Keywords

Pinda concanensis; Phytochemicals; Antibacterial activity; Gram-positive bacteria; Gram-negative bacteria

Introduction

Phytochemicals are the chemicals produces by various parts of the plants. These bioactive constituents of plants are phenolics, flavonoids, steroids, terpenoids, carotenoids, alkaloids, tannins and glycosides. These compounds have various activities such as antibacterial some have been reported to exhibit heamolytic and foaming activity reported [1]. According to Fransworth and Morris, the majority of natural products are secondary metabolites, of which unevenly 12,000 have been identified thus far [2]. Plants use these products to protect themselves against microbes, insects and herbivores. The use of crude extracts and dry powder samples from medicinal and aromatic plants for the creation and manufacturing of alternative traditional medicines and food additives is becoming increasingly popular. For the sake of public health, it is crucial to utilize preservatives and antibacterial substances to restrict the growth of dangerous microorganisms in food [3]. Plants from family Apiaceae are commonly used for flavoring of foods and in medicine. The plant under present investigation Pinda concanensis (Dalzell) belonging to family Apiaceae is a medicinal plant from Western Ghats of India. The synonym of Pinda is Heracleum pinda (Dalzell and Gibson). It is an annual herb with tuberous roots, which are eaten raw by local folklore. This plant is heavily exploited for its medicinal uses, especially for the essential oil extracted from its seeds [4]. The plant Pinda concanensis has the potential of antioxidant, antimicrobial activity. There are very few studies on phytochemical investigation of Pinda concanensis. The current work used distilled water and methanol as solvents to extract secondary metabolites such as phenolic, flavonoid, saponins, alkaloid and terpenoids from Pinda concanensis and its antibacterial activity against gram positive and gram negative bacteria like Bacillus cereus and Escherichia coli respectively and quantitative analysis [5].

MATERIALS AND METHODS

Collection of plant material

The plant Pinda concanensis were collected from Kas plateau, Satara latitude-17°43′12.58″N to 17°72'01.61"N and longitude 73°49′ 22.05″E to73°82' 27.92"E. The plants were identified, germplasm of these plants were maintained in the lead botanical garden, department of botany, Shivaji University, Kolhapur. A voucher specimen (KSW 001) was deposited in the herbarium of department of botany, Shivaji University, Kolhapur [6].

Preparation of extracts

The fresh and dry plant material extract was prepared by using two solvents; methanol and distilled water. The different parts of plant (25 g) were ground using laboratory grinder. The extracts were then filtered through Whatman filter paper no.1 by using Buchner’s funnel and final volume was adjusted to100 ml with respective solvents. For each plant part same extraction procedure with these two solvents was adopted [7]. All the plant extracts (25%) were stored in refrigerator at 4°C and were used for further analysis.

Analysis of total phenolic

Total Phenolic Contents (TPC) of the plant extracts were assessed using Folin-Ciocalteu method with slight modifications. The reaction mixture was prepared by mixing an aliquot of extracts (12.5 μl) with distilled water (0 μl) and Folin-Ciocalteu reagent (12.5 μl) incubate 10 minutes and 125 μl of saturated Na₂CO₃ solution. Reaction mixture was further incubated for 90 minutes at room temperature in dark [8]. The absorbance was measured at 760 nm in 96 well microplate. The assays were prepared in triplicates for each analysis and the mean values of absorbance were obtained [9]. The readings were compared with a standard phenolic compound i.e. tannic acid and were expressed as milligram Tannic Acid Equivalents per gram Fresh Weight/Dry weight (mg TAE of DW or FW) [10].

Analysis of total flavonoids

Total Flavonoids Content (TFC) of the different plant parts were analyzed by using modified colorimetric method. The reaction mixture was prepared by mixing 150 μl 2% methanolic AlCl₃ with 150 μl plant extract. Incubation of 10 minutes at room temperature was done. After incubation the absorbance was measured at 368 nm in 96 well microplates [11]. The assays were prepared in triplicates for each analysis and the mean values of absorbance were obtained. The values were expressed as milligram Quercetin equivalents per gram Fresh Weigh/Dry Weight (mg QE/g of DW or FW) [12].

