin have been isolated from S. delayayi Swertia Linn. () is an annual or Franch5. Swertiamarin; mangiferin; oleanolic acid; perennial herb comprising more than 170 species, 1,5,8-trihydroxy-3-methoxyxanthone;1,8-dihydroxy-3, found at an altitude of 1200 to 3000 m from Kashmir 7-dimeth-oxyxanthone;1,8-dihydroxy-3, 5-dimethoxy- to Nepal, China and also distributed in Bhutan, Khasi xanthone have been reported from S. mussotti Franch.6. hills and Sikkim1-4. Swertia has an erect, about 1 m S. mileensis T. N. Ho & W. L. Shi was reported long stem, leaves are opposite to each other without to contain swerilactones C and D7. Swertia cordata leaf stalks and have smooth margins. The flowers (G. Don) Clarke aerial parts were reported to contain have four or five united petals, which may be deeply ursolic acid and mangiferin8. Atta-ur-Rehman et al divided. The petals overlap and are twisted in the bud. (1994)9 isolated 1-hydroxy-3,5,7,8-tetramethoxy- Nectar-secreting pits are found at the base of the xanthone and 1,7-dihydroxy-3, 5, 8-trimethoxy petals and sepals in some species. Approximately xanthone from this plant. S. cordata is used in 20 species of this genus are used in Chinese medicines as an alternative febrifuge, antihelmintic traditional medicine to treat hepatic, choleric and and as a bitter tonic1,8,9. anti-inflammatory diseases. The genus contains Previous studies have determined concentrations of different bitter principles, iridoids and secoiridoids, mineral elements in a few species of Swertia10-12 but for example amarogentin, swertiamarin, sweroside, the mineral composition in S. cordata has not been amaroswerin, and gentiopicroside, which are mainly reported so far. Additionally this is the first responsible for its pharmacological activity1. antimicrobial activity report of S. cordata aerial parts Various chemical constituents have been and roots extract. isolated from different species of Swertia. Materials and Methods Oleanolic acid; gentiopicroside; swertiamarin; daucosterol; swertiadecoraxanthone-II; isovitexin; Plant material Fresh samples of Swertia cordata (G. Don) Clarke —————— were collected in the month of October, 2009 *Correspondent author: Phone: +919568296342 from village Vinayak near Pangot (29°25’25”N E-mail: [email protected] 79°25’37”E) 15 km away from Nainital, India. 274 INDIAN J NAT PROD RESOUR, SEPTEMBER 2013

The plant identification was confirmed by the steroids were performed on the methanol extract of Botanical Survey of India (BSI), Dehra Dun. S. cordata aerial parts and roots. A voucher specimen (No.112968) was deposited in Microorganisms the Herbarium Section at BSI, Dehra Dun, India. Three Gram positive, three Gram negative and two

Mineral analysis fungi were used for antimicrobial activity studies. Mineral content in plant was estimated by wet Gram positive bacteria were Staphylococcus aureus digestion method13. Aerial parts and roots of the plant (MTCC 3160), Bacillus subtilis (MTCC 441) and were air-dried and ground into fine powder. 1.0 g Bacillus mycoides (MTCC 645). Gram negative powdered plant material was first digested with conc. bacteria were Escherichia coli (MTCC 406), Pseudomonas aeruginosa (MTCC 424) and Proteus HNO 3 (5 mL each), followed by application of vulgaris (MTCC 426). Yeast like fungi used were 15 mL of tri-acid mixture (HNO3, HClO4 and H2SO4, 10:4:1, v/v) heated at 200°C and reduce to 1 mL. Candida albicans (MTCC 227) and Aspergillus The residue after digestion was dissolved in double niger (MTCC 404). Required microorganisms were distilled water, filtered and diluted to 100 mL. This procured from Institute of Microbial Technology, solution was used for the estimation of minerals. Chandigarh, India.