Quantification of total saponin

Saponin content in Pinda concanensis was determined according to the method described with slight modification. Total Saponins also known as vanillin-sulfuric acid assay. In this, reaction mixture consists of 50 μl of plant extract, 50 μl of 8% vanillin to which 500 μl of 72% H₂SO₄ was added. Reaction mixture was incubated for 10 min at 60°C in water bath [13]. Allow cooling and absorbance was measured at 544 nm (Multiskan sky spectrophotometer, thermo scientific) wavelength. The total saponin content was calculated from the calibration curve and the results were expressed as mg of diosgenin equivalent per gm fresh or dry weight [14].

Quantification of total alkaloids

Total Alkaloid Content (TAC) of the extracts was assessed using 1, 10-phenanthroline method described by Singh. The assay mixture was prepared by using 100 μl plant extract 100 μl 1,10-phenanthroline and 100 μl FeCl₃ in 0.5 M HCl, make a total volume 1 ml by using distilled water and incubated for 30 minutes in water bath maintained at 70 ± 2°C (Till orange color appear) [15]. Above reaction mixture excluding 1% plant extract, substituted by distilled water served as a blank. The samples were prepared in triplicates for each analysis and the mean value of absorbance was obtained. The absorbance was measured at 510 nm in 96 well microplate against reagent blank. (Multiskan sky spectrophotometer, thermo scientific). The OD measurements were compared to standard curve of colchicines (a standard alkaloid) and expressed as milligrams Colchicine Equivalent (CE) per gram of Fresh Weight (FW) or Dry Weight (DW) of respective plant parts of Pinda concanensis [16].

Quantification of total terpenoid

Total triterpenoid content was determined by colorimetry using the following procedure, 25 μl of plant extract was mixed with 37.5 μl vanillin-glacial acetic acid solution (5% w/v) and 125 μl of perchloric acid [17]. Then samples were heated for 45 min at 60°C and cooled on ice bath. Absorbance was measured at 548 nm wavelength after the addition of 562.5 μl of glacial acetic acid, using 96 well plate reader (Multiskan sky spectrophotometer, thermo scientific). The total terpenoid content was calculated from the calibration curve of ursolic acid and the results were expressed as mg of ursolic acid equivalent per gm fresh or dry weight [18].

Antibacterial activity

Preparation of nutrient broth: The nutrient broth was prepared by using nutrient powder without agar containing only nutrient ingredients are peptone (5 gm./liter), sodium chloride (5 gm/liter), HM peptone B#(1.50 gm./liter), yeast extract (1.50 gm./liter) (# equivalent to beef extract) final PH (25°C) 7.4 ± 0.2. For bacterial growth 13 grams dissolved in 1000 ml distilled water. Heat if necessary to dissolve the medium completely [19]. Dispense into tubes or flasks as desired. Sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Bacterial culture: Escherichia coli, (Gram -ve) Bacillus cereus (Gram +ve). Strains were collected from National Center for Microbial Resource (NCMR); all bacterial strains were retrieved for fresh culture in nutrient broth [20]. For growth curve study each freshly grown strain was inoculated in to nutrient broth and incubated for 12 hours at 37°C. Absorbance was recorded after every 30 min.

Anti-microbial activity: The antimicrobial activity of Pinda concanensis was carried out by the following method. In 96 well plate 200 μl nutrient broth was distributed into each well, 25 μl of plant samples with different concentrations (1%, 2%, 3%, 4% and 5%) to determine MIC and 20 μl bacterial culture was supplemented to each well [21]. Plate was incubated at 37°C for 10 hours in a spectrophotometer (Multiskan sky 96 well plate reader, thermo scientific). Absorbance was recorded at 600 nm. After incubation, the growth curve was plotted for the analysis of plant samples inhibitory activity [22]. Gentamicin (20 μg/ml to 100 μg/ml) was used as standard positive control while methanol and water as negative control. Percentage of inhibition was calculated by,

Percentage of inhibition (%)={(A₀–A₁)/A₀} × 100

Whereas, A₀ =Absorbance of bacterial growth.
               A₁=Absorbance of sample inhibiting bacterial growth.