Macro minerals, viz. Na, K, Ca and Li were estimated Determination of zone of inhibition by AIMIL, Flame Photometer while micro The disc diffusion method14 was used to evaluate elements, viz. Fe, Cu, Mn, Zn and Co were estimated the antimicrobial activity. The test solutions of aerial by Atomic Absorption Spectrophotometer, model parts and roots methanol extract at the concentrations 4129, Electronic Corporation of India Ltd. Estimation of 4000, 2000, 1000, 500 and 250 µg/mL were of each element was carried out 3 times and the mean prepared by dissolving the extracts in values were reported and used. For macro elements, dimethylsulphoxide (DMSO). 0.1mL of test solutions standard blank and sample solutions were passed were injected into sterilized discs of 5 mm in through flame photometer and concentrations were diameter. Chloramphenicol (25 µg/disc), ampicillin determined in µg/mL. Total amount of macro (25 µg/disc) and fluconazole (25 µg/mL) were used as elements in mg/kg dry weight of samples were positive controls. As a negative control, a blank disc calculated by the formula: impregnated with DMSO was used. The test discs, standard discs and blank discs were placed in petri Macro element mg/kg = Y/W × 50 (or trace element dish with a particular microorganism. The petri dishes mg/kg =Y/100) were then incubated at 37°C for 24 h for bacterial where Y= flame photometer reading and W= weight growth and at 27°C for 48 h for the growth of yeast. of sample. Nutrient agar and potato dextrose agar medium The concentrations of micro elements were were used for the growth of bacteria and yeast, calculated by the same formula, where Y= reading respectively. Each extract was analyzed in triplicate. of atomic absorption spectrophotometer and W= Results and Discussion weight of sample. Mineral content of samples Extraction procedure The mineral profiles of S. cordata aerial parts and The aerial parts and roots of the plant S. cordata roots are shown in Table 1. Among all the elements, were shade dried, powdered and extracted separately highest concentration of K was recorded in both aerial with 70% methanol using Soxhlet apparatus. parts and roots of S. cordata. The concentration of After extraction each filtrate was concentrated on other elements in aerial parts decreased in the order a rotary evaporator under vacuum at 20°C till a Ca>Na>Fe>Zn>Mn>Cu>Li>Co while in roots the residual mass was obtained. Each extract was then decreasing order of elements was Ca>Fe>Na>Zn> stored separately in screw capped vials under Mn>Cu>Li>Co (Fig. 1). refrigeration, till needed. The analysis showed that both aerial parts and roots have low amount of sodium with relatively high Phytochemical screening concentration of potassium. K/Na ratio in diet is Various tests for the presence of alkaloids, an important factor in prevention of hypertension and flavanoids, glycosides, terpenoids, xanthones and arterosclerosis since K depresses and Na enhances JOSHI et al: ELEMENTAL AND ANTIMICROBIAL ANALYSIS OF SWERTIA CORDATA 275

11 Table 1—Mineral composition of Swertia cordata reported in S. speciosa while lesser than S. chirayita aerial parts and roots (dry wt mg/100g) and S. paniculata10,12. As shown in Table 1, roots have S. No. Minerals Aerial parts Roots higher Zn content than aerial parts while Li was found 1 Sodium (Na) 18.29 32.92 in equal proportion. Cobalt was found in traces in 2 Potassium (K) 385.6 421.3 both aerial parts and roots. 3 Calcium (Ca) 277.00 166.20 4 Lithium (Li) 4.00 4.00 Antimicrobial activity 5 Iron (Fe) 21.6 140.2 Antibacterial and antifungal activity of S. cordata 6 Cobalt (Co) 0.1 0.7 aerial parts and roots methanol extract are shown 7 Zinc (Zn) 5.2 14.5 in Table 2. Antimicrobial potential of the extracts 8 Manganese (Mn) 5.1 6.9 were evaluated by measuring the diameter of zones of 9 Copper (Cu) 4.3 4.1 inhibition (mm), including the diameter of disc and

the results (mm of zone of inhibition) were expressed as average values. The zone of inhibition markedly decreased on decreasing the concentration of the extracts for all the strains used for study. The results showed that S. cordata aerial parts exhibited good antibacterial activity (Fig. 2) while roots were found to possess good antifungal activity (Fig. 3). Aerial parts showed good antibacterial activity against B. mycoides while E. coli was found insensitive for roots. Roots exhibited very good antifungal activity against C. albicans while aerial parts showed moderate activity against both the fungal strains. Antifungal activity of roots was found even comparable with Fluconazole while antibacterial activity for both aerial

parts and roots was found low as compared to the Fig. 1—Mineral composition of Swertia cordata aerial parts and controls (Ampicillin, Chloramphenicol). roots The inhibitory effects of S. cordata methanol extract against tested microorganisms might be blood pressure15. Calcium is associated with growth related to the role of mineral contents21, active and and maintenance of bones, teeth and muscles16,17. effective phytochemical constituents like flavonoids22, Comparatively S. cordata aerial parts have higher xanthones23, alkaloids24, etc. The presence of Ca content than roots, however, this Ca content micronutrients and biologically active constituents in was found lesser than those in S. chirayita (Roxb. plant extract usually interfere with growth and ex Fleming) Karsten, S. speciosa D. Don and metabolism of microorganisms to destroy them25. S. paniculata Wall.10-12. Iron is an essential trace It has been reported in many studies that the element for haemoglobin formation, normal antimicrobial property of plant extracts is partly functioning of central nervous system and in the contributed by minerals. The antibacterial effect oxidation of carbohydrates, proteins and fats18. of zinc on Streptococci and Staphylococcus was From the results, aerial parts of S. cordata have lower described as early as 1949(Ref.26). It has been Fe content than roots. In human body copper exists documented that the basic mechanism that lies behind as an integral part of Cu proteins ceruloplasmin, the antibacterial activity of zinc ion depends on its which is concerned with the release of iron from the ability to bind to the membranes of microorganism, cells into the plasma and is involved in energy thereby prolonging the lag phase of growth cycle metabolism19,20. The Cu content in S. cordata roots and increasing the generation time of the organisms and aerial parts were present in equal amounts. which takes for each organism more time to complete Manganese is another micro element essential for cell division27. Relying upon the results of the present human nutrition and it acts as activator of many investigation it could be suggested that these findings enzymes19. The Mn content in S. cordata aerial may be exploited for the remedies of a wide range parts and roots was found comparable with the values of microbial diseases of as well as animals. 276 INDIAN J NAT PROD RESOUR, SEPTEMBER 2013

Table 2—Antimicrobial activity of Swertia cordata aerial parts and roots Zone of Inhibition (mm) Microbes Aerial Parts (µg/mL) Roots (µg/mL) Control 4000 2000 1000 500 250 4000 2000 1000 500 250 AM CP FZ S. aureus 12 9 7 6 - 9 8 7 6 - 32 12 - B. mycoides 14 13 10 8 6 9 7 6 - - 25 - B. subtilis 12 10 8 7 - 9 8 7 6 - 20 30 - P. vulgaris 11 10 8 7 - 9 8 7 6 - 16 14 - E. coli 13 11 9 7 6 - - - - - 21 23 - P. aeruginosa 12 7 6 - - 7 6 - - - 23 10 - C. albicans 13 10 8 7 6 20 17 14 10 8 - - 15 A. niger 12 10 9 8 7 13 12 9 7 6 - - 13 AM = Ampicillin (25 µg/disc), CP = Chloramphenicol (25 µg/disc), FZ= Fluconazole (25 µg/mL)

Fig. 2—Antimicrobial activity of S. cordata aerial parts

Fig. 3—Antimicrobial activity of S. cordata roots JOSHI et al: ELEMENTAL AND ANTIMICROBIAL ANALYSIS OF SWERTIA CORDATA 277

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